Tumor angiogenesis factor. Speculations on an approach to cancer chemotherapy

Tumor angiogenesis factor (TAF) and its importance in determining a strategy for cancer chemotherapy are discussed. It is suggested that inhibition of RNA synthesis or increased RNA catabolism might interfere with the metabolism of solid tumor cells more so than in normal cells, and thus hinder angiogenesis and pursuant tumor growth by preventing the synthesis of the RNA component of TAF. An attempt is made to indicate potential models for anti-angiogenesis agents of this type. The drugs offered as initial prototypes for investigations along these lines are actinomycin D (which likely has antimetabolite and anti-angiogenesis activities), polyriboinosinic-polyribocytidylic acid (which likely has adjuvant and anti-angiogenesis activities) and ribonuclease (which in theory might be a purely anti-angiogenetic agent). It is noted that these models may turn out to be less than ideal as therapeutic agents due to problems of toxicity, metabolism, potency, or distribution, but nonetheless might serve to yield insights into the design of new cancer chemotherapeutic drugs. In addition, some evidence is cited suggesting that actinomycin D may be more effective against certain tumors when employed in lower, chronic dosages rather than its present use in “loading” dosages.The concept of anti-angiogenesis agents as fundamentally “tumoristatic” therapies is discussed, and the likelihood that such agents might be effectively “tumoricidal” in immunocompetent hosts is mentioned. The main promise of an anti-angiogenetic strategy is efficacy against presently intractable slowly growing human cancers when used in combination with other treatment modalities. In summary, a strategy of cancer chemotherapy predicated upon interference with RNA synthesis or increase in RNA catabolism is offered as a potential mechanism for establishing anti-angiogenesis, and as a promising alternative and adjunct to present methods.     

Cancer-linked targets modulated by curcumin

In spite of major advances in oncology, the World Health Organization predicts that cancer incidence will double within the next two decades. Although it is well understood that cancer is a hyperproliferative disorder mediated through dysregulation of multiple cell signaling pathways, most cancer drug development remains focused on modulation of specific targets, mostly one at a time, with agents referred to as “targeted therapies,” “smart drugs,” or “magic bullets.” How many cancer targets there are is not known, and how many targets must be attacked to control cancer growth is not well understood. Although more than 90% of cancer-linked deaths are due to metastasis of the tumor to vital organs, most drug targeting is focused on killing the primary tumor. Besides lacking specificity, the targeted drugs induce toxicity and side effects that sometimes are greater problems than the disease itself. Furthermore, the cost of some of these drugs is so high that most people cannot afford them. The present report describes the potential anticancer properties of curcumin, a component of the Indian spice turmeric (Curcuma longa), known for its safety and low cost. Curcumin can selectively modulate multiple cell signaling pathways linked to inflammation and to survival, growth, invasion, angiogenesis, and metastasis of cancer cells. More clinical trials of curcumin are needed to prove its usefulness in the cancer setting.     

Historical Development of Origins Research

Following the publication of the Origin of Species in 1859, many naturalists adopted the idea that living organisms were the historical outcome of gradual transformation of lifeless matter. These views soon merged with the developments of biochemistry and cell biology and led to proposals in which the origin of protoplasm was equated with the origin of life. The heterotrophic origin of life proposed by Oparin and Haldane in the 1920s was part of this tradition, which Oparin enriched by transforming the discussion of the emergence of the first cells into a workable multidisciplinary research program.On the other hand, the scientific trend toward understanding biological phenomena at the molecular level led authors like Troland, Muller, and others to propose that single molecules or viruses represented primordial living systems. The contrast between these opposing views on the origin of life represents not only contrasting views of the nature of life itself, but also major ideological discussions that reached a surprising intensity in the years following Stanley Miller’s seminal result which showed the ease with which organic compounds of biochemical significance could be synthesized under putative primitive conditions. In fact, during the years following the Miller experiment, attempts to understand the origin of life were strongly influenced by research on DNA replication and protein biosynthesis, and, in socio-political terms, by the atmosphere created by Cold War tensions.The catalytic versatility of RNA molecules clearly merits a critical reappraisal of Muller’s viewpoint. However, the discovery of ribozymes does not imply that autocatalytic nucleic acid molecules ready to be used as primordial genes were floating in the primitive oceans, or that the RNA world emerged completely assembled from simple precursors present in the prebiotic soup. The evidence supporting the presence of a wide range of organic molecules on the primitive Earth, including membrane-forming compounds, suggests that the evolution of membrane-bounded molecular systems preceded cellular life on our planet, and that life is the evolutionary outcome of a process, not of a single, fortuitous event.It is generally assumed that early philosophers and naturalists appealed to spontaneous generation to explain the origin of life, but in fact, the possibility of life emerging directly from nonliving matter was seen at first as a nonsexual reproductive mechanism. This changed with the transformist views developed by Erasmus Darwin, Georges Louis Leclerc de Buffon, and, most importantly, by Jean-Baptiste de Lamarck, all of whom invoked spontaneous generation as the mechanism that led to the emergence of life, and not just its reproduction. “Nature, by means of of heat, light, electricity and moisture”, wrote Lamarck in 1809, “forms direct or spontaneous generation at that extremity of each kingdom of living bodies, where the simplest of these bodies are found”.Like his predecessors, Charles Darwin surmised that plants and animals arose naturally from some primordial nonliving matter. As early as 1837 he wrote in his Second Notebook that “the intimate relation of Life with laws of chemical combination, & the universality of latter render spontaneous generation not improbable.” However, Darwin included few statements about the origin of life in his books. He avoided the issue in the Origin of Species, in which he only wrote “… I should infer from analogy that probably all organic beings which have ever lived on this Earth have descended from some one primordial form, into which life was first breathed” (Peretó et al. 2009).Darwin added few remarks on the origin of life his book, and his reluctance surprised many of his friends and followers. In his monograph on the radiolaria, Haeckel wrote “The chief defect of the Darwinian theory is that it throws no light on the origin of the primitive organism—probably a simple cell—from which all the others have descended. When Darwin assumes a special creative act for this first species, he is not consistent, and, I think, not quite sincere …” (Haeckel 1862).Twelve years after the first publication of the Origin of Species, Darwin wrote the now famous letter to his friend Hooker in which the idea of a “warm little pond” was included. Mailed on February 1st, 1871, it stated that “It is often said that all the conditions for the first production of a living organism are now present, which could ever have been present. But if (and Oh! what a big if!) we could conceive in some warm little pond with all sorts of ammonia and phosphoric salts—light, heat, electricity &c. present, that a proteine compound was chemically formed, ready to undergo still more complex changes, at the present day such matter w^d be instantly devoured, or absorbed, which would not have been the case before living creatures were formed.” Although Darwin refrained from any further public statements on how life may have appeared, his views established the framework that would lead to a number of attempts to explain the origin of life by introducing principles of historical explanation (Peretó et al. 2009). Here I will describe this history, and how it is guiding current research into the question of life’s origins.     

