Amino acids and Ca2+ stimulate different patterns of Ca2+ oscillations through the Ca2+-sensing receptor

We determined the effect of aromatic aminoacid stimulation of the human extracellular Ca²⁺-sensingreceptor (CaR) on intracellular Ca²⁺ concentration([Ca²⁺]_i) in single HEK-293 cells. Additionof L-phenylalanine or L-tryptophan (at 5 mM)induced [Ca²⁺]_i oscillations from a restingstate that was quiescent at 1.8 mM extracellular Ca²⁺concentration ([Ca²⁺]_e). Each[Ca²⁺]_i peak returned to baseline values, andthe average oscillation frequency was ~1 min¹ at37°C. Oscillations were not induced or sustained if the[Ca²⁺]_e was reduced to 0.5 mM, even in thecontinued presence of amino acid. Average oscillation frequency inresponse to an increase in [Ca²⁺]_e (from 1.8 to 2.5-5 mM) was much higher (~4 min¹) than thatinduced by aromatic amino acids. Oscillations in response to[Ca²⁺]_e were sinusoidal whereas those inducedby amino acids were transient. Thus both amino acids andCa²⁺, acting through the same CaR, produce oscillatoryincreases in [Ca²⁺]_i, but the resultantoscillation pattern and frequency allow the cell to discriminate whichagonist is bound to the receptor.

     

Furosemide and Ca2+ affect 86Rb+ efflux from pancreatic beta-cells by different mechanisms

The interaction between furosemide, calcium and D-glucose on the 86Rb+ efflux from beta-cell-rich mouse pancreatic islets was investigated in a perifusion system with high temporal resolution. Raising the glucose concentration from 4 to 20 mM induced an initial decrease in 86Rb+ efflux, which was followed by a steep increase and then a secondary decrease. Removal of extracellular calcium increased the 86Rb+ efflux at 4 mM D-glucose but reduced it at 20 mM. The initial biphasic changes in 86Rb+ efflux induced by 20 mM D-glucose were inhibited by calcium deficiency. Furosemide (100 microM) reduced the 86Rb+ efflux rate both at 4 and 20 mM D-glucose and the magnitudes appeared to be similar at either glucose concentration. Furosemide (100 microM) reduced the glucose-induced (10 mM) 45Ca+ uptake but did not affect the basal (3 mM D-glucose) 45Ca+ uptake. However, the ability of furosemide (100 microM) to reduce the 86Rb+ efflux at a high glucose concentration (20 mM) was independent of extracellular calcium. The inhibitory effects of furosemide and calcium deficiency on the 86Rb+ efflux rate appeared to be additive. It is concluded that the effect of furosemide on 86Rb+ efflux is not secondary to reduced calcium uptake and that the effects of furosemide and calcium deficiency are mediated by different mechanisms. The effect of furosemide is compatible with inhibition of loop diuretic-sensitive co-transport of Na+, K+ and Cl- and the effect of calcium deficiency with reduced activity of calcium-regulated potassium channels.     

Cation-pi (Na+-Trp) interactions in the crystal structure of tetragonal lysozyme.

Experimental evidence of a cation-pi interaction between a sodium cation (Na+) and the indole ring of residue Trp123 in a structure (2.0 A) of hen egg-white lysozyme is presented. The geometry of the metal ion-pi interaction observed in the protein structure (distance between the aromatic plane and the cation approximately 4 A) is consistent with geometries observed among small molecules crystal structures and quantum chemistry ab initio calculations. The present crystal structure of lysozyme provides unique structural information about the geometry of binding of cations to pi systems in proteins. It shows that the metal ion-pi interaction within proteins is not significantly different from similar bindings found in small molecules and that it can be modeled by theoretical methods.     

