Basal fat oxidation and after a peak oxygen consumption test in obese women with a beta2 adrenoceptor gene polymorphism

The Glu27Glu genotype in the beta2-adrenergic receptor (ADRB2) has been linked to a higher fat deposition and obesity in females. Also, in our population, it has been described that physically active women carrying the Glu allele had a higher BMI as compared to non-carriers performing the same level of activity. Since exercise may counterbalance a gene predisposition to obesity, we tested the hypothesis of a potential different metabolic response among ADRB2 Gln27Gln versus Glu27Glu obese women when submitted to a peak oxygen consumption test on a treadmill. In our study, 10 obese women with the Gln27Gln genotype were compared to 9 matched obese women bearing the Glu27Glu genotype. The ADRB2 polymorphism was identified by PCR-RFLP, fat oxidation was determined by indirect calorimetry and blood measurements were carried out following conventional procedures. The ADRB2 Glu27Glu subjects had lower plasma glycerol levels (P = 0.026), while plasma triglycerides (P <0.001) and the insulin:glucose ratio were higher (P = 0.046) as compared to the Gln27Gln group along the peak oxygen consumption trial intervention. There was a significantly lower fat oxidation (P = 0.024) in the Glu27Glu obese women during the recovery compared to Gln27Gln obese individuals. These data suggest that exercise would not benefit equally the two ADRB2 polymorphism homozygous groups, since both lipolysis and fat oxidation promoted by a peak oxygen consumption test appear to be blunted in the polymorphic Glu27Glu obese group.     

Rational design and directed evolution of a bacterial-type glutaminyl-tRNA synthetase precursor

Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNA(Gln) is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNA(Gln), and an amidotransferase converts Glu-tRNA(Gln) to Gln-tRNA(Gln). The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNA(Gln). It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Persistent depolarization and Glu uptake inhibition operate distinct osmoregulatory mechanisms in the mammalian brain

The ways of coupling neuronal with glial compartments in natural physiology was investigated in microdialysis experiments by monitoring extracellular concentration of amino acids in the brain of anaesthetized rats. We hypothesized that extracellular [Glu], [Gln] and [Tau] patterns would be state-dependent. This was tested by stimulation of N-methyl-D-aspartate (NMDA) receptors, by inhibition of Glu uptake or by local depolarization with a high-K(+) dialysate, coupled with the addition of Co(2+) to block Ca(2+) influx. The results showed that (1) extracellular [Gln] was low whereas [Glu] and [Tau] were high during infusion of NMDA (0.5-1.0 mM) or high-K(+) (80 mM) in the hippocampus and ventrobasal thalamus, (2) hippocampal extracellular [Glu], [Gln] and [Tau] were increased in response to the Glu uptake inhibitor, L-trans-pyrrolidine-2, 4-dicarboxilic acid (tPDC, 0.5-3.0 mM), in a concentration-dependent manner, (3) high-K(+)-induced increase of extracellular [Glu] was partially blocked by the addition of 10 mM CoCl(2) with the high-K(+) dialysate in the hippocampus. Searching for main correlations between changes in [Glu], [Gln] and [Tau] by calculating partial correlations and with the use of factor analyses we found, the primary response of the mammalian brain to persistent depolarization is the neuronal uptake of [Gln] and release of [Tau] thereupon, acting independently of Glu changes. When glial and neuronal uptake of Glu is blocked, releases of Tau occur from neuronal as well as glial compartments accompanied by increases of [Gln] in the mammalian brain.     

