Evidence that Dopamine is Acting at α-Adrenoceptors of the Rat Vas Deferens

Abstract

In the rat vas deferens, a vast number of experiments have shown that the α-adrenoceptors present are of two types: α₁ and α₂. This series of experiments with the isolated rat vas deferens was designed to probe by pharmacological means, the nature of the responses elicited by neurogenic transmural stimulation and also those responses evoked by exogenous NE and DA. The methodology required the production of chemical denervation, neurotransmitter depletion, and the use of specific adrenoceptor blockers. The results obtained with the blocking agents, yohimbine or prazosin versus NE and DA, were pA2 values that were virtually interchangeable. The effects of chemical alteration with 6-OH-DA or reserpine point to a certain similarity and interdependence of the mechanism of action for the two neurotransmitters. Therefore, it is suggested that these two transmitters act at the same receptor site or share a common receptive microenvironment in the rat vas deferens.     

A conformational rationale for the origin of the mechanism of nucleicacid-directed protein synthesis of ‘living’ organisms

The physical basis for the natural evolution of a primitive decoding system is presented using the concepts of molecular interactions. Oligoribonucleotides of five residues havingU at the 5-end, a purine at the 3-end and any combination of three bases in the middle is taken as a primitive tRNA (PIT). From conformational considerations PIT is expected to haveU-turn conformation wherein, N₃–H₃ of baseU hydrogen-bonds with phosphate, three residues ahead leaving triplet bases called primitive anticodons (PAC) into a helical conformation, and this creates a cleft betweenU and PAC. An amino acid can be comfortably nestled into the cleft with the amide hydrogens and carboxyl oxygen hydrogen-bonded to the last purine and the first uridine, while the side-chain can interact with the cleft side of PAC. The other side of PAC is free to base-pair with triplet codons on a longer RNA. Also two PACs can recognize consecutive triplet codons, and this leads to a dynamic interaction in which the amino and carboxyl ends are brought into proximity, making the formation of peptide bond feasible.The cleft formed by different anticodon triplets, broadly speaking, shows preferences for the corresponding amino acids of the presently known codon assignment.Thus the nucleicacid-directed protein synthesis, which is a unique feature of all living organisms is shown to be a natural consequence of a particular way of favourable interaction between nucleic acids and amino acids, and our model provides the missing link between the chemical evolution of small organic molecules and biological evolution through the process of mutations in nucleicacids and nucleicacid-directed protein synthesis.Contribution No. 507 from this department.     

‘Manipulation’ without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites

Previous studies have suggested that Plasmodium parasites can manipulate mosquito feeding behaviours such as probing, persistence and engorgement rate in order to enhance transmission success. Here, we broaden analysis of this ‘manipulation phenotype’ to consider proximate foraging behaviours, including responsiveness to host odours and host location. Using Anopheles stephensi and Plasmodium yoelii as a model system, we demonstrate that mosquitoes with early stage infections (i.e. non-infectious oocysts) exhibit reduced attraction to a human host, whereas those with late-stage infections (i.e. infectious sporozoites) exhibit increased attraction. These stage-specific changes in behaviour were paralleled by changes in the responsiveness of mosquito odourant receptors, providing a possible neurophysiological mechanism for the responses. However, we also found that both the behavioural and neurophysiological changes could be generated by immune challenge with heat-killed Escherichia coli and were thus not tied explicitly to the presence of malaria parasites. Our results support the hypothesis that the feeding behaviour of female mosquitoes is altered by Plasmodium, but question the extent to which this is owing to active manipulation by malaria parasites of host behaviour.     

