Small changes in essential amino acid concentrations alter diet selection in amino acid-deficient rats

A new plate designed specifically to address complex wrist pathology was used for the internal fixation of 22 complex fractures of the distal radius in 22 patients in a prospective multicenter trial. The majority of fractures were group C2- and C3-type fractures according to the Comprehensive Classification of Fractures. No plate failures, loss of reduction, nonunions, or infections occurred. Within the average follow-up time of 14 months, the functional results (including an average motion of 76% and an average grip strength of 56% of the contralateral side) were comparable to those reported for similar fractures in previous investigations. Five patients had irritation of the tendons in the second dorsal compartment. This trial serves both as a verification of the safety and efficacy of this distal radius plate as well as a demonstration of its utility in the treatment of complex fractures of the distal radius.     

Positive affect, negative affect, and social interaction.

Two studies explored the relations of positive and negative affect (PA and NA) to social interaction. In Study 1, unacquainted dyads were surreptitiously videotaped as they participated in a 6-min interaction. Participants then evaluated the quality of the interaction. Independent observers also rated the videotaped interactions. Trait PA was positively related to both participant and observer evaluations of interaction quality. In Study 2, undergraduates kept diaries of their social interactions for 1 week, PA was again related to interaction quality. Both PA and NA were positively related to the number of interactions in which participants engaged, and the amount of time spent engaged in social contact, although different types of social encounters produced these relations. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Loss of the signature six carboxyl amino acid tail from ovine interferon-tau does not affect biological activity

Interferon-tau (IFN-tau) is a type I IFN that is secreted from conceptuses of Bovidae (sheep, cattle, and related ruminant ungulates) for a few days during early pregnancy. It acts to prolong the life span of the corpus luteum. All secreted forms of IFN-tau, like the related IFN-omega, are 172 amino acids in length and differ from IFN-alpha and -beta by the presence of six additional amino acids at their carboxyl termini. The aim of this study was to determine whether this carboxyl tail was important for biological activity of IFN-tau, particularly for its antiluteolytic function in ewes. Full-length ovine IFN-tau (p3) and a mutated form truncated by six amino acids at its carboxyl terminal (p3Trn6, 166 amino acids) were produced in Escherichia coli. Both proteins had similar antiviral activities (2.12 +/- 0.92 x 10(8) IU/mg for p3; 1.96 +/- 0.58 x 10(8) IU/mg for p3Trn6) when tested on Madin-Darby bovine kidney (MDBK) cells. Antiproliferative activity, as measured on human Daudi cells by determining the protein concentration required to inhibit growth by 50%, was slightly higher (p < 0.05) for p3Trn6 (7.36 +/- 0.46 pM) than for p3 (13.99 +/- 0.85 pM). Most importantly, p3 and p3Trn6 were equally capable of prolonging the life span of the corpus luteum of nonpregnant ewes when the proteins were administered at doses of either 60 or 300 microg/day into the uterine lumen through indwelling uterine cannulae from Day 10 to Day 18 postestrus. Therefore, the carboxyl-terminal amino acid extension for IFN-tau does not appear to serve a functional role in the action of these proteins.     

The Plasmodium falciparum-CD36 interaction is modified by a single amino acid substitution in CD36

CD36 is an 88-kD glycoprotein involved in the cytoadherence of Plasmodium falciparum-parasitized erythrocytes (PE) to endothelial cells. The molecular mechanisms involved in CD36-dependent cytoadherence were examined by expressing three CD36 homologues (human, murine, and rat) in COS-7 cells and observing their PE-binding characteristics over a pH range of 6.0 to 7.4 and following iodination of these receptors. PE binding to human CD36 was pH dependent, with peak binding at pH 6.8 to 7.0, and binding was unaffected by iodination. In contrast, PE adherence to murine and rat CD36 was insensitive to changes in pH, and iodination significantly reduced binding. We further show that the differences observed in the binding phenotype of human and rodent CD36 can be attributed to a single residue. Site-directed mutagenesis of the histidine at position 242 of human CD36 to tyrosine (found in rodent CD36) conferred the binding phenotype of rodent CD36 onto human CD36. Furthermore, substitution of the tyrosine at position 242 of rat CD36 for histidine conferred the binding phenotype of human CD36 onto rat CD36. These findings suggest that residue 242 is part of, or important to the conformation of, the PE-binding domain of CD36.     