Mutagenic and enzymological studies of the hydratase and isomerase activities of 2-enoyl-CoA hydratase-1

Structural and enzymological studies have shown the importance of Glu144 and Glu164 for the catalysis by 2-enoyl-CoA hydratase-1 (crotonase). Here we report about the enzymological properties of the Glu144Ala and Glu164Ala variants of rat mitochondrial 2-enoyl-CoA hydratase-1. Size-exclusion chromatography and CD spectroscopy showed that the wild-type protein and mutants have similar oligomerization states and folding. The kcat values of the active site mutants Glu144Ala and Glu164Ala were decreased about 2000-fold, but the Km values were unchanged. For study of the potential intrinsic Delta3-Delta2-enoyl-CoA isomerase activity of mECH-1, a new assay using 2-enoyl-CoA hydratase-2 and (R)-3-hydroxyacyl-CoA dehydrogenase as auxiliary enzymes was introduced. It was demonstrated that rat wild-type mECH-1 is also capable of catalyzing isomerization with the activity ratio (isomerization/hydration) of 1/5000. The kcat values of isomerization in Glu144Ala and Glu164Ala were decreased 10-fold and 1000-fold, respectively. The data are in line with the proposal that Glu164 acts as a protic amino acid residue for both the hydration and the isomerization reaction. The structural factors favoring the hydratase over the isomerase reaction have been addressed by investigating the enzymological properties of the Gln162Ala, Gln162Met, and Gln162Leu variants. The Gln162 side chain is hydrogen bonded to the Glu164 side chain; nevertheless, these mutants have enzymatic properties similar to that of the wild type, indicating that catalytic function of the Glu164 side chain in the hydratase and isomerase reaction does not depend on the interactions with the Gln162 side chain.     

Analysis of Glutamine Accumulation in Rat Brain Mitochondria in the Presence of a Glutamine Uptake Inhibitor,Histidine, Reveals Glutamine Pools With a Distinct Access to Deamidation

Rat cerebral nonsynaptic mitochondria were incubated in medium containing 2 mM glutamine (Gln) or 2 mM glutamate (Glu), in the presence of a Gln uptake inhibitor histidine (His) as well as other basic amino acids, lysine and arginine (Lys, Arg) not inhibiting Gln uptake. Subsequently, the mitochondrial contents of Glu and Gln were determined by HPLC. Incubation in the presence of Glu alone increased the Glu content from 3.5 to 15 nmol/mg protein, without affecting the Gln content. On the other hand, incubation with Gln increased the content of Gln from 1.5 to 12 nmol/mg, and that of Glu to 10 nmol/mg. As expected, addition of His did not alter the Glu and Gln content resulting from incubation with Glu. However, His significantly decreased to almost the preincubation level the content of Glu in mitochondria incubated with Gln, without affecting the content of Gln. No other amino acid had any effect on these parameters. The results point to the existence of distinct Gln pools, one of which is accessible to external Gln via a His-sensitive transporter and is accessible for deamidation in the mitochondria.Special issue dedicated to Dr. Lawrence F. Eng.     

Roles of glutamine in neurotransmission

Glutamine (Gln) is found abundantly in the central nervous system (CNS) where it participates in a variety of metabolic pathways. Its major role in the brain is that of a precursor of the neurotransmitter amino acids: the excitatory amino acids, glutamate (Glu) and aspartate (Asp), and the inhibitory amino acid, γ-amino butyric acid (GABA). The precursor-product relationship between Gln and Glu/GABA in the brain relates to the intercellular compartmentalization of the Gln/Glu(GABA) cycle (GGC). Gln is synthesized from Glu and ammonia in astrocytes, in a reaction catalyzed by Gln synthetase (GS), which, in the CNS, is almost exclusively located in astrocytes (Martinez-Hernandez et al., 1977). Newly synthesized Gln is transferred to neurons and hydrolyzed by phosphate-activated glutaminase (PAG) to give rise to Glu, a portion of which may be decarboxylated to GABA or transaminated to Asp. There is a rich body of evidence which indicates that a significant proportion of the Glu, Asp and GABA derived from Gln feed the synaptic, neurotransmitter pools of the amino acids. Depolarization-induced-, calcium- and PAG activity-dependent releases of Gln-derived Glu, GABA and Asp have been observed in CNS preparations in vitro and in the brain in situ. Immunocytochemical studies in brain slices have documented Gln transfer from astrocytes to neurons as well as the location of Gln-derived Glu, GABA and Asp in the synaptic terminals. Patch-clamp studies in brain slices and astrocyte/neuron co-cultures have provided functional evidence that uninterrupted Gln synthesis in astrocytes and its transport to neurons, as mediated by specific carriers, promotes glutamatergic and GABA-ergic transmission. Gln entry into the neuronal compartment is facilitated by its abundance in the extracellular spaces relative to other amino acids. Gln also appears to affect neurotransmission directly by interacting with the NMDA class of Glu receptors. Transmission may also be modulated by alterations in cell membrane polarity related to the electrogenic nature of Gln transport or to uncoupled ion conductances in the neuronal or glial cell membranes elicited by Gln transporters. In addition, Gln appears to modulate the synthesis of the gaseous messenger, nitric oxide (NO), by controlling the supply to the cells of its precursor, arginine. Disturbances of Gln metabolism and/or transport contribute to changes in Glu-ergic or GABA-ergic transmission associated with different pathological conditions of the brain, which are best recognized in epilepsy, hepatic encephalopathy and manganese encephalopathy.     

