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The shark GABA-benzodiazepine receptor: further evidence for a not so late phylogenetic appearance of the benzodiazepine receptor.

Whilst the brain-specific benzodiazepine receptor has been assumed to show a late evolutionary appearance, we present evidence for the presence of a central benzodiazepine binding site in sharks, which shows a high affinity for [3H]Ro 15-1788. However, the receptor density and the affinities of several benzodiazepine receptor ligands are lower than in mammals, thus presumably explaining why the benzodiazepine binding sites had previously escaped detection in elasmobranchs. Additionally, radio- and immunohistochemistry were performed to localize the radioligand binding sites and the antigenic sites of the shark gamma-aminobutyric acid (GABA)-benzodiazepine receptor. In cerebellum, the granular layer reveals a high density of [3H]muscimol binding sites. The immunoreaction obtained with the beta-subunit-specific monoclonal antibody bd-17 seemingly parallels the distribution of high-affinity GABA binding sites. In contrast, [3H]Ro 15-1788 binding sites are evenly distributed in the molecular and granular layers, thus the results are similar to those previously described for rat cerebellum. Apparently, the respective distributions in this brain region are well conserved throughout vertebrate evolution.

Animals↗

Polymerase-chain-reaction-based semi-quantification of hepatitis D viraemia in patients treated with high doses of alpha 2b interferon.

To study the antiviral efficacy of high doses of alpha 2b interferon (alpha 2b-IFN) for chronic hepatitis D treatment, we used polymerase-chain-reaction(PCR)-based semi-quantitative detection of HDV RNA. The semi-quantification method used was based on the appearance of a positive amplification signal as a function of the number of PCR cycles. By amplifying dilutions (10(-1)-10(-8)) of an HDV-positive woodchuck liver RNA, we confirmed that exponential amplification efficacy occurred at between 20 and 30 cycles. Positive signals were observed from dilution 10(-2) (gel electrophoresis after 20 cycles of PCR) to dilution 10(-7) (hybridization after 30 cycles of PCR). To characterize the HDV RNA level in sera of 8 patients treated with alpha 2b-IFN (10 MU/3 times a week) for 1 year, we extracted RNA from serum samples taken every 6 months. All samples were amplified in parallel for 20 and 30 PCR cycles. Analysis of HDV cDNA after ethidium bromide/agarose gel electrophoresis and after molecular hybridization (100 times more sensitive than gel analysis), enabled us to grade the signals observed from negative to positive as 1+, 2+, 3+ and 4+, with all results being positive. Three types of evolution of HDV viraemia were evidenced among the 8 treated patients. HDV replication continued to occur at a high level at the 6th and 12th month in 2 patient sera. For 2 other patients, an HDV RNA decrease or disappearance was evidenced in the serum at the 6th month; however, viral replication recurred at a higher level at the 12th month.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Molecular evolution of tetracycline-resistance plasmids carrying TetM found in Neisseria gonorrhoeae from different countries.

High level tetracycline resistant strains of Neisseria gonorrhoeae (TRNG) have been shown to carry a 40.6 kb (25.2 MDa) conjugative plasmid with a Class M tetracycline resistance determinant. Restriction endonuclease analysis mapping showed that there were at least two different TRNG plasmid types which were found in geographically distinct locations. The physical maps of these two plasmids were compared to a gonococcal conjugative plasmid which did not encode tetracycline resistance. The plasmid type which is endemic in the Netherlands was found to be closely related to the gonococcal conjugative plasmid, which supports the established hypothesis that the 40.6 kb plasmid has evolved by transposition of the TetM determinant into the conjugative plasmid. The plasmid found in the United States has either evolved by substantial divergent evolution or it results from a different transposition event. In the UK there have been isolations of TRNGs carrying either of the two plasmid types reflecting a flow of people both across the Atlantic and in Europe. It is possible that further TetM-containing plasmids will be found in N. gonorrhoeae paralleling the family of TEM beta-lactamase encoding plasmids already described.

Bacterial Proteins↗

Double layers and ion phase-space holes in the auroral upward-current region.

