Relationship of Cell Envelope Stability to Substrate Capture in a Marine Psychrophilic Bacterium

Cells of a psychrophilic marine bacterium were found to take up a variety of amino acids from seawater. Some of the amino acids that were taken up were released when the cells were exposed to a hypotonic salt solution. The proportion that was released varied according to the amino acid. A pool of the amino acid arginine that was formed during very short periods of exposure of cells to the exogenously supplied amino acid was particularly sensitive to reductions in salinity. In general, exposure to hypotonic salt solutions also resulted in reduced amino acid uptake by the cells. Complete removal of seawater salts (SE treatment) produced obvious structural alterations in the cell envelope, resulting in an even greater reduction in amino acid uptake. Under these conditions, amino acid-binding components were released by the cells. Differential centrifugation and fluorescent antibody studies indicated that arginine-binding components are located on or near the surface of intact cells. The data suggest that substrate receptors were sensitive to reductions in seawater salt concentrations and that lesions at this level affected the organism's substrate uptake and retention capabilities.     

Carrier-mediated Uptake of Arginine and Urea by Chlamydomonas reinhardtii

Chlamydomonas reinhardtii possesses a high affinity, highly specific carrier involved in uptake of exogenous arginine. Carrier-mediated uptake of other amino acids cannot be detected, even in cultures maintained on amino acids as a nitrogen source or starved for nitrogen. This fact may contribute to the difficulty of isolating strains auxotrophic for amino acids other than arginine; conventional selection media may not supply adequate quantities of amino acids to permit growth of auxotrophs. A urea carrier is also present in C. reinhardtii but is readily distinguished from the arginine carrier on the basis of kinetic properties and sensitivity to a range of structural analogs. Ammonia appears to play a major role in regulating (depressing) activity of the arginine uptake system. Activity of the urea uptake system is elevated in nitrogen-starved cultures and elevated even further in the presence of urea or arginine. Extensive, independent fluctuations in the two uptake systems observed in semisynchronous cultures suggest that both are subject to modulation by a complex set of interacting endogenous and exogenous factors.     

Role of glutamine synthetase activity in the uptake and metabolism of arginine and proline in the cyanobacterium Anabaena cycadeae

Abstract The uptake of arginine and proline and their assimilation as nitrogen source have been studied in the cyanobacterium Anabaena cycadeae and its glutamine auxotropic mutant lacking glutamine synthetase activity. The uptake pattern of arginine and proline was found to be biphasic in both wild-type and mutant strains, consisting of an initial fast phase lasting up to 60 s followed by a slower second phase. The uptake activities of both the amino acids were also found to be similar in both the strains. The wild-type strain, having normal glutamine synthetase activity, utilized arginine and proline as sole nitrogen source, whereas the mutant strain lacking glutamine synthetase activity could not do so. These results suggest that: (1) glutamine synthetase activity is necessarily required for the assimilation of arginine and proline as nitrogen source, but it is not required for the uptake of these amino acids; and (2) glutamine synthetase serves as the sole ammonia-assimilating enzyme as well as glutamine-forming route in heterocystous cyanobacteria.     

Nitrogen nutrition in the cyanobacterium Nostoc ANTH, a symbiotic isolate from Anthoceros: uptake and assimilation of inorganic-n and amino acids

Amino acid uptake and utilization of various nitrogen sources (amino acids, nitrite, nitrate and ammonia) were studied in Nostoc ANTH and i ts mu tant (Het(-)Nif(-)) isolate defective in heterocyst formation and N2-fixation. Both parent and its mutant grew at the expense of glutamine, asparagine and arginine as a source of fixed-nitrogen. Growth was better in glutamine-and asparagine-media as compared to that in arginine media. Glutamine and asparagine repressed heterocyst formation, N2-fixation and nitrate reduction in Nostoc ANTH, but arginine did so only partially. The poor growth in arginine-medium was not due to poor uptake rates, since the uptake rates were not significantly different from those for glutamine or asparagine. The glutamine synthetase activity remained unaffected during cultivation in media containing any one of the three amino acids tested. The uptake of amino acids was substrate-inducible, energy-dependent and required de novo protein synthesis. Nitrate and ammonium repressed ammonium uptake, but did not repress uptake of amino acids. In N2-medium (BG-11(0)), the uptake of ammonium and amino acids in the mutant was significantly higher than its parent strain. This was apparently due to nitrogen limitation since the mutant was unable to fix N2 and the growth medium lacked combined-N.     