Polymorphisms in base-excision repair and nucleotide-excision repair genes in relation to lung cancer risk

Polymorphisms in DNA repair genes may be associated with differences in DNA repair capacity, thereby influencing the individual susceptibility to smoking-related cancer. We investigated the association of 10 base-excision and nucleotide-excision repair gene polymorphisms (XRCC1 -77 T/C, Arg194Trp, Arg280His and Arg399Gln; APE1 Asp148Glu; OGG1 Ser326Cys; XPA -4 G/A; XPC PAT; XPD Asp312Asn and Lys751Gln) with lung cancer risk in Caucasians. Genotypes were determined by PCR-RFLP and PCR-single base extension assays in 110 lung cancer patients and 110 age- and sex-matched controls, and the results were analyzed using logistic regression adjusted for relevant covariates. A significant association between the APE1 Asp148Glu polymorphism and lung cancer risk was found, with adjusted odds ratios (OR) of 3.38 (p=0.001) for the Asp/Glu genotype and 2.39 (p=0.038) for the Glu/Glu genotype. Gene-smoking interaction analyses revealed a statistically significant interaction between cumulative cigarette smoking and the XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms: these polymorphisms were significantly associated with lung cancer in nonsmokers and light smokers (<25 PY; OR=4.92, p=0.021 for XRCC1 399 Gln/Gln; OR=3.62, p=0.049 for XPD 751 Gln/Gln), but not in heavy smokers (> or =25 PY; OR=0.68, p=0.566 for XRCC1 399 Gln/Gln; OR=0.46, p=0.295 for XPD 751 Gln/Gln). Both the XRCC1 Arg194Trp and Arg280His as well as the OGG1 Ser326Cys heterozygous genotypes were associated with a significantly reduced risk for lung cancer (OR=0.32, p=0.024; OR=0.25, p=0.028; OR=0.51, p=0.033, respectively). No associations with lung cancer risk were found for the XRCC1 -77 T/C, the XPA -4 G/A and the XPC PAT polymorphisms. In conclusion, the APE1 Asp148Glu polymorphism is highly predictive for lung cancer, and cumulative cigarette smoking modifies the associations between the XRCC1 Arg399Gln and the XPD Lys751Gln polymorphisms and lung cancer risk.     

Glutamate transport and cellular glutamine metabolism: regulation in LLC-PK1 vs. LLC-PK1-F+ cell lines