‘A river that is cutting its own bed’: the serology of syphilis between laboratory,society and the law

This paper focuses on the role of regulation in the shaping new scientific facts. Fleck chose to study the origins of a diagnostic test for a disease seen as a major public health problem, that is, a ‘scientific fact’ that had a direct and immediate influence outside the closed universe of fundamental scientific research. In 1935, when Fleck wrote his book, Genesis and development of a scientific fact, he believed that the tumultuous early history of the Wassermann reaction had come to an end, and that this reaction was successfully stabilized through the standardization of laboratory practices and thanks to the rise of a specific professional segment—the serologists. He could not have predicted that in the 15 years that followed the publication of his book, regulatory measures—barely metioned in his historical narrative—would play a key role in the destabilization of the original meaning of this reaction. The introduction of mass screening for syphilis—mainly via legislation that introduced obligatory premarital tests and promoted the testing of pregnant women—weakened in fine the link between Wassermann serology and infection by the etiological agent of syphilis, the bacterium Treponema pallidum. Fleck elected to study the Wassermann reaction because of its novelty, its complexity, and because it became the focus of a controversy regarding its origins. However, the Wassermann reaction was also one the first examples of a medical technology regulated by the state and incorporated into legal dispositions. It may therefore be seen as an exemplary case of the close intertwining of scientific investigations, their practical applications and regulatory practices.     

Biogeography of culturable marine bacteria from both poles reveals that ‘everything is not everywhere’ at the genomic level

Based on 16S rRNA gene analyses, the same bacterial operational taxonomic units (OTUs) are common to both the Arctic and Antarctic oceans, supporting the concept ‘everything is everywhere’. However, whether the same OTUs from both poles have identical genomes, i.e. whether ‘everything is still everywhere’ at the genomic level has not yet been examined systematically. Here, we isolated, sequenced and compared the genomes of 45 culturable marine bacteria belonging to three genera of Salinibacterium, Psychrobacter and Pseudoalteromonas from both polar oceans. The bacterial strains with identical 16S rRNA genes were common to both poles in every genus, and four identical genomes were detected in the genus Salinibacterium from the Arctic region. However, no identical genomes were observed from opposite poles in this study. Our data, therefore, suggest that ‘everything is not everywhere’ at the genomic level. The divergence time between bacteria is hypothesized to exert a strong impact on the bacterial biogeography at the genomic level. The geographical isolation between poles was observed for recently diverged, highly similar genomes, but not for moderately similar genomes. This study thus improves our understanding of the factors affecting the genomic-level biogeography of marine microorganisms isolated from distant locations.     

What is the role of thermodynamics on protein stability?

The most challenging and emerging field of biotechnology is the tailoring of proteins to attain the desired characteristic properties. In order to increase the stability of proteins and to study the function of proteins, the mechanism by which proteins fold and unfold should be known. It has been debated for a long time how exactly the linear form of a protein is converted into a stable 3-dimensional structure. The literature showed that many theories support the fact that protein folding is a thermodynamically controlled process. It is also possible to predict the mechanism of protein deactivation and stability to an extent from thermodynamic studies. This article reviewed various theories that have been proposed to explain the process of protein folding after its biosynthesis in ribosomes. The theories of the determination of the thermodynamic properties and the interpretation of thermodynamic data of protein stability are also discussed in this article.     

Vaccination against shigellosis: is it the path that is difficult or is it the difficult that is the path?

Following several decades of research, there is not yet a convincing vaccine against shigellosis. It is still difficult, in spite of the breadth of strategies (i.e. live attenuated oral, killed oral, subunit parenteral) to select an optimal option. Two approaches are clearly emerging: (i) live attenuated deletion mutants based on rational selection of genes that are key in the pathogenic process, and (ii) conjugated detoxified polysaccharide parenteral vaccines, or more recently conjugated synthetic carbohydrates. Some of these approaches have already undergone phase I and II clinical trials with promising results, but important issues have also emerged, particularly the discrepancy between colonization and immunogenic potential of live attenuated vaccine candidates depending upon the population concerned (i.e. non endemic vs. endemic areas). Efforts are needed to definitely establish the proof of concept of these approaches, and thus the need for clinical trials which should also soon explore the possibility to associate different serotypes, in response to serotype specific protection against shigellosis. More basic research is also required to improve what we can still consider as first-generation vaccines, and to explore possible new paradigms including the search for cross-protective antigens.     

