Evolving concepts of tumor heterogeneity

Past and recent findings on tumor heterogeneity have led clinicians and researchers to broadly define cancer development as an evolving process. This evolutionary model of tumorigenesis has largely been shaped by seminal reports of fitness-promoting mutations conferring a malignant cellular phenotype. Despite the major clinical and intellectual advances that have resulted from studying heritable heterogeneity, it has long been overlooked that compositional tumor heterogeneity and tumor microenvironment (TME)-induced selection pressures drive tumor evolution, significantly contributing to tumor development and outcomes of clinical cancer treatment. In this review, we seek to summarize major milestones in tumor evolution, identify key aspects of tumor heterogeneity in a TME-dependent evolutionary context, and provide insights on the clinical challenges facing researchers and clinicians alike.     

Evolving concepts of thyroid hormone action.

The past 25 years have witnessed dramatic changes in our concepts of thyroid hormone action. Progress in this area was made possible by the recognition of the central role of triiodothyronine in mediating thyroid hormone action and the recognition of specific nuclear receptors in target tissues as demonstrated by displacement studies. The cloning of the receptors and receptor variants has enabled investigators to undertake detailed analyses of the biochemical events which underlie the physiological and pathological action of thyroid hormone.     

The dissociation of human deoxyhemoglobin and mixed state hemoglobin into monomer.

The experimental hybridizations between fully deoxygenated human and canine hemoglobins and between half-ligated human hemoglobin and canine cyanomethemoglobin show that new two hybrids in addition to the parent hemoglobins were clearly formed in the mixtures at the high concentration of KI. Thus, human deoxyhemoglobin under the present conditions is in an equilibrium with three species, tetramer in equilibrium dimer in equilibrium monomer. This means that the deoxyhemoglobin is in R-T equilibrium, and shifts considerably toward the R state under the present conditions. On the other hand, the half-ligated hemoglobin in 1.5 M KI becomes much more dissociable than the deoxy T state and appears to be completely transformed into the R state. Nevertheless, the co-operativity, n, is still high (n = 2.0).     

Evolving concepts in cancer therapy through targeting sphingolipid metabolism

Traditional methods of cancer treatment are limited in their efficacy due to both inherent and acquired factors. Many different studies have shown that the generation of ceramide in response to cytotoxic therapy is generally an important step leading to cell death. Cancer cells employ different methods to both limit ceramide generation and to remove ceramide in order to become resistant to treatment. Furthermore, sphingosine kinase activity, which phosphorylates sphingosine the product of ceramide hydrolysis, has been linked to multidrug resistance, and can act as a strong survival factor. This review will examine several of the most frequently used cancer therapies and their effect on both ceramide generation and the mechanisms employed to remove it. The development and use of inhibitors of sphingosine kinase will be focused upon as an example of how targeting sphingolipid metabolism may provide an effective means to improve treatment response rates and reduce associated treatment toxicity. This article is part of a Special Issue entitled Tools to study lipid functions.     