The glutamate (Glu) transporter may modulate cellular glutamine(Gln) metabolism by regulating both the rates of hydrolysis andsubsequent conversion of Glu to -ketoglutarate andNH⁺₄. By delivering Glu, a competitiveinhibitor of Gln for the phosphate-dependent glutaminase (PDG) as wellas an acid-load activator of glutamate dehydrogenase (GDH) flux, thetransporter may effectively substitute extracellularly generated Glufrom the -glutamyltransferase for that derived intracellularly fromGln. We tested this hypothesis in two closely related porcine kidneycell lines, LLC-PK₁ and LLC-PK₁-F⁺,the latter selected to grow in the absence of glucose, relying on Glnas their sole energy source. Both cell lines exhibited PDG suppressionas the result of Glu uptake while disrupting the extracellularL-Glu uptake, withD-aspartate-acceleratedintracellular Glu formation coupled primarily to the ammoniagenicpathway (GDH). Conversely, enhancing the extracellular Glu formationwith p-aminohippurate and Glu uptakesuppressed intracellular Gln hydrolysis whileNH⁺₄ formation from Glu increased. Thus theseresults are consistent with the transporter's dual role in modulatingboth PDG and GDH flux. Interestingly, PDG flux was actually higher inthe Gln-adapted LLC-PK₁-F⁺cell line because of a two- to threefold enhancement in Gln uptake despite greater Glu uptake than in the parentalLLC-PK₁ cells, revealing theimportance of both Glu and Gln transport in the modulation of PDG flux.Nevertheless, when studied at physiological Gln concentration, PDG fluxfalls under tight Glu transporter control as Gln uptake decreases,suggesting that cellular Gln metabolism may indeed be under Glutransporter control in vivo.

     

Evidence that (Glu4)-neurotensin is the naturally occurring neurotensin in plasma

Neurotensin (NT) is a tridecapeptide (less than Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH). It has been suggested that the naturally occurring neurotensin in (Gln4)-NT and that it can be hydrolysed to (Glu4)-NT during extraction. The aim of the present study was to determine whether the neurotensin found in human and rat plasma after fat ingestion is (Gln4)-NT or (Glu4)-NT. Two neurotensin antisera are described that have high avidity for (Glu4)-NT but low avidity for (Gln4)-NT. (Gln4)-NT could be separated from (Glu4)-NT by ion-exchange chromatography. The plasma samples were analyzed without prior extraction to avoid hydrolysis of the naturally occurring peptide. The results indicate that neurotensin in unextracted human and rat plasma is present as (Glu4)-NT, as originally proposed by Carraway and Leeman [4].     

Glutamine decreases lipopolysaccharide-induced intestinal inflammation in infant rats

Using a gastrostomy-fed (GF) rat infant "pup-in-a-cup" model, the effects of protein deprivation and supplemental glutamine (Gln) and glutamate (Glu) were examined to test the hypothesis that Gln decreases the proinflammatory response induced by LPS in the developing infant rat small intestine. Four groups of 6- to 7-day-old pups were fed a rat milk substitute (RMS), one providing 100% and three providing 25% of normal protein intake for another 6 days. Two of the 25% protein-fed groups received supplemental Gln or Glu. GF and LPS treatment blunted body growth and intestinal villus height and increased intestinal cytokine-induced neutrophil chemoattractant (CINC) mRNA in the protein-deprived, non-Gln-treated group compared with mother-fed pups (P < 0.05). Gln blunted intestinal CINC mRNA (P < 0.05), but Glu did not. Intestinal CINC peptide in the LPS-treated pups provided 100 and 25% protein was elevated approximately 13-fold compared with the mother-reared pups (P < 0.001). Gln and Glu decreased intestinal CINC peptide by 73 and 80%, respectively. GF, LPS-treated pups also had a higher level of plasma CINC peptide (P < 0.05). Gln but not Glu decreased plasma CINC peptide (P < 0.05). An approximate sixfold elevation of intestinal MPO activity in the GF, LPS-treated rats was decreased by Gln and Glu by 92% (P < 0.001) and 54% (P < 0.05), respectively. Intestinal and plasma TNF-alpha were increased in GF, LPS-treated pups (P < 0.01), and Gln and Glu both blunted this increase (P < 0.05) in the intestine but not in the plasma. The results indicate that Gln decreases the LPS-induced inflammatory response in infant rat intestine under different conditions of protein intake.     