General properties and regulation of arginine transporting systems in Saccharomyces cerevisiae

The transport of arginine-¹⁴C by exponentially growing cellsof Saccharomyces cerevisiae (ATCC 9763) was studied in the presenceof various amino acids, ammonium and urea. Arginine transportwas inhibited when the cells were preincubated with these compoundsfor 1 hr. Little or no inhibition of transport occurred whenthe preincubation period was omitted. Kinetics studies revealedthat arginine was transported by two distinct systems havinghigh and low affinities for this amino acid. At given arginineconcentrations the high affinity system was capable of transportingarginine molecules at approximately seventy times the rate ofthe low affinity system. The general requirements for arginine transport revealed energyand temperature dependencies in addition to sensitivity to anumber of metabolic inhibitors. Transfer of cells to N-freemedium was accompanied by increased rates of transport. Thisincrease was shown for the uptake of ten different amino acids.For L-arginine, this increase was prevented by addition of cycloheximide. Analyses of amino acid pools, after various experimental treatments,failed to reveal any consistent correlation between transportrates and the concentrations of individual amino acids or ammonium. It is concluded that arginine transport of S. cerevisiae isregulated by inhibition and repression. In this respect theavailability of ammonium would appear to be of prime importancein the development of transport activity. (Received December 5, 1975; )     

Study of amino acids as nitrogen source in Chlamydomonas reinhardtii

All five L‐amino acids tested (L‐serine, L‐lysine, L‐leucine, L‐cysteine and L‐arginine) were used by Chlamydomonas reinhardtii as sole nitrogen source. Among these, L‐Cys was special as it has not been reported before. While these amino acids could be used in the dark only in the presence of acetic acid, in conditions of light they could support the growth of C. reinhardtii without the supplementation of acetic acid. When cultured in the TAP‐N medium, the chlorophyll content was found to be lower in the dark, but higher in the light for the cells grown with L‐Arg than with other four amino acids. Exogenously supplied L‐Ser and L‐Lys did not accumulate in the cells, demonstrating that they were used by supplying ammonium to the cells from the activity of an extracellular deaminase. Further results showed that the induction of the extracellular deaminase activity required a period of nitrogen starvation, regardless of the medium containing acetic acid or not. Results also showed that the uptake of L‐Cys was similar to L‐Leu, most likely via passive diffusion. When L‐Cys and L‐Leu were supplied together to the nitrogen‐starved cells, the absorption of L‐Cys did not affect the uptake of L‐Leu.     

保守的第52位色氨酸突变引起的胰高血糖素样肽1受体N端片段活性丧失

通过易错PCR方法建立了一个鼠肺不同长度的nGLP-1R(从第21个氨基酸开始到第145个氨基酸)的噬菌体随机突变展示肽库,通过噬菌体表面展示技术检测胰高血糖素样肽1受体N端片段(nGLP-1R)在缺失一段或两段基因后是否还具有结合Exendin-4的活性.经ELISA分析发现了一株无结合活性的突变株,命名为EP16.经测序比对,发现EP16缺失了前20个和后10个氨基酸,且第52位色氨酸突变为精氨酸.为确定EP16与Exendin-4无结合活性的原因,重新构建了无前20个和后10个氨基酸的EP16野生型及第52位色氨酸变为精氨酸的全长nGLP-1Rw52R与EP16进行对比分析.结果表明,EP16的活性丧失是由保守的第52位色氨酸突变为精氨酸引起的,缺失的前20个和后10个氨基酸没有影响其生物学活性.关键位点单个氨基酸残基的突变可以改变胰高血糖素样肽1受体N端片段整个蛋白质的生物学活性.     