Evolving concepts in plant glycolysis: two centuries of progress

Glycolysis, the process responsible for the conversion of monosaccharides to pyruvic acid, is a ubiquitous feature of cellular metabolism and was the first major biochemical pathway to be well characterized. Although the majority of glycolytic enzymes are common to all organisms, the past quarter of a century has revealed that glycolysis in higher plants possesses numerous distinctive features. Research in the nineteenth century established convincingly that plants carry out alcoholic fermentation under anaerobic conditions. In 1878, Wilhelm Pfeffer asserted that a non-oxygen-requiring ‘intramolecular respiration’ was involved in the aerobic respiration of plants. Between 1900 and 1950 it was demonstrated that plants metabolize sugar and starch by a glycolytic pathway broadly similar to that of yeasts and muscle tissue. In 1948, the first purification and characterization of a plant glycolytic enzyme, aldolase, was published by Paul Stumpf. By 1960 the presence of each of the 10 enzymes of glycolysis, presumed at the time to be located in the cytosol, had been confirmed in higher plants. Shortly after 1960 it was shown that the mechanism of glycolytic regulation in plants had features in common with that of animals and yeasts, especially as regards the important role played by the enzyme phosphofructokinase; but important regulatory properties peculiar to plants were soon demonstrated. In the last 30 years, higher-plant glycolysis has been found to exhibit a number of additional characteristics peculiar to plant systems. One conspicuous feature of plant glycolysis, discovered in the 1970s, is the presence of a complete or nearly complete sequence of glycolytic enzymes in plastids, distinct and spatially separated from the glycolytic enzymes located in the cytosol. Plastidic and cytosolic isoenzymes of glycolysis have been shown to differ in their kinetic and regulatory properties, suggesting that the two pathways are independently regulated. Since about 1980 it has become increasingly clear that the cytosolic glycolysis of plants may make use of several enzymes other than the conventional ones found in yeasts, muscle tissue and plant plastids: these enzymes include a pyrophosphate-dependent phosphofructokinase, a non-reversible and nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, a phosphoenolpyruvate phosphatase (vacuolar location) and a three-enzyme sequence able to produce pyruvate from phosphoenolpyruvate avoiding the pyruvate-kinase step. These non-conventional enzymes may catalyze glycolysis in the plant cytosol especially under conditions of metabolic stress. Experiments on transgenic plants possessing significantly elevated or reduced (reduced to virtually nil in some cases) levels of glycolytic enzymes are currently playing an important part in improving our understanding of the regulation of plant glycolysis; such experiments illustrate an impressive degree of flexibility in the pathway's operation. Plant cells are able to make use of enzymes bypassing or substituting for several of the conventional enzymic steps in the glycolytic pathway; the extent and conditions under which these bypasses operate are the subject of current research. The duplication of the glycolytic pathway in plants and the flexible nature of the pathway have possibly evolved in relation to the crucial biosynthetic role played by plant glycolysis beyond its function in energy generation; both functions must proceed if a plant is to survive under varying and often stressful environmental or nutritional conditions.     

Evolving concepts on the pathogenic mechanisms of aniridia related keratopathy

Heterozygosity for PAX6 deficiency (PAX6+/-) results in aniridia. Corneal changes in aniridia-related keratopathy (ARK) include corneal vascular pannus formation, conjunctival invasion of the corneal surface, corneal epithelial erosions and epithelial abnormalities, which eventually result in corneal opacity and contribute to visual loss. Corneal changes in aniridia have been attributed to congenital deficiency of corneal limbal stem cells. The aim of this paper is to review the potential mechanisms that may underlie the pathogenesis of aniridia related keratopathy. Current evidence, based on clinical observations and an animal model of aniridia suggest that the proliferative potential of the corneal limbal stem cells may not primarily be impaired. The corneal changes in aniridia may be related to an abnormality within the limbal stem cell niche. The mechanisms underlying progressive corneal pathology in aniridia appear multi-factorial and include: (1) abnormal corneal healing responses secondary to anomalous extracellular matrix metabolism; (2) abnormal corneal epithelial differentiation leading to fragility of epithelial cells; (3) reduction in cell adhesion molecules in the PAX6 heterozygous state, rendering the cells susceptible to natural shearing forces; and (4) conjunctival and corneal changes leading to the presence of cells derived from conjunctiva on the corneal surface.     

On the reality of transition state dissociation constants

Transition state dissociation constants are currently considered, utilizing stopped flow equipment. The underlying theory is briefly reviewed, relating the ideas to steady state kinetics of enzyme systems. The ideas are further analyzed under the consideration of chemical relaxation. Test conditions are described which would allow an investigation of the concepts of transition state dissociation constants by chemical relaxation techniques. A discussion concerning the way in which the concepts of transition state dissociation constants relate to other theories which assume short-lived, but real, dissociation constants is included. The theory is rigorously analyzed (in a second part), revealing the nature of the assumption of a transition state dissociation constant: While they may be written in a formal manner, they are not based on reality—on kinetic grounds direct interconversions between transition states are practically impossible. This applies also to transition state dissociation constants involving protons.     

