Strain differences in amiloride inhibition of NaCl responses in mice,Mus musculus

Summary The effects of lingual treatment with amiloride, an inhibitor of salt taste responses in several mammalian species, on NaCl responses of the chorda tympani nerve were compared between four inbred strains of mouse (BALB/cCrSlc, DBA/2CrSlc, C57BL/6CrSlc and C3H/HeSlc). In C57BL and C3H mice amiloride significantly suppressed responses of the chorda tympani nerve to NaCl at a concentration 0.1 M or more whereas in BALB and DBA mice the drug did not significantly affect the responses to NaCl at any concentration, suggesting a lack of the amiloride-sensitive receptor component for NaCl in the latter two strains.A two-bottle preference test demonstrated that all strains of mouse usually showed no preference for NaCl at any concentration and avoided NaCl at 0.3 M or more, although some differences were observed in that C57BL and C3H mice showed aversive responses to 0.1 and 0.15 M NaCl, whereas BALB and DBA mice were indifferent to these solutions.The results suggest that there exist prominent differences between mouse strains in the amiloride-sensitive component of their salt receptor systems. However, in mice the taste information derived from the amiloride-sensitive receptor component probably has no remarkable effect on behavioral responses to NaCl except for a possible contribution to decreasing aversion thresholds for NaCl by increasing overall taste information about NaCl.     

D-Amino acids in chronic renal failure and the effects of dialysis and urinary losses

Summary Total D-amino acids were measured in plasma for 20 non-dialysed patients (creatinine clearance < 12 ml/minute), 20 on CAPD, 20 on haemodialysis and 20 normals. Plasma D-tyrosine and D-phenylalanine were measured in 8 of each group by HPLC. Total D-amino acids, D-tyrosine and D-phenylalanine were significantly greater for patients than normals. D-amino acids and D-tyrosine correlated with creatinine and were decreased during HD. During dialysis, the mean losses for D-tyrosine and D-phenylalanine were similar, about 0.2 mg/CAPD exchange and 3 mg/4 hour haemodialysis (i.e. 2% of the total amino acid, as in plasma). Clearance was unaffected by stereochemical configuration. Urinary losses/24 hour in the non-dialysed patients were 0.35 mg D-tyrosine and 0.25 mg D-phenylalanine. Clearance for D-phenylalanine was greater than for the L-enantiomer. Increases in D-amino acids in renal failure are probably due to depletion of D-amino acid oxidase, but may be enhanced by a D-amino acid rich diet, peptide antibiotics and D-amino acid oxidase inhibiting drugs and metabolites. Possible toxic effects need further investigation.     

Effect of genetic background on establishment of mouse embryonic stem cells.

We established 13 embryonic stem (ES) cell lines from 542 embryos crossed between various strains of mice: 10 lines from 129/Sv-ter embryos (10/48, 20.8%) and 3 lines out of other 9 combinations of intra- or inter-strain matings (1 from intracross of C57BL/6CrSlc, 1 from B6D2F1 x C57BL/6CrSlc, 1 from Yok:ddY x Slc:ICR). No ES cell line from 129/Sv-ter x Slc:ICR embryos suggests that ICR strain might have inhibitory genetic factor(s) for the ES cell formation. Some ES cell lines could be obtained from hybrids even if none or few lines from their parental strains, suggesting a heterosis effect can be expected for establishing ES cell lines in mice.     

Mechanistic aspects of the transamination reactions catalyzed by D-amino acid transaminase from Haliscomenobacter hydrossis