9种氨基酸对甲醛捕获能力的研究

本文研究了9种氨基酸对甲醛的捕获效果,筛选出了对甲醛捕获效果较好的4种氨基酸,即精氨酸、赖氨酸、半胱氨酸盐酸盐、组氨酸,并研究了这4种氨基酸在不同温度和时间下对甲醛的捕获规律。结果表明,半胱氨酸盐酸盐的甲醛捕获效果最好,捕获率可达100％,而且受温度影响最小;其次是精氨酸、赖氨酸、组氨酸,捕获效果均在50％左右,但受温度影响较大;精氨酸、赖氨酸在低温下对甲醛生成有促进作用,高温下有捕获作用;组氨酸在高温下对甲醛的捕获率显著提高,可达87．99％。     

Free amino acid levels and the regulation of nitrate uptake in maize cell suspension cultures

The ability of individual amino acids to regulate nitrate uptakeand induction was studied in a Zea mays embryo cell line grownin suspension culture. The maize cells exhibited a marked preferencefor absorbing amino acids over nitrate when both were presentin culture medium. The addition of an individual amino acid(2 mM glutamine, glycine, aspartic acid, or arginine) to theculture medium with 1 mM nitrate completely inhibited nitrateuptake and resulted in a cycle of low levels of nitrate influxfollowed by efflux to the growth medium. Glutamine was readilyabsorbed by the cells and was particularly effective in supportingoptimum cell growth in the absence of an inorganic nitrogensource as compared to the three other amino acids evaluated.However, neither glutamine nor any of the remaining 19 proteinaceousamino acids appeared to be solely responsible for regulationof nitrate uptake and induction. The ability of amino acidsto regulate nitrate uptake and assimilation appears to be morerelated to their overall levels in the cell rather than to anaccumulation of a specific amino acid. Key words: Amino acids, nitrate uptake, maize, regulation, cell suspension culture     

Blue-light-control of the uptake of amino acids and of ammonia in Chlorella mutants

In non-photosynthetic, yellow or colourless mutant cells of Chlorella kessleri , grown with nitrate as sole nitrogen source, blue light inhibited the uptake of the amino acids glycine, proline and arginine and of ammonia in growing cells, while it enhanced the uptake of these amino acids in resting cells. On the other hand, in cells grown with ammonia as the only nitrogen source without nitrate reductase activity, blue light did not influence the uptake of amino acids and of ammonia in growing cells, while it enhanced the uptake of amino acids in resting cells. Addition of methionine sulphoximine, a potent inhibitor of glutamine synthetase, to growing cells, resulted in intracellular ammonia-accumulation and inhibition of uptake of glycine and of ammonia. For the colourless mutant, blue light was shown to activate purified nitrate reductase. These results indicate that in the mutant cells of Chlorella examined, uptake of ammonia seems to be influenced by nitrate reductase and the uptake of amino acids was influenced by both nitrate reductase and an unknown blue-light-receptor(s). The uptake of urea in mutant cells is not influenced by the irradiation with blue light. Uptake of glycine was also increased after addition of glucose (hexose) in the dark. Because blue light is known to enhance the breakdown of starch, a reaction producing glucose for oxidative degradation in the algae used, the role of glucose (hexose) in the blue light-affected uptake of amino acids is discussed.     

A quantitative assessment of the contribution of individual plasma amino acids to the synthesis of milk proteins by the goat mammary gland

1. Arteriovenous differences of plasma free amino acids across the lactating mammary glands of six goats have been measured. 2. In four experiments, measurements of blood flow, amino acid arteriovenous differences, milk yield and milk nitrogen showed that the uptake of nitrogen in the form of amino acids was sufficient to provide all the nitrogen of the milk proteins synthesized in the mammary gland. 3. In the same four experiments the uptake from the plasma and output into the milk of individual amino acids per unit time were compared. The uptakes of essential amino acids and glutamic acid were approximately equal to the corresponding output figures. The uptake of serine was consistently less than the output, and the uptake of other non-essential amino acids was very variable, in some experiments being approximately equal to the output figures and in others being considerably less. 4. As in cows, there was an uptake of ornithine in all experiments, though ornithine is absent from milk. In goats, though not in cows, the uptake of arginine was consistently greatly in excess of the requirement for arginine residues in milk protein. 5. The possible significance of the uptakes of arginine and ornithine for the synthesis of serine and other non-essential amino acids in the mammary gland is discussed. 6. The importance of clamping the external pudic vein, when sampling mammary venous blood from the caudal superficial epigastric vein, is indicated.     

