Liquid chromatographic assay of phenothiazine, thioxanthene and butyrophenone neuroleptics and antihistamines in blood and plasma with conventional and radial compression columns and UV and electrochemical detection

An assay strategy for determining a wide range of phenothiazine, thioxanthene and butyrophenone neuroleptics and antihistamines both alone and in combination in blood and plasma is described. The general method employs liquid chromatography with both conventional and radial compression nitrile bonded columns. Detection is by ultraviolet absorption spectrophotometry or by amperometry depending on the concentrations to be measured. Ultraviolet absorption is suitable down to 10 ng/ml. Below this level amperometry is preferable. The various compounds are used as internal standards for each other. The lower limit of detection is approximately 0.1 ng ml⁻¹ with a 10-ml sample. The within-run coefficient of variation is a maximum of 7.3%.     

HIFs and tumors--causes and consequences

For most organisms oxygen is essential fo life. When oxygen levels drop below those required to maintain the minimum physiological oxygen requirement of an organism or tissue it is termed hypoxia. To counter act possible deleterious effects of such a state, an immediate molecular response is initiated causing adaptation responses aimed at cell survival. This response is mediated by the hypoxia-inducible factor-1 (HIF-1), which is a heterodimer consisting of an alpha- and a beta-subunit. HIF-1 alpha protein is stabilized under hypoxic conditions and therefore confers selectivity to this response. Hypoxia is characteristic of tumors, mainly because of impaired blood supply resulting from abnormal growth. Over the past few years enormous progress has been made in the attempt to understand how the activation of the physiological response to hypoxia influences neoplastic growth. In this review some aspects of HIF-1 pathway activation in tumors and the consequences for pathophysiology and treatment of neoplasia are discussed.     

Mechanisms and function of flower and inflorescence reversion

Flower and inflorescence reversion involve a switch from floral development back to vegetative development, thus rendering flowering a phase in an ongoing growth pattern rather than a terminal act of the meristem. Although it can be considered an unusual event, reversion raises questions about the nature and function of flowering. It is linked to environmental conditions and is most often a response to conditions opposite to those that induce flowering. Research on molecular genetic mechanisms underlying plant development over the last 15 years has pinpointed some of the key genes involved in the transition to flowering and flower development. Such investigations have also uncovered mutations which reduce floral maintenance or alter the balance between vegetative and floral features of the plant. How this information contributes to an understanding of floral reversion is assessed here. One issue that arises is whether floral commitment (defined as the ability to continue flowering when inductive conditions no longer exist) is a developmental switch affecting the whole plant or is a mechanism which assigns autonomy to individual meristems. A related question is whether floral or vegetative development is the underlying default pathway of the plant. This review begins by considering how studies of flowering in Arabidopsis thaliana have aided understanding of mechanisms of floral maintenance. Arabidopsis has not been found to revert to leaf production in any of the conditions or genetic backgrounds analysed to date. A clear-cut reversion to leaf production has, however, been described in Impatiens balsamina. It is proposed that a single gene controls whether Impatiens reverts or can maintain flowering when inductive conditions are removed, and it is inferred that this gene functions to control the synthesis or transport of a leaf-generated signal. But it is also argued that the susceptibility of Impatiens to reversion is a consequence of the meristem-based mechanisms controlling development of the flower in this species. Thus, in Impatiens, a leaf-derived signal is critical for completion of flowering and can be considered to be the basis of a plant-wide floral commitment that is achieved without accompanying meristem autonomy. The evidence, derived from in vitro and other studies, that similar mechanisms operate in other species is assessed. It is concluded that most species (including Arabidopsis) are less prone to reversion because signals from the leaf are less ephemeral, and the pathways driving flower development have a high level of redundancy that generates meristem autonomy even when leaf-derived signals are weak. This gives stability to the flowering process, even where its initiation is dependent on environmental cues. On this interpretation, Impatiens reversion appears as an anomaly resulting from an unusual combination of leaf signalling and meristem regulation. Nevertheless, it is shown that the ability to revert can serve a function in the life history strategy (perenniality) or reproductive habit (pseudovivipary) of many plants. In these instances reversion has been assimilated into regular plant development and plays a crucial role there.     

Convection and diffusion in tissues and tissue cultures

The relative importance of transport by diffusion and convection in permeable tissues is investigated in three-dimensional structures. The transport of a solute takes place from a number of sources embedded in the tissue, to a number of sinks similarly embedded. An enhancement factor E is defined to be the ratio of the transport rate in the presence of a pressure difference between the sources and sinks, to the transport rate without a pressure difference. It is shown that E is a unique function of a parameter W, which characterizes the properties of the tissue and the pressure difference. This relation is independent of the number or the geometries of the sources and sinks.This relation is compared with experimental data obtained in a hollow fiber tissue culture device, with two sets of hollow fibers kept at different pressures. This relation is also used to estimate the importance of convection in vivo for a wide range of mammalian tissues and solute molecules.     

