The adaptive regulation of amino acid transport system A is associated to changes in ATA2 expression

The activity of transport system A for neutral amino acids is adaptively stimulated upon amino acid starvation. In cultured human fibroblasts this treatment causes an increase in the expression of the ATA2 system A transporter gene. ATA2 mRNA increase and transport stimulation are suppressed by system A substrates, but they are unaffected by other amino acids. Supplementation of amino acid-starved cells with substrates of system A causes a decrease in both ATA2 mRNA and system A transport activity. These results suggest a direct relationship between ATA2 expression and system A transport activity.     

Effects of 5-azacytidine, sodium butyrate, and phorbol esters on amino acid transport system A in a kidney epithelial cell line, MDCK: evidence for multiple mechanisms of regulation

Neutral amino acid transport by system A was investigated in the epithelial cell lines MDCK and MDCK-T1. The latter line is a chemically induced, oncogenically transformed line derived from MDCK. Inducers of differentiation, sodium butyrate and 5-azacytidine, and a tumor promoter, TPA, were used as probes to delineate pathways of regulation involved in system A response to a variety of physiological conditions and agents. Azacytidine, an inhibitor of DNA methylation, and butyrate, an enhancer of histone acetylation, inhibited expression of system A, had little effect on system ASC, and slightly stimulated system L. Inhibition of system A expression by butyrate and azacytidine occurred under different conditions. Increases in system A activity due to amino acid starvation or transformation were inhibited by butyrate but not by azacytidine. Repressed system A activity, normally observed in the presence of high levels of amino acids, was more sensitive to azacytidine than to butyrate. The tumor promoter, TPA, stimulated system A activity in MDCK cells under normal growth conditions but did not stimulate activity in amino acid-starved MDCK cells or in MDCK-T1 cells. Stimulation of system A activity by TPA was prevented by prior exposure to butyrate but not to azacytidine. These results suggest 1) that system A expression observed in growing amino-acid-repressed MDCK cells is modulated by an azacytidine-sensitive mechanism and 2) that the elevated expression of system A activity induced by amino acid starvation, by chemical transformation to MDCK-T1, and by TPA is modulated by a butyrate-sensitive mechanism.     

Comparative assessment of the resistance of the unstirred water layer to solute transport between two different intestinal perfusion systems

The resistance of the unstirred water layer to solute transport was estimated in two different intestinal single-pass perfusion systems for a comparative study, using D-glucose as a model compound. One is a well established perfusion system in anesthetized rats as a standard (system A). The other is the one in unanesthetized rats for comparison (system B). It was demonstrated that in system B as well as in system A the resistance of the unstirred water layer to D-glucose transport should be taken into account and this resistance, accordingly, the effective thickness of the unstirred water layer (delta) which is assumed to be in proportion to its resistance, could be described as a function of the perfusion rate by using a film model. The delta decreased with increasing perfusion rate and was larger in system A than in system B at each perfusion rate; 785 microns in system A versus 319 microns in system B at the perfusion rate of 0.16 ml/min and 337 microns versus 184 micron at that of 2.95 ml/min. Thus in system B the effective thickness, accordingly, the resistance, of the unstirred water layer was reduced to about 50% of that in system A, but the resistance of the unstirred water layer could still account for 85% of the total resistance at the maximum as far as D-glucose absorption was concerned, while 93% in system A. These results suggest that, compared with perfusion experiments in anesthetized rats (system A), the resistance of the unstirred water layer is reduced but cannot be left out of consideration even if perfusion experiments are performed in unanesthetized rats (system B). And the lower resistance of the unstirred water layer in system B was attributed to a turbulent flow in contrary to a laminar flow in system A.     

Development of an inducible gene expression system for Lactobacillus sakei

AIM: To develop an inducible gene expression system for Lactobacillus sakei, based on the regulatory system of sakacin A production. METHODS AND RESULTS: A Lactobacillus/Escherichia coli shuttle vector; pKRV3, was constructed including the signal transducing system genes of the bacteriocin sakacin A. The gusA gene fused to PsapA promoter, cloned in this vector allowed for inducible beta-glucuronidase expression in L. sakei and L. plantarum following the addition of the sakacin A inducing peptide. PsapA appeared to be a strong and tightly controlled promoter when compared with known promoters. CONCLUSION: The pKRV3 system can be used as an inducible gene expression system in lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel, inducible gene expression system has been developed for lactic acid bacteria relevant in food fermentations.     

Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results.     

5-fluorotryptophan resistant mutants affecting the A and L transport systems in the mouse L cell line A9

Tryptophan transport has been examined in A9 and in mutants resistant to 5-fluorotryptophan (5-FT). Evidence indicates that in A9 cells two systems are present for tryptophan transport, which are analogous to the A and L systems found in Ehrlich ascites cells differing, however, in terms of amino acid specificity. Tryptophan uptake via the L system, a high affinity, low capacity system, is Na⁺ independent and occurs by a counter transport mechanism, while uptake via the A system, a low affinity, high capacity system, is Na⁺ dependent. Alanine, arginine, lysine, proline, asparagine, and aspartate (listed in order of decreasing inhibitory effect) inhibit tryptophan uptake via the A system from approximately 80-50% while having no inhibitory effect on the L system. In addition, glutamine which inhibits tryptophan uptake by 80% via the L system only inhibits to the extent of 20% via the A system. Previous kinetic studies of 5FT resistant clone FT^r37 indicated system A was altered while the analysis of the effects of the mutation on system L was inconclusive. However, in these studies Na⁺ independent uptake was not altered in FT^r 37 indicating system L was not affected. Amino acid competition studies confirmed this observation and suggested that a change in the specificity of system A had occurred in FT^r 37. The amino acid competition studies in FT^r 23, indicated that the specificities of both systems differed from A9. The possibility that this change may be due to a single mutational event is discussed.     

Selective up-regulation of system a transporter mRNA in diabetic liver.

The transport of alanine by system A is an important source of carbons for the synthesis of glucose in the liver. Here, we show that the mRNA encoding the ubiquitously expressed isoform of the rat system A transporter (SAT2) is dramatically increased in liver following streptozotocin-induced diabetes. This increase in SAT2 mRNA is intensified in the gluconeogenic periportal hepatocytes and also in hepatocytes surrounding the central vein. SAT3, the more abundant system A mRNA isoform present in liver, is restricted to perivenous hepatocytes and is also increased following this treatment but to a much lesser extent than SAT2 mRNA. SN1, an abundant system N mRNA isoform expressed in both perivenous and periportal hepatocytes, is not affected by streptozotocin treatment. A pharmacological dose of glucagon also increased both SAT2 and SAT3 mRNA levels in liver while SN1 mRNA levels remained unaffected. These results indicate that the increase in system A activity observed in liver following experimentally induced diabetes or glucagon treatment is due to the selective increase in mRNAs encoding system A transporters.     

Evidence for a dual pattern of cranial venous sinuses on the endocranial cast of Taung (Australopithecus africanus)

According to published accounts, an enlarged occipital-marginal sinus system is absent in Australopithecus africanus, although it occurs in high frequencies in A. robustus, A. Boisei, and Hadar hominids commonly designated A. afarensis. In this report, we describe, for the first time, an enlarged occipital-marginal sinus system on the endocranial cast of the Taung specimen, which is part of the holotype of A. africanus. In addition, well-developed right transverse and sigmoid sinuses are represented on the Taung endocast. The various components of the dual venous sinus system on the Taung endocast are measured, and the system is compared to those of other fossil hominids. The compresence of a lateral sinus system and enlarged occipital and marginal sinuses occurs in two Hadar specimens, 2 specimens of A. robustus crassidens, 1 A. boisei specimen, and several early H. sapiens crania. Hence, the presence of strong transverse sinus impressions in a fragmentary specimen may not be interpreted as an indication that an enlarged occipital-marginal sinus system was not present in the original specimen. Conversely, lack of transverse sinus grooves in a fragmentary specimen does provide indirect evidence than an enlarged occipital-marginal system would probably have been present in the whole specimen, as in 2 specimens of A. boisei. Including Taung, enlarged occipital and marginal sinuses occur in 1 out of 5, or 20%, of A. africanus specimens. This figure compares well with the range of mean frequencies in modern human cranial series (1.5 to 28%), but is much lower than are the frequencies for A. boisei, A. robustus, and the Hadar hominids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cortisol stimulates system A amino acid transport and SNAT2 expression in a human placental cell line (BeWo)