From discovering to understanding

Friedrich Miescher''s attempts to uncover the function of DNAIt might seem as though the role of DNA as the carrier of genetic information was not realized until the mid-1940s, when Oswald Avery (1877–1955) and colleagues demonstrated that DNA could transform bacteria (Avery et al, 1944). Although these experiments provided direct evidence for the function of DNA, the first ideas that it might have an important role in processes such as cell proliferation, fertilization and the transmission of heritable traits had already been put forward more than half a century earlier. Friedrich Miescher (1844–1895; Fig 1), the Swiss scientist who discovered DNA in 1869 (Miescher, 1869a), developed surprisingly insightful theories to explain its function and how biological molecules could encode information. Although his ideas were incorrect from today''s point of view, his work contains concepts that come tantalizingly close to our current understanding. But Miescher''s career also holds lessons beyond his scientific insights. It is the story of a brilliant scientist well on his way to making one of the most fundamental discoveries in the history of science, who ultimately fell short of his potential because he clung to established theories and failed to follow through with the interpretation of his findings in a new light.…a brilliant scientist well on his way to making one of the most fundamental discoveries in the history of science […] fell short of his potential because he clung to established theories…Open in a separate windowFigure 1Friedrich Miescher (1844–1895) and his wife, Maria Anna Rüsch. © Library of the University of Basel, Switzerland.It is a curious coincidence in the history of genetics that three of the most decisive discoveries in this field occurred within a decade: in 1859, Charles Darwin (1809–1882) published On the Origin of Species by Means of Natural Selection, in which he expounded the mechanism driving the evolution of species; seven years later, Gregor Mendel''s (1822–1884) paper describing the basic laws of inheritance appeared; and in early 1869, Miescher discovered DNA. Yet, although the magnitude of Darwin''s theory was realized almost immediately, and at least Mendel himself seems to have grasped the importance of his work, Miescher is often viewed as oblivious to the significance of his discovery. It would be another 75 years before Oswald Avery, Colin MacLeod (1909–1972) and Maclyn McCarthy (1911–2005) could convincingly show that DNA was the carrier of genetic information, and another decade before James Watson and Francis Crick (1916–2004) unravelled its structure (Watson & Crick, 1953), paving the way to our understanding of how DNA encodes information and how this is translated into proteins. But Miescher already had astonishing insights into the function of DNA.Between 1868 and 1869, Miescher worked at the University of Tübingen in Germany (Figs 2,​,3),3), where he tried to understand the chemical basis of life. A crucial difference in his approach compared with earlier attempts was that he worked with isolated cells—leukocytes that he obtained from pus—and later purified nuclei, rather than whole organs or tissues. The innovative protocols he developed allowed him to investigate the chemical composition of an isolated organelle (Dahm, 2005), which significantly reduced the complexity of his starting material and enabled him to analyse its constituents.Open in a separate windowFigure 2Contemporary view of the town of Tübingen at about the time when Miescher worked there. The medieval castle housing Hoppe-Seyler''s laboratory can be seen atop the hill at the right. © Stadtarchiv Tübingen, Germany.Open in a separate windowFigure 3The former kitchen of Tübingen castle, which formed part of Hoppe-Seyler''s laboratory. It was in this room that Miescher worked during his stay in Tübingen and where he discovered DNA. After his return to Basel, Miescher reminisced how this room with its shadowy, vaulted ceiling and its small, deep-set windows appeared to him like the laboratory of a medieval alchemist. Photograph taken by Paul Sinner, Tübingen, in 1879. © University Library Tübingen.In carefully designed experiments, Miescher discovered DNA—or “Nuclein” as he called it—and showed that it differed from the other classes of biological molecule known at that time (Miescher, 1871a). Most notably, nuclein''s elementary composition with its high phosphorous content convinced him that he had discovered a substance sui generis, that is, of its own kind; a conclusion subsequently confirmed by Miescher''s mentor in Tübingen, the eminent biochemist Felix Hoppe-Seyler (1825–1895; Hoppe-Seyler, 1871; Miescher, 1871a). After his initial analyses, Miescher was convinced that nuclein was an important molecule and suggested in his first publication that it would “merit to be considered equal to the proteins” (Miescher, 1871a).Moreover, Miescher recognized immediately that nuclein could be used to define the nucleus (Miescher, 1870). This was an important realization, as at the time the unequivocal identification of nuclei, and hence their study, was often difficult or even impossible to achieve because their morphology, subcellular localization and staining properties differed between tissues, cell types and states of the cells. Instead, Miescher proposed to base the characterization of nuclei on the presence of this molecule (Miescher, 1870, 1874). Moreover, he held that the nucleus should be defined by properties that are related to its physiological activity, which he believed to be closely linked to nuclein. Miescher had thus made a significant first step towards defining an organelle in terms of its function rather than its appearance.Importantly, his findings also showed that the nucleus is chemically distinct from the cytoplasm at a time when many scientists still assumed that there was nothing unique about this organelle. Miescher thus paved the way for the subsequent realization that cells are subdivided into compartments with distinct molecular composition and functions. On the basis of his observations that nuclein appeared able to separate itself from the “protoplasm” (cytoplasm), Miescher even went so far as to suggest the “possibility that [nuclein can be] distributed in the protoplasm, which could be the precursor for some of the de novo formations of nuclei” (Miescher, 1874). He seemed to anticipate that the nucleus re-forms around the chromosomes after cell division, but unfortunately did not elaborate on under which conditions this might occur. It is therefore impossible to know with certainty to which circumstances he was referring.Miescher thus paved the way for the subsequent realization that cells are subdivided into compartments with distinct molecular composition and functionsIn this context, it is interesting to note that in 1872, Edmund Russow (1841–1897) observed that chromosomes appeared to dissolve in basic solutions. Intriguingly, Miescher had also found that he could precipitate nuclein by using acids and then return it to solution by increasing the pH (Miescher, 1871a). At the time, however, he did not make the link between nuclein and chromatin. This happened around a decade later, in 1881, when Eduard Zacharias (1852–1911) studied the nature of chromosomes by using some of the same methods Miescher had used when characterizing nuclein. Zacharias found that chromosomes, such as nuclein, were resistant to digestion by pepsin solutions and that the chromatin disappeared when he extracted the pepsin-treated cells with dilute alkaline solutions. This led Walther Flemming (1843–1905) to speculate in 1882 that nuclein and chromatin are identical (Mayr, 1982).Alas, Miescher was not convinced. His reluctance to accept these developments was at least partly based on a profound scepticism towards the methods—and hence results—of cytologists and histologists, which, according to Miescher, lacked the precision of chemical approaches as he applied them. The fact that DNA was crucially linked to the function of the nucleus was, however, firmly established in Miescher''s mind and in the following years he tried to obtain additional evidence. He later wrote: “Above all, using a range of suitable plant and animal specimens, I want to prove that Nuclein really specifically belongs to the life of the nucleus” (Miescher, 1876).Although the acidic nature of DNA, its large molecular weight, elementary composition and presence in the nucleus are some of its central properties—all first determined by Miescher—they reveal nothing about its function. Having convinced himself that he had discovered a new type of molecule, Miescher rapidly set out to understand its role in different biological contexts. As a first step, he determined that nuclein occurs in a variety of cell types. Unfortunately, he did not elaborate on the types of tissue or the species his samples were derived from. The only hints as to the specimens he worked with come from letters he wrote to his uncle, the Swiss anatomist Wilhelm His (1831–1904), and his parents; his father, Friedrich Miescher-His (1811–1887), was professor of anatomy in Miescher''s native Basel. In his correspondence, Miescher mentioned other cell types that he had studied for the presence of nuclein, including liver, kidney, yeast cells, erythrocytes and chicken eggs, and hinted at having found nuclein in these as well (Miescher, 1869b; His, 1897). Moreover, Miescher had also planned to look for nuclein in plants, especially in their spores (Miescher, 1869c). This is an intriguing choice given his later fascination with vertebrate germ cells and his speculation on the processes of fertilization and heredity (Miescher, 1871b, 1874).Another clue to the tissues and cell types that Miescher might have examined comes from two papers published by Hoppe-Seyler, who wanted to confirm his student''s results, which he initially viewed with scepticism, before their publication. In the first, another of Hoppe-Seyler''s students, Pal Plósz, reported that nuclein is present in the nucleated erythrocytes of snakes and birds but not in the anuclear erythrocytes of cows (Plósz, 1871). In the second paper, Hoppe-Seyler himself confirmed Miescher''s findings and reported that he had detected nuclein in yeast cells (Hoppe-Seyler, 1871).In an addendum to his 1871 paper, published posthumously, Miescher stated that the apparently ubiquitous presence of nuclein meant that “a new factor has been found for the life of the most basic as well as for the most advanced organisms,” thus opening up a wide range of questions for physiology in general (Miescher, 1870). To argue that Miescher understood that DNA was an essential component of all forms of life is probably an over-interpretation of his words. His statement does, however, clearly show that he believed DNA to be an important factor in the life of a wide range of species.In addition, Miescher looked at tissues under different physiological conditions. He quickly noticed that both nuclein and nuclei were significantly more abundant in proliferating tissues; for instance, he noted that in plants, large amounts of phosphorous are found predominantly in regions of growth and that these parts show the highest densities of nuclei and actively proliferating cells (Miescher, 1871a). Miescher had thus taken the first step towards linking the presence of phosphorous—that is, DNA in this context—to cell proliferation. Some years later, while examining changes in the bodies of salmon as they migrate upstream to their spawning grounds, he noticed that he could, with minimal effort, purify large amounts of pure nuclein from the testes, as they were at the height of cell proliferation in preparation for mating (Miescher, 1874). This provided additional evidence for a link between proliferation and the presence of a high concentration of nuclein.Miescher''s most insightful comments on this issue, however, date from his time in Hoppe-Seyler''s laboratory in Tübingen. He was convinced that histochemical analyses would lead to a much better understanding of certain pathological states than would microscopic studies. He also believed that physiological processes, which at the time were seen as similar, might turn out to be very different if the chemistry were better understood. As early as 1869, the year in which he discovered nuclein, he wrote in a letter to His: “Based on the relative amounts of nuclear substances [DNA], proteins and secondary degradation products, it would be possible to assess the physiological significance of changes with greater accuracy than is feasible now” (Miescher, 1869c).Importantly, Miescher proposed three exemplary processes that might benefit from such analyses: “nutritive progression”, characterized by an increase in the cytoplasmic proteins and the enlargement of the cell; “generative progression”, defined as an increase in “nuclear substances” (nuclein) and as a preliminary phase of cell division in proliferating cells and possibly in tumours; and “regression”, an accumulation of lipids and degenerative products (Miescher, 1869c).When we consider the first two categories, Miescher seems to have understood that an increase in DNA was not only associated with, but also a prerequisite for cell proliferation. Subsequently, cells that are no longer proliferating would increase in size through the synthesis of proteins and hence cytoplasm. Crucially, he believed that chemical analyses of such different states would enable him to obtain a more fundamental insight into the causes underlying these processes. These are astonishingly prescient insights. Sadly, Miescher never followed up on these ideas and, apart from the thoughts expressed in his letter, never published on the topic.…Miescher seems to have understood that an increase in DNA was not only associated with, but also a prerequisite for cell proliferationIt is likely, however, that he had preliminary data supporting these views. Miescher was generally careful to base statements on facts rather than speculation. But, being a perfectionist who published only after extensive verification of his results, he presumably never pursued these studies to such a satisfactory point. It is possible his plans were cut short by leaving Hoppe-Seyler''s laboratory to receive additional training under the supervision of Carl Ludwig (1816–1895) in Leipzig. While there, Miescher turned his attention to matters entirely unrelated to DNA and only resumed his work on nuclein after returning to his native Basel in 1871.Crucially for these subsequent studies of nuclein, Miescher made an important choice: he turned to sperm as his main source of DNA. When analysing the sperm from different species, he noted that the spermatozoa, especially of salmon, have comparatively small tails and thus consist mainly of a nucleus (Miescher, 1874). He immediately grasped that this would greatly facilitate his efforts to isolate DNA at much higher purity (Fig 4). Yet, Miescher also saw beyond the possibility of obtaining pure nuclein from salmon sperm. He realized it also indicated that the nucleus and the nuclein therein might play a crucial role in fertilization and the transmission of heritable traits. In a letter to his colleague Rudolf Boehm (1844–1926) in Würzburg, Miescher wrote: “Ultimately, I expect insights of a more fundamental importance than just for the physiology of sperm” (Miescher, 1871c). It was the beginning of a fascination with the phenomena of fertilization and heredity that would occupy Miescher to the end of his days.Open in a separate windowFigure 4A glass vial containing DNA purified by Friedrich Miescher from salmon sperm. © Alfons Renz, University of Tübingen, Germany.Miescher had entered this field at a critical time. By the middle of the nineteenth century, the old view that cells arise through spontaneous generation had been challenged. Instead, it was widely recognized that cells always arise from other cells (Mayr, 1982). In particular, the development and function of spermatozoa and oocytes, which in the mid-1800s had been shown to be cells, were seen in a new light. Moreover, in 1866, three years before Miescher discovered DNA, Ernst Haeckel (1834–1919) had postulated that the nucleus contained the factors that transmit heritable traits. This proposition from one of the most influential scientists of the time brought the nucleus to the centre of attention for many biologists. Having discovered nuclein as a distinctive molecule present exclusively in this organelle, Miescher realized that he was in an excellent position to make a contribution to this field. Thus, he set about trying to better characterize nuclein with the aim of correlating its chemical properties with the morphology and function of cells, especially of sperm cells.His analyses of the chemical composition of the heads of salmon spermatozoa led Miescher to identify two principal components: in addition to the acidic nuclein, he found an alkaline protein for which he coined the term ‘protamin''; the name is still in use today; protamines are small proteins that replace histones during spermatogenesis. He further determined that these two molecules occur in a “salt-like, not an ether-like [that is, covalent] association” (Miescher, 1874). Following his meticulous analyses of the chemical composition of sperm, he concluded that, “aside from the mentioned substances [protamin and nuclein] nothing is present in significant quantity. As this is crucial for the theory of fertilization, I carry this business out as quantitatively as possible right from the beginning” (Miescher, 1872a). His analyses showed him that the DNA and protamines in sperm occur at constant ratios; a fact that Miescher considered “is certainly of special importance,” without, however, elaborating on what might be this importance. Today, of course, we know that proteins, such as histones and protamines, bind to DNA in defined stoichiometric ratios.Miescher went on to analyse the spermatozoa of carp, frogs (Rana esculenta) and bulls, in which he confirmed the presence of large amounts of nuclein (Miescher, 1874). Importantly, he could show that nuclein is present in only the heads of sperm—the tails being composed largely of lipids and proteins—and that within the head, the nuclein is located in the nucleus (Miescher, 1874; Schmiedeberg & Miescher, 1896). With this discovery, Miescher had not only demonstrated that DNA is a constant component of spermatozoa, but also directed his attention to the sperm heads. On the basis of the observations of other investigators, such as those of Albert von Kölliker (1817–1905) concerning the morphology of spermatozoa in some myriapods and arachnids, Miescher knew that the spermatozoa of some species are aflagellate, that is, lack a tail. This confirmed that the sperm head, and thus the nucleus, was the crucial component. But, the question remained: what in the sperm cells mediated fertilization and the transmission of hereditary traits from one generation to the next?On the basis of his chemical analyses of sperm, Miescher speculated on the existence of molecules that have a crucial part in these processes. In a letter to Boehm, Miescher wrote: “If chemicals do play a role in procreation at all, then the decisive factor is now a known substance” (Miescher, 1872b). But Miescher was unsure as to what might be this substance. He did, however, strongly suspect the combination of nuclein and protamin was the key and that the oocyte might lack a crucial component to be able to develop: “If now the decisive difference between the oocyte and an ordinary cell would be that from the roster of factors, which account for an active arrangement, an element has been removed? For otherwise all proper cellular substances are present in the egg,” he later wrote (Miescher, 1872b).Owing to his inability to detect protamin in the oocyte, Miescher initially favoured this molecule as responsible for fertilization. Later, however, when he failed to detect protamin in the sperm of other species, such as bulls, he changed his mind: “The Nuclein by contrast has proved to be constant [that is, present in the sperm cells of all species Miescher analysed] so far; to it and its associations I will direct my interest from now on” (Miescher, 1872b). Unfortunately, however, although he came tantalizingly close, he never made a clear link between nuclein and heredity.The final section