Learning and imprinting in stationary and non-stationary environment

The performance of the extended Bush-Mosteller learning and imprinting scheme developed previously is studied for stationary and non-stationary stochastic environments. As a performance criterion the average missing information level is chosen. For a stationary environment the approximate time course of the latter is derived and discussed, an exact symmetry in the performance of learning and imprinting schemes is proved, and the biological advantage of imprinting processes, with respect to energy consumption, is pointed out. For a non-stationary environment the performance of proper learning schemes is shown to be superior to imprinting processes, as the adaptability of the latter to novel environmental properties decreases exponentially in time. The optimal memory range of a learning system is calculated as a function of the time span during which the environment changes significantly and of the mean amplitude with which these changes occur.Supported by the Deutsche Forschungsgemeinschaft and the Humboldt Foundation.Fellow of the Humboldt Foundation; on leave of absence from the University of Poona, India.     

SPR技术与功能基因组和蛋白质组研究

表面等离子体共振(surface plasmon resonance,SPR)依据光学—介质相互作用原理建立,属于实时和非标记的测试方法。SPR方法在研究分子间相互作用方面具有其独特的优势,其非标记和实时检测以及可以进行动力学分析的特点,给研究生物大分子的相互作用提供了诱人的解决方案。近来,随着SPR成像技术和SPR芯片制备技术的进展,将为功能基因组学和蛋白质组学研究提供重要的新的技术平台。     

Sentience and sensation

When animals are used in a biomedical research activity that may result in more than mild or momentary pain or distress, humanity, federal regulations and common sense direct us to use the least sentient species that can fulfill the aims of the research. The use of a less-sentient species is in line with the concept of Replacement, one of the well-known 3Rs of laboratory animal use. But what is a less-sentient species? Is a chimpanzee less sentient than a human; is a dog less sentient than a chimpanzee; and is a mouse less sentient than a dog? Does 'less sentient' imply that a species is less able to experience pain, is less intelligent or has less self-awareness? This essay will explore some of the relationships between sentience, pain and vertebrate phylogeny.     

Biosynthesis and degradation of anandamide and 2-arachidonoylglycerol and their possible physiological significance

N -arachidonoylethanolamine (anandamide) was the first endogenous cannabinoid receptor ligand to be discovered. Dual synthetic pathways for anandamide have been proposed. One is the formation from free arachidonic acid and ethanolamine, and the other is the formation from N -arachidonoyl phosphatidylethanolamine (PE) through the action of a phosphodiesterase. These pathways, however, do not appear to be able to generate a large amount of anandamide, at least under physiological conditions. The generation of anandamide from free arachidonic acid and ethanolamine is catalyzed by a degrading enzyme anandamide amidohydrolase/fatty acid amide hydrolase operating in reverse and requires large amounts of substrates. As for the second pathway, arachidonic acids esterified at the 1-position of glycerophospholipids, which are mostly esterified at the 2-position, are utilized for the formation of N -arachidonoyl PE, a stored precursor form of anandamide. In fact, the actual levels of anandamide in various tissues are generally low except in a few cases. 2-Arachidonoylglycerol (2-AG) was the second endogenous cannabinoid receptor ligand to be discovered. 2-AG is a degradation product of arachidonic acid-containing glycerophospholipids such as inositol phospholipids. Several investigators have demonstrated that 2-AG is produced in a variety of tissues and cells upon stimulation. 2-AG acts as a full agonist at the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating and indicates that 2-AG is the most efficacious endogenous natural ligand for the cannabinoid receptors.In this review, we summarize the tissue levels, biosynthesis, degradation and possible physiological significance of two endogenous cannabimimetic molecules, anandamide and 2-AG.     

Synthesis and biological evaluation of cepharadiones A and B and related dioxoaporphines

Described herein is the first total synthesis and structural confirmation of cepharadione A, a naturally occurring DNA damaging agent. Also reported is the synthesis of cepharadione B, a closely related natural product, as well as the biological evaluation of both natural products. Finally, the preparation and biological evaluation of novel dioxoaporphine analogues is described.     

Nitrogen supply and the control of expansive growth and function in leaves and roots

In plants which have acclimatized to limiting supplies of nitrogen (steady-state nutrition), leaf expansion (numbers and sizes of leaves and cells) is under tight control. Over a wide range of nitrogen supplies, the control of leaf growth is associated with a narrow band of photosynthetic rate per leaf area (measured at the growth climate) and, at limiting supplies, a carbon uptake which is in excess of immediate carbon usage in structural growth.For every increment of nitrogen absorbed, root extension is greater at limited nitrate supply, but V_max values (per root dry weight) for nitrate absorption are typically less. However, the capacity of the whole root system for nitrate uptake at limited supply is sufficient to allow for maximum growth, should nitrate supply be increased.It is concluded that a better understanding at the cellular level of the mechanisms which result in a greater inhibition of the expansion of single leaves than of root extension would contribute to an understanding of differences in carbon sink strength among plant organs. This may be a crucial step towards a more physiologically-based appreciation of plant dry matter distribution among organs in plants experiencing different nitrogen supplies.     

