Expression of human argininosuccinate synthetase after retroviral-mediated gene transfer

The cDNA sequence for human argininosuccinate synthetase (AS) was introduced into plasmid expression vectors with an SV40 promoter or Rous sarcoma virus promoter to construct pSV2-AS and pRSV-AS, respectively, and human enzyme was synthesized after gene transfer into Chinese hamster cells. The functional cDNA was inserted into the retroviral vectors pZIP-NeoSV(X) and pZIP-NeoSV(B). Ecotropic AS retrovirus was produced after calcium-phosphate-mediated gene transfer of these constructions into the packaging cell line -2, and viral liters up to 10⁵ CFU/ml were obtained. Recombinant AS retrovirus was evaluated by detecting G-418-resistant colonies after infection of the rodent cells, XC, NRK, and 3T3. Colonies were also obtained when infected XC cells were selected in citrulline medium for expression of AS activity. Southern blot analysis of infected cells demonstrated that the recombinant retroviral genome was not altered grossly after infecting some rodent cells, while other cells showed evidence of rearrangement. A rapid assay for detecting AS retrovirus was developed based on the incorporation of [¹⁴C]citrulline into protein by intact 3T3 cells or XC cells.     

Control of argininosuccinate synthetase by arginine in human lymphoblasts

Human lymphoblasts in long-term culture have the enzyme activities necessary to convert citrulline to arginine: argininosuccinate synthetase and argininosuccinate lyase. Upon transfer from arginine-supplemented to citrulline-supplemented medium, lymphoblasts exhibit a lag period before resuming exponential growth. During this lag the specific activity of argininosuccinate synthetase increases an average of 60-fold. Argininosuccinate lyase activity remains unchanged. If normal lymphoblasts are starved in arginine-deficient medium without citrulline or if argininosuccinate lyase-deficient lymphoblasts are transferred to citrulline-containing medium, argininosuccinate synthetase activity increases linearly for several days and reaches even higher levels. Cycloheximide blocks the increase in enzyme activity. Cells grown in citrulline medium and pulse labeled with³⁵S-methionine incorporate more³⁵S-methionine into argininosuccinate synthetase protein than cells grown in arginine; the rate of disappearance of radioactively labeled enzyme is the same in citrulline- and arginine-grown cells. Arginine or a closely related metabolite thus appears to repress the synthesis of argininosuccinate synthetase of human lymphoblasts in culture.     

Mutations and polymorphisms in the human argininosuccinate synthetase (ASS1) gene

Citrullinemia type I is an autosomal recessive disorder that is caused by a deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS1). Deficiency of ASS1 shows various clinical manifestations encompassing severely affected patients with fatal neonatal hyperammonemia as well as asymptomatic individuals with only a biochemical phenotype. This is a comprehensive report of all 87 mutations found to date in the ASS1 gene on chromosome 9q34.1. A large proportion of the mutations (n=27) are described here for the first time. Mutations are distributed throughout exons 3 to 15, most of them being identified in exons 5, 12, 13, and 14. The mutation G390R in exon 15 is the single most common mutation in patients with the classical phenotype. Certain mutations clearly link to specific clinical courses but the clinical phenotype cannot be anticipated in all patients. This update presents a survey of the correlation between mutations in the ASS1 gene and the respective clinical courses as described so far. It also sheds light on the geographic incidence of the mutations. Enzymatic studies have been done in bacterial and human cell systems. However, the prognostic value of genetic aberrations with respect to their effect on protein function and clinical manifestation remains uncertain. Hum Mutat 0, 1–8, 2008. © 2008 Wiley‐Liss, Inc.     

Expression of human argininosuccinate synthetase in murine hematopoietic cells in vivo

As part of an effort to develop a model system for somatic gene therapy, a human argininosuccinate synthetase (AS) cDNA has been introduced into two gag⁺ retroviral vectors, and high titer clones were obtained for both constructs. The presence of proviral DNA sequences was detected in individual spleen colonies after infection of primary murine marrow cells with each virus. Mice were reconstituted for long-term survival with marrow infected with one virus, and they demonstrated elevated levels of AS expression in peripheral blood for up to eight weeks posttransplantation.     