of his 1874 paper on the occurrence and properties of nuclein in the spermatozoa of different vertebrate species is of particular interest because Miescher tried to correlate his chemical findings about nuclein with the physiological role of spermatozoa. He had realized that spermatozoa represented an ideal model system to study the role of DNA because, as he would later put it, “[f]or the actual chemical–biological problems, the great advantage of sperm [cells] is that everything is reduced to the really active substances and that they are caught just at the moment when they exert their greatest physiological function” (Miescher, 1893a). He appreciated that his data were still incomplete, yet wanted to make a first attempt to pull his results together and integrate them into a broader picture to explain fertilization.At the time, Wilhelm Kühne (1837–1900), among others, was putting forward the idea that spermatozoa are the carriers of specific substances that, through their chemical properties, achieve fertilization (Kühne, 1868). Miescher considered his results of the chemical composition of spermatozoa in this context. While critically considering the possibility of a chemical substance explaining fertilization, he stated that: “if we were to assume at all that a single substance, as an enzyme or in any other way, for instance as a chemical impulse, could be the specific cause of fertilization, we would without a doubt first and foremost have to consider Nuclein. Nuclein-bodies were consistently found to be the main components [of spermatozoa]” (Miescher, 1874).With hindsight, these statements seem to suggest that Miescher had identified nuclein as the molecule that mediates fertilization—a crucial assumption to follow up on its role in heredity. Unfortunately, however, Miescher himself was far from convinced that a molecule (or molecules) was responsible for this. There are several reasons for his reluctance, although the influence of his uncle was presumably a crucial factor as it was he who had been instrumental in fostering the young Miescher''s interest in biochemistry and remained a strong influence throughout his life. Indeed, when Miescher came tantalizingly close to uncovering the function of DNA, His''s views proved counterproductive, probably preventing him from interpreting his findings in the context of new results from other scientists at the time. Miescher thus failed to take his studies of nuclein and its function in fertilization and heredity to the next level, which might well have resulted in recognizing DNA as the central molecule in both processes.One specific aspect that diverted Miescher from contemplating the role of nuclein in fertilization was a previous study in which he had erroneously identified the yolk platelets in chicken oocytes as a large number of nuclein-containing granules (Miescher, 1871b). This led him to conclude that the comparatively minimal quantitative contribution of DNA from a spermatozoon to an oocyte, which already contained so much more of the substance, could not have a significant impact on the latter''s physiology. He therefore concluded that, “not in a specific substance can the mystery of fertilization be concealed. […] Not a part, but the whole must act through the cooperation of all its parts” (Miescher, 1874).It is all the more unfortunate that Miescher had identified the yolk platelets in oocytes as nuclein-containing cells because he had realized that the presumed nuclein in these granules differed from the nuclein (that is, DNA) he had isolated previously from other sources, notably by its much higher phosphorous content. But influenced by His''s strong view that these structures were genuine cells, Miescher saw his results in this light. Only several years later, based on results from his contemporaries Flemming and Eduard A. Strasburger (1844–1912) on the morphological properties of nuclei and their behaviour during cell divisions, and Albrecht Kossel''s (1853–1927) discoveries about the composition of DNA (Portugal & Cohen, 1977), did Miescher revise his initial assumption that chicken oocytes contain a large number of nuclein-containing granules. Instead, he finally conceded that the molecules comprising these granules were different from nuclein (Miescher, 1890).Another factor that prevented Miescher from concluding that nuclein was the basis for the transmission of hereditary traits was that he could not conceive of how a single substance might explain the multitude of heritable traits. How, he wondered, could a specific molecule be responsible for the differences between species, races and individuals? Although he granted that “differences in the chemical constitution of these molecules [different types of nuclein] will occur, but only to a limited extent” (Miescher, 1874).And thus, instead of looking to molecules, he—like his uncle His––favoured the idea that the physical movement of the sperm cells or an activation of the oocyte, which he likened to the stimulation of a muscle by neuronal impulses, was responsible for the process of fertilization: “Like the muscle during the activation of its nerve, the oocyte will, when it receives appropriate impulses, become a chemically and physically very different entity” (Miescher, 1874). For nuclein itself, Miescher considered that it might be a source material for other molecules, such as lecithin––one of the few other molecules with a high phosphorous content known at the time (Miescher, 1870, 1871a, 1874). Miescher clearly preferred the idea of nuclein as a repository for material for the cell—mainly phosphorous—rather than as a molecule with a role in encoding information to synthesize such materials. This idea of large molecules being source material for smaller ones was common at the time and was also contemplated for proteins (Miescher, 1870).The entire section of Miescher''s 1874 paper in which he discusses the physiological role of nuclein reads as though he was deliberately trying to assemble evidence against nuclein being the key molecule in fertilization and heredity. This disparaging approach towards the molecule that he himself had discovered might also be explained, at least to some extent, by his pronounced tendency to view his results so critically; tellingly, he published only about 15 papers and lectures in a career spanning nearly three decades.The modern understanding that fertilization is achieved by the fusion of two germ cells only became established in the final quarter of the nineteenth century. Before that time, the almost ubiquitous view was that the sperm cell, through mere contact with the egg, in some way stimulated the oocyte to develop—the physicalist viewpoint. His was a key advocate of this view and firmly rejected the idea that a specific substance might mediate heredity. We can only speculate as to how Miescher would have interpreted his results had he worked in a different intellectual environment at the time, or had he been more independent in the interpretation of his results.We can only speculate as to how Miescher would have interpreted his results had he worked in a different intellectual environment at the time…Miescher''s refusal to accept nuclein as the key to fertilization and heredity is particularly tragic in view of several studies that appeared in the mid-1870s, focusing the attention of scientists on the nuclei. Leopold Auerbach (1828–1897) demonstrated that fertilized eggs contain two nuclei that move towards each other and fuse before the subsequent development of the embryo (Auerbach, 1874). This observation strongly suggested an important role for the nuclei in fertilization. In a subsequent study, Oskar Hertwig (1849–1922) confirmed that the two nuclei—one from the sperm cell and one from the oocyte—fuse before embryogenesis begins. Furthermore, he observed that all nuclei in the embryo derive from this initial nucleus in the zygote (Hertwig, 1876). With this he had established that a single sperm fertilizes the oocyte and that there is a continuous lineage of nuclei from the zygote throughout development. In doing so, he delivered the death blow to the physicalist view of fertilization.By the mid-1880s, Hertwig and Kölliker had already postulated that the crucial component of the nucleus that mediated inheritance was nuclein—an idea that was subsequently accepted by several scientists. Sadly, Miescher remained doubtful until his death in 1895 and thus failed to appreciate the true importance of his discovery. This might have been an overreaction to the claims by others that sperm heads are formed from a homogeneous substance; Miescher had clearly shown that they also contain other molecules, such as proteins. Moreover, Miescher''s erroneous assumption that nuclein occurred only in the outer shell of the sperm head resulted in his failure to realize that stains for chromatin, which stain the centres of the heads, actually label the region where there is nuclein; although he later realized that virtually the entire sperm head is composed of nuclein and associated protein (Miescher, 1892a; Schmiedeberg & Miescher, 1896).Unfortunately, not only Miescher, but the entire scientific community would soon lose faith in DNA as the molecule mediating heredity. Miescher''s work had established DNA as a crucial component of all cells and inspired others to begin exploring its role in heredity, but with the emergence of the tetranucleotide hypothesis at the beginning of the twentieth century, DNA fell from favour and was replaced by proteins as the prime candidates for this function. The tetranucleotide hypothesis—which assumed that DNA was composed of identical subunits, each containing all four bases—prevailed until the late 1940s when Edwin Chargaff (1905–2002) discovered that the different bases in DNA were not present in equimolar amounts (Chargaff et al, 1949, 1951).Unfortunately, not only Miescher, but the entire scientific community would soon lose faith in DNA as the molecule mediating heredityJust a few years before, in 1944, experiments by Avery and colleagues had demonstrated that DNA was sufficient to transform bacteria (Avery et al, 1944). Then in 1952, Al Hershey (1908–1997) and Martha Chase (1927–2003) confirmed these findings by observing that viral DNA—but no protein—enters the bacteria during infection with the T2 bacteriophage and, that this DNA is also present in new viruses produced by infected bacteria (Hershey & Chase, 1952). Finally, in 1953, X-ray images of DNA allowed Watson and Crick to deduce its structure (Watson & Crick, 1953) and thus enable us to understand how DNA works. Importantly, these experiments were made possible by advances in bacteriology and virology, as well as the development of new techniques, such as the radioactive labelling of proteins and nucleic acids, and X-ray crystallography—resources that were beyond the reach of Miescher and his contemporaries.In later years (Fig 5), Miescher''s attention shifted progressively from the role of nuclein in fertilization and heredity to physiological questions, such as those concerning the metabolic changes in the bodies of salmon as they produce massive amounts of germ cells at the expense of muscle tissue. Although he made important and seminal contributions to different areas of physiology, he increasingly neglected to explore his most promising line of research, the function of DNA. Only towards the end of his life did he return to this question and begin to reconsider the issue in a new light, but he achieved no further breakthroughs.Open in a separate windowFigure 5Friedrich Miescher in his later years when he was Professor of Physiology at the University of Basel. In this capacity he also founded the Vesalianum, the University''s Institute for Anatomy and Physiology, which was inaugurated in 1885. This photograph is the frontispiece on the inside cover of a collection of Miescher''s publications and some of his letters, edited and published by his uncle Wilhelm His and colleagues after Miescher''s death. Underneath the picture is Miescher''s signature. © Ralf Dahm.One area, however, where he did propose intriguing hypotheses—although without experimental data to support them—was the molecular underpinnings of heredity. Inspired by Darwin''s work on fertilization in plants, Miescher postulated, for instance, how information might be encoded in biological molecules. He stated that, “the key to sexuality for me lies in stereochemistry,” and expounded his belief that the gemmules of Darwin''s theory of pangenesis were likely to be “numerous asymmetric carbon atoms [present in] organic substances” (Miescher, 1892b), and that sexual reproduction might function to correct mistakes in their “stereometric architecture”. As such, Miescher proposed that hereditary information might be encoded in macromolecules and how mistakes could be corrected, which sounds uncannily as though he had predicted what is now known as the complementation of haploid deficiencies by wild-type alleles. It is particularly tempting to assume that Miescher might have thought this was the case, as Mendel had published his laws of inheritance of recessive characteristics more than 25 years earlier. However, there is no reference to Mendel''s work in the papers, talks or letters that Miescher has left to us.Miescher proposed that hereditary information might be encoded in macromolecules and how mistakes could be corrected…What we do know is that Miescher set out his view of how hereditary information might be stored in macromolecules: “In the enormous protein molecules […] the many asymmetric carbon atoms allow such a colossal number of stereoisomers that all the abundance and diversity of the transmission of hereditary [traits] may find its expression in them, as the words and terms of all languages do in the 24–30 letters of the alphabet. It is therefore completely superfluous to see the sperm cell or oocyte as a repository of countless chemical substances, each of which should be the carrier of a special hereditary trait (de Vries Pangenesis). The protoplasm and the nucleus, that my studies have shown, do not consist of countless chemical substances, but of very few chemical individuals, which, however, perhaps have a very complex chemical structure” (Miescher, 1892b).This is a remarkable passage in Miescher''s writings. The second half of the nineteenth century saw intense speculation about how heritable characteristics are transmitted between the generations. The consensus view assumed the involvement of tiny particles, which were thought to both shape embryonic development and mediate inheritance (Mayr, 1982). Miescher contradicted this view. Instead of a multitude of individual particles, each of which might be responsible for a specific trait (or traits), his results had shown that, for instance, the heads of sperm cells are composed of only very few compounds, chiefly DNA and associated proteins.He elaborated further on his theory of how hereditary information might be stored in large molecules: “Continuity does not only lie in the form, it also lies deeper than the chemical molecule. It lies in the constituent groups of atoms. In this sense I am an adherent of a chemical model of inheritance à outrance [to the utmost]” (Miescher, 1893b). With this statement Miescher firmly rejects any idea of preformation or some morphological continuity transmitted through the germ cells. Instead, he clearly seems to foresee what would only become known much later: the basis of heredity was to be found in the chemical composition of molecules.To explain how this could be achieved, he proposed a model of how information could be encoded in a macromolecule: “If, as is easily possible, a protein molecule comprises 40 asymmetric carbon atoms, there will be 2⁴⁰, that is, approximately a trillion isomerisms [sic]. And this is only one of the possible types of isomerism [not considering other atoms, such as nitrogen]. To achieve the incalculable diversity demanded by the theory of heredity, my theory is better suited than any other. All manner of transitions are conceivable, from the imperceptible to the largest differences” (Miescher, 1893b).Miescher''s ideas about how heritable characteristics might be transmitted and encoded encapsulate several important concepts that have since been proven to be correct. First, he believed that sexual reproduction served to correct mistakes, or mutations as we call them today. Second, he postulated that the transmission of heritable traits occurs through one or a few macromolecules with complex chemical compositions that encode the information, rather than by numerous individual molecules each encoding single traits, as was thought at the time. Third, he foresaw that information is encoded in these molecules through a simple code that results in a staggeringly large number of possible heritable traits and thus explain the diversity of species and individuals observed.Miescher''s ideas about how heritable characteristics might be transmitted and encoded encapsulate several important concepts that have since been proven to be correctIt is a step too far to suggest that Miescher understood what DNA or other macromolecules do, or how hereditary information is stored. He simply could not have done, given the context of his time. His findings and hypotheses that today fit nicely together and often seem to anticipate our modern understanding probably appeared rather disjointed to Miescher and his contemporaries. In his day, too many facts were still in doubt and too many links tenuous. There is always a danger of over-interpreting speculations and hypotheses made a long time ago in today''s light. However, although Miescher himself misinterpreted some of his findings, large parts of his conclusions came astonishingly close to what we now know to be true. Moreover, his work influenced others to pursue their own investigations into DNA and its function (Dahm, 2008). Although DNA research fell out of fashion for several decades after the end of the nineteenth century, the information gleaned by Miescher and his contemporaries formed the foundation for the decisive experiments carried out in the middle of the twentieth century, which unambiguously established the function of DNA.As such, perhaps the most tragic aspect of Miescher''s career was that for most of his life he firmly believed in the physicalist theories of fertilization, as propounded by His and Ludwig among others, and his reluctance to combine the results from his rigorous chemical analyses with the ‘softer'' data generated by cytologists and histologists. Had he made the link between nuclein and chromosomes and accepted its key role in fertilization and heredity, he might have realized that the molecule he had discovered was the key to some of the greatest mysteries of life. As it was, he died with a feeling of a promising career unfulfilled (His, 1897), when, in actual fact, his contributions were to outshine those of most of his contemporaries.…he died with a feeling of a promising career unfulfilled […] when, in actual fact, his contributions were to outshine those of most of his contemporariesIt is tantalizing to speculate the path that Miescher''s investigations—and biology as a whole—might have taken under slightly different circumstances. What would have happened had he followed up on his preliminary results about the role of DNA in different physiological conditions, such as cell proliferation? How would his theories about fertilization and heredity have changed had he not been misled by the mistaken identification of what appeared to him to be a multitude of small nuclei in the oocyte? Or how would he have interpreted his findings concerning nuclein had he not been influenced by the views of his uncle, but also those of the wider scientific establishment?There is a more general lesson in the life and work of Friedrich Miescher that goes beyond his immediate successes and failures. His story is that of a brilliant researcher who developed innovative experimental approaches, chose the right biological systems to address his questions and made ground-breaking discoveries, and who was nonetheless constrained by his intellectual environment and thus prevented from interpreting his findings objectively. It therefore fell to others, who saw his work from a new point of view, to make the crucial inferences and thus establish the function of DNA.? Open in a separate windowRalf Dahm     