The biological effects and mode of action of L-canavanine, a structural analogue of L-arginine.

Many of the 200 or so non-protein amino acids synthesized by higher plants are related structurally to the constituents of common proteins. L-Canavanine, the guanidinooxy structural analogue of L-arginine, is representative of this group. It has provided valuable insight into the biological effects and the mode of action of non-protein amino acids which acts as analogues of the protein amino acids. The arginyl-tRNA synthetases of numerous canavanine-free species charge canavanine, and canavanine is subsequently incorporated into the nascent polypeptide chain. Production of canavanine-containing proteins ultimately can disrupt critical reactions of RNA and DNA metabolism as well as protein synthesis. Canavanine also affects regulatory and catalytic reactions of arginine metabolism, arginine uptake, formation of structural components, and other cellular precesses. In these ways, canavanine alters essential biochemical reactions and becomes a potent antimetabolite of arginine in a wide spectrum of species. These deleterious properties of canavanine render it a highly toxic secondary plant constituent that probably functions as an allelochemic agent that deters the feeding activity of phytophagous insects and other herbivores.     

Some Factors Which Affect Amino Acid Uptake by Saccharomyces carlsbergensis

When fully grown cells of Saccharomyces carlsbergensis were suspended in a solution of glucose and labeled amino acids, there was a lag phase before rapid uptake of certain amino acids. During this lag, significant amounts of sugar were utilized. The lag phase varied in length, depending upon the amino acid under study, but could be shortened by aeration of the cells and eliminated by their preincubation in glucose solution. Divalent metal ions, especially Ca²⁺ added during the early stages of the lag phase, increased the length of the lag, an effect that could be reversed by washing with ethylenediaminetetraacetate, but amino acids which normally showed little or no lag before uptake were insensitive to Ca²⁺. The rate of uptake of amino acids or of sugar was essentially unaffected by Ca²⁺, whereas 2,4-dinitrophenol caused an overall decrease in the rate of uptake of all amino acids tested. The relevance of these observations to commercial brewing practice is shown.     

Isolation and Characterization of Strains Defective in Vacuolar Ornithine Permease inNeurospora crassa

Vacuolar uptake of ornithine and lysine was characterized inNeurospora crassausing a cupric ion permeabilization system. Michaelis constants were measured as 1.4 mM for lysine and 11.0 mM for ornithine, and maximal velocities were determined. Vacuolar lysine uptake was shown to be inhibited competitively byl-arginine and histidine while ornithine uptake was inhibited by a variety of amino acids. Strains defective in the vacuolar ornithine permease were isolated using a filtration enrichment method. Two isolates—RSC-39 and RSC-63—had a reduced ability to accumulate ornithine. Vacuolar uptake of amino acids was measured using cupric ion-permeabilized mycelia; both strains had reduced ornithine uptake while lysine uptake and arginine uptake were normal. For both isolates, both the Michaelis constant and the maximal velocity for ornithine uptake were reduced compared to those of wild type. These results suggest that both strains are defective in the gene which encodes the vacuolar ornithine permease.     

A herpes simplex virus recombinant that exhibits a single-chain antibody to HER2/neu enters cells through the mammary tumor receptor, independently of the gD receptors

