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ABSTRACT The American lobster [Homarus americanus (Edwards, 1837)] is an omnivore decapod with generalist and opportunistic feeding habits, in which gastric fluid cysteine and aspartic peptidases are the main proteolytic [...]

Aspartate key to proton selectivity Voltage-gated proton channels are considered completely selective for protons -- no evidence exists for the permeation of ions other than H.sup.+. A study of the underlying mechanism of the high selectivity of the human voltage-gated proton channel reveals an Asp112 residue as the selectivity filter. When this residue was changed to a neutral amino acid, the mutant channel lost proton selectivity and either became anion-selective or did not conduct any ions. The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel.sup.1, H.sub.V1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes.sup.2,3,4, histamine secretion by basophils.sup.5, sperm capacitation.sup.6, and airway pH.sup.7. The most selective ion channel known, H.sub.V1 shows no detectable permeability to other ions.sup.1. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity.sup.1,8,9,10,11, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions.sup.12. Here we identify aspartate 112 as a crucial component of the selectivity filter of H.sub.V1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H.sub.V1 requires an acidic group at the selectivity filter., Author(s): Boris Musset [sup.1] , Susan M. E. Smith [sup.2] , Sindhu Rajan [sup.3] , Deri Morgan [sup.1] , Vladimir V. Cherny [sup.1] , Thomas E. DeCoursey [sup.1] Author Affiliations: [...]

Many of the marine microorganisms which are adapted to grow at temperatures above 80[degrees]C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakarensis (formerly Thermococcus kodakaraensis), manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and a-glutamate. Under heat stress the level of DIP increased 20-fold compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, over-expression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85[degrees]C) and supraoptimal (93.7[degrees]C) temperatures for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakarensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptatian of (hyper)thermophilic organisms. doi: 10.1128/JB.01115-09