Cerulein-induced changes in plasma amino acid concentrations are not a valid test for pancreatic insufficiency

OBJECTIVES: There is controversy about the cholecystokinin (CCK)-induced decrease of plasma amino acids for the diagnosis of pancreatic insufficiency in that it is unclear whether the conflicting results are due to different degrees of stimulation by CCK. We have therefore evaluated the amino acid consumption test by examining the dose-response relation among increasing doses of cerulein and plasma amino acids compared with the release of pancreatic polypeptide in six healthy volunteers and six patients with severe pancreatic insufficiency proven by a pathological para-aminobenzoic acid test. METHODS: Stepwise increasing doses of cerulein (10-80 pmol/kg/h) were given i.v., each for 60 min with a secretin background (1 CU/kg/h). In the volunteer group, an additional experiment with infusion of placebo (saline 0.9%) was performed. RESULTS: CCK-induced amino acid changes were small in volunteers (maximum decrease: 8.4 +/- 0.9%; mean +/- SEM), tended to be more pronounced in patients (maximum decrease: 13.8 +/- 2.8%; NS), and did not permit a distinction among volunteers and patients. There was no dose-response relationship between CCK and plasma amino acids in either group. In contrast, pancreatic polypeptide levels increased markedly and dose-dependently in volunteers and patients and tended to be lower in patients with pancreatic insufficiency. CONCLUSIONS: The decrease of plasma amino acid levels in response to cerulein does not reflect pancreatic function and does not permit the diagnosis of pancreatic insufficiency.     

Specific and generalized expectancies in marital interaction.

40 married couples engaged in 2 laboratory interactions. The Marital Agendas Protocol was used to assess relational efficacy, and spouses' expectancies for partner behaviors during each interaction were elicited. Subsequent to the interactions, spouses' perceptions of their partners' actual behaviors were indicated, and spouses chose situational or dispositional attributions for each behavior. Results showed that distressed couples expect more negative and fewer positive behaviors and that spouses with high relational efficacy choose relationship-enhancing attributions more often than do low-efficacy spouses. The low-efficacy group showed strong preferences for distress-maintaining attributions. The results are interpreted as supporting the concept of sentiment override as well as the usefulness of the specific operationalization of relational efficacy utilized. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid

The gamma-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (alpha1-6, beta1-3, gamma1-3, delta1, and epsilon1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the beta subunit subtype present within the receptor. Receptors containing beta2- or beta3-, but not beta1 subunits, are highly sensitive to the agent. Here, chimeric beta1/beta2 subunits coexpressed in Xenopus laevis oocytes with human alpha6 and gamma2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the beta3 subunit to Ser (the homologous residue in beta1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the beta1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk-) cells stably expressing the alpha6beta3gamma2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.     

Longitudinal changes of brain amino acid content occurring before, during and after epileptic activity

In cats affected with cortical epileptogenic foci induced by penicillin application to and cobalt implantation into the pericruciate area, the brain amino acids contents were determined in the focus as well as in extrafocal areas. In different groups of animals, brain removal for biochemical determinations was performed at different times before, during and after epilepsy and the values compared to controls. The only amino acid to show a significant change before appearance of spikes in both types of epilepsy was taurine, which decreased. Cobalt epilepsy was accompanied by changes in a larger number of amino acids than penicillin epilepsy: in the former the brain content of taurine, GABA, aspartate, glutamate, serine, threonine, glycine and alanine was altered. The changes were proportional to the severity of epilepsy and more prominent in the focus area. After disappearance of spikes the levels of most amino acids returned to normal except for some amino acids, previously unaffected by penicillin epilepsy, which were decreased. It is proposed that the decrease in brain taurine, occurring before the appearance of penicillin and cobalt epilepsy, could increase the excitability of a certain neuronal population and thus, by potentiating the effects on neurons of penicillin and cobalt, contribute to the initiation of epilepsy.     

