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.     

Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line

We have recently isolated TSC-22 (transforming growth factor-beta-stimulated clone-22) cDNA as an anticancer, drug-inducible (with vesnarinone) gene in a human salivary gland cancer cell line, TYS. We have also reported that TSC-22 negatively regulates the growth of TYS cells and that down-regulation of TSC-22 in TYS cells plays a major role in salivary gland tumorigenesis (Nakashiro et al, 1998). In this study, we transfected TYS cells with an expression vector encoding the TSC-22-GFP (green fluorescent protein) fusion protein, and we established TSC-22-GFP-expressing TYS cell clones. Next, we examined (a) the subcellular localization of the fusion protein, (b) the sensitivity of the transfectants to several anticancer drugs (5-fluorouracil, cis-diaminedichloroplatinum, peplomycin), and (c) induction of apoptotic cell death in the transfectants by 5-fluorouracil treatment. The TSC-22-GFP fusion protein was clearly localized to the cytoplasm, but not to the nucleus. Over-expression of the TSC-22-GFP fusion protein did not affect cell growth, but significantly increased the sensitivity of the cells to the anticancer drugs (p < 0.01; one-way ANOVA). Furthermore, over-expression of the TSC-22-GFP fusion protein markedly enhanced 5-fluorouracil-induced apoptosis. These findings suggest that over-expression of TSC-22-GFP protein in TYS cells enhances the chemosensitivity of the cells via induction of apoptosis.     

Adult-onset type II citrullinemia and idiopathic neonatal hepatitis caused by citrin deficiency: involvement of the aspartate glutamate carrier for urea synthesis and maintenance of the urea cycle

Citrin is a mitochondrial aspartate glutamate carrier primarily expressed in the liver, heart, and kidney. We found that adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin. In this report, we describe the frequency of SLC25A13 mutations, the roles of citrin as a member of the urea cycle and as a member of the malate-aspartate shuttle, the relationship between its functions and symptoms of citrin deficiency, and therapeutic issues.     

Energy expenditure on household, childcare and occupational activities of women from urban poor households

This present study attempts to measure the energy cost of activities of women from the poor socio-economic group in India. Women in the age group of 18-40 years (n 98) either working for incomes or classified as homemakers were randomly selected. Time disposition studies were conducted by a 24 h observation of their activities on a typical day. Predominant activities were identified from the activity profiles and standardized for posture and duration. The BMR (Douglas bag method) and energy cost of the activities (Kofranyi-Michaelis meter) were measured by indirect calorimetry. The energy consumption during these activities ranged from 2.94-12.51 kJ/min. The tasks were divided into standard, household, childcare, occupational and other activities. Using the World Health Organization/Food and Agriculture Organization/United Nations University (1985) criteria, attempts were made to categorize the activities into light, moderate and heavy. It was significant that except for walking, the standard activities and occupational work could be classified into the light category (< 2.2 BMR). Most of the household and childcare activities except cooking were classified into the moderate to heavy (2.2-> 2.8 BMR). The energy expenditure of activities did not differ significantly between women with different occupations. This present study provides an important database on energy costs of activities for computing energy requirements of women involved in similar activity patterns.     

Diabetes in the Goto-Kakizaki rat is accompanied by impaired insulin-mediated myosin-bound phosphatase activation and vascular smooth muscle cell relaxation