The uptake of branched-chain amino acids (BCAA) from plasma by lactating porcine mammary gland substantially exceeds their output in milk, whereas glutamine output is 125% greater than its uptake from plasma. In this study, we tested the hypothesis that BCAA are catabolized for glutamine synthesis in mammary tissue. Mammary tissue slices from sows on d 28 of lactation were incubated at 37[degrees]C for 1 h in Krebs buffer containing 0.5 or 2 mmol/L L-[1-[.sup.14]C]- or L-[U-[.sup.14]C]-labeled leucine, isoleucine, or valine. Rates of BCAA transport and degradation in mammary tissue were high, with ~60% of transaminated BCAA undergoing oxidative decarboxylation and the remainder being released as branched-chain [alpha]ketoacids (BCKA). Most (~70%) of the decarboxylated BCAA were oxidized to C[O.sub.2]. Rates of net BCAA transamination were similar to rates of glutamate, glutamine, aspartate, asparagine, and alanine synthesis. Consistent with the metabolic data, mammary tissue expressed BCAA aminotransferase (BCAT), BCKA decarboxylase, glutamine synthetase (GS), glutamate-oxaloacetate aminotransferase, glutamate-pyruvate aminotransferase, and asparagine synthetase, but no phosphate-activated glutaminase, activity. Western blot analysis indicated relatively high levels of mitochondrial and cytosolic isoforms of BCAT, as well as BCKA dehydrogenase and GS proteins in mammary tissue. Our results demonstrate that glutamine and aspartate (abundant amino acids in milk protein) were the major nitrogenous products of BCAA catabolism in lactating porcine mammary tissue and provide a biochemical basis to explain an enrichment of glutamine and aspartate in sow milk. J. Nutr. 139: 1502-1509, 2009.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ajhg.2009.04.017 Byline: Joy Armistead (1)(5), Sunita Khatkar (1)(5), Britta Meyer (6), Brian L. Mark (2), Nehal Patel (1)(5), Gail Coghlan (3)(5), Ryan E. Lamont (1)(5), Shuangbo Liu (1), Jill Wiechert (1)(5), Peter A. Cattini (4)(5), Peter Koetter (6), Klaus Wrogemann (1)(3)(5), Cheryl R. Greenberg (1)(3)(5), Karl-Dieter Entian (6), Teresa Zelinski (1)(3)(5), Barbara Triggs-Raine (1)(3)(5) Abstract: Bowen-Conradi syndrome (BCS) is an autosomal-recessive disorder characterized by severely impaired prenatal and postnatal growth, profound psychomotor retardation, and death in early childhood. Nearly all reported BCS cases have been among Hutterites, with an estimated birth prevalence of 1/355. We previously localized the BCS gene to a 1.9 Mbp interval on human chromosome 12p13.3. The 59 genes in this interval were ranked as candidates for BCS, and 35 of these, including all of the best candidates, were sequenced. We identified variant NM_006331.6:c.400A[right arrow]G, p.D86G in the 18S ribosome assembly protein EMG1 as the probable cause of BCS. This mutation segregated with disease, was not found in 414 non-Hutterite alleles, and altered a highly conserved aspartic acid (D) residue. A structural model of human EMG1 suggested that the D86 residue formed a salt bridge with arginine 84 that would be disrupted by the glycine (G) substitution. EMG1 mRNA was detected in all human adult and fetal tissues tested. In BCS patient fibroblasts, EMG1 mRNA levels did not differ from those of normal cells, but EMG1 protein was dramatically reduced in comparison to that of normal controls. In mammalian cells, overexpression of EMG1 harboring the D86G mutation decreased the level of soluble EMG1 protein, and in yeast two-hybrid analysis, the D86G substitution increased interaction between EMG1 subunits. These findings suggested that the D-to-G mutation caused aggregation of EMG1, thereby reducing the level of the protein and causing BCS. Author Affiliation: (1) Department of Biochemistry and Medical Genetics, University of Manitoba, 745 Bannatyne Ave., Winnipeg, MB, Canada R3R 0J9 (2) Department of Microbiology, University of Manitoba, 745 Bannatyne Ave., Winnipeg, MB, Canada R3R 0J9 (3) Department of Pediatrics & Child Health, University of Manitoba, 745 Bannatyne Ave., Winnipeg, MB, Canada R3R 0J9 (4) Department of Physiology, University of Manitoba, 745 Bannatyne Ave., Winnipeg, MB, Canada R3R 0J9 (5) Centre for the Investigation of Genetic Disease, Manitoba Institute for Child Health, Winnipeg, MB, Canada R3E 3P4 (6) Excellence Center: Macromolecular Complexes, Institute for Molecular Biosciences, Johann Wolfgang Goethe University, Marie Curie St. 9, 60439 Frankfurt/Main, Germany Article History: Received 9 March 2009; Revised 15 April 2009; Accepted 27 April 2009 Article Note: (miscellaneous) Published online: May 21, 2009

AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of L-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of L-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, L-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes L-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a functional unit.

NMR isotopic filiation of [sup.13]C-labelled aspartate and glutamate was used to explore the tricarboxylic acid (TCA) pathway in Saccharomyces cerevisiae during anaerobic glucose fermentation. The assimilation of [[3-.sup.13]]C]aspartate led to the formation of [2,[3-.sup.13]C]malate and [2,[3-.sup.13]C]succinate, with equal levels of [sup.13]C incorporation, whereas site-specific enrichment on C-2 and C-3 of succinate was detected only with [[3-.sup.13]C]glutamate. The non-random distribution of [sup.13]C labelling in malate and succinate demonstrates that the TCA pathway operates during yeast fermentation as both an oxidative and a reductive branch. The observed [sup.13]C distribution suggests that the succinate dehydrogenase (SDH) complex is not active during glucose fermentation. This hypothesis was tested by deleting the SDH1 gene encoding the flavoprotein subunit of the SDH complex. The growth, fermentation rate and metabolite profile of the sdh1 mutant were similar to those of the parental strain, demonstrating that SDH was indeed not active. Filiation experiments indicated the reductive branch of the TCA pathway was the main pathway for succinate production if aspartate was used as the nitrogen source, and that a surplus of succinate was produced by oxidative decarboxylation of 2-oxoglutarate if glutamate was the sole nitrogen source. Consistent with this finding, a kgd1 mutant displayed lower levels of succinate production on glutamate than on other nitrogen sources, and higher levels of oxoglutarate dehydrogenase activity were observed on glutamate. Thus, the reductive branch generating succinate via fumarate reductase operates independently of the nitrogen source. This pathway is the main source of succinate during fermentation, unless glutamate is the sole nitrogen source, in which case the oxidative decarboxylation of 2-oxoglutarate generates additional succinate.