The dynamic evolution of the boundary between the ionosphere and auroral cavity is studied using 1D and 2D kinetic Vlasov simulations. The initial distributions of three singly ionized species (H+, O+, e-) are determined from space-based observations on both sides of an inferred strong double layer. The kinetic simulations reproduce features of parallel electric fields, electron distributions, ion distributions, and wave turbulence seen in satellite observations in the auroral upward-current region and, for the first time, demonstrate that auroral acceleration can be driven by a parallel electric field supported, in part, by a quasistable, strong double layer. In addition, the simulations verify that the streaming interaction between accelerated O+ and H+ populations continuously replenished by the double layer provides the free energy for the persistent formation of ion phase-space holes.

Journal Article↗

Treatment of acute hepatic encephalopathy in cirrhotics with a branched-chain amino acids enriched versus a conventional amino acids mixture. A controlled study of 70 patients.

Acute hepatic encephalopathy in 70 cirrhotic patients was monitored during parenteral administration of amino acids between January 1979 and January 1984. The diagnosis of cirrhosis was confirmed by needle biopsy, and HE by conventional clinical and EEG parameters. The infusion of AA solutions was initiated 48 h after admission and during a 5-day period: 34 patients received a control aminoacid solution, a commercially available AA mixture (Azonutril), and 36 patients a modified solution enriched in BAA prepared from crystallized AA dissolved in distilled water. The calorie intake for both groups was 1600 calories per day from glucose and lipid emulsion. No significant difference was noted based on clinical evolution, even though the plasma AAA/BAA ratio was corrected using the modified AA solution. Of the 34 patients in Group 1: 10 improved, 14 were unchanged, 10 deteriorated and 7 died. Of the 36 patients in Group 2: 12 improved, 14 were unchanged, 10 deteriorated and 7 died. EEG tracing evolved in parallel fashion. The authors conclude that modified AA solutions are ineffective in the treatment of acute hepatic encephalopathy in cirrhotic patients.

Acute Disease↗

The developmental fate of the cephalic mesoderm in quail-chick chimeras.

The developmental fate of the cephalic paraxial and prechordal mesoderm at the late neurula stage (3-somite) in the avian embryo has been investigated by using the isotopic, isochronic substitution technique between quail and chick embryos. The territories involved in the operation were especially tiny and the size of the transplants was of about 150 by 50 to 60 microns. At that stage, the neural crest cells have not yet started migrating and the fate of mesodermal cells exclusively was under scrutiny. The prechordal mesoderm was found to give rise to the following ocular muscles: musculus rectus ventralis and medialis and musculus oblicus ventralis. The paraxial mesoderm was separated in two longitudinal bands: one median, lying upon the cephalic vesicles (median paraxial mesoderm--MPM); one lateral, lying upon the foregut (lateral paraxial mesoderm--LPM). The former yields the three other ocular muscles, contributes to mesencephalic meninges and has essentially skeletogenic potencies. It contributes to the corpus sphenoid bone, the orbitosphenoid bone and the otic capsules; the rest of the facial skeleton is of neural crest origin. At 3-somite stage, MPM is represented by a few cells only. The LPM is more abundant at that stage and has essentially myogenic potencies with also some contribution to connective tissue. However, most of the connective cells associated with the facial and hypobranchial muscles are of neural crest origin. The more important result of this work was to show that the cephalic mesoderm does not form dermis. This function is taken over by neural crest cells, which form both the skeleton and dermis of the face. If one draws a parallel between the so-called "somitomeres" of the head and the trunk somites, it appears that skeletogenic potencies are reduced in the former, which in contrast have kept their myogenic capacities, whilst the formation of skeleton and dermis has been essentially taken over by the neural crest in the course of evolution of the vertebrate head.

Animals↗

Tackling regional health inequalities in france by resource allocation : a case for complementary instrumental and process-based approaches?