Evolving concepts of the epidemiology, diagnosis, and therapy of Mycobacterium tuberculosis infection

Tuberculosis in the United States is evolving in nearly all respects--epidemiology, diagnosis, treatment, and prophylaxis. Today a relatively larger segment of the population has predisposing factors to infection with tuberculosis. There is a greater percentage of people who are elderly, who have immigrated from countries endemic for tuberculosis, or who are immunosuppressed due to medications necessary for other conditions, because of malignancies, or because of infection with HIV. Skin test classifications have been revised to give different meanings to different-sized areas of induration at the injection site for defined populations. More sensitive, more specific, and faster diagnostic laboratory tests for tuberculosis are being developed. Short-course chemotherapy of from six to nine months is now accepted as standard treatment, regardless of exactly which of the proven regimens of antibiotics or accepted lengths of therapy is used. Patient compliance is improved with the shorter courses both for treatment and for prophylaxis. Better compliance with therapy results in better treatment outcomes of infections with Mycobacterium tuberculosis.     

Temperature-induced dissociation of protein aggregates: accessing the denatured state

The thermal denaturation of lysozyme and ribonuclease A (RNase A) under reducing and nonreducing conditions at neutral pH has been monitored by Fourier transform infrared spectroscopy. In the absence of the reductant, lysozyme and RNase A undergo apparent three- and two-state denaturation, respectively, as observed from the conformation-sensitive amide I' band. For both proteins the hydrogen-deuterium exchange takes place at lower temperatures than the main denaturation temperatures, suggesting that a transient denaturation mechanism occurs. The observed transition at 51.2 degrees C during the denaturation of lysozyme is attributed to this transient effect, rather than to the loss of tertiary structure. Under reducing conditions lysozyme aggregates during the heating phase, whereas RNase A shows only a minor aggregation, which further increases during the cooling step. The reduced stability of both proteins can be correlated with the transient denaturation behavior, which is also suggested to be involved in protein aggregation at physiologically relevant temperatures. In addition, it is shown that when the temperature is further increased, the amorphous aggregates dissociate. Comparison of the dissociated states with the denatured states obtained under nonreducing conditions indicates that these states have the same conformation. By using a two-dimensional correlation analysis we were able to show that the dissociation is preceded by a conformational change. It is argued that this extends to other types of perturbation.     

Current concepts in reprogramming somatic cells to pluripotent state

Recently considerable interests have been roused in nuclear reprogramming by somatic cell nuclear transfer using an egg cytoplasm and/or by other means, such as fusion, cell extracts treatment and genes transfections. However, the very mechanism of reprogramming still remains elusive. Epigenetic modifications, which play a significant role in normal mammalian development in vivo is also involved in the process of reprogramming in vitro. The latter shares some of the other features observed in nuclear reprogramming in vivo. In this review, we discuss the main epigenetic changes involved in nuclear reprogramming and currently available approaches to achieve nuclear reprogramming in vitro and its future prospects.     

Evolving concepts of the intrarenal renin-angiotensin system in health and disease: contributions of molecular biology.

Previous physiological and biochemical studies suggest the existence of an endogenous renin-angiotensin system (RAS) in the kidney. However, these data cannot exclude the contribution of the circulating RAS. Proof of the local synthesis of RAS components in the kidney has been obtained recently through the use of molecular biological techniques. Using Northern blot analysis, we have demonstrated the intrarenal expression of renin, angiotensinogen, and angiotensin-converting enzyme messenger RNAs. Employing in situ hybridization histochemistry, we have localized the intrarenal tissue sites of renin and angiotensinogen messenger RNA synthesis. Renin gene expression was found in cells of the juxtaglomerular apparatus. Angiotensinogen mRNA was primarily produced in the proximal convoluted tubule with lesser amounts in glomerular tufts and vasculature. These findings led us to hypothesize that the proximal tubule is a major site of renal Ang II synthesis and that locally synthesized Ang II might directly modulate tubular function. Both genes are subject to feedback regulation. Our studies showed that Ang II exerted a stimulatory effect on angiotensinogen but a negative feedback effect on renin gene expression. Dietary NaCl restriction stimulated the expression of both genes, although the onset of renin gene activation required more prolonged sodium chloride restriction. Furthermore, our data indicated that the sodium cation, irrespective of the anion, was primarily important in regulating renal angiotensinogen mRNA levels. Our studies also showed altered intrarenal renin or angiotensinogen expressions in pathophysiological states, e.g. in experimental heart failure and the spontaneously hypertensive rat. Taken together, these data support the existence of a intrarenal RAS and suggest its potential roles in the regulation of renal function in health and disease.     