Generation of a mouse model for citrullinemia by targeted disruption of the argininosuccinate synthetase gene

Argininosuccinate synthetase (ASS) is a urea cycle enzyme that forms argininosuccinate from citrulline and aspartate. Mutations at the ASS locus in man cause the inherited disease, citrullinemia. Citrullinemia is inherited as an autosomal recessive trait and is characterized, biochemically, by elevated levels of blood citrulline and ammonia and often results in early neonatal death if untreated. We have used homologous recombination in embryonic stem cells to generate a line of mice having a targeted disruption of theAss gene. Homozygous mutant animals develop high levels of blood citrulline, become hyperammonemic, and die within one or two days after birth. Because the phenotype of the mutant mice closely resembles that of humans who lack the ASS enzyme, we expect that these mice will serve as a useful model for exploring new treatments for citrullinemia including somatic gene therapy.     

锂诱导肿瘤细胞凋亡及其机理初探

研究微量元素锂在体外对肿瘤细胞系凋亡的诱导作用。方法在细胞培养体系中加入ＬｉＣｌ进行诱导，以流式细胞分析，ＤＮＡ电流分析及细胞形态变为指标检测凋亡，并用ｍＲＮＡ狭缝杂交和Ｗｅｓｔｅｒｎｂｌｏｔ测定凋亡相关基因的改变。加入大分子合成抑制或第二信使相关药物观察对锂诱凋亡的影响。     

Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.     

Elevated gene expression of argininosuccinate synthetase in peripheral lymphocytes from systemic lupus erythematosus (SLE) patients.

Argininosuccinate synthetase (ASS) is a rate-limiting enzyme of urea cycle and functions primarily in the liver, whereas ASS activity is hardly detected in normal lymphocytes. In this study, we examined the level of ASS gene expression in peripheral blood lymphocytes (PBL) from human SLE patients by amplification of reverse-transcribed mRNA using the polymerase chain reaction. We have demonstrated that (a) approximately 40% of SLE patients exhibited 2.5 to 5 times higher expression of ASS gene in PBL than those of healthy PBL and (b) the elevation of ASS gene expression of PBL significantly correlates with the active pathogenesis of SLE patients according to the criteria of Japanese Ministry of Health and Welfare (p < 0.001 by student's two-tailed t-test). Thus, it is suggested that ASS gene expression is a promising marker of hyperactivated lymphocytes uniquely generated in patients with systemic autoimmune disease.     

人核受体hLRH-1不同变异体在多种肿瘤组织和细胞系中的表达

目的:初步分析人核受体hLRH-1不同变异体在多种肿瘤组织和细胞系中的表达。方法:常规RT-PCR法分析hLRH-1v1和hLRH-1在肝癌等多种肿瘤组织和HepG2等肿瘤细胞系中的表达情况，实时定量PCR法分析hLRH-1v1，hLRH-1和hOct4在HepG2，SMMC-7721和BEL-7402三株肝癌细胞系的表达水平。结果:hLRH-1v1的表达见于所有检测的12种类型肿瘤和8种恶性肿瘤细胞系，hLRH-1的表达则仅在12种类型肿瘤中的6种肿瘤组织中可检测到，但8种恶性肿瘤细胞系均为hLRH-1阳性表达；在HepG2，SMMC-7721和BEL-7402三株肝癌细胞系中hLRH-1v1和hOct4的表达呈正相关。结论:hLRH-1v1在肿瘤中广泛表达，可能在维持肿瘤干细胞的自我更新中发挥重要作用。     

On the genetic mechanism of induction of CD3gamma-negative human T cell variants

Invariant CD3gamma molecules are important components in TCR complex formation and function. Both CD3gamma alleles seem to be expressed co-dominantly. In the present report, we present experimental data which indicate that the induction of CD3gamma(-) Jurkat variants occurs with a frequency similar to that of TCRalpha(-) or TCRbeta(-) Jurkat cells. CD3delta(-), CD3epsilon(-) or CD3zeta(-) Jurkat variants were never obtained despite extensive efforts. Our data suggest a possible explanation for this genetic puzzle: the human CD3gamma gene has a mutational hot spot in a nucleotide sequence of nine adenosines (9A) in the exon 3 encoding most of the external CD3gamma domain. Thus, both CD3gamma alleles are easily mutated to either 8A or 10A sequences. Furthermore, absence of CD3gamma molecules in Jurkat T cells causes severe defects in TCR / CD3 assembly and function.     