Exploring the world of phospholipids and their interactions with proteins: the work of William Dowhan

The Gene Encoding the Phosphatidylinositol Transfer Protein Is Essential for Cell Growth (Aitken, J. F., van Heusden, G. P., Temkin, M., and Dowhan, W. (1990) J. Biol. Chem. 265, 4711–4717)A Phospholipid Acts as a Chaperone in Assembly of a Membrane Transport Protein (Bogdanov, M., Sun, J., Kaback, H. R., and Dowhan, W. (1996) J. Biol. Chem. 271, 11615–11618)William Dowhan''s curiosity about the connections between phospholipids and proteins associated with them goes back as far as his days as a graduate student with Esmond Snell at the University of California, Berkeley. In these two JBC Classics, his group''s ability to manipulate biochemical and molecular genetics tools to answer fundamental questions about lipid biology shines through. “William Dowhan and his research group have made many contributions to the biochemistry of phospholipid metabolism and membrane biogenesis,” says Robert Simoni at Stanford University.Open in a separate windowBill Dowhan (right) is shown here with the late Chris Raetz (left), who was a longtime collaborator and friend, and his former postdoctoral advisor, the late Gene Kennedy, on the occasion of Kennedy''s 90th birthday in 2009 (photo courtesy of William Dowhan).The first paper, published in 1990, documented the importance of phosphatidylinositol/phosphatidylcholine transfer proteins in vivo. Dowhan''s group, which has been based at the University of Texas Medical School since 1972, used a combination of biochemistry and genetics to clone the protein''s gene. Dowhan had first heard of phospholipid transfer proteins in 1969, when he began his postdoctoral training with Eugene (Gene) Kennedy at Harvard Medical School. At his very first Kennedy lab meeting, the discussion centered around a publication that had just come out (1). The paper described “one of the first observations of proteins in the soluble phase that transferred lipids between bilayers,” recalls Dowhan. “No one could figure out what these proteins really did in vivo, but they knew the proteins had this function” of transferring lipids between membranes.As he moved through his career, Dowhan focused on cloning and characterizing genes and purifying enzymes responsible for phospholipid metabolism in Escherichia coli. Then came a sabbatical in 1983 with Gottfried (Jeff) Schatz at the Biozentrum of the University of Basel in Switzerland, that expanded Dowhan''s research directions into yeast genetics. He says the opportunity to work with Schatz “got me into the possibility of looking for this phosphatidylinositol/phosphatidylcholine transfer protein (PI-TP) in yeast, which I probably would have never done if I hadn''t taken this sabbatical.”Fresh from his sabbatical, Dowhan started tracking down the protein and its gene in vivo. “I submitted a grant at that time with some preliminary data that we had begun to purify to homogeneity the PI-TP from yeast, which had never been done before. Fortunately, we got the grant,” he says.The Dowhan group managed to purify PI-TP from yeast. “The most important part was using basic biochemistry and understanding how to purify proteins before the advent of genetically tagging proteins for affinity chromatography,” explains Dowhan.For the next step in the process of finding the gene for the protein, Dowhan and colleagues had to apply reverse genetics because the yeast genome was not available in the late 1980s. They sequenced the amino terminus of the protein, made the corresponding oligonucleotide probes, tested yeast cDNA libraries with those probes, and pulled out the gene. “We still didn''t know the role PI-TP played in cell function. But now we had the sequence of the gene and the knock-out mutant was not viable,” notes Dowhan. “So we published” the findings.At the same time, Vytas Bankaitis, now at the University of North Carolina, had been working on cloning the SEC14 gene in yeast, which is necessary for vesicular transport. “It turns out we had missed the DNA sequence,” Dowhan says. From Bankaitis'' work, it was obvious that “PI-TP was the product of the SEC14 gene. It all came together in a joint report in Nature. Now we had a function associated with the SEC14 gene, which we didn''t have before,” Dowhan explains (2). “We had a phenotype of a mutant lacking this phospholipid transfer protein, which then stopped vesicular transport.”This initial link between phospholipid metabolism and vesicular transport opened up the field to characterization of the Sec14 protein superfamily in a broad range of biological systems. These proteins contain lipid-binding domains, which sense membrane lipid composition and integrate lipid metabolism and lipid-mediated signaling with an array of cellular processes.The second JBC Classic focused on a different feature of phospholipids: their role in protein folding. Dowhan was fascinated by membrane proteins ever since he was a graduate student and had gone to the Kennedy laboratory as a postdoctoral fellow, intending to purify the membrane component expressed by the lac operon for lactose transport in E. coli. He was unsuccessful because, at that time, the necessary detergents were not available. Once the lactose permease was purified (3), Dowhan noticed in the literature that other researchers mentioned that when the protein was reconstituted in liposomes missing phosphatidylethanolamine, the protein was defective in energy-dependent uphill transport. Dowhan recalls that he wondered, “Was that an artifact of the liposome system or was that also true in vivo?”To get to the bottom of this observation, Dowhan''s group used E. coli to generate null mutants of what were considered to be absolutely essential genes for phospholipid synthesis and cell viability. They created a null mutant of the pssA gene, which encodes the committed step to the synthesis of the major phospholipid, phosphatidylethanolamine. By establishing conditions in which bacterial cells lacking phosphatidylethanolamine remained viable, the investigators were able to identify and characterize different cell phenotypes caused by the missing phospholipid both in vivo and in vitro. In collaboration with Ronald Kaback at UCLA, Dowhan''s group showed that phosphatidylethanolamine was essential for the proper folding of an epitope of lactose permease that was also necessary to support the energy-dependent uphill transport of lactose. “Studies by others have since shown a similar chaperone role for lipids in other bacteria, plants and mammalian cells,” notes Simoni.To obtain their data, the investigators developed a new technique, the Eastern-Western blot. In this method, membrane proteins were delipidated and partially denatured by SDS. The proteins underwent gel electrophoresis and then were transferred to a solid support by Western blotting. A series of individual lipids were then laid over the proteins at a 90° angle so that the investigators could see, after incubating with conformation-specific antibodies, which lipids helped which membrane proteins regain proper conformation.This technique was used to establish that phosphatidylethanolamine was necessary in a late step of folding of lactose permease, but was not necessary to maintain the final folded state. This observation suggested that lipids act as molecular chaperones in helping protein maturation. “This paper set the stage for understanding how lipids affect the topological organization of wild-type proteins in the membrane,” notes Dowhan.Dowhan and his collaborator Mikhail Bogdanov have continued using bacterial mutants in phospholipid metabolism to systematically manipulate the native membrane lipid compositions during the cell cycle. They have analyzed the transmembrane domain orientation of membrane proteins to establish the molecular basis for lipid-dependent organization of lactose permease and other secondary transporters (4).Dowhan says his work has two take-home messages. One is that “Lipids aren''t just glorified biological detergents,” he says. “They have specific roles” in the cell. The other message is in the power of numbers. Dowhan says the more techniques applied to solve a biological mystery, the more likely the mystery will be successfully solved.     