DNA ligase IV is thought to be involved in DNA double-strand break repair and DNA non-homologous end-joining pathways, but these mechanisms are still unclear. To investigate the roles of DNA ligase IV from a biologically functional viewpoint, the authors studied its relationship to meiosis in a basidiomycete, Coprinus cinereus, which shows a highly synchronous meiotic cell cycle. The C. cinereus cDNA homologue of DNA ligase IV (CcLIG4) was successfully cloned. The 3.2 kb clone including the ORF encoded a predicted product of 1025 amino acid residues with a molecular mass of 117 kDa. A specific inserted sequence composed of 95 amino acids rich in aspartic acid and glutamic acid could be detected between tandem BRCT domains. The inserted sequence had no sequence identity with other eukaryotic counterparts of DNA ligase IV or with another aspartic acid and glutamic acid rich sequence inserted in C. cinereus proliferating cell nuclear antigen (CcPCNA), although the length and the percentages of aspartic and glutamic acids were similar. In addition, the recombinant CcLIG4 protein not only showed ATP-dependent ligase activity, but also used [(dT)].sub.16]/poly(dA) and [(dT).sub.16]/poly(rA) as substrates, and had double-strand ligation activity, like human DNA ligase IV. Northern hybridization analysis and in situ hybridization indicated that CcLIG4 was expressed not only at the pre-meiotic S phase but also at meiotic prophase I. Intense signals were observed in leptotene and zygotene. Based on these observations, the possible role(s) of C. cinereus DNA ligase IV during meiosis are discussed.

Extracts of a multiply peptidase-deficient (pepNABDPQTE iadA iaaA) Salmonella enterica serovar Typhimurium strain contain an aspartyl dipeptidase activity that is dependent on M[n.sup.2+]. Purification of this activity followed by N-terminal sequencing of the protein suggested that the M[n.sup.2+]-dependent peptidase is DapE (N-succinyl-L,L-diaminopimelate desuccinylase). A dapE chromosomal disruption was constructed and transduced into a multiply peptidase-deficient (MPD) strain. Crude extracts of this strain showed no aspartyl peptidase activity, and the strain failed to utilize Asp-Leu as a leucine source. The dapE gene was cloned into expression vectors in order to overproduce either the native protein (DapE) or a hexahistidine fusion protein (DapE-[His.sub.6]). Extracts of a strain carrying the plasmid overexpresssing native DapE in the MPD dapE background showed a 3,200-fold elevation of M[n.sup.2+]-dependent aspartyl peptidase activity relative to the MPD dap[E.sup.+] strain. In addition, purified DapE-[His.sub.6] exhibited M[n.sup.2+]-dependent peptidase activity toward aspartyl dipeptides. Growth of the MPD strain carrying a single genomic copy of dapE on Asp-Leu as a Leu source was slow but detectable. Overproduction of DapE in the MPD dapE strain allowed growth on Asp-Leu at a much faster rate. DapE was found to be specific for N-terminal aspartyl dipeptides: no N-terminal Glu, Met, or Leu peptides were hydrolyzed, nor were any peptides containing more than two amino acids. DapE is known to bind two divalent cations: one with high affinity and the other with lower affinity. Our data indicate that the form of DapE active as a peptidase contains Z[n.sup.2+] in the high-affinity site and M[n.sup.2+] in the low-affinity site.

