Inhibition by excitatory sulphur amino acids of the high-affinityl-glutamate transporter in synaptosomes and in primary cultures of cortical astrocytes and cerebellar neurons

A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[³H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K _m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K _iK _m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K _i>10–1000 times the appropriateK _m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme     

Interactions between fish,grazing invertebrates and algae in a New Zealand stream: a trophic cascade mediated by fish-induced changes to grazer behaviour?

Experiments in laboratory stream channels compared the behaviour of Deleatidium mayfly nymphs in the absence of fish with that in the presence of either native common river galaxias (Galaxias vulgaris Stokell) or introduced brown trout (Salmo trutta L.). Galaxias present similar predation risks to prey during day and night but are more active at night. Whereas, trout present a higher predation risk during the day. Deleatidium maintained a fixed nocturnal drift periodicity that is characteristic of streams containing visually feeding fish regardless of the nature of the predation regime presented in the laboratory. However, the number on the substratum surface, and therefore able to graze algae, was lower when fish were present than when they were absent. The number was lower during the day in the presence of trout, when they present the highest predation risk, and lower during the night compared to the day in trials with galaxias when galaxias activity disturbs Deleatidium from the substratum. Increases in the probability of Deleatidium leaving a patch, reductions in the proportion of mayflies on high quality patches and reductions in the distance travelled from refuge also reflected variations in the predation regime. Similar differences in positioning were observed under the same predation regimes in in situ channels in the Shag River and these were associated with differences in algal biomass. Algal ash-free dry mass (AFDM) and chlorophyll a (chl a) were higher on the tops of cobbles when fish were present. Fish also affected the biomass and the distribution of algae on cobbles as AFDM and chl a were higher on the sides of cobbles from channels with trout compared to those with galaxias. Changes in grazing behaviour, caused by predator avoidance, are likely to have been responsible for differences in algal biomass because no significant differences were detected between treatments in the biomass of Deleatidium or of total invertebrates.     

The functional units of a peptostreptococcal protein L

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.     

X chromosome linkage studies in familial Rett syndrome

Four families, each with two individuals affectecd by Rett Syndrome (RS), were analysed using restriction fragment lenght polymorphisms and microsatellite markers from the X chromosome. In two of the families, X-linked dominant inheritance of the RS defect from a germinally mosaic mother could be assumed. Therefore, maternal X chromosome markers showing discordant inheritance were used to exclude regions of the X chromosome as locations of the RS gene. Much of the short arm could be excluded, including regions containing three candidate genes, OTC, synapsin 1 and synaptophysin. Although most of the long arm was inherited in common it was possible to exclude a centromeric region. Inheritance of X chromosome markers is also presented for two families with affected aunt-niece pairs, one of which has not been previously studied at the DNA level.     

Effect of heat stress on glucose kinetics during exercise

Hargreaves, Mark, Damien Angus, Kirsten Howlett, Nelly MarmyConus, and Mark Febbraio. Effect of heat stress on glucose kinetics during exercise. J. Appl.Physiol. 81(4): 1594-1597, 1996.To identify themechanism underlying the exaggerated hyperglycemia during exercise inthe heat, six trained men were studied during 40 min of cyclingexercise at a workload requiring 65% peak pulmonary oxygen uptake(O_{2 peak}) on twooccasions at least 1 wk apart. On one occasion, the ambient temperaturewas 20°C [control (Con)], whereas on the other, it was40°C [high temperature (HT)]. Rates ofglucose appearance and disappearance were measured by using a primedcontinuous infusion of[6,6-²H]glucose. Nodifferences in oxygen uptake during exercise were observed betweentrials. After 40 min of exercise, heart rate, rectal temperature,respiratory exchange ratio, and plasma lactate were all higher in HTcompared with Con (P < 0.05). Plasmaglucose levels were similar at rest (Con, 4.54 ± 0.19 mmol/l; HT,4.81 ± 0.19 mmol/l) but increased to a greater extent duringexercise in HT (6.96 ± 0.16) compared with Con (5.45 ± 0.18;P < 0.05). This was the result of ahigher glucose rate of appearance in HT during the last 30 min ofexercise. In contrast, the glucose rate of disappearance and metabolicclearance rate were not different at any time point during exercise.Plasma catecholamines were higher after 10 and 40 min of exercise in HTcompared with Con (P < 0.05),whereas plasma glucagon, cortisol, and growth hormone were higher in HTafter 40 min. These results indicate that the hyperglycemia observedduring exercise in the heat is caused by an increase in liver glucoseoutput without any change in whole body glucoseutilization.

     

The Total Chemical Synthesis of Monocyte Chemotactic Protein-1 (MCP-1)

The affinity-based N^α-amino protecting group tetrabenzo [a,c,g,i]fluorenyl-17-methoxycarbonyl (Tbfmoc) has been utilized as a hydrophobic probe to allow the simple, quick and highly effective isolation of a 76 residue cysteine-containing protein (MCP-1). The base-labile Tbfmoc group can be removed under very mild conditions, which preserve the thiol-con taining protein in the reduced state. Oxidative folding was then used to furnish the biologically active β-chemokine MCP-1.     

Regions of DNA sequence homology between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens

Summary Despite the fact that pTiC58 and pTiB6S3 functionally, have been shown to date to have only tumorigenicity and phage AP1 exclusion in common, many restriction fragments of the plasmids contain DNA sequences common to both. The bulk of this homologous DNA is concentrated in a few restriction endonuclease fragments and the remainder is organized in short discontinuous regions spread over many fragments. In pTiB6S3 the bulk of the homology is distributed throughout a 29x10⁶ dalton segment comprising 8 Sma I fragments. This region includes those sequences which are transferred to and transcribed in tumorigenic plant cells induced by B6-806 or closely related strains. The pattern of homology within this portion of the plasmid shows a region of low sequence homology (Sma I Fragment 3 b) apparently corresponding to the gene or genes coding for octopine synthesis in the plant tumor cells, surrounded by regions of high sequence homology. The extent of inter-plasmid homology then decreases with increasing distance from fragment 3b. The remainder of the homology is distributed throughout a segment of maximum size 21.5x10⁶ daltons comprising two Sma I fragments and cannot yet be definitely linked with any specific plasmid function.     

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.