Biphasic effect of IL-1beta on the activity of argininosuccinate synthetase in Caco-2 cells. Involvement of nitric oxide production

The expression of the argininosuccinate synthetase gene (ASS), the limiting enzyme of arginine synthesis, was previously shown to be rapidly induced by a short-term (4 h) exposure to IL-1beta in Caco-2 cells [Biochimie, 2005, 403-409]. The present report shows that, by contrast, a long-term (24 h) exposure to IL-1beta inhibited the ASS activity despite an increase in both specific mRNA level and protein amount, demonstrating a post-translational effect. Concerning the mechanism involved, we demonstrate that the inhibiting effect is linked to the production of nitric oxide (NO) induced by IL-1beta. Indeed, the inhibiting effect of IL-1beta was totally blocked in the presence of l-NMMA, an inhibitor of the inducible nitric oxide synthase, or by culturing the cells in an arginine-deprived medium. Moreover, a decrease in the ASS activity was induced by culturing the cells in the presence of SNAP, a NO donor. Conversely, blocking the action of NO by antioxidant agents, the stimulatory effect of IL-1beta on ASS activity was restored, as measured at 24 h. Finally, such an inhibiting effect of NO on ASS activity may be related, at least in part, to S-nitrosylation of the protein. The physiological relevance of the antagonistic effects of IL-1beta and NO on ASS is discussed.     

RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells

Background  

Sensitivity of cancer cells to recombinant arginine deiminase (rADI) depends on expression of argininosuccinate synthetase (AS), a rate-limiting enzyme in synthesis of arginine from citrulline. To understand the efficiency of RNA interfering of AS in sensitizing the resistant cancer cells to rADI, the down regulation of AS transiently and permanently were performed in vitro, respectively.     

mRNA降解在植物发育以及抗性反应中的作用

植物mRNA的降解对于维持其生化和细胞学的功能都是必需的,而且这种降解要根据植物发育和外界环境的变化进行及时的调整。与酵母和哺乳动物相比,人们对植物mRNA的降解机制了解较少。对近几年来该领域的研究进展进行总结,包括参与植物mRNA降解的酶类和基因芯片技术的应用,以及mRNA降解的生物学意义等。     

The glutamine synthetase gene family of Arabidopsis thaliana: light-regulation and differential expression in leaves, roots and seeds.

Summary Glutamine synthetase (GS) plays an important role in the assimilation of nitrogen by higher plants. We present here a molecular analysis of the GS polypeptides, mRNAs, and genes of Arabidopsis thaliana. Western blot analysis of leaf and root protein extracts revealed at least two distinct GS polypeptides; 43 kDa and 39 kDa GS polypeptides were present in leaves, while only a 39 kDa GS was detected in roots. The 43 kDa GS polypeptide is light-inducible. In etiolated seedlings only the 39 kDa GS was detected. However, upon greening the 43 kDa GS increased to levels comparable to those observed in light-grown plants. Four distinct GS cDNA clones, Atgsl1, Atgsrl, Atgsr2 and Atk6 were isolated and characterized. Their complete nucleotide and deduced amino acid sequences are presented. The coding sequences of the four clones are 70–88% similar while their 5 and 3 untranslated regions exhibit less than 50% similarity. Northern blots of leaf, root and germinated seed RNA revealed that the four cDNAs hybridize to mRNAs which are differentially expressed in the organs of Arabidopsis thaliana. Atgsl1 is leaf-specific and hybridizes to a 1.6 kb mRNA. Both Atgsr1 and Atgskb6 hybridize to 1.4 kb mRNAs which are expressed in both roots and germinated seeds. Atgsr2 hybridizes to a 1.4 kb mRNA, which is primarily expressed in roots with low levels of expression in seeds and leaves. Atgsl1, which represents the leaf-specific mRNA, is induced by light. Atgsl1 mRNA levels increase during the greening of etiolated seedlings while Atgsr1 levels remain constant. Southern blot analysis indicated that the Arabidopsis genome contains at least four and possibly five distinct GS genes.     