神经酰胺介导粒细胞凋亡机理初探

目的 观察神经酰胺对ＨＬ－６０及中性粒细胞（ＰＭＮ）凋亡产生的影响,探讨其在粒细胞发生过程中可能的调节作用。方法：采用ＤＮＡ凝胶电泳,流式细胞术,光镜及ＲＴ－ＰＣＲ等技术对细胞进行凋亡鉴定及相关基因ｂｃｌ－２,ｃ－ｍｙｃ,ｍＲＮＡ表达分析。结果 神经酰胺（ｃ２－ｃｅｒａｍｉｄｅ）肿瘤坏死因子（ＴＮＦα）及长春新碱（Ｖｉｎｃｒｉｓｔｉｎｅ）均可不同程度地诱导ＨＬ－６０及成熟ＰＭＮ产生凋亡,ＣＰＫ激活     

Induction ofSpodoptera littoralis nuclear polyhedrosis virus in cell cultures

Summary A nuclear polyhedrosis virus (NPV) infection was initiated in aSpodoptera littoralis cell line when haemocytes from granulosis virus (GV)-infectedS. littoralis larvae and ultra-violet-inactivated Sendai virus were added.With 1 Figure     

Regulation of human natural killer (NK) cell function: Induction of killing of an NK-resistant renal carcinoma cell line

Natural killer (NK)-like activity against a renal carcinoma cell line, Cur, was assessed. There was no spontaneous killing of Cur cells by human peripheral blood mononuclear cells in 4-hr assays. Cur killing was observed in 18-hr assays, but the magnitude of killing was variable and always markedly less than that against K562. Cur killing was mediated by a nonadherent, nonphagocytic lymphocyte, the activity of which could be modulated both positively and negatively by monocytes or their products. Preincubation of effectors with monocyte supernatant, interleukin 1 (IL-1), -interferon (IFN), or interleukin 2 (IL-2) greatly increased the magnitude of Cur killing and accelerated the kinetics of lysis. The addition of prostaglandin E₂ (PGE₂) duringin vitro activation of NK by IL-2 profoundly inhibited subsequent Cur lysis, whereas only minimal inhibition of K562 lysis was noted. However, following activation with IL-2, lysis of Cur targets was less sensitive to the inhibitory effects of PGE₂. Removal of Leu 11b(+), OKM1(+), orl-leucylleucine methyl ester-sensitive cells markedly decreased both Cur and K562 lysis. Moreover, CD16(+) cells purified with the fluorescence-activated cell sorter were found to mediate Cur killing. Whereas Cur and K562 lysis is mediated by phenotypically similar effector cells, the present studies demonstrate that the cytotoxic functions defined by the ability to lyse these two targets differ in response to a variety of immunoregulatory stimuli.     

Induction of nuclear aberrations by smokeless tobacco in epithelial cells of human oral mucosa

Cytologic and cytogenetic studies were performed to assess the prevalence of somatic cell genetic damage in 48 young adults equally divided to represent users and nonusers of smokeless tobacco. Exposure was ascertained by measuring saliva cotinine using capillary gas chromatography. Squamous epithelial cells sampled from the oral mucosa demonstrated significant cytologic alterations associated with tobacco exposure. The frequency of micronucleated cells was significantly (P less than .01) higher in the labial mucosa of exposed (2.22%) compared to unexposed (0.27%) individuals. The frequency of micronuclei varied widely between exposed subjects but was higher in heavily (2.48%) compared to lightly (1.29%) exposed individuals as measured by saliva cotinine levels. Morphologic classification of epithelial cell nuclei showed that the frequency of cells with normal nuclear structure was significantly (P less than .01) reduce in exposed individuals. Analysis of oral epithelial cells of five additional nonusers of smokeless tobacco but wearers of orthodontic appliances to stimulate abrasion demonstrated no difference from the nonexposed control group. Unlike the case with cigarette smokers, peripheral lymphocyte sister-chromatid exchange frequency was not affected by exposure to smokeless tobacco. The oral cytology data, however, support an interpretation of exposure-dependent nuclear alterations, including micronuclei, in the oral epithelium associated with the use of smokeless tobacco. Altogether, results suggest that use of smokeless tobacco may cause genetic damage to cells in the oral epithelium.