The human epidermal growth factor receptor 2/neuregulin (HER2/neu) receptor is overexpressed in highly malignant mammary and ovarian tumors and correlates with a poor prognosis. It is a target for therapy; humanized monoclonal antibodies to HER2 have led to increased survival of patients with HER2/neu-positive breast cancer. As a first step in the design of an oncolytic herpes simplex virus able to selectively infect HER2/neu-positive cells, we constructed two recombinants, R-LM11 and R-LM11L, that carry a single-chain antibody (scFv) against HER2 inserted at residue 24 of gD. The inserts were 247 or 256 amino acids long, and the size of the gD ectodomain was almost doubled by the insertion. We report the following. R-LM11 and R-LM11L infected derivatives of receptor-negative J or CHO cells that expressed HER2/neu as the sole receptor. Entry was dependent on HER2/neu, since it was inhibited in a dose-dependent manner by monoclonal antibodies to HER2/neu and by a soluble form of the receptor. The scFv insertion in gD disrupted the ability of the virus to enter cells through HVEM but maintained the ability to enter through nectin1. This report provides proof of principle that gD can tolerate fusion to a heterologous protein almost as large as the gD ectodomain itself without loss of profusion activity. Because the number of scFv's to a variety of receptors is continually increasing, this report makes possible the specific targeting of herpes simplex virus to a large collection of cell surface molecules for both oncolytic activity and visualization of tumor cells.     

Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms.

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors.     

Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides

Protein D2 of Pseudomonas aeruginosa outer membrane is known to facilitate the specific permeation of imipenem (N-formimdoylthienamycin) across this membrane barrier. We have characterized the binding site in the protein D2 channel by studying the competitive inhibition, by various solutes, of imipenem diffusion into the periplasm. We found that basic amino acids, lysine, arginine, histidine, and ornithine, were effective inhibitors. L- and D-lysine were found to be competitive inhibitors with approximate Ki values of 0.6 and 0.3 mM, respectively. Peptides containing L-lysine at the carboxyl terminus, as well as dipeptides containing L-lysine at the amino terminus, were also able to inhibit the transport. Wild type cells transported tripeptide Thr-Ser-Lys into the periplasm three to four times as rapidly as the mutant cells lacking the D2 protein. These results suggest that protein D2 plays a physiologically significant role in the uptake of basic amino acids and peptides containing these amino acids across the outer membrane of P. aeruginosa.     

Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.     

Crustacean peptide and peptide-like pheromones and kairomones

Crustacean peptide pheromones, kairomones, and substituted amino sugar kairomones are reviewed from a historical perspective. These crustacean information molecules are secondary functions of structural polymers. They are partial hydrolysis products, generated usually by the action of trypsin-like enzymes on proteins, and glycosidase enzymes on glycoproteins and proteoglycans. Structure-function studies based upon synthetic mimics of peptide information molecules show neutral amino acids with a basic carboxyl terminal are active in modifying physiological and or behavioral responses. Behaviorally active substituted amino sugar mimics are disaccharide hydrolysis products of heparin and chondroitin sulfate. Similar molecules are also used as information molecules by a variety of other marine organisms indicating they are a common biological theme.     

Multiple pathways for cationic amino acid transport in rat seminiferous tubule cells

Arginine and ornithine are known to be important for various biological processes in the testis, but the delivery of extracellular cationic amino acids to the seminiferous tubule cells remains poorly understood. We investigated the activity and expression of cationic amino acid transporters in isolated rat Sertoli cells, peritubular cells, pachytene spermatocytes, and early spermatids. We assessed the l-arginine uptake kinetics, Na(+) dependence of transport, profiles of cis inhibition of uptake by cationic and neutral amino acids, and sensitivity to trans stimulation of cationic amino acid transporters, and studied the expression of the genes encoding them by RT-PCR. Our data suggest that l-arginine is taken up by Sertoli cells and peritubular cells, principally via system y(+)L (SLC3A2/SLC7A6) and system y(+) (SLC7A1 and SLC7A2), with system B(0+) making a minor contribution. By contrast, system B(0+), associated with system y(+)L (SLC3A2/SLC7A7 and SLC7A6), made a major contribution to the transport of cationic amino acids in pachytene spermatocytes and early spermatids. Sertoli cells had higher rates of l-arginine transport than the other seminiferous tubule cells. This high efficiency of arginine transport in Sertoli cells and the properties of the y(+)L system predominating in these cells strongly suggest that Sertoli cells play a key role in supplying germ cells with l-arginine and other cationic amino acids. Furthermore, whereas cytokines induce nitric oxide (NO) production in peritubular and Sertoli cells, little or no upregulation of arginine transport by cytokines was observed in these cells. Thus, NO synthesis does not depend on the stimulation of arginine transport in these somatic tubular cells.