This article aims to evaluate the results of two different approaches underlying the attempts to reduce health inequalities in France. In the 'instrumental' approach, resource allocation is based on an indicator to assess the well-being or the quality of life associated with healthcare provision, the argument being that additional resources would respond to needs that could then be treated quickly and efficiently. This governs the distribution of regional hospital budgets. In the second approach, health professionals and users in a given region are involved in a consensus process to define those priorities to be included in programme formulation. This 'procedural' approach is employed in the case of the regional health programmes. In this second approach, the evaluation of the results runs parallel with an analysis of the process using Rawlsian principles, whereas the first approach is based on the classical economic model.At this stage, a pragmatic analysis based on both the comparison of regional hospital budgets during the period 1992-2003 (calculated using a 'RAWP [resource allocation working party]-like' formula) and the evolution of regional health policies through the evaluation of programmes for the prevention of suicide, alcohol-related diseases and cancers provides a partial assessment of the impact of the two types of approaches, the second having a greater effect on the reduction of regional inequalities.

Budgets↗

Application of the Schlieren pulsed method for the observation of simple and multiple scattering of ultrasonic waves.

The Schlieren pulsed method uses short-term lighting triggered by an acoustic pulse. This allows for an observation of elastic deformation fields in pulsed regime and for an evaluation of the evolution of the pulse in the interior of homogenous and heterogeneous media. In this paper we apply the Schlieren pulsed method to determine the conditions of the change from simple to multiple scattering. An ultrasound transducer put in water emits a wide-band pulse. The pulse goes through a region of parallel cylindrical wires, uniformly spaced and perpendicular to the acoustic beam. Varying the width of the zone of the scatterer cylinders, it is possible to optically observe the transmitted acoustic field and to calculate the transmission coefficient. By studying the behavior of this coefficient in function of the medium width, we obtained the zone in which the transition between simple and multiple scattering happens.

Letter↗

Long-term experimental evolution in Escherichia coli. IV. Targets of selection and the specificity of adaptation.

This study investigates the physiological manifestation of adaptive evolutionary change in 12 replicate populations of Escherichia coli that were propagated for 2000 generations in a glucose-limited environment. Representative genotypes from each population were assayed for fitness relative to their common ancestor in the experimental glucose environment and in 11 novel single-nutrient environments. After 2000 generations, the 12 derived genotypes had diverged into at least six distinct phenotypic classes. The nutrients were classified into four groups based upon their uptake physiology. All 12 derived genotypes improved in fitness by similar amounts in the glucose environment, and this pattern of parallel fitness gains was also seen in those novel environments where the limiting nutrient shared uptake mechanisms with glucose. Fitness showed little or no consistent improvement, but much greater genetic variation, in novel environments where the limiting nutrient differed from glucose in its uptake mechanisms. This pattern of fitness variation in the novel nutrient environments suggests that the independently derived genotypes adapted to the glucose environment by similar, but not identical, changes in the physiological mechanisms for moving glucose across both the inner and outer membranes.

Adaptation, Physiological↗

Mitochondrial restriction fragment length polymorphism (RFLP) and sequence variation among closely related avian species and the genetic characterization of hybrid Dendroica warblers.

To address several interconnected goals, we used mitochondrial DNA (mtDNA) sequences to explore evolutionary relationships among four potentially hybridizing taxa in a North American avian superspecies (Dendroica occidentalis, D. townsendi, D. virens, and D. nigrescens). We first compared the results of a previous restriction fragment length polymorphism (RFLP)-based study with 1453 nucleotides from the mitochondrial cytochrome oxidase subunit I (COI), ATP-synthase 6 (ATPase 6), and ATP-synthase 8 (ATPase 8) genes. Separate phylogenetic analyses of the RFLP and sequence data provided identical and well-supported hierarchical species-level reconstructions that grouped occidentalis and townsendi as sister taxa. We then explored several general features of mitochondrial evolution via a comparison of the RFLP and sequence data sets. Qualitative rate differences that seemed evident in highly autocorrelated comparisons of RFLP vs. sequence pairwise distances were not supported when autocorrelation was removed. We also noted a high variance in corresponding RFLP and sequence distances after the removal of autocorrelation effects. This variance suggests that caution should be used when combining RFLP and sequence-based data in studies that require the large-scale synthesis of divergence estimates drawn from sources employing different molecular techniques. Finally, we used our parallel RFLP and sequence data to design and validate a rapid and inexpensive polymerase chain reaction-RFLP (PCR-RFLP) protocol for determining species-specific mitochondrial haplotypes. This PCR-RFLP technique will be applied in ongoing studies of the occidentalis/townsendi hybrid zone, where the historic and geographical complexity of the interbreeding populations necessitates the genotyping of thousands of individual warblers.