Ontogeny and phylogeny of endothermy and torpor in mammals and birds

Endothermic thermoregulation in small, altricial mammals and birds develops at about one third to half of adult size. The small size and consequently high heat loss in these young should result in more pronounced energetic challenges than in adults. Thus, employing torpor (a controlled reduction of metabolic rate and body temperature) during development would allow them to save energy. Although torpor during development in endotherms is likely to occur in many species, it has been documented in only a few. In small, altricial birds (4 orders) and marsupials (1 order), which are poikilothermic at hatching/birth, the development of competent endothermic thermoregulation during cold exposure appears to be concurrent with the capability to display torpor (i.e. poikilothermy is followed by heterothermy), supporting the view that torpor is phylogenetically old and likely plesiomorphic. In contrast, in small, altricial placental mammals (2 orders), poikilothermy at birth is followed first by a homeothermic phase after endothermic thermoregulation is established; the ability to employ torpor develops later (i.e. poikilothermy-homeothermy-heterothermy). This suggests that in placentals torpor is a derived trait that evolved secondarily after a homeothermic phase in certain taxa perhaps as a response to energetic challenges. As mammals and birds arose from different reptilian lineages, endothermy likely evolved separately in the two classes, and given that the developmental sequence of torpor differs between marsupials and placentals, torpor seems to have evolved at least thrice.     

Problems and paradigms: Morphogens and pattern formation

Morphogen gradient theories have enjoyed considerable popularity since the beginning of this century, but conclusive evidence for a role of morphogens in controlling multicellular development has been elusive. Recently, work on three secreted signalling proteins, Activin in Xenopus, and Wingless and Dpp in Drosophila, has stongly suggested that these proteins function as morphogens. In order to define a factor as a morphogen, it is necessary to show firstly, that it has a direct effect on target cells and secondly, that it affects the development of target cells in a concentration-dependent manner. With these criteria in mind, the evidence available for a variety of proposed morphogens is discussed. While the evidence is not conclusive in most of the cases considered, there is a strong case in favour of the three proteins mentioned above, which suggests that morphogens are potentially of general importance in controlling the development of multicellular organisms.     

Sex selection and restricting abortion and sex determination

Sex selection in India and China is fostered by a limiting social structure that disallows women from performing the roles that men perform, and relegates women to a lower status level. Individual parents and individual families benefit concretely from having a son born into the family, while society, and girls and women as a group, are harmed by the widespread practice of sex selection. Sex selection reinforces oppression of women and girls. Sex selection is best addressed by ameliorating the situations of women and girls, increasing their autonomy, and elevating their status in society. One might argue that restricting or prohibiting abortion, prohibiting sex selection, and prohibiting sex determination would eliminate sex selective abortion. But this decreases women's autonomy rather than increases it. Such practices will turn underground. Sex selective infanticide, and slower death by long term neglect, could increase. If abortion is restricted, the burden is placed on women seeking abortions to show that they have a legally acceptable or legitimate reason for a desired abortion, and this seriously limits women's autonomy. Instead of restricting abortion, banning sex selection, and sex determination, it is better to address the practice of sex selection by elevating the status of women and empowering women so that giving birth to a girl is a real and positive option, instead of a detriment to the parents and family as it is currently. But, if a ban on sex selective abortion or a ban on sex determination is indeed instituted, then wider social change promoting women's status in society should be instituted simultaneously.     

Construction and analysis of parallel and antiparallel Holliday junctions

The Holliday junction is a four-stranded DNA intermediate that arises during recombination reactions. We have designed and constructed a set of Holliday junction analogs that model each of the ideal conformations available to a 2-fold symmetric four-arm junction. The strategy used is to connect two arms of a junction molecule with a short tether of thymidines. These DNA molecules share a common core sequence but have different arms that are connected so that each molecule is constrained in either an antiparallel or a parallel structure. For tethered antiparallel molecules the identity of the crossover strands is determined by which arms are connected. Different arm connections gave molecules representing each of the two antiparallel crossover isomers. Two parallel molecules that differ in the length and position of the tether exhibit opposite biases in their choice of crossover strands. Thus, a physical constraint applied at a distance from the branch point can determine the conformation of a junction.     