Pyridoxal-5′-phosphate-(PLP-) dependent D-amino acid transaminases (DAATs) catalyze stereoselective reversible transfer of the amino group between D-amino acids and keto acids. In vivo DAATs are commonly known to synthesize D-glutamate for cell wall peptidoglycans. Today DAATs meet increasing attention for application in the synthesis of D-amino acids, whereas little is known about the mechanism of substrate recognition and catalytic steps of the D-amino acids conversion by DAATs. In this work, the pre-steady-state kinetics of the half-reactions of DAAT from Haliscomenobacter hydrossis with D-glutamate, D-alanine, D-leucine, and D-phenylalanine was examined at two wavelengths, 416 and 330 nm, using a stopped-flow technique. Monophasic kinetics was observed with specific substrates D-glutamate and D-alanine, whereas half-reactions with D-leucine and D-phenylalanine exhibited biphasic kinetics. All half-reactions proceeded until the complete conversion of PLP due to the release of the pyridoxamine-5′-phosphate form of cofactor from the holoenzyme . Comparison of kinetic parameters of half-reactions and the overall transamination reactions for D-leucine, D-phenylalanine revealed the increase in the rates of deamination of these substrates in the overall reaction with α-ketoglutarate. In the overall transamination reaction, the catalytic turnover rates for D-leucine and D-phenylalanine increased by 260 and 60 times, correspondingly, comparing with the slowest step rate constants in the half-reactions. We suggested the activating effect by a specific substrate α-ketoglutarate in the overall transamination reaction. The study of half-reactions helped to quantify the specificity of DAAT from H. hydrossis for D-amino acids with different properties. The results obtained are the first detailed analysis of half-reactions catalyzed by DAAT.     

Genetic influences on oestrous cyclicity in mice: evidence that cycle length and frequency are differentially regulated.

Studies in C57BL/6J, DBA/2J and C3H/HeJ mice and in two F1 hybrid strains (B6D2F1 and B6C3HF1) 2-5 months old revealed marked genotypic differences among inbred strains. C57 mice had three times as many regular (3-6 days) cycles as DBA and C3H mice, due largely to fewer pseudopregnant-like (7-14 day) cycles. C57 had longer regular cycles than DBA and C3H mice. Although the frequencies of regular cycles of DBA and C3H mice were similar, the cycles of C3H mice were shorter than those of DBA mice. The results indicated that the genetic determinants of the frequency of regular cycles differ from those specifying cycle length. Frequency of regular cycles of F1 hybrids was either intermediate between the parent strains (B6D2F1) or similar to the C57 strain (B6C3HF1), suggesting that regular cycle frequency shows additive genetic variation in the former crosses, but mostly dominant variance in the latter background. Regular cycles were either shorter than in both parent strains (B6D2F1) or similar to one of them (B6C3HF1), indicating heterosis and dominance for genes specifying short cycles. Although the lack of reciprocal crosses meant that maternal effects and possible genomic imprinting effects could not be assessed, these results reveal marked genetic influences on cycle length and frequency and suggest that some of the genes specifying these two traits differ.     

Effects of isoproterenol treatment on gustatory neural responses in three inbred strains of mice

1. Treatment of a beta-agonist, isoproterenol, for 5 days reduced chorda tympani responses to sucrose by about 40% of the control without affecting responses to other taste stimuli, such as NaCl, HCl and quinine HCl, in balb CrSlc mice whereas such reduction of sucrose responses was not observed in C57BL/6-CrSlc and C3H/HeSlc mice, although in the latter two strains long-lasting off-responses to quinine HCl appeared after the treatment. 2. In BALB mice, the magnitude of reduction of sucrose responses by isoproterenol increased with prolonging the treatment from 1 to 5 days, although it reached almost its maximum level by the 3 days treatment. 3. BALB mice with the removal of the submandibular glands showed slightly greater control responses of the chorda tympani nerve to sucrose than BALB mice with the sham-operation or the removal of the sublingual glands, and showed no significant reduction of sucrose responses by isoproterenol treatment. 4. These results suggest that isoproterenol probably did not act directly on sweetener receptors of taste cell membranes but affect them through the submandibular salivary system.     

Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure D-amino acids

Two recombinant reaction systems for the production of optically pure D-amino acids from different D,L-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were D-hydantoinase and D-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. The D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest level of synthesis of the enzyme, thus avoiding intermediate accumulation, which decreases the reaction rate. Both systems were able to produce 100% conversion and 100% optically pure D-methionine, D-leucine, D-norleucine, D-norvaline, D-aminobutyric acid, D-valine, D-phenylalanine, D-tyrosine, and D-tryptophan from the corresponding hydantoin racemic mixture. For the production of almost all D-amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2.     