Adenosine Triphosphatases↗

Gene duplication of coagulation factor V and origin of venom prothrombin activator in Pseudonaja textilis snake.

The origin and evolution of venom toxins is a mystery that has evoked much interest. We have recently shown that pseutarin C, a prothrombin activator from Pseudonaja textilis venom, is structurally and functionally similar to mammalian coagulation factor Xa-factor Va complex. Its catalytic subunit is homologous to factor Xa while the nonenzymatic subunit is homologous to factor Va. P. textilis therefore has two parallel prothrombin activator systems: one expressed in its venom gland as a toxin and the other expressed in its liver and released into its plasma as a haemostatic factor. Here we report the complete amino acid sequence of factor V (FV) from its liver determined by cDNA cloning and sequencing. The liver FV shows 96% identity to pseutarin C nonenzymatic subunit. Most of the functional sites involved in its interaction with factor Xa and prothrombin are conserved. However, many potential sites of post-translational modifications and one critical cleavage site for activated protein C are different. The absence of the latter cleavage site makes pseutarin C nonenzymatic subunit resistant to inactivation and enhances its potential as an excellent toxin. By PCR and real-time quantitative analysis, we show that pseutarin C nonenzymatic subunit gene is expressed specifically in the venom gland at approximately 280 fold higher than that of FV gene in liver. These two are thus encoded by two separate genes that express in a highly tissue-specific manner. Our results imply that the gene encoding pseutarin C nonenzymatic subunit was derived by the duplication of plasma FV gene and they have evolved to perform distinct functions.

Amino Acid Sequence↗

Parallel evolutionary paths to mycoheterotrophy in understorey Ericaceae and Orchidaceae: ecological evidence for mixotrophy in Pyroleae.

Several forest understorey achlorophyllous plants, termed mycoheterotrophs (MHs), obtain C from their mycorrhizal fungi. The latter in turn form ectomycorrhizas with trees, the ultimate C source of the entire system. A similar nutritional strategy occurs in some green forest orchids, phylogenetically close to MH species, that gain their C via a combination of MH and photosynthesis (mixotrophy). In orchid evolution, mixotrophy evolved in shaded habitats and preceded MH nutrition. By generalizing and applying this to Ericaceae, we hypothesized that green forest species phylogenetically close to MHs are mixotrophic. Using stable C isotope analysis with fungi, autotrophic, mixotrophic and MH plants as comparisons, we found the first quantitative evidence for substantial fungi-mediated mixotrophy in the Pyroleae, common ericaceous shrubs from boreal forests close to the MH Monotropoideae. Orthilia secunda, Pyrola chlorantha, Pyrola rotundifolia and Chimaphila umbellata acquired between 10.3 and 67.5% of their C from fungi. High N and 15N contents also suggest that Pyroleae nutrition partly rely on fungi. Examination of root fungal internal transcribed spacer sequences at one site revealed that 39 species of mostly endophytic or ectomycorrhizal fungi, including abundant Tricholoma spp., were associated with O. secunda, P. chlorantha and C. umbellata. These fungi, particularly ectomycorrhizal associates, could thus link mixotrophic Pyroleae spp. to surrounding trees, allowing the C flows deduced from isotopic evidence. These data suggest that we need to reconsider ecological roles of understorey plants, which could influence the dynamics and composition of forest communities.

Adaptation, Biological↗

Phylogenetic relationships among monogenean gill parasites (Dactylogyridea, Ancyrocephalidae) infesting tilapiine hosts (Cichlidae): systematic and evolutionary implications.