The [S.sub.N]2 displacement of [Cl.sup.-] from 1,2-dichloroethane by acetate (C[H.sub.3]C[O.sup.-.sub.2]) in water and by the carboxylate of the active site aspartate in the haloalkane dehalogenase of Xanthobacter autothropicus have been compared by using molecular dynamics simulations. In aqueous solution, six families of contact-pair structures (I-VI) were identified, and their relative concentrations and dissociation rate constants were determined. The near attack conformers (NACs) required for the [S.sub.N]2 displacement reaction are members of the IV (C[H.sub.3]CO[O.sup.-]***C[H.sub.2](Cl)C[H.sub.2]Cl) family and are formed in the sequence II [right arrow] III [right arrow] IV [right arrow] NAC. The NAC subclass is defined by the--CO[O.sup.-]***C--Cl contact distance of [less than or equal to] 3.41 [Angstrom] and the--CO[O.sup.-]***C--Cl angle of 157-180[degrees]. The mole percentage of NACs is 0.16%, based on the 1 M standard state. This result may be compared with 13.4 mole percentage of NACs in the Michaelis complex in the enzyme. It follows that NAC formation in the enzyme is favored by 2.6 kcal/mol. Because reaction coordinates from S to TS, both in water and in the enzyme, pass via NAC (i.e., S [right arrow] NAC [right arrow] TS), the reduction in the S [right arrow] NAC barrier by 2.6 kcal/mol accounts for [approximately equal to] 25% of the reduction of total barrier in the S [right arrow] TS (10.7 kcal/mol). The remaining 75% of the advantage of the enzymatic reaction revolves around the efficiency of NAC [right arrow] TS step. This process, based on previous studies, is discussed briefly.

The N-methyl-D-aspartate (NMDA) glutamate receptor (NMDAR), long implicated in developmental plasticity, shows decay time kinetics that shorten postnatally as NR2A subunits are added to the receptor. Neither the mechanism nor immediate effect of this change is known. We studied developing NMDAR currents by using visual neurons in slices from NR2A knockout (NR2AKO) and WT mice. Both strains show increased dendritic levels of synaptic density scaffolding protein PSD-95 with age. Dendritic levels of NR2A increased at the same time in WT and immunoprecipitated with PSD-95. PSD-95/NMDAR binding was significantly decreased in the NR2AKO. Moreover, NMDAR miniature currents (minis) were lost and rise times of NMDAR evoked currents increased in mutant mice. Age-matched WT cells showed NR2A-rich receptors predominating in minis, yet slow NR2B mediated currents persisted in evoked currents. Disrupting photoreceptor activation of retinal ganglion cells eliminated increases in PSD-95 and NR2A in superior collicular dendrites of WT mice and slowed the loss of miniature NMDAR currents in NR2AKOs. These data demonstrate that NMDARs that respond to single quantal events mature faster during development by expressing the NR2A subunit earlier than NMDARs that respond to evoked release. We hypothesize that NR2A-rich NMDARs may be localized to the center of developing synapses by an activity-dependent process that involves the targeting of PSD-95 to the postsynaptic density. Neonatal receptors become restricted to perisynpatic or extrasynaptic sites, where they participate primarily in evoked currents.

Mutagenesis was used to probe the interface between the small GTPase Cdc42p and the CRIB domain motif of Ste20p. Members of a cluster of hydrophobic residues of Cdc42p were changed to alanine and/ or arginine. The interaction of the wild-type and mutant proteins was measured using the two-hybrid assay; many, but not all, changes reduced interaction between Cdc42p and the target CRIB domain. Mutations in conserved residues in the CRIB domain were also tested for their importance in the association with Cdc42p. Two conserved CRIB domain histidines were changed to aspartic acid. These mutants reduced mating, as well as responsiveness to pheromone-induced gene expression and cell cycle arrest, but did not reduce in vitro the kinase activity of Ste20p. GFP-tagged mutant proteins were unable to localize to sites of polarized growth. In addition, these point mutants were synthetically lethal with disruption of CLA4 and blocked the Ste20p-Cdc42p two-hybrid interaction. Compensatory mutations in Cdc42p that reestablished the two-hybrid association with the mutant Ste20p CRIB domain baits were identified. These mutations improved the pheromone responsiveness of cells containing the CRIB mutations, but did not rescue the lethality associated with the CRIB mutant CLA4 deletion interaction. These results suggest that the Ste20p-Cdc42p interaction plays a direct role in Ste20p kinase function and that this interaction is required for efficient activity of the pheromone response pathway.