The dissociation of insulin from human insulin antibodies

The dissociation of insulin from human insulin antibodies has been investigated using a technique that is rapid and does not require addition of excess unlabelled insulin. A slow (k₁ = 2·1^?3 min^?1 and a fast (k₂ = 4·10^?2 min^?1) dissociating antibody component were identified in all studies. These have been shown to correspond, respectively, to the high and low affinity antibody components of equilibrium binding studies. The range of k₁ and k₂ values and their response to temperature change is small. Insulin resistance and stability of diabetes are not related to properties of antibody dissociation. Dissociation is faster in the presence of high (6–850 nM) insulin concentration due to increased binding to the fast dissociating component without change in the dissociation rate constants. When incubation time is increased beyond achivement of maximal binding there is a time-dependent rise in binding to the slow dissociating component, with a concomitant fall in k₁. The traditional concept that equilibrium is established at maximum binding requires further examination.     

Functional protection of human haemoglobin against protein dissociation

The spectroscopic and functional properties of human adult haemoglobin are clearly disrupted by concentrations of urea > 0.4 M. This disruption of structure and function is completely obviated by the presence of 0.2 M trimethylamine N-oxide (TMAO). Spectroscopic data suggest that TMAO prevents urea-induced production of high-spin haem. Functional analysis shows that TMAO exerts its influence by counteracting urea-induced destabilisation of the T state of the haemoglobin protein. Further studies show, however, that TMAO is not able to exert any such stabilising influences in the presence of high concentrations of typical organic solvent denaturants.     

Transient state kinetic analysis of the ATP-induced dissociation of the dynein-microtubule complex

The kinetics of ATP-induced dissociation of dynein from the dynein-microtubule complex has been investigated by stopped flow light scattering methods. The addition of ATP to the dynein-microtubule complex induced a large, rapid decrease in light scattering followed by a smaller and much slower decrease. The fast light scattering change was shown to be a measure of the ATP-induced dissociation of dynein from the dynein-microtubule complex and was distinguished from microtubule disassembly by several criteria. (i) The fast reaction occurred over a period of milliseconds and the rate was a function of the ATP concentration, whereas, the slow reaction occurred over a period of several seconds and was independent of ATP concentration; (ii) the amplitude of the fast reaction was directly proportional to the amount of dynein bound to the microtubule lattice; and (iii) only the slow phase was inhibited by the addition of the microtubule-stabilizing drug, taxol. The rate of ATP-induced dissociation of dynein from the microtubule increased linearly with increasing ATP concentration to give an apparent second order rate constant for ATP binding equal to k1 = 4.7 X 10(6) M-1 s-1 according to the following pathway: (formula; see text) where M X D represents the dynein-microtubule complex and D represents dynein. The loss of signal amplitude at high ATP concentration provided a minimum estimate for the rate of dissociation of the ternary complex (M X D X ATP) equal to kd greater than 1000 s-1. Thus, the dynein-microtubule system is similar to actomyosin in that ATP induces an extremely rapid dissociation of dynein from the microtubule.     