We studied the systematics of 14 species of monogenean (Ancyrocephalidae) gill parasites from West African tilapiine hosts (Cichlidae) using both morphological and genetic data. With these tools, we were able to: (i) confirm the validity of the previously described morphological parasite species and of the genus Scutogyrus; (ii) propose that some stenoxenous species (i.e., parasite species with more than one host) may be composed of sister species (e.g., Cichlidogyrus tilapiae); (iii) state that the use of the morphology of the haptoral sclerites is more suitable to infer phylogenetic relationships than the morphology of the genitalia (which seems to be more useful to resolve species-level identifications, presumably because of its faster rate of change). These results imply that: (i) the specificity of these monogenean parasites is greater than initially supposed (what were thought to be stenoxenous species may be assemblages of oïoxenous sister species); (ii) related species groups (i.e., "tilapiae," "halli," and "tiberianus") have to be, as genus Scutogyrus, validated within the 54 ancyrocephalid species described from 18 species of tilapiine hosts in West Africa, (iii) the group "tilapiae," due to its morphology and host range, have to be considered as being the most primitive; (iv) the occurrence of lateral transfers and parallel speciation processes are necessary to describe the repartition of the newly described parasite groups on the three host genera studied (Tilapia, Oreochromis, and Sarotherodon).

Animals↗

Lateral transfer at the gene and subgenic levels in the evolution of eukaryotic enolase.

Enolase genes from land plants and apicomplexa (intracellular parasites, including the malarial parasite, Plasmodium) share two short insertions. This observation has led to the suggestion that the apicomplexan enolase is the product of a lateral transfer event involving the algal endosymbiont from which the apicomplexan plastid is derived. We have examined enolases from a wide variety of algae, as well as ciliates (close relatives of apicomplexa), to determine whether lateral transfer can account for the origin of the apicomplexan enolase. We find that lateral gene transfer, likely occurring intracellularly between endosymbiont and host nucleus, does account for the evolution of cryptomonad and chlorarachniophyte algal enolases but fails to explain the apicomplexan enolase. This failure is because the phylogenetic distribution of the insertions--which we find in apicomplexa, ciliates, land plants, and charophyte green algae--directly conflicts with the phylogeny of the gene itself. Protein insertions have traditionally been treated as reliable markers of evolutionary events; however, these enolase insertions do not seem to reflect accurately the evolutionary history of the molecule. The lack of congruence between insertions and phylogeny could be because of the parallel loss of both insertions in two or more lineages, or what is more likely, because the insertions were transmitted between distantly related genes by lateral transfer and fine-scale recombination, resulting in a mosaic gene. This latter process would be difficult to detect without such insertions to act as markers, and such mosaic genes could blur the "tree of life" beyond the extent to which whole-gene lateral transfer is already known to confound evolutionary reconstruction.

Amino Acid Sequence↗

Sequence similarities in (alpha/beta)8-barrel enzymes revealed by conserved regions of alpha-amylase.

The parallel (alpha/beta)8-barrel is a frequently occurring protein-folding motif. Although the arrangement of secondary structural elements along the barrel is very similar in different (alpha/beta)8-barrel enzymes, there is a very low mutual amino acid sequence homology among the enzymes, contributing in part to the hazy view of their evolution. Here an approach to identifying at least the rough of evolutionarily conserved (alpha/beta)8-barrel sequence is presented. Based on the idea that highly conserved sequence regions of a particular enzyme should be more or less conserved in the sequences of the other evolutionary related enzymes, five sequence similarities of ten different (alpha/beta)8-barrel enzymes were revealed, using the five conserved regions of the amino acid sequence of the alpha-amylase (alpha/beta)8-barrel as the templates.

Amino Acid Sequence↗

Biophysical studies of photosystem II-related recovery processes after a heat pulse in barley seedlings (Hordeum vulgare L.).