Evidence that expression of SpαI/65 hereditary elliptocytosis is compounded by a genetic factor that is linked to the homologous α-spectrin allele

Summary Many cases of hereditary elliptocytosis (HE) result from mutated spectrin -chains. It has repeatedly been observed that the amount of a mutant -chain is different in various affected individuals, resulting in clinical pictures of variable severity. The different levels are thought to result from different percentages of the spectrin allele in trans. Such percentages, in turn, could be under genetic control. We tested this hypothesis in a large Algerian family with Sp^I/65 HE. In an informative sibship, we found three persons with a distinctly high level of expression of the Sp^I/65 variant, suggesting the existence, in trans, of a low percentage -allele. The -spectrin gene haplotype associated with the latter was constantly – +–, based on the XbaI, PvuII and MspI polymorphic sites. In contrast, a basal level of expression of the Sp^I/65 variant in the same sibship indicated, in trans, the existence of a normal percentage -allele. The haplotype corresponding to this other -allele was + – +. Study of another generation of the family showed, however, that the – + – haplotype could also be linked to a normal percentage -allele. These results are consistent with the view that the expression level of ^I/65 spectrin (and of other types of -variants) is compounded by a genetic factor that is linked to the normal -allele in trans. The low percentage allele itself remains silent in the simple heterozygous state.     

A case of ‘tachy-brady syndrome’: What is the mechanism?

A young male presented with incessant narrow QRS tachycardia and left ventricular dysfunction. 24-Holter monitoring revealed multiple episodes of sustained and nonsustained episodes of tachycardia with prolonged sinus pauses at termination. The analysis of the electrocardiogram, followed by an invasive electrophysiological study, suggested an unusual mechanism for this tachy-brady syndrome.     

The C-terminal tail of human neuronal calcium sensor 1 regulates the conformational stability of the Ca²⁺₋ activated state

Neuronal calcium sensor 1 (NCS-1) and orthologs are expressed in all organisms from yeast to humans. In the latter, NCS-1 plays an important role in neurotransmitter release and interacts with a plethora of binding partners mostly through a large solvent-exposed hydrophobic crevice. The structural basis behind the multispecific binding profile is not understood. To begin to address this, we applied NMR spectroscopy to determine the solution structure of calcium-bound human NCS-1. The structure in solution demonstrates interdomain flexibility and, in the absence of a binding partner, the C-terminal tail residues occupy the hydrophobic crevice as a ligand mimic. A variant with a C-terminal tail deletion shows lack of a defined structure but maintained cooperative unfolding and dramatically reduced global stability. The results suggest that the C-terminal tail is important for regulating the conformational stability of the Ca(2+)-activated state. Furthermore, a single amino acid mutation that was recently diagnosed in a patient with autistic spectrum disorder was seen to affect the C-terminal tail and binding crevice in NCS-1.     

Evidence for the presence of volume-sensitive KCl transport in ‘young’ human red cells

‘Young’ human red cells are shown to possess a specific K⁺ pathway which is dependent on Cl⁻ and sensitive to cell volume. This system was latent in ‘mature’ cells but was revealed by high hydrostatic pressure. This suggests the pathway is functionally active in ‘young’ cells but becomes masked with cell maturation.     