Research has been conducted on the dopamine D1-like receptors. Experimental results demonstrate that these receptors modulate N-methyl-D-aspartate glutamate receptor-mediated functions via direct protein-protein interactions.

Research has been conducted on Pyrococcus furiosus ferredoxin with the disulfide bond. The solution molecular model of this ferredoxin constructed via the use of NMR spectral parameters, distance geometry and simulated annealing protocols is described.

Research has been conducted on the malic enzyme reaction. The substrates for this reactions have been identified and characterized with respect to the reaction catalyzed and reaction's kinetics and the results have been compared to those observed with L-malate.

Research has been conducted on the arginine substitutions in the DRY motif (aspartic acid, arginine and tyrosine). The effect of these substitutions in alpha (sub)1B -adrenergic receptors and angiotensin II type 1A receptors on their localization, signaling and desensitization has been studied and the results are presented.

Research has been conducted on the receptor methylation in Escherichia coli chemotaxis. The role of this receptor in adaptation has been investigated and the details are reported.

Antagonists to the N-methyl-D-aspartate (NMDA) receptor bind to various extraneuronal tissues. We therefore assessed the expression of the main NMDA subunit, NR1, in various tissues. We demonstrate that NR1 appears to be most abundant in the rat kidney and heart. NR1 is present in total rat kidney, cortex, and medulla. Of the NR2 subunits, only the NR2C subunit protein is present in the kidney. The abundance of the NR1 subunit protein increases with kidney development. Both NR1 and NR2C are present in opossum kidney, Madin-Darby canine kidney, and LLC-P[K.sub.1] cells. Immunohistochemistry studies show that the NR1 subunit is present in the renal proximal tubule. NR1 is abundant in the atrium and ventricle but is also expressed in the aorta and pulmonary artery. The NR2 subunits are not expressed in the heart. NR1 subunit protein expression is constant throughout heart development. Finally, the NR1 subunit protein is expressed in heart cells (H9c2) grown in culture. These studies reveal the presence of the NMDA receptor in the kidney and the cardiovascular system. N-methyl-D-aspartate receptor; development

Amyloid [beta]-peptide (A[beta]) is generated by the consecutive cleavages of [beta]-and [gamma]-secretase. The intramembraneous [gamma]-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with [gamma]-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of A[beta] production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the [gamma]-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with [gamma]-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by [gamma]-secretase inhibitors results in delayed reinternalization of the [beta]-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in [gamma]-secretase processing and in trafficking.

Although [Zn.sup.2+] is contained in large amounts in the synaptic terminals of hippocampal mossy fibers (MFs), its physiological role in synaptic transmission is poorly understood. By using the newly developed high-sensitivity [Zn.sup.2+] indicator ZnAF-2, the spatiotemporal dynamics of [Zn.sup.2+] was monitored in rat hippocampal slices. When high-frequency stimulation was delivered to the MFs, the concentration of extracellular [Zn.sup.2+] was immediately elevated in the stratum lucidum, followed by a mild increase in the stratum radiatum adjacent to the stratum lucidum, but not in the distal area of stratum radiatum. The [Zn.sup.2+] increase was insensitive to a non-N-methyl-D-aspartate (NMDA) receptor antagonist but was efficiently attenuated by tetrodotoxin or [Ca.sup.2+]-free medium, suggesting that [Zn.sup.2+] is released by MF synaptic terminals in an activity-dependent manner, and thereafter diffuses extracellularly into the neighboring stratum radiatum. Electrophysiological analyses revealed that NMDA receptor-mediated synaptic responses in CA3 proximal stratum radiatum were inhibited in the immediate aftermath of MF activation and that this inhibition was no longer observed in the presence of a [Zn.sup.2+]-chelating agent. Thus, [Zn.sup.2+] serves as a spatiotemporal mediator in imprinting the history of MF activity in contiguous hippocampal networks. We predict herein a novel form of metaplasticity, i.e., an experience-dependent non-Hebbian modulation of synaptic plasticity.

Research has been conducted on the 2Fe and 4Fe iron (super)- cluster centers in eukaryotic organisms. The biosynthetic apparatus for these cluster center assembly has been investigated and the results indicate that the assembly of the clusters can occur only in the mitochondrion with the proteins imported from the nuclear transcribed mRNA.