Terbium luminescence in synthetic peptide loops from calcium-binding proteins with different energy donors

Fourteen 14-mer peptides corresponding to a consensus sequence of metal-binding loops from proteins of the calmodulin family were synthesized. The effect of varying both the position in the binding loop, and the type of aromatic side chains as energy donors for enhancement of terbium luminescence, was studied. It was concluded that tryptophan in loop position 7 gave optimal luminescence enhancement, and that the additional inclusion of a tyrosine in the loop at positions 2 or 4 could further boost emission from the bound terbium. In all further cases energy transfer from aromatic residues at positions other than 7 was markedly less efficient. These results suggest that the peptides assume a configuration which allows a hexadentate ligand structure around the bound terbium ion. This is consistent with a Dexter-type electron exchange model of energy transfer.     

Expression cloning of a Na+-independent aromatic amino acid transporter with structural similarity to H+/monocarboxylate transporters

A cDNA was isolated from rat small intestine by expression cloning which encodes a novel Na+-independent transporter for aromatic amino acids. When expressed in Xenopus oocytes, the encoded protein designated as TAT1 (T-type amino acid transporter 1) exhibited Na+-independent and low-affinity transport of aromatic amino acids such as tryptophan, tyrosine, and phenylalanine (Km values: approximately 5 mm), consistent with the properties of classical amino acid transport system T. TAT1 accepted some variations of aromatic side chains because it interacted with amino acid-related compounds such as l-DOPA and 3-O-methyl-DOPA. Because TAT1 accepted N-methyl- and N-acetyl-derivatives of aromatic amino acids but did not accept their methylesters, it is proposed that TAT1 recognizes amino acid substrates as anions. Consistent with this, TAT1 exhibited sequence similarity (approximately 30% identity at the amino acid level) to H+/monocarboxylate transporters. Distinct from H+/monocarboxylate transporters, however, TAT1 was not coupled with the H+ transport but it mediated an electroneutral facilitated diffusion. TAT1 mRNA was strongly expressed in intestine, placenta, and liver. In rat small intestine TAT1 immunoreactivity was detected in the basolateral membrane of the epithelial cells suggesting its role in the transepithelial transport of aromatic amino acids. The identification of the amino acid transporter with distinct structural and functional characteristics will not only facilitate the expansion of amino acid transporter families but also provide new insights into the mechanisms of substrate recognition of organic solute transporters.     

Comparative analysis of the volatile fraction of fruit juice from different Citrus species

The volatile composition of fruit from four Citrus varieties (Powell Navel orange, Clemenules mandarine, and Fortune mandarine and Chandler pummelo) covering four different species has been studied. Over one hundred compounds were profiled after HS-SPME-GC-MS analysis, including 27 esters, 23 aldehydes, 21 alcohols, 13 monoterpene hydrocarbons, 10 ketones, 5 sesquiterpene hydrocarbons, 4 monoterpene cyclic ethers, 4 furans, and 2 aromatic hydrocarbons, which were all confirmed with standards. The differences in the volatile profile among juices of these varieties were essentially quantitative and only a few compounds were found exclusively in a single variety, mainly in Chandler. The volatile profile however was able to differentiate all four varieties and revealed complex interactions between them including the participation in the same biosynthetic pathway. Some compounds (6 esters, 2 ketones, 1 furan and 2 aromatic hydrocarbons) had never been reported earlier in Citrus juices. This volatile profiling platform for Citrus juice by HS-SPME-GC-MS and the interrelationship detected among the volatiles can be used as a roadmap for future breeding or biotechnological applications.     