Functional investigation of conserved membrane-embedded glutamate residues in the proton-coupled peptide transporter YjdL

Proton-dependent oligopeptide transporters (POTs) are secondary active symporters that utilize the proton gradient to drive the inward translocation of di- and tripeptides. We have mutated two highly conserved membraneembedded glutamate residues (Glu20 and Glu388) in the E. coli POT YjdL to probe their possible functional roles, in particular if they were involved/implicated in recognition of the substrate N-terminus. The mutants (Glu20Asp, Glu20Gln, Glu388Asp, and Glu388Gln) were tested for substrate uptake, which indicated that both the negative charge and the side chain length were important for function. The IC50 values of dipeptides with lack of or varying N-terminus (Ac-Lys, Gly- Lys, β-Ala-Lys, and 4-GABA-Lys), showed that Gly-Lys and β-Ala-Lys ranged between ~0.1 to ~1.0 mM for wild type and Glu20 mutants. However, for Glu388Gln the IC50 increased to ~2.0 and > 10 mM for Gly-Lys and β-Ala-Lys, respectively, suggesting that Glu388, and not Glu20, is able to sense the position of the N-terminus and important for the interaction. Furthermore, uptake as a function of pH showed that the optimum at around pH 6.5 for wild type YjdL shifted to 7.0-7.5 for the Glu388Asp/Gln mutants while the Glu20Asp retained the wild type optimum. Uptake by the Glu20Gln on the other hand was completely unaffected by the bulk pH in the range tested, which indicated a possible role of Glu20 in proton translocation.     

RNASEL Asp541Glu and Arg462Gln polymorphisms in prostate cancer risk: evidences from a meta-analysis

Epidemiological studies have evaluated the association between RNASEL Asp541Glu and Arg462Gln polymorphisms and prostate cancer (PCa) risk. However, the results remain inconclusive. To derive a more precise estimation of the association between RNASEL polymorphisms and PCa risk, we performed a meta-analysis based on nineteen case?Ccontrol studies. We used odds ratios (ORs) with 95% confidence intervals (CIs) to assess the strength of the association. Overall, we found that both Asp541Glu and Arg462Gln polymorphisms were not associated with PCa risk (for Asp541Glu polymorphism: Glu/Glu vs. Asp/Asp: OR 1.17, 95% CI: 0.95?C1.45, P?=?0.13; Glu/Asp vs. Asp/Asp: OR 1.02, 95% CI: 0.92?C1.14, P?=?0.70; for Arg462Gln polymorphism: Gln/Gln vs. Arg/Arg: OR 0.98, 95% CI: 0.88?C1.08, P?=?0.62; Gln/Arg vs. Arg/Arg: OR 0.97, 95% CI: 0.91?C1.04, P?=?0.53). The insignificant association was maintained in the dominant and the recessive genetic models. In subgroup analyses, the significant association was not detected in Caucasian populations. However, we found the significant association of RNASEL Asp541Glu polymorphism with sporadic PCa (Glu/Glu vs. Asp/Asp: OR 1.29, 95% CI: 1.04?C1.59, P?=?0.02; Glu/Asp vs. Asp/Asp: OR 1.24, 95% CI: 1.03?C1.50, P?=?0.03). In conclusion, we found that these RNASEL polymorphisms were not related to overall PCa risk, especially in Caucasians. However, in subgroup analyses we found a suggestion that RNASEL 541Gln allele might be a low-penetrent risk factor for sporadic PCa.     

A maximal effort trial in obese women carrying the beta2-adrenoceptor Gln27Glu polymorphism

The aim of this study was to observe the relationship of the beta2-adrenoceptor (ADRB2) gene polymorphism Gln27Glu in a group of obese female subjects submitted to an acute physical activity test. Six Glu27/Glu27 obese women were compared with six Gln27/Gln27 obese women (coupled by age, BMI, waist circumference, percentage of fat mass and absence of smoking). These groups were selected after having genotyped 91 obese subjects (BMI>30 kg/m2, age 20-60). Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) were used to genotype the obese population. The physical activity challenge consisted of a Maximal effort trial (VO2 max) following the Bruce protocol on a walking treadmill. The frequency for the Glu allele was 0.42, which is similar to other Caucasian populations. In basal conditions, subjects with the Glu27/Glu27 genotype showed a higher value of plasma insulin, while circulating glucose levels were similar in both groups. Also, diastolic arterial pressure was higher in the Glu27/Glu27 obese women. After the VO2 max trial, the Glu27 group had a significantly higher respiratory exchange ratio than the Gln27 group suggesting a lower post- exercise fat oxidation. These results provide evidence about a possible impact of the Gln27Glu polymorphism in the ADRB2 gene concerning a better response to exercise in obese Gln27 female subjects, in which basal insulinemia may play a role.     