There is no ‘Conundrum’ of InsP6

Indirect assays have claimed to quantify phytate (InsP₆) levels in human biofluids, but these have been based on the initial assumption that InsP₆ is there, an assumption that our more direct assays disprove. We have shown that InsP₆ does not and cannot (because of the presence of an active InsP₆ phosphatase in serum) exist in mammalian serum or urine. Therefore, any physiological effects of dietary InsP₆ can only be due either to its actions in the gut as a polyvalent cation chelator, or to inositol generated by its dephosphorylation by gut microflora.We are grateful to Dr Vucenik for bringing up a number of interesting points.It is true that we have not quantified the dietary intakes of our human donors any more (but also hardly any less) than has been done by those groups claiming that InsP₆ is present in bodily fluids. As a qualitative observation we should point out that in fact all our donors for ref. [1] do have a regular intake of dietary cereals and indeed, one is a strict vegetarian on a high cereal diet. But it is quantification that reveals this to be a specious issue. The limits of detection in our two relevant publications [1,2] for InsP₆ in plasma and urine were, respectively, around two and three orders of magnitude lower than the levels claimed to be present by Grases et al. [3] in the fluids of experimentally phytate-deprived human subjects. These numbers make the argument that we could not detect any InsP₆ simply because we chose donors on the ‘wrong’ diet untenable.So how have those many claims that InsP₆ is present in body fluids come about? For most of them, the simple answer appears to be that the assays used are indirect and are based entirely on the assumption that InsP₆ is present in the first place. Thus, for example, Valiente and co-workers [4,5] and Chen and co-workers [6,7] measured organic phosphate remaining after a series of fractionations of urine samples and simply assumed it was due to InsP₆, as did March et al. measuring inorganic phosphate after a similar protocol [8]. Grases co-workers [9] have used extensively a less indirect assay, which, after initial ion chromatography and dephosphorylation by a phytase, measures myo-inositol by mass spectrometry, but nevertheless the assay starts with the assumption that InsP₆ is there and that this is what they are quantifying. More recently, direct quantification of InsP₆ in plasma by mass spectrometry has been claimed [10] on the basis that there are peaks in plasma at m/z 624 running near where InsP₆ standards elute in two different HPLC separations [10,11]. But no evidence is presented to show even that these peaks are the same compound, let alone any data to establish firmly that InsP₆ is present, e.g. a minimal requirement of m/z quantified to two decimal places with allowance for C₁₃ content or a full disintegration fingerprint (see also [12]). Any quantified misidentification is likely to have a stochastic element to it, and it is noteworthy that Perelló & Grases have stated [11, p. 255]: ‘…we have found some humans and rats having undetectable [InsP₆], probably depending on their diet or other unknown factors’. In the light of the preceding discussion, we can offer a simpler explanation: the InsP₆ was never there in the first place.In contrast to these claims we have, using two entirely independent specific and sensitive assays with quantified spiking recovery, unambiguously shown that InsP₆ is not present in plasma or urine. This is crucial and central to the whole debate about the actions of dietary InsP₆, because it means that InsP₆ never enters the blood. It is only absorbed after being dephosphorylated, principally to inositol (see [1,2] for further discussion). Ironically, the most direct evidence for this lies in Dr Vucenik''s own data in experiments examining the fate of radioactive InsP₆ fed to animals, in which only inositol was detected in the blood [13]. This particular study was, as Dr Vucenik points out in her letter, conducted on mice. However, exactly the same conclusion (i.e. InsP₆ does not enter the circulation from the gut) is equally clear in her earlier study [14], which she did not cite and which was indeed on rats; does this omission ‘reflect poorly’ on Dr Vucenik''s own ‘report and the author''s credibility in culling scientific data’?In short, dietary InsP₆ can have only two fates: it can stay in the gut, ultimately to be defecated [15], and while it is there it can chelate metal ions to alter their uptake from the gut into the body. This is no ‘straw-man’ and is certainly the most likely explanation for all of the effects of InsP₆ on cultured cells, which comprise the majority of the reports cited by Dr Vucenik. Alternatively, InsP₆ can be converted to inositol (principally by the gut microflora [15]) and be taken up as such into the circulation; were any InsP₆ to get into the blood it would in any case be rapidly dephosphorylated by the phosphatase activity we have shown to be present in human plasma [1].For animal studies, we have raised the possibility [1,2] that it is the inositol so generated (Vitamin Bh, harmless as far as we know) that is the active mediator of any reported beneficial effects of dietary InsP₆. We note that most of the websites touting InsP₆ as a dietary supplement advocate inositol as an important (essential?) co-supplement; that the only human cancer study highlighted as important by Dr Vucenik that we could examine [16] did not administer InsP₆ alone, but only in conjunction with inositol; and that in the few studies where the separate contributions of inositol and InsP₆ have been considered, there are data suggesting that it may be the inositol that matters (e.g. fig. 1 of [17]). Moreover, we are not the only ones to suggest this idea. In the Discussion of their paper (on mice) in which InsP₆ was shown not to enter the blood from the gut [13], Dr Vucenik and her colleagues state: ‘Inositol may be responsible for the antitumor actions observed in both chemopreventitive and efficacy studies of IP₆ … A question remains as to whether the activity of IP₆ in animal models can be replicated by administration of inositol alone because only inositol was detected in plasma and tumor after oral gavage’. Precisely.Finally, returning to InsP₆ itself, which, incidentally, is officially classified by the FDA as a ‘fake’ cancer cure (http://www.fda.gov/drugs/guidancecomplianceregulatoryinformation/enforcementactivitiesbyfda/ucm171057.htm), our data lead inevitably to the conclusion that while InsP₆ might impact on the gut environment and thus indirectly on its microflora [2,12], its only plausible direct action on the body will be to inhibit cation uptake from the diet. Although InsP₆ binds trivalent cations with a higher affinity than divalents [18], it is nevertheless comparatively non-specific in this action. Administering chemicals to the diet to manipulate ion uptake is not unknown in modern medicine; for treatment of iron disorders such as haemochromatosis, as an alternative to injection of Desferral, oral administration of the closely related chelator Deferasirox is now sometimes recommended [19]. But Deferasirox is a highly iron-specific chelator, administered under close medical supervision for a directly iron-related pathology. Recommending unmonitored, widespread administration of InsP₆ to address a veritable multitude of different pathologies [20] seems to us to be an entirely different matter.In a well-fed human, where the cation to InsP₆ ratio in the diet is high, InsP₆ may very well do no harm (it is, after all, a natural component of our diet) and there is much evidence to support this idea, as argued by Dr Vucenik. But if InsP₆ is not impacting on cation uptake from the diet to do any harm it is difficult to understand how at exactly the same time it can impact on the same uptake to do good. (See reference [21] for the studies Dr Vucenik requested ‘unequivocally demonstrating the toxicity of pure Ca-Mg-InsP₆ as it occurs naturally’ in humans with low dietary cation uptake.) In the light of the above discussion and our rigorous data, we stand unreservedly by our original closing statement [1]: ‘…that chronically altering cation absorption from the gut by artificially loading the diet with a non-specific chelator … in the hope that it might impact indirectly on cancer or other pathologies seems highly inadvisable’.     