Parasite sulphur amino acid metabolism

This paper reviews current knowledge regarding the metabolism of the sulphur-containing amino acids methionine and cysteine in parasitic protozoa and helminths. Particular emphasis is placed on the unusual aspects of parasite biochemistry which may present targets for rational design of antiparasite drugs. In general, the basic pathways of sulphur amino acid metabolism in most parasites resemble those of their mammalian hosts, since the enzymes involved in (a) the methionine cycle and S-adenosylmethionine metabolism, (b) the trans-sulphuration sequence, (c) the transminative catabolism of methionine, (d) the oxidative catabolism of cysteine and (e) glutathione synthesis have been demonstrated variously in several helminth and protozoan species. Despite these common pathways, there also exist numerous differences between parasite and mammalian metabolism. Some of these differences are relatively subtle. For example, the biochemical properties (and primary amino acid structures) of certain parasite methionine cycle enzymes and S-adenosylmethionine decarboxylases differ from those of the corresponding mammalian enzymes, and nematodes and trichomonads possess a novel, non-mammalian form of the trans-sulphuration enzyme cystathionine beta-synthase. The most profound differences between parasite and mammalian biochemistry relate to a number of unusual enzymes and thiol metabolites found in parasitic protozoa. In certain protozoa the pathway for methionine recycling from 5'-methylthioadenosine differs markedly from the mammalian route, and involves 2 exclusively microbial enzymes. Trypanosomatid protozoa contain the non-mammalian antioxidant thiol compounds ovothiol A and trypanothione, together with unique trypanothione-linked enzymes. Specific anaerobic protozoa possess another exclusively microbial enzyme, methionine gamma-lyase, which catabolises methionine (and homocysteine); the physiological significance of these non-mammalian activities is not fully understood. These unusual features offer opportunities for chemotherapeutic exploitation, and in some cases represent metabolic similarities with bacteria. Additionally, some anaerobic protozoa contain unidentified thiols and this implies the presence of further unusual enzymes/pathways in these organisms. So far, no truly unique targets for chemotherapy have been found in helminth sulphur amino acid metabolism, and to some degree this reflects the relative lack of detailed study in the area.     

Ionic and haemodynamic changes influence the release of the excitatory amino acid glutamate in the posterior hypothalamus

Argyrophilic and tau-positive abnormal structures occurring in glial cells are called glial fibrillary tangles. In the astrocyte, a conspicuous tau-positive structure is known to appear in progressive supranuclear palsy (PSP). In this report, another type of argyrophilic and tau-positive astrocytes is reported. The morphology of this new type is quite different from that of the previously reported tau-positive astrocyte in PSP and they are designated here as thorn-shaped astrocytes (TSA). TSA have an apparently argyrophilic cytoplasm with a few short processes and often have a small eccentric nucleus, whose appearance resembles that of a reactive astrocyte. Immunohistochemically, TSA are positive to anti-tau antibodies but are negative for ubiquitin. Simultaneous immunostaining revealed the coexistence of tau and glial fibrillary acidic protein epitopes in the same cytoplasm. Electron microscopically, bundles of 15-nm straight tubules were included in the cytoplasm together with abundant glial filaments. In the vicinity of a cluster of TSA, related structures of perivascular or subpial tau-positive linings, which correspond to astrocytic end-feet, are sometimes observed. In almost all cases, a few TSA are generally located in a confined area of subpial and subependymal regions. Although TSA appear to be intimately associated with some diseases, they are also found in a wide range of cytoskeletal disorders including the aged brain with neurofibrillary tangles. TSA are presumed to be a secondary induced product in relation to astrocytic reaction.     