MicroRNAs and drug modulation in cancer: an intertwined new story

MicroRNAs (miRNAs) are endogenous small non-coding RNAs (ncRNAs) which play important regulatory roles in physiological processes such as cellular differentiation, proliferation, development, apoptosis and stem cell self-renewal. An increasing number of papers have clearly claimed their involvement in cancer, providing, in some cases, also the molecular mechanisms implicated. Several studies led to the conclusion that miRNAs can be effectively used as anticancer agents alone or in combination with existing anticancer drugs. In particular, miRNAs can be effectively used to overcome drug resistance, one of the main factors responsible for anticancer treatment insuccess. One of the main questions remains how to modulate the expression of miRNAs in cancer cells. Interestingly, a few studies have shown that the expression of miRNAs is affected by drugs (including some drugs currently used as anticancer agents), therefore providing the rationale for an intertwined scenario in which miRNAs can be modulated by drugs and, in turn, can affect drug sensitivity of cancer cells.     

Gradients and the Specification of Planar Polarity in the Insect Cuticle

In addition to specifying cell fate, there is a wealth of evidence that molecular gradients are also primarily responsible for specifying cell polarity, particularly in the plane of epithelial sheets (“planar polarity”). The first compelling evidence of a role for gradients in specifying planar polarity came from transplantation experiments in the insect cuticle. More recent molecular genetic analyses in the fruit fly Drosophila have begun to give insights into the molecular nature of the gradients involved, and how they are interpreted at the cellular level.Development requires the coordinated specification of at least three attributes: cell fate, tissue size, and cell polarity. In both theory and practice, all three can be specified by the action of gradients. This article examines the experimental evidence for gradients acting to specify cell polarity in developing tissues, considers the mechanisms by which they are thought to act, and discusses what remains unknown. The problem of how cell polarity is specified in the plane of a tissue (“planar polarity”) is addressed. The tissues discussed are all formed from epithelial sheets that also show apicobasal cell polarity.For more than half a century, the preeminent system for studying the regulation of planar polarity in epithelia has been the insect cuticle. This lends itself to the study of the problem by virtue of often being adorned by structures such as hairs, scales, ridges, or other protrusions that reveal the polarity of the underlying cells. However, the lack of polarized structures on the surface of other epithelial-derived tissues should not be taken as evidence that the cells are not planar polarized, because often such polarity is cryptically expressed and only becomes apparent when the cells participate in a polarized process, such as cell division or cell intercalation.     

Mechanisms of resistance to alkylating agents

Alkylating agents are the most widely used anticancer drugs whose main target is the DNA, although how exactly the DNA lesions cause cell death is still not clear. The emergence of resistance to this class of drugs as well as to other antitumor agents is one of the major causes of failure of cancer treatment. This paper reviews some of the best characterized mechanisms of resistance to alkylating agents. Pre- and post-target mechanisms are recognized, the former able to limit the formation of lethal DNA adducts, and the latter enabling the cell to repair or tolerate the damage. The role in the pre-target mechanisms of reduced drug accumulation and the increased detoxification or activation systems (such as DT-diaphorase, metallothionein, GST/GSH system, etc...) are discussed. In the post-target mechanisms the different DNA repair pathways, tolerance to alkylation damage and the ‘downstream’ effects (cell cycle arrest and/or apoptosis) are examined. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ligand Binding in Hemoglobin: the Work of Quentin H. Gibson

Oxygen Binding and Subunit Interaction of Hemoglobin in Relation to the Two-state Model(Gibson, Q. H., and Edelstein, S. J. (1987) J. Biol. Chem. 262, 516–519)Ligand Recombination to the α and β Subunits of Human Hemoglobin(Olson, J. S., Rohlfs, R. J., and Gibson, Q. H. (1987) J. Biol. Chem. 262, 12930–12938)Quentin Howieson Gibson was born in Aberdeen, Scotland in 1918. He attended Queen''s University Belfast and received his M.B., Ch.B. degree in 1941, his M.D. in 1944, and his Ph.D. in 1946. After graduating, he became a lecturer in the Department of Physiology at the University of Sheffield and worked his way up to become Professor and Head of the Department of Biochemistry by 1955.Open in a separate windowQuentin H. GibsonIn 1963, Gibson came to the U.S. and joined the faculty of the Graduate School of Medicine at the University of Pennsylvania as a Professor of Physiology. He remained at Penn until 1965 when he became the Greater Philadelphia Professor of Biochemistry, Molecular and Cell Biology at Cornell University. In 1996, Gibson joined the Department of Biochemistry and Cell Biology at Rice University.Gibson is probably best known for his research on the structure and function of hemoglobin. The hemoglobin molecule consists of four globular protein subunits, each of which contains a heme group that can bind to one molecule of oxygen. The binding of oxygen to hemoglobin is cooperative, the first bound oxygen alters the shape of the molecule to increase the binding affinity of the additional subunits. Conversely, hemoglobin''s oxygen binding capacity is decreased in the presence of carbon monoxide because both gases compete for the same binding sites on hemoglobin, carbon monoxide binding preferentially in place of oxygen.Gibson started his hemoglobin studies in graduate school, submitting a thesis titled “Methaemoglobin,” in which he studied the form of hemoglobin where the iron in the heme group is in the Fe³⁺ state rather than the Fe²⁺ state and is thus unable to carry oxygen. He followed this up with research on familial idiopathic methemoglobinemia, a hereditary hematological disease in which hemoglobin is unable to bind to oxygen, causing dyspnea and fatigue after physical exertion. He was able to identify the pathway involved in the reduction of methemoglobin (1), thereby describing the first hereditary disorder involving an enzyme deficiency. As a result, the disease was named “Gibson''s syndrome.” Since then, Gibson has made numerous additional contributions to the study of hemoglobin, some of which are detailed in the two Journal of Biological Chemistry (JBC) Classics reprinted here.In the first Classic, Gibson and Stuart J. Edelstein look at the oxygen binding and subunit interaction in hemoglobin. In 1965, Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux proposed a model which stated that proteins that exhibit cooperativity can exist in only two conformational states, and the equilibrium between these two states is modified by binding of a ligand, oxygen in the case of hemoglobin (2). This became known as the “concerted” or the “MWC” model, for Monod, Wyman, and Changeux. (More information on Wyman''s research on protein chemistry and allosterism can be found in his JBC Classic (3).)By the mid-1980s, several groups had found evidence that challenged this model as it related to the mechanistic basis of ligand binding by hemoglobin. For example, Frederick C. Mills and Gary K. Ackers reported that the subunit interactions of hemoglobin decreased on binding of the fourth molecule of oxygen to hemoglobin (4). The effect, which they called “quaternary enhancement,” was incompatible with the two-state MWC allosteric model. In the first Classic, Gibson and Edelstein measured the free energy of binding of the fourth oxygen molecule and compared their result of −8.6 kcal/mol with Mills and Acker''s result of −9.3 kcal/mol. Gibson''s smaller value was consistent with other values found in the literature, and it also allowed reasonable representation of the equilibrium curve using the two-state model without invoking quaternary enhancement.In the second JBC Classic, Gibson looks at ligand binding in human hemoglobin. This paper was an extension of an analysis Gibson had done the previous year on ligand rebinding to sperm whale myoglobin (5). In the paper reprinted here, Gibson and his colleagues explored the rebinding of CO, O₂, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to the isolated α- and β-chains of hemoglobin as well as the intact molecule. From these experiments the researchers were able to determine the differences between the overall rate constants of the two hemoglobin subunits as well as the differences in binding of the various ligands.In recognition of his contributions to science, Gibson has earned many honors including memberships in the Royal Society of London, the National Academy of Sciences, and the American Association for the Advancement of Science. He served as an Associate Editor for the JBC from 1975 to 1994.     

Combined Drug Action of 2-Phenylimidazo[2,1-b]Benzothiazole Derivatives on Cancer Cells According to Their Oncogenic Molecular Signatures

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by “RTK swapping” by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation.     