Leaves of 7-day-old barley seedlings were subjected to heat pulses at 50 degrees C for 20 or 40s to inhibit partially or fully the oxygen evolution without inducing visible symptoms. By means of biophysical techniques, we investigated the time course and mechanism of photosystem II (PSII) recovery. After the heat treatment, the samples were characterized by typical heat stress symptoms: loss of oxygen evolution activity, strong decrease of Fv/Fm, induction of the K-step in the fluorescence induction transient, emergence of the AT-thermoluminescence-band and a dramatic increase in membrane permeability. In the first 4h in the light following the heat pulse, the AT-band and the K-step disappeared in parallel, indicating the loss of this restricted activity of PSII. This phase was followed by a recovery period, during which PSII-activity was gradually restored in the light. In darkness, no recovery, except for the membrane permeability, was observed. A model is presented that accounts for (i) the damage induced by the heat pulse on the membrane architecture and on the PSII donor side, (ii) the light-dependent removal of the impaired reaction centers from the disorganized membrane, and (iii) the subsequent light-independent restoration of the membrane permeability and the de novo synthesis of the PSII reaction centers in the light.

Ascorbate Peroxidases↗

The recovery of acute pancreatitis depends on the enzyme amount stored in zymogen granules at early stages.

Little is known about the changes in pancreatic enzyme storage in acute pancreatitis. We have performed flow cytometric studies of zymogen granules from rats with acute pancreatitis induced by hyperstimulation with caerulein. A comparison was made with rats treated with hydrocortisone (10 mg/kg/day) over 7 days before inducing pancreatitis in order to find out whether the amount of enzymes stored in the pancreas plays a key role in the development of pancreatitis. The potentially therapeutic effect of L-364,718 (0.1 mg/kg/day, for 7 days), a CCK receptor antagonist, was assayed in the rats with caerulein-induced pancreatitis which had previously received the hydrocortisone treatment. A significant increase in the intragranular enzyme content was observed 5 h after hyperstimulation with caerulein. The highest values were reached in the rats previously treated with hydrocortisone. The greatest pancreatic enzyme load was parallel to the highest values in plasma amylase, edema and haematocrit observed. Acute pancreatitis was reversed seven days later. At this stage smaller granules appeared in the pancreas whose enzyme content was similar to that of controls when no treatment was applied after pancreatitis. In contrast, L-364,718 administration prevented the favourable evolution of pancreatitis since the antagonism exerted on CCK receptors induced a blockade of secretion of the large amounts of enzymes stored in the pancreas. Moreover, the enzyme content in zymogen granules was below normal values since the stimulatory CCK action on enzyme synthesis can be inhibited by L-364,718. Our results suggest that the efficiency of CCK antagonists, as potential therapy, would also depend on the load of enzymes in the pancreas when acute pancreatitis is produced.

Acute Disease↗

;Low-waves' in chlorophyll fluorescence kinetics indicate deprivation of bicarbonate.

A brief reversible lowering of chlorophyll fluorescence yield (so called low-waves) immediately after application of a saturating light pulse in parallel with a short-time enhancement of the P700 oxidation level was observed in the green alga Haematococcus pluvialis. The phenomenon occurred in the steady-state time region of fluorescence induction kinetics under mild acidic conditions, and was eliminated by bicarbonate. Shortly after expression of low-waves, the photosynthetic oxygen evolution rate decreased and the non-photochemical chlorophyll fluorescence quenching component increased. The enhancement of the non-photochemical chlorophyll fluorescence quenching component was nigericin-sensitive indicating its dependence on the transthylakoid proton gradient. On the other hand, the formation of low-waves was not removed by the uncoupler. Only when bicarbonate was applied additionally, the reversible short-term decrease in fluorescence yield following each saturating light flash was abolished. Dimethyl-4-nitroso-aniline as an artificial electron acceptor of Photosystem I did not limit the brief drops in fluorescence. However, formate as a competitive inhibitor of bicarbonate binding in Photosystem II induced low-wave formation. Therefore, our results suggest that low-waves in chlorophyll fluorescence kinetics indicate deprivation of bicarbonate in the reaction centre of Photosystem II.

Journal Article↗