Differential mRNA expression levels and gene sequences of carboxylesterase in both deltamethrin resistant and susceptible strains of the cotton aphid, Aphis gossypii

Extensive use of insecticides on cotton has prompted resistance development in the cotton aphid, Aphis gossypii (Glover) in China. A deltamethrin‐selected population of cotton aphids from Xinjiang Uygur Autonomous Region, China with 228.59‐fold higher resistance to deltamethrin was used to examine how carboxylesterase conferred resistance to this pyrethroid insecticide. The carboxylesterase activity in the deltamethrin‐resistant strain was 3.67‐, 2.02‐ and 1.16‐fold of the susceptible strain when using α‐naphthyl acetate (α‐NA), β‐naphthyl acetate (β‐NA) and α‐naphthyl butyrate (α‐NB) as substrates, respectively. Carboxylesterase cDNA was cloned and sequenced from both deltamethrin‐resistant and susceptible strains. The cDNA contained 1581 bp open reading frames (ORFs) coding a 526 amino acid protein. Only one amino acid substitution (Val⁸⁷‐Ala) was observed between deltamethrin‐resistant and susceptible strains but it is not genetically linked to resistance by the catalytic triad and signature motif analysis. The real‐time polymerase chain reaction analysis indicated that the resistant strain had a 6.61‐fold higher level of carboxylesterase mRNA than the susceptible strain. The results revealed that up‐regulation of the carboxylesterase gene, not modified gene structure, may be responsible for the development of resistance in cotton aphids to deltamethrin.     

Compared expression levels of ornithine transcarbamylase and carbamylphosphate synthetase in liver and small intestine of normal and mutant mice

Enzymatic assay, electrophoretic immunoblotting and RNA dot-blot techniques were employed to investigate the expression of the ornithine transcarbamylase (OTC) gene in liver and small intestine of Sparse fur mice with abnormal skin and hair (Spf-ash) and Sparse fur mice (Spf) which exhibit an X-linked OTC deficiency. We found a reduced OTC activity in these two tissues. We now show that this reduction is less pronounced in the intestine than in the liver of the Spf-ash strain. During the first 2 weeks of life, the deficiency appears to be less severe than in the adult mice. The enzymatic activity of carbamylphosphate synthetase I (CPS), another enzyme of the urea cycle, is significantly modified in the Spf mutant strain only.     

Saturation of transgene protein synthesis from mRNA in cells producing a large number of transgene mRNA

Experimental results have suggested that transgene expression can be saturated when large amounts of plasmid vectors are delivered into cells. To investigate this saturation kinetic behavior, cells were transfected with monitoring and competing plasmids using cationic liposomes. Even although an identical amount of a monitoring plasmid expressing firefly luciferase (FL) was used for transfection, transgene expression from the plasmid was greatly affected by the level of transgene expression from competing plasmids expressing renilla luciferase (RL). Similar results were obtained by exchanging the monitoring and competing plasmids. The competing plasmid‐dependent reduction in transgene expression from the monitoring plasmid was also observed in mouse liver after hydrodynamic injection of plasmids. On the other hand, the mRNA and protein expression level of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), an endogenous gene, in the liver hardly changed even when transgene expression process is saturated. The expression of FL from a monitoring plasmid was significantly restored by siRNA‐mediated degradation of RL mRNA that was expressed from a competing plasmid. These results suggest that the efficiency of protein synthesis from plasmid vectors is reduced when a large amount of mRNA is transcribed with no significant changes in endogenous gene expression. Biotechnol. Bioeng. 2011;108: 2380–2389. © 2011 Wiley Periodicals, Inc.     

mRNA出核转运及其偶联网络

真核细胞中,编码蛋白质基因的表达是一个复杂的、分步骤进行的过程,这个过程从转录和新生pre-mRNA的核内加工开始,经过正确加工的成熟mRNA从加工位点释放,出核转运后在细胞质内翻译成蛋白质。mRNA出核转运是基因表达中的关键步骤,由进化上高度保守的特定蛋白质介导完成。mRNA出核与转录和mRNA加工步骤密切偶联,这样的偶联可以提高基因表达的有效性和准确性。     