Probing of DNA-binding sites of Escherichia coli RecA protein utilizing 1-anilinonaphthalene-8-sulfonic acid

RecA protein of Escherichia coli plays an essential role in homologous recombination of DNA strands. To analyze the interaction of RecA with single-stranded DNA (ssDNA), we performed a fluorescence competition assay employing 1-anilinonaphthalene-8-sulfonic acid (ANS) as an extrinsic fluorescent probe. ANS bound to RecA at three sites, leading to enhancement of ANS fluorescence. Addition of synthetic polynucleotides to the RecA-ANS complex in the absence of a nucleotide quenched the ANS fluorescence, indicating displacement of ANS molecules by ssDNA. Less effective quenching by poly(dA) suggests that the nucleoprotein filament on poly(dA) may differ from those on poly(dT) and poly(dC). A titration experiment with poly(dT) and poly(dA) showed clear stoichiometric binding of 3.5 nucleotides per protein. The site size for poly(dC) was 7.0, which could be explained by the formation of a double helix of poly(dC). ATP and other nucleotides also displaced the ANS. To identify ANS-binding sites, ANS was incorporated into RecA by UV irradiation, and fluorescent peptides were isolated from the proteolytic digest. Sequence analysis suggested that ANS binds to or near the ATP-binding region. These results suggest that the fluorescence quenching and photoincorporation assay using ANS may be useful for the analysis of the interaction of a protein and its ligand.     

Conformational changes affect binding and catalysis by ester-hydrolysing antibodies

D2.3, D2.4 and D2.5 are ester-hydrolysing antibodies raised against a phosphonate transition state analogue (TSA). All three antibody-TSA binding kinetics, as monitored by fluorescence quenching, indicate an "induced-fit" mechanism: fast bimolecular association followed by a unimolecular isomerisation (k=1-7 s-1). Isomerisation leads to a 30-170-fold increase in affinity towards the TSA and, consequently, to higher catalytic rates. Antibody D2.3 exhibits a complex three-step binding mechanism, in which the last step is a "very slow" isomerisation (k<0.02 s-1). This very slow isomerisation is limiting the rate of catalysis by D2.3, as indicated by the kinetics of product release which show characteristics of enzyme "conformational memory" or "hysteresis". The results support a mechanism consisting of pre-equilibrium between "nether-active" (low affinity) and "active" (high affinity) antibody conformers (prior to ligand addition) as well as induced-fit, i.e. isomerisation of the nether-active ligand-antibody complex to give the active complex. Crystal structures of these antibodies, free and complexed, have previously indicated that their conformation does not change upon binding. Here, we show that the buffer used to crystallise the antibodies, and in particular its polyethylene glycol component, alters the pre-equilibrium in favour of the active conformer, leading to its crystallisation both in the presence and in the absence of the TSA.     

Excitatory amino acid receptors and puberty

Glutamate is an important excitatory signal in the hypothalamus for the steroid-mediated preovulatory gonadotropin surge. Steroids may exert this action by regulating glutamate receptor levels or glutamate release, or both. Work in our laboratory found no changes in NMDA and kainate receptor binding in the hypothalamus of castrated or castrated plus steroid-replaced male and female rats. Likewise, we found that NMDA and kainate binding did not change over the onset of puberty in the female rat. A competitive quantitative RT-PCR assay using exogenous internal standards was used to measure NMDAR1, GluR1, and beta-actin mRNAs levels. NMDAR1 and GluR1 expression was examined in the preoptic hypothalamic area and in the medial basal hypothalamus at Postnatal Days 10, 15, 20, 25, 30, 32, 34, 36, 40, and 63. A transient increase in GluR1 mRNA levels in the preoptic hypothalamic area was observed on Day 20, with all other time points showing comparable levels. NMDAR1 levels in the POA and medial basal hypothalamus did not change significantly at any of the time points; in contrast, however, AMPA receptor binding levels were increased in the hypothalamus at the time of puberty in the female rat. Thus, in addition to the previously reported elevation of glutamate release rates in the hypothalamus at the time of puberty, AMPA receptors may also be elevated and play a role in mediating glutamate regulatory effects on the timing of puberty in the female rat.     

Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination

The recA locus of pathogenic mycobacteria differs from that of non-pathogenic species in that it contains large intervening sequences termed protein introns or inteins that are excised by an unusual protein-splicing reaction. In addition, a high degree of illegitimate recombination has been observed in the pathogenic Mycobacterium tuberculosis complex. Homologous recombination is the main mechanism of integration of exogenous nucleic acids in M. smegmatis, a non-pathogenic mycobacterium species that carries an inteinless RecA and is amenable to genetic manipulations. To investigate the function of recA in mycobacteria, recA- strains of M. smegmatis were generated by allelic exchange techniques. These strains are characterized (i) by increased sensitivity towards DNA-damaging agents [ethylmethylsulphonate (EMS), mitomycin C, UV irradiation] and (ii) by the inability to integrate nucleic acids by homologous recombination. Transformation efficiencies using integrative or replicative vectors were not affected in recA- mutants, indicating that in mycobacteria RecA does not affect plasmid uptake or replication. Complementation of the recA- mutants with the recA from M. tuberculosis restored resistance towards EMS, mitomycin C and UV irradiation. Transformation of the complemented strains with suicide vectors targeting the pyrF gene resulted in numerous allelic exchange mutants. From these data, we conclude that the intein apparently does not interfere with RecA function, i.e. with respect to competency for homologous recombination, the RecAs from pathogenic and non-pathogenic mycobacteria are indistinguishable.     

Rapid postmortem changes in the cellular localisation of amino acid transmitters in the retina as assessed by immunocytochemistry

We have assessed by means of immunocytochemistry, the cellular distributions of the amino acid transmitters GABA, glycine and glutamate, and the free-radical scavenger taurine, in the retinae of adult rabbits at various times after death. Within 10 min of death, horizontal cells began to display immunoreactivity for GABA, whilst displaced amacrine cells began to display immunoreactivity for glycine. By 40 min postmortem, GABA was present in glial cells. Glutamate, which is not normally detectable in retinal glia, was detected in such glia by 20 min postmortem. By contrast immunocytochemically detectable glycine did not accumulate in glia. There was a gradual diminution of immunoreactivity for taurine in glial cells and photoreceptors. By 2 h postmortem, most immunoreactivity had disappeared from the retina. We conclude that amino acid transmitters show rapid changes in their distributions immediately after death, which may be related to changes in the patterns of transmitter release and uptake, and changes in degradation mechanisms. The rapid changes in cellular localisation of amino acid immunoreactivity illustrated in this study, indicate that the fixation of nervous tissues must be performed rapidly. Moreover, the massive loss of immunoreactivity by 2 h postmortem suggests that any assays for content of these transmitters at this, and subsequent time-points, will bear little resemblance to the values obtained at the time of death.     

The effect of amino acids and amino acid derivatives on cell proliferation

The effect of certain amino acids and amino acid derivatives on cell proliferation have been studied in the author's Institute for more than 25 years. The optically active forms of arginine, lysine, aspartic acid and glutamic acid influence the growth of transplantable rat tumors. L-arginine, D-lysine, L-aspartic acid and D-glutamic acid promoted; D-aspartic acid, L-glutamic acid, D-arginine and L-lysine inhibited tumour growth. E-amino trimethyl-lysine (TML) stimulated cell proliferation in various cell systems (bone marrow, small intestine, cultured lymphocytes). When administered simultaneously with high doses of Cyclophosphamide, Vincristin or Doxorubicin to tumour-bearing mice, TML decreased the toxicity of the antitumour drugs, resulting in a higher rate of survivors. L-leucine methyl ester caused cell death of mouse peritoneal macrophages by inducing disruption of macrophages.