Both placental system A activity and fetal plasma cortisol concentrations are associated with intrauterine growth retardation, but it is not known if these factors are mechanistically related. Previous functional studies using hepatoma cells and fibroblasts produced conflicting results regarding the regulation of system A by cortisol. Using the b30 BeWo choriocarcinoma cell line, we investigated the regulation of system A by cortisol. System A function was analyzed using methyl amino isobutyric acid (MeAIB) transcellular transport studies. Transporter expression [system A transporter (SNAT)1/2] was studied at the mRNA and protein levels using Northern and Western blotting, respectively. Localization was carried out using immunocytochemistry. The [(14)C]MeAIB transfer rate across BeWo monolayers after preincubation with cortisol for 24 h was significantly increased compared with control. This was associated with a relocalization of the SNAT2 transporter at lower cortisol levels and significant upregulation of mRNA and protein expression levels at cortisol levels >1 microM. This is the first study to show functional and molecular regulation of system A by cortisol in BeWo cells. It is also the first study to identify which system A isoform is regulated. These results suggest that cortisol may be involved in upregulation of system A in the placenta to ensure sufficient amino acid supply to the developing fetus.     

The role of system A for neutral amino acid transport in the regulation of cell volume

System A is a secondary active, sodium dependent transport system for neutral amino acids. Strictly coupled with Na,K-ATPase, its activity determines the size of the intracellular amino acid pool, through a complex network of metabolic reaction and exchange fluxes. Many hormones and drugs affect system A activity in specific cell models or tissues. In all the cell models tested thus far the activity of the system is stimulated by amino acid starvation, cell cycle progression, and the incubation under hypertonic conditions. These three conditions produce marked alterations of cell volume. The stimulation of system A activity plays an important role in cell volume restoration, through an expansion of the intracellular amino acid pool. Under normal conditions, system A substrates represent a major fraction of cell compatible osmolytes, organic compounds that exert a protein stabilizing effect. It is, therefore, likely that the activation of system A represents a portion of a more complex response triggered by exposure to stresses of various nature. Since system A transporters have been recently cloned, the molecular bases of these regulatory mechanisms will probably be elucidated in a short time.     

The role of system A for neutral amino acid transport in the regulation of cell volume

System A is a secondary active, sodium dependent transport system for neutral amino acids. Strictly coupled with Na,KATPase, its activity determines the size of the intracellular amino acid pool, through a complex network of metabolic reaction and exchange fluxes. Many hormones and drugs affect system A activity in specific cell models or tissues. In all the cell models tested thus far the activity of the system is stimulated by amino acid starvation, cell cycle progression, and the incubation under hypertonic conditions. These three conditions produce marked alterations of cell volume. The stimulation of system A activity plays an important role in cell volume restoration, through an expansion of the intracellular amino acid pool. Under normal conditions, system A substrates represent a major fraction of cell compatible osmolytes, organic compounds that exert a protein stabilizing effect. It is, therefore, likely that the activation of system A represents a portion of a more complex response triggered by exposure to stresses of various nature. Since system A transporters have been recently cloned, the molecular bases of these regulatory mechanisms will probably be elucidated in a short time.     

检测SortaseA酶催化蛋白质连接效率的新型报道系统

利用分子进化技术对Sortase A酶的催化活性进行定向改造已经成为当前的研究热点和重点,为了高效筛选特定催化活性的Sortase A酶突变体,需要建立一种能够快速准确鉴定Sortase A酶活性的方法。为此,设计了由GFP-LPETG蛋白和GGGYK-Biotin组成的新型报道底物系统。利用重组基因工程技术,成功制备GFP-LPETG蛋白,野生型Sortase A酶及近期报道的一个高活性突变型Sortase A酶。以GFP-LPETG及GGGYK-Biotin为报道底物建立连接体系,结果显示,连接反应动力学可直接通过SDS-PAGE凝胶电泳法精确测定。用此连接体系比较野生型与突变型Sortase A酶催化的连接反应效率,证实高活性突变酶具有野生酶无法比拟的高催化活性。此报道系统简单快速灵敏,可应用于系统的筛选,为后续进一步定向优化Sortase A酶奠定了基础。     

Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A

We investigated the molecular mechanism involved in the adaptive regulation of the amino acid transport system A, a process in which amino acid starvation induces the transport activity. These studies were done with rat C6 glioma cells. System A activity in these cells is mediated exclusively by the system A subtype, amino acid transporter A2 (ATA2). The other two known system A subtypes, ATA1 and ATA3, are not expressed in these cells. Exposure of these cells to an amino acid-free medium induces system A activity. This process consists of an acute phase and a chronic phase. Laser-scanning confocal microscopic immunolocalization of ATA2 reveals that the acute phase is associated with recruitment of preformed ATA2 from an intracellular pool to the plasma membrane. In contrast, the chronic phase is associated with an induction of ata2 gene expression as evidenced from the increase in the steady-state levels of ATA2 mRNA, restoration of the intracellular pool of ATA2 protein, and blockade of the induction by cycloheximide and actinomycin D. The increase in system A activity induced by amino acid starvation is blocked specifically by system A substrates, including the non-metabolizable alpha-(methylamino)isobutyric acid.     

Modification of system A amino acid carrier by diethyl pyrocarbonate

Sodium-dependent alanine transport in plasma membrane vesicles from rat liver was inactivated in a time- and concentration-dependent fashion by prior treatment of membranes with the acylating reagent diethyl pyrocarbonate (DEPC). Both components of Na+/alanine cotransport (systems A and ASC) were inhibited. Exposure of vesicles to p-bromophenacyl bromide and methyl p-nitrobenzenesulfonate, which share with DEPC reactivity against histidine residues, also led to inhibition of alanine transport through systems A and ASC. The presence of Na+ (100 mM NaCl) and L-alanine (10 mM) during exposure to vesicles to DEPC protected against inactivation of system A (but not system ASC) transport activity. This protective effect was specific and required the presence of L-alanine since the presence of L-phenylalanine alone (10 mM) or L-phenylalanine plus Na+ (100 mM NaCl) did not cause any detectable protection. This overall pattern of protection is opposite to that previously found against specific sulfhydryl reagents (i.e. N-ethylmaleimide), where protection of system ASC was nearly maximal. The pH profile for DEPC-dependent inhibition of system A transport activity suggests modification of amino acid residue(s) with a pKr of approximately 7, most likely histidine(s), in close parallel with the pH dependence of system A transport activity. Our results suggest the presence of critical histidine residues on the system A carrier that may be responsible for the pH dependence of system A transport activity.     

A well-characterized polycistronic-like gene expression system in yeast

Efficient expression of multiple genes is critical to yeast metabolic engineering for the bioproduction of bulk and fine chemicals. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. 2A viral peptides enable the cotranslation of multiple proteins from a single mRNA by ribosomal skipping. However, the wide adaptation of 2A viral peptides for polycistronic-like gene expression in yeast awaits in-depth characterizations. Additionally, a one-step assembly of such a polycistronic-like system is highly desirable. To this end, we have developed a modular cloning (MoClo) compatible 2A peptide-based polycistronic-like system capable of expressing multiple genes from a single promoter in yeast. Characterizing the bi-, tri-, and quad-cistronic expression of fluorescent proteins showed high cleavage efficiencies of three 2A peptides: E2A from equine rhinitis B virus, P2A from porcine teschovirus-1, and O2A from Operophtera brumata cypovirus-18. Applying the polycistronic-like system to produce geraniol, a valuable industrial compound, resulted in comparable or higher titers than using conventional monocistronic constructs. In summary, this highly-characterized polycistronic-like gene expression system is another tool to facilitate multigene expression for metabolic engineering in yeast.     

Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.     

Synthesis and degradation of poly(A) in permeable cells of Escherichia coli.