Our laboratory has demonstrated that insulin rapidly stimulates myosin-bound phosphatase (MBP) activity in vascular smooth muscle cells (VSMCs). In this study, we examined whether diabetes is accompanied by alterations in MBP activation and elucidated the components of the signaling pathway that mediate the effects of diabetes. VSMCs isolated from Goto-Kakizaki (GK) diabetic rats (a model for type 2 diabetes) exhibited marked impairment in MBP activation by insulin that was accompanied by failure of insulin to decrease the phosphorylation of a regulatory myosin-bound subunit (MBS) of MBP and inhibit Rho kinase activity resulting in increased myosin light-chain (MLC)20 phosphorylation and VSMC contraction. In VSMCs isolated from control rats, insulin inactivates Rho kinase and decreases MBS phosphorylation, leading to MBP activation. In addition to this pathway, insulin also appears to activate MBP by stimulating the phosphatidylinositol (PI) 3-kinase/nitric oxide (NO)/cGMP signaling pathway because treatment with the synthetic inhibitors of PI 3-kinase, NO synthase (NOS), and cGMP all blocked insulin's effect on MBP activation, whereas cGMP agonists and sodium nitroprusside (SNP) mimicked insulin's effect on MBP activation. VSMCs from diabetic GK rats exhibit reductions in insulin-mediated induction of inducible NOS protein expression and cGMP generation but normal MBP activation in response to SNP and cGMP agonist. This observation led us to examine the effect of diabetes on the activation status of the upstream insulin-signaling components. Although GK diabetes did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor or its content, insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation was severely impaired. This was accompanied by marked reductions in IRS-1-associated PI 3-kinase activity. We conclude that insulin stimulates MBP via its regulatory subunit, MBS, by inactivating Rho kinase and stimulating NO/cGMP signaling via PI 3-kinase as part of a complex signaling network that controls MLC20 phosphorylation and VSMC contraction. Defective signaling via Rho kinase and the IRS-1/PI 3-kinase/NOS/cGMP pathway may mediate the inhibitory effects of hyperglycemia and diabetes on MBP activation in this experimental model.     

Quantitative methylation-specific polymerase chain reaction gene patterns in urine sediment distinguish prostate cancer patients from control subjects.

PURPOSE: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of prostate cancers and is a promising marker for cancer detection. We sought to develop a test for prostate cancer based on a quantitative methylation-specific polymerase chain reaction (QMSP) of multiple genes in urine sediment DNA. PATIENTS AND METHODS: We tested urine sediment DNA for aberrant methylation of nine gene promoters (p16INK4a, p14(ARF), MGMT, GSTP1, RARbeta2, CDH1 [E-cadherin], TIMP3, Rassf1A, and APC) from 52 patients with prostate cancer and 21 matched primary tumors by quantitative fluorogenic real-time polymerase chain reaction. We also analyzed urine sediments from 91 age-matched individuals without any history of genitourinary malignancy as controls. RESULTS: Promoter hypermethylation of at least one of the genes studied was detected in urine samples from all 52 prostate cancer patients. Urine samples from the 91 controls without evidence of genitourinary cancer revealed no methylation of the p16, ARF, MGMT, and GSTP1 gene promoters, whereas methylation of RARbeta2, TIMP3, CDH1, Rassf1A, and APC was detected at low levels. CONCLUSION: Overall, methylation found in urine samples matched the methylation status in the primary tumor. A combination of only four genes (p16, ARF, MGMT, and GSTP1) would theoretically allow us to detect 87% of prostate cancers with 100% specificity. Our data support further development of the noninvasive QMSP assay in urine DNA for early detection and surveillance of prostate cancer.     

Allogeneic stem-cell transplantation using a reduced-intensity conditioning regimen has the capacity to produce durable remissions and long-term disease-free survival in patients with high-risk acute myeloid leukemia and myelodysplasia.

PURPOSE: The toxicity of allogeneic stem-cell transplantation can be substantially reduced using a reduced-intensity conditioning (RIC) regimen. This has increased the proportion of patients with myeloid malignancies eligible for allogeneic transplantation. However, the capacity of RIC allografts to produce durable remissions in patients with acute myeloid leukemia (AML) and myelodysplasia (MDS) has not yet been defined, and consequently, the role of RIC allografts in the management of these diseases remains conjectural. PATIENTS AND METHODS: Seventy-six patients with high-risk AML or MDS received an allograft using a fludarabine/melphalan RIC regimen incorporating alemtuzumab. The median age of the cohort was 52 years (range, 18 to 71 years). RESULTS: The 100-day transplantation-related mortality rate was 9%, and no patient developed greater than grade 2 graft-versus-host disease. With a median follow-up of 36 months (range, 13 to 70 months), 27 patients were alive and in remission, with 3-year actuarial overall survival (OS) and disease-free survival (DFS) rates of 41% and 37%, respectively. The 3-year OS and DFS rates of patients with AML in complete remission at the time of transplantation were 48% and 42%, respectively. Disease relapse was the most common cause of treatment failure and occurred at a median time of 6 months after transplantation. All but one patient destined to relapse did so within 24 months of transplantation. CONCLUSION: The extended follow-up in this series identifies a high risk of early disease relapse but provides evidence that RIC allografts can produce sustained DFS in a significant number of patients with AML who would be ineligible for allogeneic transplantation with myeloablative conditioning.