Research has been conducted on the N-terminal zinc finger of Sp1. The role of amino acid in DNA binding affinity and base specificity has been investigated and the results indicate that recognition rule for the secondary strand's base can be used as the basis for the zinc finger protein formation.

Research has been conducted on Bacillus subtilis relaxed aspartate-auxotrophic strain. The ability of the strain to develop genetic competence and the role of the RelA protein in the competence development induction under certain physiological conditions have been investigated and discussed.

To differentiate multiple activities of presenilin 1 (PS1), we generated transgenic mice expressing two human PS1 alleles: one with the aspartate to alanine mutation at residue 257 (hPS1D257A) that impairs the proteolytic activity of PS1, and the other deleting amino acids 340-371 of the hydrophilic loop sequence (hPS1[DELTA]cat) essential for [beta]-catenin interaction. We show here that although hPS1[DELTA]cat is fully competent in rescuing the PS1-null lethal phenotype, hPS1D257A does not exhibit developmental activity. hPS1D257A also leads to the concurrent loss of the proteolytic processing of Notch and [beta]-amyloid precursor protein (APP) and the generation of [beta]-amyloid peptides (A[beta]). Further, by measuring the levels of endogenous A[[beta].sub.x-40] and A[[beta].sub.x-42] in primary neuronal cultures, we confirmed the concept that PS1 is indispensable for the production of secreted A[beta].

The [Ca.sup.2+] affinity and permeation of the epithelial [Ca.sup.2+] channel (ECaC1) were investigated after expression in Xenopus oocytes. ECaC1 displayed anomalous mole-fraction effects. Extra-cellular [Ca.sup.2+] and [Mg.sup.2+] reversibly inhibited ECaC1 whole cell [Li.sup.+] currents: I[C.sub.50] = 2.2 [+ or -] 0.4 [micro]M (n = 9) and 235 [+ or -] 35 [micro]M (n = 10), respectively. These values compare well with the [Ca.sup.2+] affinity of the L-type voltage-gated [Ca.sup.2+] ([Ca.sub.v]1.2) channel measured under the same conditions, suggesting that high-affinity [Ca.sup.2+] binding is a well-conserved feature of epithelial and voltage-gated [Ca.sup.2+] channels. Neutralization of D550 and E535 in the pore region had no significant effect on [Ca.sup.2+] and [Mg.sup.2+] affinities. In contrast, neutralization of D542 significantly decreased [Ca.sup.2+] affinity (I[C.sub.50] = 1.1 [+ or -] 0.2 mM, n = 6) and [Mg.sup.2+] affinity (I[C.sub.50] > 25 [+ or -] 3 mM, n = 4). Despite a 1,000-fold decrease in [Ca.sup.2+] affinity in D542N, [Ca.sup.2+] permeation properties and the [Ca.sup.2+]-to-[Ba.sup.2+] conductance ratio remained comparable to values for wild-type ECaCi. Together, our observations suggest that D542 plays a critical role in [Ca.sup.2+] affinity but not in [Ca.sup.2+] permeation in ECaCi. Xenopus oocytes; structure function; site-directed mutagenesis; single channel; distal tubule; kidney; selectivity

Research has been conducted on the bacterial toxins, colicin N and A. The pore-forming behavior of the colicins' domains has been investigated in the acid conditions and the results indicate that colicin A is a protein relying on the acid sensitive interactions for its stability at the neutral pH.