Evidence for ‘dawn’ and ‘dusk’ oscillators in the Nasonia photoperiodic clock

Females of Nasonia vitripennis were maintained in light cycles from 12 to 72 hr in length, with 4 to 28 hr photoperiods, and their offspring examined for larval diapause. This ‘resonance’ technique revealed periodic maxima of diapause induction, about 24 hr apart. The ‘ascending slopes’ of these maxima appeared to obtain their principal time cue from dusk and the ‘descending slopes’ from dawn. This suggests that two independent—dawn and dusk—oscillators are involved in the Nasonia photoperiodic clock. The results are interpreted in terms of ‘internal coincidence’.N. vitripennis was shown to be able to distinguish between 12 and 18 hr of red light (>600 nm) in the photoperiodic sense. A ‘positive’ resonance experiment using such a red light was also performed. This shows that the spectral sensitivity of the pigment coupling the circadian system to the environmental light cycle extends into the red end of the spectrum.     

Evidence that the “waxy” protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase

The aim of this work was to identify the starch-granule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with α-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of thewaxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starch-synthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between “waxy” proteins and starch-granule-bound starch synthases generally. I am grateful to my colleagues Kay Denyer, Ian Dry (CSIRO, Adelaide, Australia), Rob Ireland (Mount Allison University, New Brunswick, Canada), Cathie Martin and Steve Rawsthorne for useful discussions during the course of this work, Cliff Hedley for the gift of pea seeds, and Ian Bedford for preparing pea starch and gels of starch-granule-bound proteins. This work was supported by the Agriculture and Food Research Council via a grant-in-aid to the John Innes Institute.