Human T lymphotrophic virus type I may not be associated with multiple sclerosis in Japan.

To study the possible involvement of human T lymphotrophic virus type I (HTLV-I) or a related retrovirus in Japanese cases of multiple sclerosis (MS), we first performed a Western blot analysis with purified Ag of HTLV-I. Ten out of 31 MS patients (32.2%), 19 of 66 patients (28.8%) with other neurologic diseases, and 2 of 64 healthy blood donors (3.1%) had antibodies reactive with Ag corresponding to the group-specific Ag (gag) proteins (p15, p19, p24) on their sera. There were no significant differences between MS and other neurologic diseases concerning the patterns and the frequency. Second, we tried to establish T cell lines from PBMC of 22 MS patients with crude IL-2 without accessory cells, because HTLV-I-infected T cells can be immortalized in a high ratio under those conditions. Only one T cell line (MS-14C), however, could be maintained in long term culture. MS-14C and cultured T cells for 3 to 5 wk derived from MS patients were examined by Southern blot analysis under both stringent and low stringent conditions with HTLV-I as a probe. No HTLV-I related bands could be detected. By polymerase chain reaction examination, we also could not detect HTLV-I provirus genome in the fresh PBMC from 20 MS patients, although some of them had gag-reactive antibodies. Our data do not favor the hypothesis of HTLV-I or an HTLV-I-related human retrovirus in the etiology of MS.     

Metabolism ofl-cysteine via transamination pathway (3-mercaptopyruvate pathway)

Summary We have studied the transamination pathway (3-mercaptopyruvate pathway) ofl-cysteine metabolism in rats. Characterization of cysteine aminotransferase (EC 2.6.1.3) from liver indicated that the transamination, the first reaction of this pathway, was catalyzed by aspartate aminotransferase (EC 2.6.1.1). 3-Mercaptopyruvate, the product of the transamination, may be metabolized through two routes. The initial reactions of these routes are reduction and transsulfuration, and the final metabolites are 3-mercaptolactate-cysteine mixed disulfide [S-(2-hydroxy-2-carboxyethylthio)cysteine, HCETC] and inorganic sulfate, respectively. The study using anti-lactate dehydrogenase antiserum proved that the enzyme catalyzing the reduction of 3-mercaptopyruvate was lactate dehydrogenase (EC 1.1.1.27). Formation of HCETC was shown to depend on low 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) activity. Results were discussed in relation to HCETC excretion in normal human subjects and patients with 3-mercaptolactate-cysteine disulfiduria. Incubation of liver mitochondria withl-cysteine, 2-oxoglutarate and glutathione resulted in the formation of sulfate and thiosulfate, indicating that thiosulfate was formed by transsulfuration of 3-mercaptopyruvate and finally metabolized to sulfate.     

SLX4–XPF mediates DNA damage responses to replication stress induced by DNA–protein interactions

The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA–protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4–XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4–ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.     

Genetic and chemical analysis of a key biosynthetic step for soyasapogenol A, an aglycone of group A saponins that influence soymilk flavor

Although certain saponins in soybean seeds have been reported to have health benefits, group A acetyl saponins cause undesirable bitter and astringent tastes in soy products. Therefore, reduction or elimination of group A saponins is an important target for soybean breeders. A wide survey of cultivated and wild soybean germplasm identified a mutant line that lacked group A saponins. The absence of soyasapogenol A, a group A saponin aglycone, is controlled by a single recessive allele, sg-5 that mapped genetically near the SSR marker, Satt117, on soybean chromosome 15 (linkage group E). The locus is epistatic to Sg-1, which controls the terminal sugar variation on the C-22 sugar chain of soyasapogenol A, and allelic differences at this locus lead to changes in the amount of DDMP saponins and their derivatives group B and E products. These findings provide a new insight into the biosynthetic pathway of soybean saponins, and identify a genetic approach that can be applied to improve the quality of foods produced from soybean.     

Differential response of methionine metabolism in two grain legumes,soybean and azuki bean,expressing a mutated form of Arabidopsis cystathionine γ-synthase

Methionine (Met) is a sulfur-containing amino acid that is essential in mammals and whose low abundance limits the nutritional value of grain legumes. Cystathionine γ-synthase (CGS) catalyzes the first committed step of Met biosynthesis, and the stability of its mRNA is autoregulated by the cytosolic concentration of S-adenosyl-l-methionine (SAM), a direct metabolite of Met. The mto1-1 mutant of Arabidopsis thaliana harbors a mutation in the AtCGS1 gene that renders the mRNA resistant to SAM-dependent degradation and therefore results in the accumulation of free Met to high levels in young leaves. To manipulate Met biosynthesis in soybean and azuki bean, we introduced the AtCGS1 mto1-1 gene into the two grain legumes under the control of a seed-specific glycinin gene promoter. Transgenic seeds of both species accumulated soluble Met to levels at least twice those apparent in control seeds. However, the increase in free Met did not result in an increase in total Met content of the transgenic seeds. In transgenic azuki bean seeds, the amount of cystathionine, the direct product of CGS, was markedly increased whereas the total content of Met was significantly decreased compared with control seeds. Similar changes were not detected in soybean. Our data suggest that the regulation of Met biosynthesis differs between soybean and azuki bean, and that the expression of AtCGS1 mto1-1 differentially affects the metabolic stability of sulfur amino acids and their metabolites in the two grain legumes.     

IEX-1 suppresses apoptotic damage in human intestinal epithelial Caco-2 cells induced by co-culturing with macrophage-like THP-1 cells

We have reported previously that apoptosis of intestinal epithelial Caco-2 cells is induced by co-culturing with human macrophage-like THP-1 cells, mainly via the action of TNFα (tumour necrosis factor α) secreted from THP-1 cells [Satsu, Ishimoto, Nakano, Mochizuki, Iwanaga and Shimizu (2006) Exp. Cell Res. 312, 3909-3919]. Our recent DNA microarray analysis of co-cultured Caco-2 cells showed that IEX-1 (immediate early-response gene X-1) is the most significantly increased gene during co-culture [Ishimoto, Nakai, Satsu, Totsuka and Shimizu (2010) Biosci. Biotechnol. Biochem. 74, 437-439]. Hence, we investigated the role of IEX-1 in the co-culture-induced damage of Caco-2 cells. We showed that IEX-1 expression induced in Caco-2 cells was suppressed by anti-TNFα antibody treatment. Experiments using IEX-1-overexpressing and -knockdown Caco-2 cells suggested that IEX-1 was involved in the suppression of Caco-2 cell damage. Increases in caspase 3 activity and TNFR1 (TNF receptor 1) mRNA expression were shown in IEX-1-knockdown Caco-2 cells, suggesting that IEX-1 plays a role in the suppression of apoptosis and protects cells by controlling sensitivity to TNFα under both normal and inflammatory conditions.