Gliotransmission modulates baseline mechanical nociception

Group II metabotropic glutamate receptors (mGluRs) couple to the inhibitory G-protein Gi. The group II mGluRs include two subtypes, mGlu2 and mGlu3, and their pharmacological activation produces analgesic effects in inflammatory and neuropathic pain states. However, the specific contribution of each one of the two subtypes has not been clarified due to the lack of selective orthosteric ligands that can discriminate between mGlu2 and mGlu3 subtypes. In this study we used mGlu2 or mGlu3 knock-out mice to dissect the specific role for these two receptors in the endogenous control of inflammatory pain and their specific contribution to the analgesic activity of mixed mGlu2/3 receptor agonists. Our results showed that mGlu2^-/- mice display a significantly greater pain response compared to their wild type littermates. Interestingly the increased pain sensitivity in mGlu2^-/- mice occurred only in the second phase of the formalin test. No differences were observed in the first phase. In contrast, mGlu3^-/- mice did not significantly differ from their wild type littermates in either phase of the formalin test. When systemically injected, a single administration of the mGlu2/3 agonist, LY379268 (3 mg/kg, ip), showed a significant reduction of both phases in wild-type mice and in mGlu3^-/- but not in mGlu2^-/- mice. However tolerance to the analgesic effect of LY379268 (3 mg/kg, ip) in mGlu3^-/- mice developed following 5 consecutive days of injection. Taken together, these results demonstrate that: (i) mGlu2 receptors play a predominant role over mGlu3 receptors in the control of inflammatory pain in mice; (ii) the analgesic activity of mixed mGlu2/3 agonists is entirely mediated by the activation of the mGlu2 subtype and (iii) the development of tolerance to the analgesic effect of mGlu2/3 agonists develops despite the lack of mGlu3 receptors.     

Evidence for the presence of two D-amino acid oxidases inPseudomonas aeruginosa PAO

By the isolation of mutants that were unable to grow on L-hydroxyproline or DL-valine, it has been possible to demonstrate the presence of two different types of D-amino acid oxidase activities inPseudomonas aeruginosa PAO. One was the D-amino acid dehydrogenase, probably involved in the oxidation of a number of D-amino acids such as D-alanine, D-phenylalanine and D-valine. The other was the inducible oxidase, specific to the oxidation of allohydroxy-D-proline formed from L-hydroxyproline during its oxidation. Thus, it has been possible to delink the involvement of the general D-amino acid dehydrogenase in the oxidative breakdown of allohydroxy-Dsproline.     

A simple and rapid method to screen for mutant mice lacking D-amino-acid oxidase activity

A simple and rapid method to screen for mutant mice (Mus musculus) lacking D-amino-acid oxidase activity has been devised. Mice were given water containing small amounts (0.02%) of either D-methionine or D-phenylalanine. Urinary levels of the D-amino acid were examined using thin-layer chromatography. Some mice excreted substantial amounts of the D-amino acid through the urine. None of them had detectable D-amino-acid oxidase activity.     

Construction of modified ribosomes for incorporation of D-amino acids into proteins

While numerous biologically active peptides contain D-amino acids, the elaboration of such species is not carried out by ribosomal synthesis. In fact, the bacterial ribosome discriminates strongly against the incorporation of D-amino acids from D-aminoacyl-tRNAs. To permit the incorporation of D-amino acids into proteins using in vitro protein-synthesizing systems, a strategy has been developed to prepare modified ribosomes containing alterations within the peptidyltransferase center and helix 89 of 23S rRNA. S-30 preparations derived from colonies shown to contain ribosomes with altered 23S rRNAs were found to exhibit enhanced tolerance for D-amino acids and to permit the elaboration of proteins containing D-amino acids at predetermined sites. Five specific amino acids in Escherichia coli dihydrofolate reductase and Photinus pyralis luciferase were replaced with D-phenylalanine and D-methionine, and the specific activities of the resulting enzymes were determined.     