A multispecific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa

Pseudomonas aeruginosa possesses dual enzymatic sequences to both L-phenylalanine and L-tyrosine, a biosynthetic arrangement further complicated by the presence of five aromatic aminotransferases. Each aminotransferase is capable of transamination in vitro with any of the three keto acid intermediates in the aromatic pathway (phenylpyruvate, 4-hydroxyphenylpyruvate, or prephenate). The fractional contribution of these aminotransferases to particular transamination reactions in vivo can best be approached through the systematic and sequential elimination of individual aminotransferase activities by mutation. A program of sequential mutagenesis has produced two aminotransferase-deficient mutations. The first mutation imposed a phenotype of bradytrophy for L-phenylalanine (doubling time of 2.4 h in minimal salts/glucose medium compared to a 1.0-h doubling time for wild type). This mutant completely lacked an enzyme denoted aminotransferase AT-2. A genetic background of aminotransferase AT-2 deficiency was used to select for a second mutation which produced a phenotype of multiple auxotrophy for L-phenylalanine, L-aspartate, and L-glutamate. The double mutant completely lacked activity for aromatic aminotransferase AT-1 in addition to the missing aminotransferase AT-2. Enzymes AT-1 (Mr = 64,000) and AT-2 (Mr = 50,000) were readily separated from one another by gel filtration and were individually characterized for pH optima, freeze-thaw stability, heat lability, and molecular weight. The phenotypic and enzymological characterizations of the aminotransferase mutants strongly support the primary in vivo role of enzyme AT-2 in L-phenylalanine and L-tyrosine biosynthesis, while enzyme AT-1 must primarily be engaged in L-aspartate and L-glutamate synthesis. The substrate specificities and possible in vivo functions for AT-3, AT-4, and AT-5 are also considered.     

Charged amino acids conserved in the aromatic acid/H+ symporter family of permeases are required for 4-hydroxybenzoate transport by PcaK from Pseudomonas putida

Charged amino acids in the predicted transmembrane portion of PcaK, a permease from Pseudomonas putida that transports 4-hydroxybenzoate (4-HBA), were required for 4-HBA transport, and they were also required for P. putida to have a chemotactic response to 4-HBA. An essential amino acid motif (DGXD) containing aspartate residues is located in the first transmembrane segment of PcaK and is conserved in the aromatic acid/H+ symporter family of the major facilitator superfamily of transporters.     

Selectivity to K+ and Na+ of protoplast fractions isolated from different regions of Aspergillus nidulans hyphae

The selectivity to K+ and Na+ of protoplast samples representing cytoplasm isolated from different regions of the hyphal filament of Aspergillus nidulans was investigated. Concentrations of both ions contained in successive protoplast fractions were measured. During lytic digestion, protoplasts were released first from apical regions and subsequently from progressively older regions of hyphae. A low K+/Na+ ratio was found in protoplasts containing primarily apical cytoplasm and a high K+/Na+ ratio was found in protoplasts originating from older regions of hyphae. The ratios were the same whether MgSO4 or mannitol was used as stabilizer. Absolute concentrations of both ions were higher in protoplasts of apical origin. Protoplasts stabilized in mannitol lost more ions than those stabilized in MgSO4 over an 8 h incubation period. Na+ losses were higher from apical protoplasts whereas K+ losses were higher from protoplasts liberated from older regions of hyphae. The addition of divalent metal cations (1.5 mM-Mn2+ or Mg2+) reduced losses of Na+ from protoplasts but did not affect loss of K+. Data obtained using protoplast samples were related to those obtained for intact mycelium. Absolute losses of both ions from mycelium were lower than for protoplasts but when compared on a protein basis the data suggested that protoplasts possess properties similar to those of intact mycelium in terms of K+ and Na+ selectivity.     

Further characterization of the polycyclic aromatic hydrocarbon binding properties of the 4S protein