Selection, subdivision and extinction and recolonization

In a subdivided population, the interaction between natural selection and stochastic change in allele frequency is affected by the occurrence of local extinction and subsequent recolonization. The relative importance of selection can be diminished by this additional source of stochastic change in allele frequency. Results are presented for subdivided populations with extinction and recolonization where there is more than one founding allele after extinction, where these may tend to come from the same source deme, where the number of founding alleles is variable or the founders make unequal contributions, and where there is dominance for fitness or local frequency dependence. The behavior of a selected allele in a subdivided population is in all these situations approximately the same as that of an allele with different selection parameters in an unstructured population with a different size. The magnitude of the quantity N(e)s(e), which determines fixation probability in the case of genic selection, is always decreased by extinction and recolonization, so that deleterious alleles are more likely to fix and advantageous alleles less likely to do so. The importance of dominance or frequency dependence is also altered by extinction and recolonization. Computer simulations confirm that the theoretical predictions of both fixation probabilities and mean times to fixation are good approximations.     

Allele-specific gene expression and epigenetic modifications and their application to understanding inheritance and cancer

Epigenetic information is characterized by its plasticity during development and differentiation as well as its stable transmission during mitotic cell divisions in somatic tissues. This duality contrasts to genetic information, which is essentially static and identical in every cell in an organism with only a few exceptions such as immunoglobulin genes in lymphocytes. Epigenetics is traditionally perceived as a means to regulate gene expression without a change in DNA sequence. This, however, does not exclude a potential role for genetic variations in providing differential backgrounds on which epigenetic modulations and their regulatory consequences are achieved. An effective approach to investigating the interplay between genetic variations and epigenetic variations is through allele-specific analysis of epigenetics and gene expression. Such studies have generated many new insights into functions of genetic variations, mechanisms of gene expression regulation, and the role of mutations and epigenetic alterations in human cancer. This article is part of a Special Issue entitled: Chromatin in time and space.     

Phylogeography and conservation of impala and greater kudu

The phylogeography of the bush habituated African bovid species impala (Aepyceros melampus) and greater kudu (Tragelaphus strepsiceros) is investigated using mitochondrial DNA (mtDNA) markers. Combined analysis of individual lineages, relationships and population genetics suggest a colonization process from Southern Africa toward Eastern regions in the greater kudu. Results are less clear for the impala, although remaining consistent with a similar pattern of historical dispersion. The study reveals a similar pattern, that is a marked divergence of lineages from South-western Africa relative to other regions. This pattern is opposed to previously published findings in other African bovid species. In the impala, the genetically isolated region is consistent with morphology because it is recognized as the subspecies A. m. petersi, the black-faced impala. In contrast, the similar split of South-western mitochondrial lineages was not expected in the greater kudu on the basis of morphology. Both species show a significant population genetic differentiation. Beyond their phylogeographical value, our results should raise conservation concerns about South-western populations of both species. The black-faced impala is categorized as vulnerable and our data show indications of hybridization with common impala A. m. melampus. The previously unrecognized genetic status of the South-western kudus could also imply conservation regulations.     

DNA and telomeres: beginnings and endings

How a cell deals with its DNA ends is a question that returns us to the very beginnings of modern telomere biology. It is also a question we are still asking today because it is absolutely essential that a cell correctly distinguishes between natural chromosomal DNA ends and broken DNA ends, then processes each appropriately - preserving the one, rejoining the other. Effective end-capping of mammalian telomeres has a seemingly paradoxical requirement for proteins more commonly associated with DNA double strand break (DSB) repair. Ku70, Ku80, DNA-PKcs (the catalytic subunit of DNA-dependent protein kinase), Xrcc4 and Artemis all participate in DSB repair through nonhomologous end-joining (NHEJ). Somewhat surprisingly, mutations in any of these genes cause spontaneous chromosomal end-to-end fusions that maintain large blocks of telomeric sequence at the points of fusion, suggesting loss or failure of a critical terminal structure, rather than telomere shortening, is at fault. Nascent telomeres produced via leading-strand DNA synthesis are especially susceptible to these end-to-end fusions, suggesting a crucial difference in the postreplicative processing of telomeres that is linked to their mode of replication. Here we will examine the dual roles played by DNA repair proteins. Our review of this rapidly advancing field primarily will focus on mammalian cells, and cannot include even all of this. Despite these limitations, we hope the review will serve as a useful gateway to the literature, and will help to frame the major issues in this exciting and rapidly progressing field. Our apologies to those whose work we are unable to include.     

Life and reference

The paper recommends a broadening of Howard Pattee's seminal distinction between a dynamic and a linguistic mode of living systems. It is observed that even the dynamic mode is always a semiotic mode although indexical and analogically coded rather than symbolic and digitally coded. The analogically coded messages corresponds to a kind of tacit knowledge hidden in macromolecular structure and shape (e.g. molecular complementarity) and in organismic architecture and communication, i.e. in the semiotic interactions of the body. It is claimed that the origin of referential processes is tied to the flow of historical singularities. The function of analog and digital codes in evolutionary systems is discussed.     

The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.