Purification, characterization, gene cloning and nucleotide sequencing of D-stereospecific amino acid amidase from soil bacterium: Delftia acidovorans

The D-amino acid amidase-producing bacterium was isolated from soil samples using an enrichment culture technique in medium broth containing D-phenylalanine amide as a sole source of nitrogen. The strain exhibiting the strongest activity was identified as Delftia acidovorans strain 16. This strain produced intracellular D-amino acid amidase constitutively. The enzyme was purified about 380-fold to homogeneity and its molecular mass was estimated to be about 50 kDa, on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active preferentially toward D-amino acid amides rather than their L-counterparts. It exhibited strong amino acid amidase activity toward aromatic amino acid amides including D-phenylalanine amide, D-tryptophan amide and D-tyrosine amide, yet it was not specifically active toward low-molecular-weight D-amino acid amides such as D-alanine amide, L-alanine amide and L-serine amide. Moreover, it was not specifically active toward oligopeptides. The enzyme showed maximum activity at 40 degrees C and pH 8.5 and appeared to be very stable, with 92.5% remaining activity after the reaction was performed at 45 degrees C for 30 min. However, it was mostly inactivated in the presence of phenylmethanesulfonyl fluoride or Cd2+, Ag+, Zn2+, Hg2+ and As3+ . The NH2 terminal and internal amino acid sequences of the enzyme were determined; and the gene was cloned and sequenced. The enzyme gene damA encodes a 466-amino-acid protein (molecular mass 49,860.46 Da); and the deduced amino acid sequence exhibits homology to the D-amino acid amidase from Variovorax paradoxus (67.9% identity), the amidotransferase A subunit from Burkholderia fungorum (50% identity) and other enantioselective amidases.     

Taste synergism between monosodium glutamate and 5'-ribonucleotide in mice.

1. Strain differences of mice were found in the taste synergism between monosodium L-glutamate (MSG) and disodium 5'-guanylate (GMP). 2. Magnitudes of chorda tympani responses to the mixture of MSG and GMP over the sum of responses to each component were greater in the order of C3H/HeSlc(C3H) greater than C57BL/6CrSlc(C57BL) greater than BALB/cCrSlc(BALB) mice. The greatest synergism was observed in response to the mixture of 0.03 M MSG and 0.1 mM GMP, to which responses were about 2.6, 1.8 and 1.4 times greater than the sum of each component in C3H, C57BL and BALB mice, respectively. 3. Magnitudes of inhibition of MSG and mixture responses by the lingual treatment of proteolytic enzyme, Pronase E, were greater in the same order of C3H greater than C57BL greater than BALB mice as that observed in magnitudes of the synergism. These results suggest that there exists quantitative differences in receptors responsible for taste synergism between MSG and GMP among three mouse strains.     

Crystal structure and functional characterization of a D-stereospecific amino acid amidase from Ochrobactrum anthropi SV3, a new member of the penicillin-recognizing proteins