The Gln27Glu beta2-adrenergic receptor variant is associated with obesity due to subcutaneous fat accumulation in Japanese men

The Trp64Arg beta3-adrenergic receptor (AR) variant is associated with visceral obesity probably due to decreased lipolysis in visceral fat (H. Kim-Motoyama et al., Diabetologia 40, 469-472, 1997). Functional alteration of beta2AR may also change fat distribution. We investigated the influence of the Gln27Glu beta2AR variant upon obesity and fat distribution. We screened 278 unrelated Japanese men and detected 249 wild-type Gln27 homozygotes, 28 Gln27/Glu27 heterozygotes, and one mutant Glu27 homozygote. The frequency of mutant Glu27 allele was significantly higher in obese subjects than in nonobese/intermediate subjects (0.11 vs 0.04, P = 0. 004). The Gln27/Glu27 heterozygotes had a significantly higher mean age-adjusted body-mass index (BMI) and mean age-adjusted subcutaneous fat area assessed by CT scan than the wild-type homozygotes but not the mean age-adjusted visceral fat areas. In summary, we have found that in Japanese men the Gln27Glu beta2AR variant is associated with obesity due to subcutaneous fat accumulation.     

The functional and structural roles of residues Gln114 and Glu117 in elongation factor Tu

The effects of substituting residues Gln114 by Glu and Glu117 by Gln, both situated in the vicinity of the guanine-nucleotide-binding pocket, were investigated in the isolated N-terminal domain (G domain) of elongation factor Tu with respect to the binding of the substrate GDP/GTP, GTPase activity and stability. The major change in the interaction with the guanine nucleotides is a lower affinity for GTP and a reduced GTPase activity when Gln114 is substituted by Glu. This mutation also abolishes most of the selective effects on the GTPase activity induced by the different monovalent cations. Substitution of Glu117 by Gln does not affect the interaction with the guanine nucleotides or the GTPase activity of the G domain in an essential way, but it reduces the stability towards denaturation of the G-domain.GDP complex. Our results therefore suggest, that Gln114 is involved in keeping a functional conformation of the guanine-nucleotide-binding pocket, whereas Glu117 participates in the regulation of the overall conformation of the G domain. Neither of these two residues appears to play a role in the actual GTPase mechanism.     

Divergent anticodon recognition in contrasting glutamyl-tRNA synthetases

The pathogenic bacterium Helicobacter pylori utilizes two essential glutamyl-tRNA synthetases (GluRS1 and GluRS2). These two enzymes are closely related in evolution and yet they aminoacylate contrasting tRNAs. GluRS1 is a canonical discriminating GluRS (D-GluRS) that biosynthesizes Glu-tRNA(Glu) and cannot make Glu-tRNA(Gln). In contrast, GluRS2 is non-canonical as it is only essential for the production of misacylated Glu-tRNA(Gln). The co-existence and evident divergence of these two enzymes was capitalized upon to directly examine how GluRS2 acquired tRNA(Gln) specificity. One key feature that distinguishes tRNA(Glu) from tRNA(Gln) is the third position in the anticodon of each tRNA (C36 versus G36, respectively). By comparing sequence alignments of different GluRSs, including GluRS1s and GluRS2s, to the crystal structure of the Thermus thermophilus D-GluRS:tRNA(Glu) complex, a divergent pattern of conservation in enzymes that aminoacylate tRNA(Glu)versus those specific for tRNA(Gln) emerged and was experimentally validated. In particular, when an arginine conserved in discriminating GluRSs and GluRS1s was inserted into Hp GluRS2 (Glu334Arg GluRS2), the catalytic efficiency of the mutant enzyme (k(cat)/K(Mapp)) was reduced by approximately one order of magnitude towards tRNA(Gln). However, this mutation did not introduce activity towards tRNA(Glu). In contrast, disruption of a glycine that is conserved in all GluRS2s but not in other GluRSs (Gly417Thr GluRS2) generated a mutant GluRS2 with weak activity towards tRNA(Glu1). Synergy between these two mutations was observed in the double mutant (Glu334Arg/Gly417Thr GluRS2), which specifically and more robustly aminoacylates tRNA(Glu1) instead of tRNA(Gln). As GluRS1 and GluRS2 are related by an apparent gene duplication event, these results demonstrate that we can experimentally map critical evolutionary events in the emergence of new tRNA specificities.     