文蛤MmASS基因克隆及生物信息学分析

利用RACE技术克隆获得文蛤精氨酸琥珀酸合成酶基因(MmASS)的cDNA序列全长,该基因全长为1 588 bp,共编码415个氨基酸,分子量为46.81 kD,理论等电点pI为5.51。预测蛋白序列包含6个保守区域,主要集中了ATP结合位点、天门冬氨酸L-Asp结合位点以及瓜氨酸L-Cit结合位点。氨基酸序列比对结果显示,MmASS蛋白序列的保守功能域与其他物种具有较高的相似度,说明该基因高度保守,可能与其他物种的ASS基因具有相似的功能。系统进化树分析结果表明,MmASS的预测蛋白序列与缢蛏、贻贝、牡蛎等双壳贝类的亲缘关系最近,符合进化规律。亚细胞定位预测结果显示,MmASS定位于细胞质的可能性最大。MmASS不同组织的表达特征结果显示,该基因在各个组织中广泛存在,在文蛤鳃组织中的表达量最高(P<0.05),其次是肝胰腺组织,由此推测MmASS参与调节文蛤各个组织的生理活动,可能在文蛤的免疫防御机制中发挥重要功能。     

Acetoacetyl-CoA synthetase gene is abundant in rat adipose, and related with fatty acid synthesis in mature adipocytes

Acetoacetyl-CoA synthetase (AACS, acetoacetate-CoA ligase, EC 6.2.1.16) is a novel cytosolic ketone body (acetoacetate)-specific ligase, the physiological role of which remains to be elucidated. We examined the expression profiles of AACS mRNA in adult rat tissues, finding that it was particularly abundant in male subcutaneous white adipose tissue after weaning. In white adipose tissue, AACS mRNA was preferentially detected in mature adipocytes but not in preadipocytes. The AACS mRNA expression in primary preadipocytes increased during the adipocyte differentiation. These expression profiles were similar to that of acetyl-CoA carboxylase-1, but not like to that of 3-hydroxy-3-methylglutaryl-CoA reductase. These results suggest that AACS in adipose tissue plays an important role in utilizing ketone body for the fatty acid-synthesis during adipose tissue development.     

Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis

Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4–5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61–71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis.     

Purification, characterization and functional analysis of asparagines synthetase encoding by ste10 gene in Ebosin biosynthesis of Streptomyces sp. 139

Ebosin produced by Streptomyces sp. 139 is a novel exopolysaccharide (EPS) with medicinal activity. This paper describes the functional study of ste10, a putative Ebosin biosynthesis gene. ste10 was cloned and expressed in Escherichia coli BL21 and the purified recombinant protein characterized. Ste10 was shown to be able of catalyzing the transfer of amide nitrogen of glutamine to the side chain of aspartate to produce asparagine. Its Km, optimum temperature and pH were determined to be 0.9 mM, 37 °C and 7.38, respectively. After ste10 gene knock-out, the monosaccharide composition of EPS-m produced by the mutant Streptomyces sp. 139 (ste10⁻) was found changed in comparison with that of Ebosin while its antagonist activity for IL-1R decreased significantly. Based on these results, it is concluded that ste10 codes for an asparagine synthetase which may function as a modificator gene of Ebosin during its biosynthesis.     

玉米蔗糖磷酸合成酶(SPS)基因的克隆及表达载体的构建

利用RT-PCR方法从玉米幼苗叶片总RNA中克隆出玉米的蔗糖磷酸合成酶(SPS)基因的全长cDNA片段。该片段与文献报道的序列具有99%的同源性。并分别构建了以双CaMV35S为启动子,以Tnos为终止子的植物双元表达载体PBISPS和以Pcab为启动子,以T35S为终止子的植物表达载体PBSPS,其中PBISPS含有NPTⅡ选择标记基因,PBSPS不含选择标记基因。