Cocultures of neurons and astrocytes from the rat striatum were used to determine whether the stimulation of neuronal receptors could affect the level of intercellular communication mediated by gap junctions in astrocytes. The costimulation of N-methyl-D-asparte (NMDA) and muscarinic receptors led to a prominent reduction of astrocyte gap junctional communication (GJC) in coculture. This treatment was not effective in astrocyte cultures, these cells being devoid of NMDA receptors. Both types of receptors contribute synergistically to this inhibitory response, as the reduction in astrocyte GJC was not observed after the blockade of either NMDA or muscarinic receptors. The involvement of a neuronal release of arachidonic acid (AA) in this inhibition was investigated because the costimulation of neuronal NMDA and muscarinic receptors markedly enhanced the release of AA in neuronal cultures and in cocultures. In addition, both the reduction of astrocyte GJC and the release of AA evoked by NMDA and muscarinic receptor costimulation were prevented by mepacrine, a phospholipase [A.sub.2] inhibitor, and this astrocyte GJC inhibition was mimicked by the exogenous application of AA. Metabolites of AA formed through the cyclooxygenase pathway seem to be responsible for the effects induced by either the costimulation of NMDA and muscarinic neuronal receptors or the application of exogenous AA because, in both cases, astrocyte GJC inhibition was prevented by indomethacin. Altogether, these data provide evidence for a neuronal control of astrocytic communication and open perspectives for the understanding of the modalities through which cholinergic interneurons and glutamatergic inputs affect local circuits in the striatum.

Differential effects of an NMDA and a non-NMDA receptor antagonist on medullary lateral tegmental field neurons. Am J Physiol Regulatory Integrative Comp Physiol 282: R100-R113, 2002.--We microiontophoresed an N-methyl-D-aspartate (NMDA) and a non-NMDA receptor antagonist onto medullary lateral tegmental field (LTF) neurons, the naturally occurring discharges of which were correlated to the cardiac-related rhythm in sympathetic nerve discharge (SND) of dialurethane-anesthetized cats. Some of these neurons were classified as sympathoexcitatory, because their firing rate decreased during baroreceptor reflex activation. Microiontophoresis of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo -[f]quinoxaline-7-sulfonamide (NBQX), a non-NMDA receptor antagonist, reduced the mean firing rates of these neurons (51 [+ or -] 8% of control, P < 0.001, n = 20) without affecting their relationship to cardiac-related SND, as indicated by the lack of significant changes in the ratio of peak to background counts in arterial pulse (AP)-triggered histograms of LTF neuronal activity and the AP-LTF coherence value at the frequency of the heartbeat. In contrast, microiontophoresis of D(-)-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist, onto LTF neurons reduced the ratio of peak to background counts in AP-triggered histograms to 57 [+ or -] 9% of control (P = 0.002, n = 16) and the AP-LTF coherence value to 25 [+ or -] 10% of control (P = 0.001, n = 10). These data support the view that non-NMDA and NMDA receptors are involved in setting the basal level of activity of LTF sympathoexcitatory neurons and in synchronizing their discharges to the AP, respectively. cardiac-related activity; excitatory amino acid neurotransmission; sympathetic nerve discharge; sympathoexcitatory neurons

Research has been conducted on the aspartate-beta-semialdehyde dehydrogenase. The structure of this dehydrogenase from Escherichia coli has been investigated via crystallography in the presence of its coenzyme and substrate analogue and the results are reported.

Research has been conducted on the aminopeptidase A, a membrane-bound zinc enzyme. The role of the Asn-353 of the conserved motif GXMEN in aminopeptidase A has been investigated by substituting this residue with the glutamine, alanine or aspartate residue via the site-directed mutagenesis and the results are reported.

Research has been conducted on the conserved aspartates in the transmembrane domain of the rat mu opioid receptor. The role of these aspartates have been investigated and the results are described.

Research has been conducted on the bacterial Na (super)+ / H (super)+ antiporter, NhaB from Vibrio alginolyticus. The pH dependence of NhaB has been investigated and the results are discussed.

Research has been conducted on the asparagine synthetases catalyzing the ATP-dependent formation of L-aspargine from L-aspartate and L-glutamine. The small-molecule inhibitors of Escherichia coli asparagine synthetase B have been investigated as a model system for treating leukemia and solid tumors.

Research has been conducted on the transmembrane domains which spontaneously fold into alpha-helices upon insertion into membrane-mimetic environments. The ability of these transmembrane helices from biological systems to retain their native secondary structures and oligomeric states has been investigated and the results are presented.

This review focuses on the major investigations into the baker's yeast made during the last decade. The comparison of the established aspartate transcarbamoylase model system with the catalytic and regulatory features of E. coli has been performed and analyzed.