K(+)-independent gastric H(+),K(+)-atpase activity. Dissociation of K(+)-independent dephosphorylation and preference for the E1 conformation by combined mutagenesis of transmembrane glutamate residues

Several mutations of residues Glu(795) and Glu(820) present in M5 and M6 of the catalytic subunit of gastric H(+),K(+)-ATPase have resulted in a K(+)-independent, SCH 28080-sensitive ATPase activity, caused by a high spontaneous dephosphorylation rate. The mutants with this property also have a preference for the E(1) conformation. This paper investigates the question of whether these two phenomena are coupled. This possibility was studied by combining mutations in residue Glu(343), present in M4, with those in residues 795 and 820. When in combined mutants Glu and/or Gln residues were present at positions 343, 795, and 820, the residue at position 820 dominated the behavior: a Glu giving K(+)-activated ATPase activity and an E(2) preference and a Gln giving K(+)-independent ATPase activity and an E(1) preference. With an Asp at position 343, the enzyme could be phosphorylated, but the dephosphorylation was blocked, independent of the presence of either a Glu or a Gln at positions 795 and 820. However, in these mutants, the direction of the E(2) <--> E(1) equilibrium was still dominated by the 820 residue: a Glu giving E(2) and a Gln giving E(1). This indicates that the preference for the E(1) conformation of the E820Q mutation is independent of an active dephosphorylation process.     

Perivenous localisation of Na-dependent glutamate transport in perfused rat liver

Periportal and perivenous hepatocytes differ in their metabolism of blood glutamate (Glu). Uncertainty about the mechanisms of Glu blood-liver exchange led us to characterise, by paired-tracer dilution, a sodium-dependent dicarboxylate transporter (resembling system X-ag) in sinusoidal membranes of perfused rat liver (Vmax = 0.18 mumol Glu/g per min, Km = 0.29 mM Glu). Tracer Glu transport was depressed 65% after necrosis of perivenous hepatocytes by acute CCl4 treatment, indicating that X-ag transporter activity is located mainly in these cells, the sites of glutamine (Gln) synthesis from glutamate and ammonia. Modulation of Glu transport may influence the extent of hepatic Gln release.     

Effect of NBS1 gene polymorphism on the risk of cervix carcinoma in a northern Indian population

Cervical cancer is one of the most common neoplastic diseases affecting women, with a worldwide incidence of almost half a million cases. A history of smoking and use of oral contraceptives have been confirmed to be risk factors for cervical cancer. Genetic susceptibility and immune response, especially impaired cellular immune response, may well be related to the development of cervical cancer. NBS1 is one of the key proteins participating in the recognition and repair of double-strand breaks that may lead to genomic instability and cancer if unrepaired. The objective of the present study was therefore to investigate NBS1 Glu185Gln gene polymorphisms and the risk of cervix cancer in a northern Indian population. We found that passive smokers having particular NBS1 genotypes (Glu/Gln, Gln/Gln or Glu/Gln + Gln/Gln)have an increased risk of developing cervix cancer (OR 5.21, p=0.000001; OR 4.60, p=0.001; OR 5.10, p=0.0000009, respectively).The risk was increased 2.4-fold in oral contraceptive users with a Glu/Gln genotype. We conclude that the risk of cervical cancer is increased in passive smokers and in users of oral contraceptives with certain NBS1 genotypes.