Poly(A) synthesis and degradation have been examined in Escherichia coli cells made permeable to nucleotides by treatment with toluene. Although newly synthesized poly(A) is normally rapidly degraded in this system, extraction of the soluble portion of the cell effectively eliminates this process without affecting poly(A) synthesis. Poly(A) synthesis in this system displays many properties associated with poly(A) synthesis by purified poly(A) polymerase in vitro including a lag in polymerization, stimulation by increased ionic strength, and a low Mg2+ optimum. As with the purified enzyme, this system uses both ADP and ATP as substrates, requires conversion of ATP to ADP, and is strongly inhibited by dADP, orthophosphate, and pyrophosphate. In contrast to the purified poly(A) polymerase, the permeable cell system displays some properties suggestive of in vivo poly(A) metabolism. Thus, the permeable cells require an endogenous RNA primer for activity, the poly(A) product remains with the cells, and the reaction is greatly stimulated by polyamines. This system should prove extremely useful for studies of poly(A) metabolism in E. coli. A surprising feature of these studies was the finding that mutant strains deficient in polynucleotide phosphorylase were unable to synthesize poly(A). The possible roles of polynucleotide phosphorylase and poly(A) in E. coli are discussed.     

Radioimmunoassay for orexin A

A radioimmunoassay for orexin A has been developed. Anti-orexin A antiserum was raised in New Zealand white rabbits immunized with a conjugate of synthetic orexin A with bovine serum albumin. This antibody did not crossreact with orexin B, hypothalamic hormones, pituitary hormones, neuropeptides or gut hormones. Radioiodination of orexin A was performed with the chloramin T method, followed by purification of radioiodinated material on Sephadex G-25 column. Orexin A was extracted from tissues using acid-acetone. The assay was performed with a double antibody system. The dilution curve of acid-acetone-extracts of rat hypothalamus in the radioimmunoassay system was parallel to the standard curve. The recovery of tissue orexin A was about 80%,and the intra-assay and inter-assay variations were 5.2% and 7.8%, respectively. Orexin A was found in the hypothalamus, cerebrum and testis. These data suggest that this assay system is suitable for the measurement of tissue orexin A and that orexin A is found in the central nervous system and testis.     

Understanding peptide competitive inhibition of botulinum neurotoxin a binding to SV2 protein via molecular dynamics simulations

Botulinum neurotoxins (BoNTs) are known as the most toxic natural substances. Synaptic vesicle protein 2 (SV2) has been proposed to be a protein receptor for BoNT/A. Recently, two short peptides (BoNT/A‐A2 and SV2C‐A3) were designed to inhibit complex formation between the BoNT/A receptor‐binding domain (BoNT/A‐RBD) and the synaptic vesicle protein 2C luminal domain (SV2C‐LD). In this article, the two peptide complex systems are studied by molecular dynamics (MD) simulations. The structural stability analysis indicates that BoNT/A‐A2 system is more stable than SV2C‐A3 system. The conformational analysis implies that the β‐sheet in BoNT/A‐A2 system maintains its secondary structure but the two β‐strands in SV2C‐A3 system have remarkable conformational changes. Based on the calculation of hydrogen bonds, hydrophobic interactions and cation‐π interactions, it is found that the internal hydrogen bonds play crucial roles in the structural stability of the peptides. Because of the stable secondary structure, the β‐sheet in BoNT/A‐A2 system establishes effective interactions at the interface and inhibits BoNT/A‐RBD binding to SV2C‐LD. In contrast, without other β‐strands forming internal hydrogen bonds, the two isolated β‐strands in SV2C‐A3 system become the random coil. This conformational change breaks important hydrogen bonds and weakens cation‐π interaction in the interface, so the complex formation is only partially inhibited by the two β‐strands. These results are consistent with experimental studies and may be helpful in understanding the inhibition mechanisms of peptide inhibitors. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 597–608, 2015.     

菰和菖蒲对富营养化水体净化效率的比较

研究了菰〔Zizania latifolia(Griseb.)Stapf〕、菖蒲(Acorus calamusL.)及它们的复合体系对富营养化水体的净化效果。结果表明,菰和菖蒲在供试富营养化水体中均能正常生长,二者单独种植体系或等量混合种植体系对富营养化水体均有一定的净化能力。单独种植的菰和菖蒲及二者的混合种植体系对供试水体中总氮的去除率分别为92.8%、92.7%和94.9%;对氨氮的去除率分别为95.5%、97.4%和96.6%;对总磷的去除率分别为83.9%、94.3%和84.7%;对CODCr的去除率分别为83.0%、85.5%和86.7%。单独种植的菖蒲对总磷的去除效果明显好于单独种植的菰和二者的混合种植体系。菰和菖蒲的混合种植体系对富营养化水体的净化效果与单独种植体系无明显差异。