Cleavage activity of the sapovirus 3C-like protease in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Summary. We recently determined the ORF1 cleavage map of Mc10, a human sapovirus (SaV) strain, as follows: NH₂–p11-p28-p35(NTPase)-p32-p14(VPg)-p70(Pro-Pol)-p60(VP1)–COOH. This cleavage was dependent on the viral encoded 3C-like protease. To identify the cleavage site of SaV ORF1, putative p70 (Pro-Pol) and p14-p70 (VPg-Pro-Pol) were expressed as N-terminal GST and C-terminal 6 × His-tag fusion proteins in Escherichia coli, and the expressed products were analyzed by SDS-PAGE and Western blotting. Our results indicated that the efficient proteolytic cleavage occurred between p14 (VPg) and p70 (Pro-Pol), and N-terminal amino acid sequencing revealed that the cleavage site was between E¹⁰⁵⁵ and A¹⁰⁵⁶. In contrast, the p70 (Pro-Pol) was not further cleaved. We also found that SaV protease cleaved the Q/G site within the rhinovirus 3C protease recognition site. Site-directed mutagenesis in a conserved GDCG motif of the protease completely abolished these proteolytic activities. This is the first report to identify the cleavage site of the SaV ORF1 polyprotein.     

A copper chelating agent suppresses carbonyl stress in diabetic rat lenses

To clarify whether transition metals are involved in carbonyl stress in diabetic tissues, we observed the effects of a metal chelating agent, trientine (TE) hydrochloride on the levels of methylglyoxal (MG), 3-deoxyglucosone (3-DG), advanced glycation end products, 8-hydroxy-2′-deoxyguanosine (8-OHdG), and polyol pathway metabolites along with semicarbazide-sensitive amine oxidase (SSAO) enzyme activity in lenses from streptozotocin-induced diabetic rats. Lens MG and 3-DG levels were significantly higher in diabetic rats than nondiabetic controls, and TE significantly restored the increase of these compounds. Lens argpyrimidine was also increased in diabetic rats as compared with controls and was significantly reduced by TE. Lens SSAO activity and 8-OHdG were also significantly elevated in diabetic rats, and TE suppressed both of them, whereas TE showed no effect on the polyol pathway metabolites. The results indicate that transition metals play a significant role in the formation of MG and 3-DG via oxidative stress and SSAO activity.     

Studies on non-surgical therapy of tubal pregnancy

8 cases of tubal pregnancy in the pre-abortional stage were detected by B-scope examination. The administration of MTX (total doses: 60-300mg) resulted in complete remission in 7 cases; the other received tubectomy because of the progression of abortion. The dose of MTX used to obtain remission is responsible for the HCG titer of urine before the treatment; 60-150mg of MTX was sufficient in cases which showed a titer below 1,000iu/l, although 75-300mg was necessary in cases over 4,000iu/l. The patency of tubes after the regimen was confirmed in 4 out of 5 cases with hystero-salpingography. Serious side effects were not observed in the present study in spite of the great efficacy; transient suppression of liver has been observed in 2 cases. Two patients had normal intra-uterine pregnancy subsequent to the treatment; one had a matured full-term female neonate and the other received D & C in 9 gestational weeks. These results suggest that the MTX regimen is practical for the treatment of patients with tubal pregnancy. This is characterized by unimpaired fertility following the treatment, although the detection of patients in the pre-abortional stage is absolutely necessary.