D-amino acid amidase (DAA) from Ochrobactrum anthropi SV3, which catalyzes the stereospecific hydrolysis of D-amino acid amides to yield the D-amino acid and ammonia, has attracted increasing attention as a catalyst for the stereospecific production of D-amino acids. In order to clarify the structure-function relationships of DAA, the crystal structures of native DAA, and of the D-phenylalanine/DAA complex, were determined at 2.1 and at 2.4 A resolution, respectively. Both crystals contain six subunits (A-F) in the asymmetric unit. The fold of DAA is similar to that of the penicillin-recognizing proteins, especially D-alanyl-D-alanine-carboxypeptidase from Streptomyces R61, and class C beta-lactamase from Enterobacter cloacae strain GC1. The catalytic residues of DAA and the nucleophilic water molecule for deacylation were assigned based on these structures. DAA has a flexible Omega-loop, similar to class C beta-lactamase. DAA forms a pseudo acyl-enzyme intermediate between Ser60 O(gamma) and the carbonyl moiety of d-phenylalanine in subunits A, B, C, D, and E, but not in subunit F. The difference between subunit F and the other subunits (A, B, C, D and E) might be attributed to the order/disorder structure of the Omega-loop: the structure of this loop cannot assigned in subunit F. Deacylation of subunit F may be facilitated by the relative movement of deprotonated His307 toward Tyr149. His307 N(epsilon2) extracts the proton from Tyr149 O(eta), then Tyr149 O(eta) attacks a nucleophilic water molecule as a general base. Gln214 on the Omega-loop is essential for forming a network of water molecules that contains the nucleophilic water needed for deacylation. Although peptidase activity is found in almost all penicillin-recognizing proteins, DAA lacks peptidase activity. The lack of transpeptidase and carboxypeptidase activities may be attributed to steric hindrance of the substrate-binding pocket by a loop comprised of residues 278-290 and the Omega-loop.     

Sweetener preference of C57BL/6ByJ and 129P3/J mice

Previous studies have shown large differences in taste responses to several sweeteners between mice of the C57BL/6ByJ (B6) and 129P3/J (129) inbred strains. The goal of this study was to compare behavioral responses of B6 and 129 mice to a wider variety of sweeteners. Seventeen sweeteners were tested using two-bottle preference tests with water. Three main patterns of strain differences were evident. First, sucrose, maltose, saccharin, acesulfame-K, sucralose and SC-45647 were preferred by both strains, but the B6 mice had lower preference thresholds and higher solution intakes. Second, the amino acids D-phenylalanine, D-tryptophan, L-proline and glycine were highly preferred by B6 mice, but not by 129 mice. Third, glycyrrhizic acid, neohesperidin dihydrochalcone, thaumatin and cyclamate did not evoke strong preferences in either strain. Aspartame was neutral to all 129 and some B6 mice, but other B6 mice strongly preferred it. Thus, compared with the 129 mice the B6 mice had higher preferences for sugars, sweet tasting amino acids and several but not all non-caloric sweeteners. Glycyrrhizic acid, neohesperidin, thaumatin and cyclamate are not palatable to B6 or 129 mice.     

Ceruletide increases threshold and tolerance to experimentally induced pain in healthy man

Previous studies suggested that ceruletide might be endowed with analgesic and sedative properties. To investigate the effects of ceruletide on experimentally induced pain and on central nervous functions, two studies, each involving 24 healthy subjects, were carried out in random double-blind fashion. Every subject participated in three experiments one week apart. In study 1, 120 and 60 ng/kg/hr ceruletide IV increased threshold and tolerance to electrically and threshold to thermally induced cutaneous pain significantly more than saline (p<0.001), the higher dose being slightly more active. Only mild sedative effects occurred. Study 2 compared the effects of 60 and 6 ng/kg/hr ceruletide IV to those of 0.4 mg/kg/hr pentazocine IV and investigated whether these effects were naloxone reversible. Both ceruletide doses, 60 ng/kg/hr slightly more than 6 ng/kg/hr, elevated threshold and tolerance to electrically induced and threshold to thermally induced pain markedly, pentazocine acted stronger and longer than ceruletide (p<0.001). Naloxone reversed the effects of pentazocine but not of ceruletide. Conclusion: ceruletide (1) exerts potent naloxone resistant analgesic effects, which, however, are inferior to those of pentazocine, and (2) produces only mild sedation.     

Relationship between the malonylation of 1-aminocyclopropane-1-carboxylic acid and D-amino acids in mung-bean hypocotyls

In sections from hypocotyls of dark-grown mung-bean (Vigna radiata L.) seedlings, D-phenylalanine and D-methionine (D-met) inhibited the formation of 1-(malonylamino)cyclopropane-1-carboxylic acid from exogenously administered 1-aminocyclopropane-1-carboxylic acid (ACC), resulting in an increase in free ACC content and stimulation of ethylene production, whereas their L-enantiomers had little or no such effect. When the hypocotyls were administered D-Met, it was mainly metabolized to N-malonylmethionine and N-malonylmethionine sulfoxide, and this malonylation process was inhibited to a greater extent by ACC and D-amino acids (phenylalanine and serine) than by L-amino acids. These results indicate that malonylation of D-amino acids and of ACC are intimately interrelated.     