Stem-cell battles. Stem-cell research in the USA is facing new legal and political challenges

The US still leads the world in stem-cell research, yet US scientists are facing yet another political and legal battle for federal funding to support research using human embryonic stem cells.Disputes over stem-cell research have been standard operating procedure since James Thompson and John Gearhart created the first human embryonic cell (hESC) lines. Their work triggered an intense and ongoing debate about the morality, legality and politics of using hESCs for biomedical research. “Stem-cell policy has caused craziness all over the world. It is a never-ending, irresolvable battle about the moral status [of embryos],” commented Timothy Caulfield, research director of the Health Law Institute at the University of Alberta in Edmonton, Canada. “We''re getting to an interesting time in history where science is playing a bigger and bigger part in our lives, and it''s becoming more controversial because it''s becoming more powerful. We need to make some interesting choices about how we decide what kind of scientific inquiry can go forward and what can''t go forward.”“Stem-cell policy has caused craziness all over the world…[i]t is a never-ending, irresolvable battle about the moral status [of embryos]”The most contested battleground for stem-cell research has been the USA, since President George W. Bush banned federal funding for research that uses hESCs. His successor, Barack Obama, eventually reversed the ban, but a pending lawsuit and the November congressional elections have once again thrown the field into jeopardy.Three days after the election, the deans of US medical schools, chiefs of US hospitals and heads of leading scientific organizations sent letters to both the House of Representatives and the Senate urging them to pass the Stem Cell Research Advancement Act when they come back into session. The implication was to pass legislation now, while the Democrats were still the majority. Republicans, boosted in the election by the emerging fiscally conservative Tea Party movement, will be the majority in the House from January, changing the political climate. The Republicans also cut into the Democratic majority in the Senate.Policies and laws to regulate stem-cell research vary between countries. Italy, for example, does not allow the destruction of an embryo to generate stem-cell lines, but it does allow research on such cells if they are imported. Nevertheless, the Italian government deliberately excluded funding for projects using hESCs from its 2009 call for proposals for stem-cell research. In the face of legislative vacuums, this October, Science Foundation Ireland and the Health Research Board in Ireland decided to not consider grant applications for projects involving hESC lines. The UK is at the other end of the scale; it has legalized both research with and the generation of stem-cell lines, albeit under the strict regulation by the independent Human Fertility and Embryology Authority. As Caulfield commented, the UK is “ironically viewed as one of the most permissive [on stem-cell policy], but is perceived as one of the most bureaucratic.”Somewhere in the middle is Germany, where scientists are allowed to use several approved cell lines, but any research that leads to the destruction of an embryo is illegal. Josephine Johnston, director of research operations at the Hastings Center in Garrison, NY, USA—a bioethics centre—said: “In Germany you can do research on embryonic stem-cells, but you can''t take the cells out of the embryo. So, they import their cells from outside of Germany and to me, that''s basically outsourcing the bit that you find difficult as a nation. It doesn''t make a lot of sense ethically.”Despite the public debates and lack of federal support, Johnson noted that the USA continues to lead the world in the field. “[Opposition] hasn''t killed stem-cell research in the United States, but it definitely is a headache,” she said. In October, physicians at the Shepherd Center, a spinal cord and brain injury rehabilitation hospital and clinical research centre in Atlanta, GA, USA, began to treat the first patient with hESCs. This is part of a clinical trial to test a stem-cell-based therapy for spinal cord injury, which was developed by the US biotechnology company Geron from surplus embryos from in vitro fertilization.Nevertheless, the debate in the USA, where various branches of government—executive, legislative and legal—weigh in on the legal system, is becoming confusing. “We''re never going to have consensus [on the moral status of fetuses] and any time that stem-cell research becomes tied to that debate, there''s going to be policy uncertainty,” Caulfield said. “That''s what''s happened again in the United States.”Johnson commented that what makes the USA different is the rules about federally funded and non-federally funded research. “It isn''t much discussed within the United States, but it''s a really dramatic difference to an outsider,” she said. She pointed out that, by contrast, in other countries the rules for stem-cell research apply across the board.The election of Barack Obama as US President triggered the latest bout of uncertainty. The science community welcomed him with open arms; after all, he supports doubling the budget of the National Institutes of Health (NIH) over the next ten years and dismantled the policies of his predecessor that barred it from funding projects beyond the 60 extant hESC lines—only 21 of which were viable. Obama also called on Congress to provide legal backing and funding for the research.The executive order had unforeseen consequences for researchers working with embryonic or adult stem cells. Sean Morrison, Director of the University of Michigan''s Centre for Stem Cell Biology (Ann Arbor, MI, USA), said he thought that Obama''s executive order had swung open the door on federal support forever. “Everybody had that impression,” he said.Leonard I. Zon, Director of the Stem Cell Program at Children''s Hospital Boston (MA, USA), was so confident in Obama''s political will that his laboratory stopped its practice of labelling liquid nitrogen containers as P (Presidential) and NP (non-Presidential) to avoid legal hassles. His lab also stopped purchasing and storing separate pipettes and culture dishes funded by the NIH and private sources such as the Howard Hughes Medical Institute (HHMI; Chevy Chase, MD, USA).But some researchers who focused on adult cells felt that the NIH was now biased in favour of embryonic cells. Backed by pro-life and religious groups, two scientists—James Sherley of the Boston Biomedical Research Institute and Theresa Deisher of AVM Biotechnology (Seattle, WA)—questioned the legality of the new NIH rules and filed a lawsuit against the Department of Health and Human Services (HHS) Secretary, Kathleen Sebelius. Deisher had founded her company to “[w]ork to provide safe, effective and affordable alternative vaccines and stem-cell therapies that are not tainted by embryonic or electively aborted fetal materials” (www.avmbiotech.com).…the debate in the USA, where various branches of government—executive, legislative and legal—weigh in on the legal system, is becoming confusingSherley argued in an Australian newspaper in October 2006 that the science behind embryonic stem-cell research is flawed and rejected arguments that the research will make available new cures for terrible diseases (Sherley, 2006). In court, the researchers also argued that they were irreparably disadvantaged in competing for government grants by their work on adult stem cells.Judge Royce C. Lamberth of the District Court of the District of Columbia initially ruled that the plaintiffs had no grounds on which to sue. However, the US District Court of Appeals for the District of Columbia overturned his decision and found that “[b]ecause the Guidelines have intensified the competition for a share in a fixed amount” of NIH funding. With the case back in his court, Lamberth reversed his decision on August 23 this year, granting a preliminary injunction to block the new NIH guidelines on embryonic stem-cell work. This injunction is detailed in the 1995 Dickey-Wicker Amendment, an appropriation bill rider, which prohibits the HHS from funding “research in which a human embryo or embryos are destroyed, discarded or knowingly subjected to risk of injury or death.” By allowing the destruction of embryos, Lamberth argued, the NIH rules violate the law.This triggered another wave of uncertainty as dozens of labs faced a freeze of federal funding. Morrison commented that an abrupt end to funding does not normally occur in biomedical research in the USA. “We normally have years of warning when grants are going to end so we can make a plan about how we can have smooth transitions from one funding source to another,” he said. Morrison—whose team has been researching Hirschsprung disease, a congenital enlargement of the colon—said his lab potentially faced a loss of US$ 250,000 overnight. “I e-mailed the people in my lab and said, ‘We may have just lost this funding and if so, then the project is over''”.Morrison explained that the positions of two people in his lab were affected by the cut, along with a third person whose job was partly funded by the grant. “Even though it''s only somewhere between 10–15% of the funding in my lab, it''s still a lot of money,” he said. “It''s not like we have hundreds of thousands of dollars of discretionary funds lying around in case a problem like that comes up.” Zon noted that his lab, which experienced an increase in the pace of discovery since Obama had signed his order, reverted to its Bush-era practices.On September 27 this year, a federal appeals court for the District of Columbia extended Lamberth''s stay to enable the government to pursue its appeal. The NIH was allowed to distribute US$78 million earmarked for 44 scientists during the appeal. The court said the matter should be expedited, but it could, over the years ahead, make its way to the US Supreme Court.The White House welcomed the decision of the appeals court in favour of the NIH. “President Obama made expansion of stem-cell research and the pursuit of groundbreaking treatments and cures a top priority when he took office. We''re heartened that the court will allow [the] NIH and their grantees to continue moving forward while the appeal is resolved,” said White House press secretary Robert Gibbs. The White House might have been glad of some good news, while it wrestles with the worst economic downturn since the Great Depression and the rise of the Tea Party movement.Even without a formal position on the matter, the Tea Party has had an impact on stem-cell research through its electoral victoriesTimothy Kamp, whose lab at the University of Wisconsin (Madison, WI, USA) researches embryonic stem-cell-derived cardiomyocytes, said that he finds the Tea Party movement confusing. “It''s hard for me to know what a uniform platform is for the Tea Party. I''ve heard a few comments from folks in the Tea Party who have opposed stem-cell research,” he said.However, the position of the Tea Party on the topic of stem-cell research could prove to be of vital importance. The Tea Party took its name from the Boston Tea Party—a famous protest in 1773 in which American colonists protested against the passing of the British Tea Act, for its attempt to extract yet more taxes from the new colony. Protesters dressed up as Native Americans and threw tea into the Boston harbour. Contemporary Tea Party members tend to have a longer list of complaints, but generally want to reduce the size of government and cut taxes. Their increasing popularity in the USA and the success of many Tea Party-backed Republican candidates for the upcoming congressional election could jeopardize Obama''s plans to pass new laws to regulate federal funding for stem-cell research.Even without a formal position on the matter, the Tea Party has had an impact on stem-cell research through its electoral victories. Perhaps their most high-profile candidate was the telegenic Christine O''Donnell, a Republican Senatorial candidate from Delaware. The Susan B. Anthony List, a pro-life women''s group, has described O''Donnell as one of “the brightest new stars” opposing abortion (www.lifenews.com/state5255.html). Although O''Donnell was eventually defeated in the 2 November congressional election, by winning the Republican primary in August, she knocked out nine-term Congressman and former Delaware governor Mike Castle, a moderate Republican known for his willingness to work with Democrats to pass legislation to protect stem-cell research.In the past, Castle and Diane DeGette, a Democratic representative from Colorado, co-sponsored the Stem Cell Research Advancement Act to expand federal funding of embryonic stem-cell research. They aimed to support Obama''s executive order and “ensure a lasting ethical framework overseeing stem cell research at the National Institutes of Health”.Morrison described Castle as “one of the great public servants in this country—no matter what political affiliation you have. For him to lose to somebody with such a chequered background and such shaky positions on things like evolution and other issues is a tragedy for the country.” Another stem-cell research advocate, Pennsylvania Senator Arlen Specter, a Republican-turned-Democrat, was also defeated in the primary. He had introduced legislation in September to codify Obama''s order. Specter, a cancer survivor, said his legislation is aimed at removing the “great uncertainty in the research community”.According to Sarah Binder, a political scientist at George Washington University in Washington, DC, the chances of passing legislation to codify the Obama executive order are decreasing: “As the Republican Party becomes more conservative and as moderates can''t get nominated in that party, it does lead you to wonder whether it''s possible to make anything happen [with the new Congress] in January.”There are a variety of opinions about how the outcome of the November elections will influence stem-cell policies. Binder said that a number of prominent Republicans have strongly promoted stem-cell research, including the Reagan family. “This hasn''t been a purely Democratic initiative,” she said. “The question is whether the Republican party has moved sufficiently to the right to preclude action on stem cells.” Historically there was “massive” Republican support for funding bills in 2006 and 2007 that were ultimately vetoed by Bush, she noted.…the debate about public funding for stem-cell research is only part of the picture, given the role of private business and states“Rightward shifts in the House and Senate do not bode well for legislative efforts to entrench federal support for stem-cell research,” Binder said. “First, if a large number of Republicans continue to oppose such funding, a conservative House majority is unlikely to pursue the issue. Second, Republican campaign commitments to reduce federal spending could hit the NIH and its support for stem-cell research hard.”Binder added that “a lingering unknown” is how the topic will be framed: “If it gets framed as a pro-choice versus pro-life initiative, that''s quite difficult for Congress to overcome in a bipartisan way. If it is framed as a question of medical research and medical breakthroughs and scientific advancement, it won''t fall purely on partisan lines. If members of Congress talk about their personal experiences, such as having a parent affected by Parkinson''s, then you could see even pro-life members voting in favour of a more expansive interpretation of stem-cell funding.”Johnson said that Congress could alter the wording of the Dickey-Wicker Amendment when passing the NIH budget for 2011 to remove the conflict. “You don''t have to get rid of the amendment completely, but you could rephrase it,” she said. She also commented that the public essentially supports embryonic stem-cell research. “The polls and surveys show the American public is morally behind there being some limited form of embryonic stem-cell research funded by federal money. They don''t favour cloning. There is not a huge amount of support for creating embryos from scratch for research. But there seems to be pretty wide support among the general public for the kind of embryonic stem-cell research that the NIH is currently funding.”In the end, however, the debate about public funding for stem-cell research is only part of the picture, given the role of private business and states. Glenn McGee, a professor at the Center for Practical Bioethics in Kansas City, MO, USA, and editor of the American Journal of Bioethics, commented that perhaps too much emphasis is being put on federal funding. He said that funding from states such as California and from industry—which are not restricted—has become a more important force than NIH funding. “We''re a little bit delusional if we think that this is a moment where the country is making a big decision about what''s going to happen with stem cells,” he said. “I think that ship has sailed.”     