A 4-S protein which specifically binds [3H]benzo(a)pyrene and other polycyclic aromatic hydrocarbons has been investigated in the rat using a hydroxylapatite assay and sucrose gradient analysis. Although there was significant interanimal variation, the specific polycyclic aromatic hydrocarbon binding activity appeared to be highest in 4-week-old male rats and declined with age. The specific [3H]benzo(a)pyrene binding activity was induced after pretreatment with either phenobarbital or isosafrole as evidenced by a 72 and 61% increase, respectively, over untreated controls. No apparent increase in specific binding activity was observed after pretreatment of animals with 3-methylcholanthrene. Pretreatment with either phenobarbital or isosafrole also resulted in the appearance of a small, nonspecific, benzo(a)pyrene binding peak at the 8- to 9-S region in the sucrose density gradients. This 8-S peak was not seen in untreated control animals and represented low affinity, high capacity binding sites. In contrast to the 8-S protein, the 4-S binding protein had low affinity for polychlorinated aromatic compounds such as tetrachlorodibenzodioxin and tetrachlorodibenzofuran. The addition of a 200-fold excess of tetrachlorodibenzofuran to incubations did not displace [3H]benzo(a)pyrene from the 4-S protein. The addition of sodium molybdate to isolation buffers, known to stabilize certain hormone receptors, did not alter the sedimentation coefficient or the specific binding activity of the 4-S protein. These experiments indicate that the 4-S protein does not appear to be a subunit of the 8-S protein. We conclude that in the rat the 4-S protein is distinct from the 8-S protein and the 4-S species may regulate the polycyclic aromatic hydrocarbon-induced expression of aryl hydrocarbon hydroxylase activity.     

Adenine + thymine content of different genes located on the broad host range plasmid RP4

The genetic map of plasmid RP4 was correlated with its adenine+thymine (AT) map. For this purpose, RP4 DNA was digested with one or both of the restriction enzymes EcoRI and HindIII and the resulting linear RP4 molecules and fragments were partially denatured, examined in the electron microscope and their AT maps were determined using a computer program. From these AT maps the EcoRI and HindIII restriction sites were located on the AT map of RP4. Since the positions of these restriction sites on the genetic map of RP4 are known, the maps could be compared. They revealed a high AT content for the Tn1 transposon and the kanamycin resistance gene. The tra-1 region is also distinguished by a sharply defined AT-rich region, whereas tra-2 and the tetracycline resistance gene have an AT content which is not distinctly different from the average AT content of RP4.     

不同氯化钠浓度对Candida etchellsii CICIM Y0600生长和生物特性的影响研究

在酱油发酵过程中增香酵母的代谢产物能显著提高酱油产品的风味和品质.为了提高增香酵母菌在酱醪中的适应性,通过逐步提高氯化钠浓度的方法对酵母菌的耐盐度进行驯化培养.通过分析表明,经过5个批次的驯化,增香酵母菌的耐氯化钠浓度由180 g/L提高到240 g/L.对驯化后的三株耐不同氯化钠浓度的增香酵母菌株进行生物特性研究.结果表明,在200 g/L氯化钠浓度的培养基中,Candida etchellsii CICIM Y0600产游离氨基酸和酸解氨基酸总量分别达到2.01g/L和7.00 g/L.和其他不同氯化钠浓度条件下比较,其产有机酸种类没有变化,挥发性酯类物质含量最高,为0.80 g/L.耐盐度的提高使增香酵母菌更好地适应了在酱油发酵中的高盐度环境.     

Competition in chemostat culture between Pseudomonas strains that use different pathways for the degradation of toluene.

Pseudomonas putida mt-2, P. cepacia G4, P. mendocina KR1, and P. putida F1 degrade toluene through different pathways. In this study, we compared the competition behaviors of these strains in chemostat culture at a low growth rate (D = 0.05 h-1), with toluene as the sole source of carbon and energy. Either toluene or oxygen was growth limiting. Under toluene-limiting conditions, P. mendocina KR1, in which initial attack is by monooxygenation of the aromatic nucleus at the para position, outcompeted the other three strains. Under oxygen limitation, P. cepacia G4, which hydroxylates toluene in the ortho position, was the most competitive strain. P. putida mt-2, which metabolizes toluene via oxidation of the methyl group, was the least competitive strain under both growth conditions. The apparent superiority of strains carrying toluene degradation pathways that start degradation by hydroxylation of the aromatic nucleus was also found during competition experiments with pairs of strains of P. cepacia, P. fluorescence, and P. putida that were freshly isolated from contaminated soil.     

Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1

Structural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding. Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport. However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein. To determine whether the other eucaryotic ABC transporters use the strategy analogous to that in some bacterial ABC transporters, the aromatic Trp653 residue in NBD1 and the Tyr1302 residue in NBD2 of human multidrug resistance-associated protein 1 (MRP1) was mutated to either a different aromatic residue or a polar cysteine residue. Substitution of the aromatic residue with a different aromatic amino acid, such as W653Y or Y1302W, did not affect ATP-dependent leukotriene C4 (LTC4) transport. In contrast, substitution of the aromatic residue with a polar cysteine residue, such as W653C or Y1302C, decreased the affinity for ATP, resulting in greatly increased Kd values for ATP binding or Km values for ATP in ATP-dependent LTC4 transport. Interestingly, although substitution of the aromatic Trp653 in NBD1 of MRP1 with a polar cysteine residue greatly decreases the affinity for ATP, the ATP-dependent LTC4 transport activities are much higher than that of wild-type MRP1, supporting our hypothesis that the increased release rate of the bound ATP from the mutated NBD1 facilitates the protein to start a new cycle of ATP-dependent solute transport.     

Differences in the aromatic domain of homologous streptococcal fibronectin-binding proteins trigger different cell invasion mechanisms and survival rates

Group A streptococci (GAS, Streptococcus pyogenes) and Group G streptococci (GGS, Streptococcus dysgalactiae ssp. equisimilis) adhere to and invade host cells by binding to fibronectin. The fibronectin-binding protein SfbI from GAS acts as an invasin by using a caveolae-mediated mechanism. In the present study we have identified a fibronectin-binding protein, GfbA, from GGS, which functions as an adhesin and invasin. Although there is a high degree of similarity in the C-terminal sequence of SfbI and GfbA, the invasion mechanisms are different. Unlike caveolae-mediated invasion by SfbI-expressing GAS, the GfbA-expressing GGS isolate trigger cytoskeleton rearrangements. Heterologous expression of GfbA on the surface of a commensal Streptococcus gordonii and purified recombinant protein also triggered actin rearrangements. Expression of a truncated GfbA (lacking the aromatic domain) and chimeric GfbA/SfbI protein (replacing the aromatic domain of SfbI with the GfbA aromatic domain) on S. gordonii or recombinant proteins alone showed that the aromatic domain of GfbA is responsible for different invasion mechanisms. This is the first evidence for a biological function of the aromatic domain of fibronectin-binding proteins. Furthermore, we show that streptococci invading via cytoskeleton rearrangements and intracellular trafficking along the classical endocytic pathway are less persistence than streptococci entering via caveolae.     

Expression of an aromatic-dependent decarboxylase which provides growth-essential CO2 equivalents for the acetogenic (Wood) pathway of Clostridium thermoaceticum.

The acetogen Clostridium thermoaceticum generates growth-essential CO2 equivalents from carboxylated aromatic compounds (e.g., 4-hydroxybenzoate), and these CO2 equivalents are likely integrated into the acetogenic pathway (T. Hsu, S. L. Daniel, M. F. Lux, and H. L. Drake, J. Bacteriol. 172:212-217, 1990). By using 4-hydroxybenzoate as a model substrate, an assay was developed to study the expression and activity of the decarboxylase involved in the activation of aromatic carboxyl groups. The aromatic-dependent decarboxylase was induced by carboxylated aromatic compounds in the early stages of growth and was not repressed by glucose or other acetogenic substrates; nonutilizable carboxylated aromatic compounds did not induce the decarboxylase. The decarboxylase activity displayed saturation kinetics at both whole-cell and cell extract levels, was sensitive to oxidation, and was not affected by exogenous energy sources. However, at the whole-cell level, metabolic inhibitors decreased the decarboxylase activity. Supplemental biotin or avidin did not significantly affect decarboxylation. The aromatic-dependent decarboxylase was specific for benzoates with a hydroxyl group in the para position of the aromatic ring; the meta position could be occupied by various substituent groups (-H, -OH, -OCH3, -Cl, or -F). The carboxyl carbon from [carboxyl-14C] vanillate went primarily to 14CO2 in short-term decarboxylase assays. During growth, the aromatic carboxyl group went primarily to CO2 under CO2-enriched conditions. However, under CO2-limited conditions, the aromatic carboxyl carbon went nearly totally to acetate, with equal distribution between the carboxyl and methyl carbons, thus demonstrating that acetate could be totally synthesized from aromatic carboxyl groups. In contrast, when cocultivated (i.e., supplemented) with CO under CO2-limited conditions, the aromatic carboxyl group went primarily to the methyl carbon of acetate.     