The use of transition state acid dissociation constants in pH-dependent enzyme kinetics

It is shown that the variation of reaction rate with pH in systems where several protonated forms of the reactants appear to be kinetically active may be expressed most economically in terms of transition state acid dissociation constants. The advantages of this approach are described in relation to the formal analysis of experimental data with regard to both simple and complex reactions and the satisfaction of the principle of microscopic reversibility. Ligand binding to metmyoglobin is used to illustrate the value of the approach in searching for detailed mechanistic explanations.     

Emerging concepts of self-organization and the living state. Special issue

This special issue present papers that examine the concept of self-organization in the origin of life. Concepts explored include chaos and order in open systems, the origin of biochemical organization, non-cellular phases of life on clay, biodynamics necessary for the emergence of energy consumers, molecular evolution, order in self-oscillators, self-organization of semi-conductor physics, self-organizing behavior of microtubules in the cytoskeleton, biogenesis and physics, perceptive funcion, and an overview of the experimental realization of artificial intelligence.     

The calcium-induced dissociation of human plasma clotting Factor XIII

1. Large quantities of human Factor XIII were prepared from ethanol precipitates of outdated human plasma. 2. Material homogeneous after chromatography on DEAE-cellulose was further resolved into two proteins, A and B, after filtration on Sepharose 6B. 3. Protein A has a molecular weight of 350000 and a subunit structure a(2)b(2) and is activated by thrombin and calcium. Protein B is inactive and probably has a subunit structure b(2). 4. Calcium causes protein A, after thrombin cleavage, to fragment to give protein B and a protein, containing only a' subunits, which is catalytically active. The latter protein slowly forms a misty precipitate which is still active and not cross-linked covalently. This confirms the suggestion of Schwartz et al. (1971) that catalytic activity is only associated with a' subunits. 5. Iodoacetate, which inhibits the enzyme, does not inhibit dissociation and aggregation of protein A. 6. The existence of two proteins and the fragmentation are possible explanations for the wide range of molecular weights given for Factor XIII in the literature.     

Kinetic analysis of amyloid protofibril dissociation and volumetric properties of the transition state

We present here the first detailed kinetic analysis of the dissociation reaction of amyloid protofibrils by utilizing pressure as an accelerator of the reaction. The experiment is carried out on an excessively diluted typical protofibril solution formed from an intrinsically denatured disulfide-deficient variant of hen lysozyme with Trp fluorescence as the reporter in the pressure range 3-400 MPa. From the analysis of the time-dependent fluorescence decay and the length distribution of the protofibrils measured on atomic force microscopy, we conclude that the protofibril grows or decays by attachment or detachment of a monomer at one end of the protofibril with a monomer dissociation rate independent of the length of the fibril. Furthermore, we find that the dissociation reaction is strongly dependent on pressure, characterized with a negative activation volume DeltaV(odouble dagger) = -50.5 +/- 1.60 ml mol(-1) at 0.1 MPa and with a negative activation compressibility Deltakappa(double dagger) = -0.013 +/- 0.001 ml mol(-1) bar(-1) or -0.9 x 10(-6) ml g(-1) bar(-1). These results indicate that the protofibril is a highly compressible high-volume state, but that it becomes less compressible and less voluminous in the transition state, most probably due to partial hydration of the existing voids. The system eventually reaches the lowest-volume state with full hydration of the monomer in the dissociated state.     

Concentration-dependent dissociation/association of human prostatic acid phosphatase

The apparent molecular mass of human prostatic acid phosphatase (PAP) was estimated over a wide range of enzyme concentrations using equilibrium centrifugation in the Airfuge tabletop ultra-centrifuge. We show that the average mass of all active PAP species steeply increases at enzyme concentrations around 100 nM. The data indicate that at lower concentrations, active monomer prevail, whereas at concentrations above 100 nM, PAP active dimers are formed. These findings were confirmed by measurements of fluorescence emission intensity as a function of enzyme concentration. A shift of the normalized PAP fluorescence intensity around 100 nM independently indicates that a major structural change of the PAP protein occurs in that range of concentrations. From these findings, we conclude that in dilute solutions, several active PAP species exist, which are involved in concentration-dependent dissociation/association equilibria.