Nodal Morphogens

Nodal signals belong to the TGF-β superfamily and are essential for the induction of mesoderm and endoderm and the determination of the left–right axis. Nodal signals can act as morphogens—they have concentration-dependent effects and can act at a distance from their source of production. Nodal and its feedback inhibitor Lefty form an activator/inhibitor pair that behaves similarly to postulated reaction–diffusion models of tissue patterning. Nodal morphogen activity is also regulated by microRNAs, convertases, TGF-β signals, coreceptors, and trafficking factors. This article describes how Nodal morphogens pattern embryonic fields and discusses how Nodal morphogen signaling is modulated.In his 1901 book “Regeneration,” Thomas Hunt Morgan speculated that “if we suppose the materials or structures that are characteristic of the vegetative half are gradually distributed from the vegetative to the animal half in decreasing amounts, then any piece of the egg will contain more of these things at one pole than the other” and “gastrulation depends on the relative amounts of the materials in the different parts of the blastula” (Morgan 1901). Although Morgan’s speculations referred to the sea urchin embryo, they foretold our current understanding of morphogen gradients in frog and fish development. Morgan’s “materials,” “structures,” and “things” are the Nodal signals that create a vegetal-to-animal activity gradient to regulate germ layer formation and patterning. This article discusses how Nodal signaling provides positional information to fields of cells. I first portray the components of the signaling pathway and describe the role of Nodal signals in mesendoderm induction and left–right axis specification. I then discuss how Nodal morphogen gradients are thought to be generated, modulated, and interpreted.     

The A to T of historical evidence

Geneticists and historians collaborated recently to identify the remains of King Richard III of England, found buried under a car park. Genetics has many more contributions to make to history, but scientists and historians must learn to speak each other''s languages.The remains of King Richard III (1452–1485), who was killed with sword in hand at the Battle of Bosworth Field at the end of the War of the Roses, had lain undiscovered for centuries. Earlier this year, molecular biologists, historians, archaeologists and other experts from the University of Leicester, UK, reported that they had finally found his last resting place. They compared ancient DNA extracted from a scoliotic skeleton discovered under a car park in Leicester—once the site of Greyfriars church, where Richard was rumoured to be buried, but the location of which had been lost to time—with that of a seventeenth generation nephew of King Richard: it was a match. Richard has captured the public imagination for centuries: Tudor-friendly playwright William Shakespeare (1564–1616) portrayed Richard as an evil hunchback who killed his nephews in order to ascend to the throne, whilst in succeeding years others have leapt to his defence and backed an effort to find his remains.The application of genetics to history is revealing much about the ancestry and movements of groups of humans, from the fall of the Roman Empire to ancient ChinaMolecular biologist Turi King, who led the Leicester team that extracted the DNA and tracked down a descendant of Richard''s older sister, said that Richard''s case shows how multi-disciplinary teams can join forces to answer history''s questions. “There is a lot of talk about what meaning does it have,” she said. “It tells us where Richard III was buried; that the story that he was buried in Greyfriars is true. I think there are some people who [will] try and say: “well, it''s going to change our view of him” […] It won''t, for example, tell us about his personality or if he was responsible for the killing of the Princes in the Tower.”The discovery and identification of Richard''s skeleton made headlines around the world, but he is not the main prize when it comes to collaborations between historians and molecular biologists. Although some of the work has focused on high-profile historic figures—such as Louis XVI (1754–1793), the only French king to be executed, and Vlad the Impaler, the Transylvanian royal whose patronymic name inspired Bram Stoker''s Dracula (Fig 1)—many other projects involve population studies. Application of genetics to history is revealing much about the ancestry and movements of groups of humans, from the fall of the Roman Empire to ancient China.Open in a separate windowFigure 1The use of molecular genetics to untangle history. Even when the historical record is robust, molecular biology can contribute to our understanding of important figures and their legacies and provide revealing answers to questions about ancient princes and kings.Medieval historian Michael McCormick of Harvard University, USA, commented that historians have traditionally relied on studying records written on paper, sheepskin and papyrus. However, he and other historians are now teaming up with geneticists to read the historical record written down in the human genome and expand their portfolio of evidence. “What we''re seeing happening now—because of the tremendous impact from the natural sciences and particularly the application of genomics; what some of us are calling genomic archaeology—is that we''re working back from modern genomes to past events reported in our genomes,” McCormick explained. “The boundaries between history and pre-history are beginning to dissolve. It''s a really very, very exciting time.”…in the absence of written records, DNA and archaeological records could help fill in gapsMcCormick partnered with Mark Thomas, an evolutionary geneticist at University College London, UK, to try to unravel the mystery of one million Romano-Celtic men who went missing in Britain after the fall of the Roman Empire. Between the fourth and seventh centuries, Germanic tribes of Angles, Saxons and Jutes began to settle in Britain, replacing the Romano-British culture and forcing some of the original inhabitants to migrate to other areas. “You can''t explain the predominance of the Germanic Y chromosome in England based on the population unless you imagine (a) that they killed all the male Romano-Celts or (b) there was what Mark called ‘sexual apartheid'' and the conquerors mated preferentially with the local women. [The latter] seems to be the best explanation that I can see,” McCormick said of the puzzle.Ian Barnes, a molecular palaeobiologist at Royal Holloway University of London, commented that McCormick studies an unusual period, for which both archaeological and written records exist. “I think archaeologists and historians are used to having conflicting evidence between the documentary record and the archaeological record. If we bring in DNA, the goal is to work out how to pair all the information together into the most coherent story.”Patrick Geary, Professor of Western Medieval History at the Institute for Advanced Study in Princeton, New Jersey, USA, studies the migration period of Europe: a time in the first millennium when Germanic tribes, including the Goths, Vandals, Huns and Longobards, moved across Europe as the Roman Empire was declining. “We do not have detailed written information about these migrations or invasions or whatever one wants to call them. Primarily what we have are accounts written later on, some generations later, from the contemporary record. What we tend to have are things like sermons bemoaning the faith of people because God''s wrath has brought the barbarians on them. Hardly the kind of thing that gives us an idea of exactly what is going on—are these really invasions, are they migrations, are they small military groups entering the Empire? And what are these ‘peoples'': biologically related ethnic groups, or ad hoc confederations?” he said.Geary thinks that in the absence of written records, DNA and archaeological records could help fill in the gaps. He gives the example of jewellery, belt buckles and weapons found in ancient graves in Hungary and Northern and Southern Italy, which suggest migrations rather than invasions: “If you find this kind of jewellery in one area and then you find it in a cemetery in another, does it mean that somebody was selling jewellery in these two areas? Does this mean that people in Italy—possibly because of political change—want to identify themselves, dress themselves in a new style? This is hotly debated,” Geary explained. Material goods can suggest a relationship between people but the confirmation will be found in their DNA. “These are the kinds of questions that nobody has been able to ask because until very recently, DNA analysis simply could not be done and there were so many problems with it that this was just hopeless,” he explained. Geary has already collected some ancient DNA samples and plans to collect more from burial sites north and south of the Alps dating from the sixth century, hoping to sort out kinship relations and genetic profiles of populations.King said that working with ancient DNA is a tricky business. “There are two reasons that mitochondrial DNA (mtDNA) is the DNA we wished to be able to analyse in [King] Richard. In the first instance, we had a female line relative of Richard III and mtDNA is passed through the female line. Fortunately, it''s also the most likely bit of DNA that we''d be able to retrieve from the skeletal remains, as there are so many copies of it in the cell. After death, our DNA degrades, so mtDNA is easier to retrieve simply due to the sheer number of copies in each cell.”Geary contrasted the analysis of modern and ancient DNA. He called modern DNA analysis “[…] almost an industrial thing. You send it off to a lab, you get it back, it''s very mechanical.” Meanwhile, he described ancient DNA work as artisanal, because of degeneration and contamination. “Everything that touched it, every living thing, every microbe, every worm, every archaeologist leaves DNA traces, so it''s a real mess.” He said the success rate for extracting ancient mtDNA from teeth and dense bones is only 35%. The rate for nuclear DNA is only 10%. “Five years ago, the chances would have been zero of getting any, so 10% is a great step forward. And it''s possible we would do even better because this is a field that is rapidly transforming.”But the bottleneck is not only the technical challenge to extract and analyse ancient DNA. Historians and geneticists also need to understand each other better. “That''s why historians have to learn what it is that geneticists do, what this data is, and the geneticists have to understand the kind of questions that [historians are] trying to ask, which are not the old nineteenth century questions about identity, but questions about population, about gender roles, about relationship,” Geary said.DNA analysis can help to resolve historical questions and mysteries about our ancestors, but both historians and geneticists are becoming concerned about potential abuses and frivolous applications of DNA analysis in their fields. Thomas is particularly disturbed by studies based on single historical figures. “Unless it''s a pretty damn advanced analysis, then studying individuals isn''t particularly useful for history unless you want to say something like this person had blue eyes or whatever. Population level studies are best,” he said. He conceded that the genetic analysis of Richard III''s remnants was a sound application but added that this often is not the case with other uses, which he referred to as “genetic astrology.” He was critical of researchers who come to unsubstantiated conclusions based on ancient DNA, and scientific journals that readily publish such papers.…both historians and geneticists are becoming concerned about potential abuses or frivolous applications of DNA analysis in their fieldsThomas said that it is reasonable to analyse a Y chromosome or mtDNA to estimate a certain genetic trait. “But then to look at the distribution of those, note in the tree where those types are found, and informally, interpretively make inferences—“Well this must have come from here and therefore when I find it somewhere else then that means that person must have ancestors from this original place”—[…] that''s deeply flawed. It''s the most widely used method for telling historical stories from genetic data. And yet is easily the one with the least credibility.” Thomas criticized such facile use of genetic data, which misleads the public and the media. “I suppose I can''t blame these [broadcast] guys because it''s their job to make the programme look interesting. If somebody comes along and says ‘well, I can tell you you''re descended from some Viking warlord or some Celtic princess'', then who are they to question.”Similarly, the historians have reservations about making questionable historical claims on the basis of DNA analysis. Geary said the use of mtDNA to identify Richard III was valuable because it answered a specific, factual question. However, he is turned off by other research using DNA to look at individual figures, such as a case involving a princess who was a direct descendant of the woman who posed for Leonardo Da Vinci''s Mona Lisa. “There''s some people running around trying to dig up famous people and prove the obvious. I think that''s kind of silly. There are others that I think are quite appropriate, and while is not my kind of history, I think it is fine,” he said. “The Richard III case was in the tradition of forensics.”…the cases in which historians and archaeologists work with molecular biologists are rare and remain disconnected in general from the mainstream of historical or archaeological researchNicola Di Cosmo, a historian at the Institute for Advanced Study, who is researching the impact of climate change on the thirteenth century Mongol empire, follows closely the advances in DNA and history research, but has not yet applied it to his own work. Nevertheless, he said that genetics could help to understand the period he studies because there are no historical documents, although monumental burials exist. “It is important to get a sense of where these people came from, and that''s where genetics can help,” he said. He is also concerned about geneticists who publish results without involving historians and without examining other records. He cited a genetic study of a so-called ‘Eurasian male'' in a prestige burial of the Asian Hun Xiongnu, a nomadic people who at the end of the third century B.C. formed a tribal league that dominated most of Central Asia for more than 500 years. “The conclusion the geneticists came to was that there was some sort of racial tolerance in this nomadic empire, but we have no way to even assume that they had any concept of race or tolerance.”Di Cosmo commented that the cases in which historians and archaeologists work with molecular biologists are rare and remain disconnected in general from the mainstream of historical or archaeological research. “I believe that historians, especially those working in areas for which written records are non-existent, ought to be taking seriously the evidence churned out by genetic laboratories. On the other hand, geneticists must realize that the effectiveness of their research is limited unless they access reliable historical information and understand how a historical argument may or may not explain the genetic data” [1].Notwithstanding the difficulties in collaboration between two fields, McCormick is excited about historians working with DNA. He said the intersection of history and genomics could create a new scientific discipline in the years ahead. “I don''t know what we''d call it. It would be a sort of fusion science. It certainly has the potential to produce enormous amounts of enormously interesting new evidence about our human past.”     