4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation.

Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-hydroxybenzoate was purified from 4-hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.     

Fate of organic carbon during decomposition of different litter types in Japan

Carbon dynamics during litter decomposition have been described in a variety of forest ecosystems and provided insights into carbon flow in soils. To quantitatively assess how decomposition processes vary between litter types, solid-state ¹³C cross-polarization and magic-angle spinning nuclear magnetic resonance (CPMAS NMR) technique was applied to analyze conifer (cedar, cypress) and hardwood (chinquapin, beech, oak, birch) litter which had degraded during a 3 year litterbag experiment throughout Japan. The results were used to identify compositional changes and estimate decomposition constants (k values) in exponential equations. Total litter and carbon type mass losses during decomposition varied significantly between litter types, being affected by the initial physicochemical litter quality. Concomitant increases and decreases in carbonyl and O/N-alkyl C compositions, respectively, were observed for all litter types, but aromatic and aliphatic C dynamics were less consistent. In hardwoods, [aromatic/aliphatic C ratio] was generally stable during decomposition, suggesting that, in hardwoods, the decomposabilities of aromatic and aliphatic C were similar. In the conifers, an increasing [aromatic/aliphatic C ratio] during decomposition suggested that aromatic C was more recalcitrant than aliphatic C. These results suggest that different decomposition processes between litter types might be related to different aromatic and aliphatic C behaviors, as affected by lignin stability and lipid leachability and biosynthesis. Variations in the k values for total litter and carbon types were not obvious between litter types, although the mass loss patterns differed significantly. The k values estimated in this study may contribute to predictions of soil carbon dynamics and the validation of carbon compartment models in forest ecosystems.     

PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida.

PcaK is a transporter and chemoreceptor protein from Pseudomonas putida that is encoded as part of the beta-ketoadipate pathway regulon for aromatic acid degradation. When expressed in Escherichia coli, PcaK was localized to the membrane and catalyzed the accumulation of two aromatic substrates, 4-hydroxybenzoate and protocatechuate, against a concentration gradient. Benzoate inhibited 4-hydroxybenzoate uptake but was not a substrate for PcaK-catalyzed transport. A P. putida pcaK mutant was defective in its ability to accumulate micromolar amounts of 4-hydroxybenzoate and protocatechuate. The mutant was also impaired in growth on millimolar concentrations of these aromatic acids. In contrast, the pcaK mutant grew at wild-type rates on benzoate. The Vmax for uptake of 4-hydroxybenzoate was at least 25 nmol/min/mg of protein, and the Km was 6 microM. PcaK-mediated transport is energized by the proton motive force. These results show that although aromatic acids in the undissociated (uncharged) form can diffuse across bacterial membranes, high-specificity active transport systems probably also contribute to the ability of bacteria to grow on the micromolar concentrations of these compounds that are typically present in soil. A variety of aromatic molecules, including naturally occurring lignin derivatives and xenobiotics, are metabolized by bacteria and may be substrates for transport proteins. The characterization of PcaK provides a foundation for understanding active transport as a critical step in the metabolism of aromatic carbon sources.