Analgesic activity of dipeptide Tyr-Pro

The effect of dipeptide Tyr-Pro, present in representatives of most families of opioid peptides, and two its analogs, Tyr-Pro-NH₂ and Tyr-Pro-OMe, on analgesic activity was studied in different tests (tail-flick test, tail pinch (Haffner’s) test, formalin test, and acetic acid writing test) describing different organization levels of pain sensitivity. Intraperitoneal administration of the dipeptide decreased the pain threshold in all above-mentioned tests. Coadministration of the dipeptide and naloxone or naloxone methiodide insignificantly decreased the dipeptide analgesic effect in the tail-flick and acetic acid writing tests. Its analogs Tyr-Pro-NH₂ and Tyr-Pro-OMe demonstrated a similar analgesic activity in the tail-flick test and a higher activity in the acetic acid writing test. Administration of individual amino acids (Tyr or Pro) or their mixture had no effect on the pain threshold.     

Effects of endomorphin-2 on arterial blood pressure and pain threshold in spontaneously hypertensive rats and modification of these effects by beta-funaltrexamine and nor-binaltorphimine

The effects of intracerebroventricular (icv) administration of endomorphin-2 (E2) on arterial blood pressure and pain threshold in spontaneously hypertensive rats (SHR) and modification of these effects by K [OP2] and mu [OP3] opioid receptors antagonists were investigated. Endomorphin-2 administrated icv in doses of 8, 16 and 32 mcg produced dose-dependent analgesic and hypotensive effect. In SHR decrease in blood pressure amounted 2.667, 4.0 and 6.534 kPa, respectively. Pain threshold increased by 1.7, 3.6 and 8.9 (g x 10). In Wistar Kyoto (WKY) strain, being the normotensive controls, E2 in doses of 8 and 16 mcg decrease in blood pressure was less pronounced and amounted 1.200 and 1.467 kPa, respectively, whereas the pain threshold increased by 7.2 and 10.4 (g x 10), respectively. Both E2 effects were antagonized by equimolar icv doses of beta-funaltrexamine (beta-FNA). Equimolar doses of nor-binaltorphimine (nor-BNI) attenuated analgesic action of E2, but were without hypotensive action produced by E2. A strong correlation between drop in blood pressure and increase in pain threshold observed in the SHR and WKY strains after icv administration of E2, indicate close interaction between systems responsible for pain perception and blood pressure control.     

The significance of D-amino acids in soil, fate and utilization by microbes and plants: review and identification of knowledge gaps

Background

D-amino acids are far less abundant in nature than L-amino acids. Both L- and D-amino acids enter soil from different sources including plant, animal and microbial biomass, antibiotics, faeces and synthetic insecticides. Moreover, D-amino acids appear in soil due to abiotic or biotic racemization of L-amino acids. Both L- and D-amino acids occur as bound in soil organic matter and as “free“ amino acids dissolved in soil solution or exchangeably bound to soil colloids. D-amino acids are mineralized at slower rates compared to the corresponding L-enantiomers. Plants have a capacity to directly take up “free“ D-amino acids by their roots but their ability to utilize them is low and thus D-amino acids inhibit plant growth.

Scope

The aim of this work is to review current knowledge on D-amino acids in soil and their utilization by soil microorganisms and plants, and to identify critical knowledge gaps and directions for future research.

Conclusion

Assessment of “free“ D-amino acids in soils is currently complicated due to the lack of appropriate extraction procedures. This information is necessary for consequent experimental determination of their significance for crop production and growth of plants in different types of managed and unmanaged ecosystems. Hypotheses on occurrence of “free“ D-amino acids in soil are presented in this review.