From DNA enzymes to cone snail venom: the work of Baldomero M. Olivera

Processivity of DNA Exonucleases (Thomas, K. R., and Olivera, B. M. (1978) J. Biol. Chem. 253, 424–429)Neuronal Calcium Channel Inhibitors. Synthesis of ω-Conotoxin GVIA and Effects on ⁴⁵Ca Uptake by Synaptosomes (Rivier, J., Galyean, R., Gray, W. R., Azimi-Zonooz, A., McIntosh, J. M., Cruz, L. J., and Olivera, B. M. (1987) J. Biol. Chem. 262, 1194–1198)The two papers being recognized here as JBC Classics speak to the journeys Baldomero “Toto” Olivera at the University of Utah has made in his life. A director of a program project funded by the National Institute of General Medical Sciences and a professor at the Howard Hughes Medical Institute, Olivera''s papers highlight how doing research in two different countries ultimately influenced his focus and contributions to molecular biology and biochemistry.Open in a separate windowBaldomero “Toto” Olivera. Photo courtesy of Olivera.Olivera began his career as a DNA biophysical chemist and enzymologist. He arrived in the United States in the 1960s to do his graduate work at the California Institute of Technology after completing a bachelor''s degree in chemistry in the Philippines. He joined the laboratory of Norman Davidson to study the biophysical chemistry of DNA. When Olivera was ready to graduate with his Ph.D. degree, Davidson suggested that Olivera go to I. Robert Lehman''s laboratory at Stanford University for his postdoctoral training. “He knew it was my intention to return to the Philippines,” recalls Olivera. Davidson felt it would be easier for Olivera to study DNA enzymology, rather than biophysical chemistry, in a Philippine academic setting because the field did not necessarily demand expensive and sophisticated instrumentation.Olivera followed his thesis advisor''s suggestion and, as a result, became an expert in DNA enzymology, including exonucleases, a large class of DNA-degrading enzymes. The first JBC paper recognized here as a Classic was published in 1978 as Olivera was starting out as an independent researcher. In it, Olivera and his first graduate student, Kirk Thomas, investigated whether or not exonuclease I, first discovered in Escherichia coli by Lehman, and other exonucleases of E. coli were processive. This was at a time when little was known about nucleic acid enzymes: restriction enzymes were just starting to gain traction, and genome sequencing was far from reality. Olivera explains that no one had given much thought to how exonucleases functioned. “The significance of this paper was that it showed that the enzymes that we examined were very different using a new parameter processivity that had never been assessed for exonucleases,” he says.Olivera and Thomas designed an assay that was based on a synthetic nucleic acid chain that contained ³H on one end and ³²P on the other. Researchers knew that exonucleases selected either the 5′- or 3′-end of the DNA to start chewing. The rationale of the Thomas and Olivera assay was that if the enzyme dissociated after every single catalytic event, one label, either the ³H or ³²P, would come off the polymer. However, if the enzyme clung to the polymer and kept chewing until the whole polymer was degraded, both radioactive labels would appear simultaneously in solution.Open in a separate windowOlivera with his first graduate student, Kirk Thomas. Photo courtesy of Olivera.Thomas and Olivera demonstrated that of the eight exonucleases they tested, only the E. coli exonuclease I and λ-exonuclease were processive, meaning that once they got started, they kept on cutting the same piece of DNA before dissociating. The others, such as the spleen and T7 exonucleases, were not processive and frequently came off the DNA.Lehman explains that at the time of this JBC paper, “methods had not yet been developed to measure quantitatively the processivity of either a DNA polymerase or a DNA exonuclease. Their paper made an important contribution to the field of DNA enzymology by describing for the first time a quantitative method for doing so and applied it to eight different DNA exonucleases, an enzymological tour de force.”The second paper highlighted as a JBC Classic was published ten years later and shows a shift in Olivera''s career. The article concerns the synthesis of a peptide found in the venom of the cone snail Conus geographus, which is indigenous to the Indo-Pacific region. All 700 types of cone snails have a special tooth that they use like a harpoon. A venom gland attached to the tooth releases the poisonous peptides to paralyze or even kill prey. These snails have to be handled with great care or not handled at all. Some can sting and cause pain like bees, but C. geographus can kill humans when it stings.There is no scientific connection between DNA enzymes and snail venom. Olivera explains that when he had returned to the Philippines as an assistant professor in the College of Medicine at the University of the Philippines, his laboratory “had absolutely no equipment. It was clear I wasn''t going to be very competitive in DNA replication [research], so we decided we''d find a project that we could start without any equipment. I collected shells as a kid, so I knew about this particular snail that killed people. I had purified enzymes as a post-doc and figured I could purify toxins by injecting them into mice, which didn''t require any equipment at all.”Olivera''s group was soon isolating and characterizing peptides from the cone snail venom. The peptides are known as conotoxins. In doing so, Olivera established the field of conotoxin research, which had a significant impact on fundamental research and medicine. For example, a peptide isolated by Olivera''s group has been approved as a drug for severe pain that cannot be relieved by morphine.Olivera had part-time appointments in the United States while maintaining his full-time position in the Philippines. He first began as a visiting associate professor at Kansas State University and later at the University at Utah. “I would spend seven or eight months in the Philippines and five or four months in the U.S,” he says. Olivera became a full-time member of the faculty at the University of Utah in the 1970s after political and economic upheaval in the Philippines over Ferdinand Marcos'' rule made Olivera decide to return full-time to the United States.Open in a separate windowConus snails. Photo courtesy of Olivera.Open in a separate windowConus snail attacks a fish. Photo courtesy of Olivera.The toxins made by the Conus snails are highly specific for particular targets in the nervous system, such as ion channels. For example, the μ-conotoxins hit sodium receptor ion channels, and ω-conotoxins (one of which, ω-GVIA, is described in this JBC Classic) bind to neuronal calcium channels to inhibit calcium uptake at the presynaptic junction and shut down biochemical signaling at certain synapses.ω-Conotoxin GVIA is a 27-amino acid peptide originally called the “shaker” peptide because it made mice shake. “A number of physiological experiments were done to suggest that it acted at synapses, potentially on calcium channels,” says Olivera. “The importance of this paper is that for the first time the peptide was chemically synthesized and became available to the whole neuroscience community.”The neuroscience community desperately needed this peptide. Up to this point, neuroscientists relied on dihydropyridines to study voltage-gated calcium channels. However, these dihydropyridines had confusing effects on neuronal voltage-gated calcium channels, which made data interpretation difficult. With ω-conotoxin GVIA as a synthetic peptide, neuroscientists now had a molecular tool that clearly targeted a very specific type of neuronal voltage-gated calcium channel.The peptide was short enough to be amenable to synthesis, and Olivera is grateful to his collaborator, Jean Rivier, who was an expert in synthesizing neuropeptides, for the successful synthesis of this peptide. The peptide had only 27 amino acids but contained three disulfide bonds, “so there were fifteen possible isomers,” recalls Olivera. “You had to get the cross-linking right to end up with the biologically active isomer.”The advantage was that Olivera and colleagues had purified the native peptide, so they could compare their synthesis attempts with the native molecule. “At the beginning, we didn''t even know what the true disulfide bonding was, so we did the work qualitatively to just show the synthetic material and native material co-eluted in a column.” The investigators later established how the disulfide bonds were arranged. Rivier, Olivera, and the rest of the team went on to show that their synthetic peptide behaved just like the natural one in inhibiting calcium entry at chicken synaptosomes and was biologically active.John Exton at Vanderbilt University says “The conotoxins have proved to be extremely important molecular probes in neuroscience in defining functional roles for many receptors and ion channels.”When the paper was published, Olivera was deluged with requests for the peptide. Rivier had been able to synthesize a sizeable amount, and because it was active at subpicomolar concentrations, a little bit of it went a long way. Olivera was able to distribute the peptide, and eventually, several commercial enterprises got into the business of producing and supplying it.“I believe there is something on the order of two thousand studies in the literature using this particular peptide,” says Olivera. “It''s interesting that there are hundreds of thousands of peptides in Conus venom that we call conotoxins. But among physiologists, if you say conotoxin, this is the peptide they think of because this is the one that''s most widely used.” In fact, points out Olivera, when the neuronal calcium channel was purified eight years later, it was actually called the conotoxin receptor.     

Use of lipid lowering drugs for primary prevention of coronary heart disease: meta-analysis of randomised trials

ObjectiveTo summarise the effect of primary prevention with lipid lowering drugs on coronary heart disease events, coronary heart disease mortality, and all cause mortality.DesignMeta-analysis.IdentificationSystematic search of the Medline database from January 1994 to June 1999 for English language studies examining drug treatment for lipid disorders (use of the MeSH terms “hyperlipidemia” and “anticholesteremic agents,” keyword searches for individual drug names, and a search strategy for identifying randomised trials to capture relevant articles); identification of older studies through systematic reviews and hand search of bibliographies.ResultsFour studies met eligibility criteria. Drug treatment reduced the odds of a coronary heart disease event by 30% (summary odds ratio 0.70, 95% confidence interval 0.62 to 0.79) but not the odds of all cause mortality (0.94, 0.81 to 1.09). When statin drugs were considered alone, no substantial differences in results were found.ConclusionsTreatment with lipid lowering drugs lasting five to seven years reduces coronary heart disease events but not all cause mortality in people with no known cardiovascular disease.