Utilization of oryzacystatin for regulating the ripening of squid shiokara, a traditional Japanese salted and fermented seafood.

Shiokara is a fermented seafood composed of sliced squid mantle muscle ripened with fresh squid liver. Preliminary sensory evaluation by using the ranking test revealed that the hardness of squid muscle in shiokara was reduced within 7 d of ripening. During the process of ripening, muscle proteins were digested by proteinases present in squid liver. The degradation of paramyosin and myosin heavy chain was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hardness of squid mantle muscle in shiokara was reduced with the degradation of paramyosin and myosin heavy chain. This degradation was mainly caused by E-64-sensitive cysteine proteinases. To control the hardness of shiokara, we used rice seed oryzacystatin, which suppresses proteolysis by papain-like cysteine proteinases. When oryzacystatin was added 4 d after the start of shiokara ripening, the muscle protein degradation stopped, without further muscle softening. These results show that oryzacystatin is useful to control the ripening of shiokara by regulating its hardness.

The plant aspartic proteinase-specific polypeptide insert is not directly related to the activity of oryzasin 1.

Many plant aspartic proteinases (APs) are different from animal and microbial APs in that they contain a polypeptide insert, approximately 100 amino acids in length, in the C-terminal region. To interpret the significance of this insert, we constructed an expression system for rice AP oryzasin 1 by linking a pro-oryzasin 1 downstream of glutathione S-transferase (GST). GST-proOS1 expressed the highest degree of hemoglobin-hydrolytic activity when treated at pH 3.3 and incubated for 24 h at room temperature. We carried out a similar experiment using an insert-lacking proOS1 mutant, GST-DeltaproOS1, as the fusion protein, and found it to show similar activity. This result indicates that the insert is not involved in the production of AP activity. We then investigated the autolysis of the two proteins by Western blot analysis. GST-proOS1 was autolyzed into 67- and 64-kDa fragments, while GST-DeltaproOS1 autolyzed to 54- and 52-kDa products. GST-DeltaproOS1 clearly produced two molecular species early in the autolytic process, and not later than 3 h from the start, but no such clear result was observed in the case of GST-proOS1. This suggests that, although the presence of the plant AP-specific insert does not influence the enzyme activity by itself, it apparently has an effect on the autolysis of OS1.

Increased expression of matrix metalloproteinase-II in experimental liver fibrosis in rats.

Matrix metalloproteinase-II (MMP-II, 72-kd type IV collagenase, or gelatinase) is one of the gene families of zinc enzymes capable of degrading extracellular matrix molecules, and specifically of degrading type IV and V collagens, gelatin, fibronectin, and elastin. In this study, we used both the liver fibrosis model and the reversibility model of experimental cirrhosis to clarify how MMP-II participates in liver fibrosis of rats. To produce fibrosis model, rats received subcutaneous injections of CCl4 twice weekly for 7, 9, or 14 weeks. For the reversibility model, rats were treated with CCl4 three times a week for 8 weeks and killed at 3, 7, 14, 28, or 42 days after discontinuation of treatment. MMP-II gene expression was studied by Northern hybridization technique, and gelatinase activity of MMP-II was examined by zymography using gelatin substrate. At the same time, an immunohistochemical study using anti-type IV collagen antibody was carried out. In liver fibrosis model, nodule formation was established at 14 weeks. Immunodeposit of type IV collagen was increased in wide fibrous septa and was clearly observed along sinusoidal wall. Gene expression of MMP-II increased up to 7 to 12 times compared with that of controls, with the expression rate being maximum at an intermediate stage of fibrosis. Zymography showed the expressions of both 65-kd latent MMP-II, which is confirmed to be activated by adding p-aminophenylmercuric acetate, and 62-kd active MMP-II during fibrosis. The expression of both forms increased 13 to 28 times as the fibrosis progressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of monkey liver cytochrome P-450 cDNAs: similarity of the primary sequences to human cytochromes P-450.

Three cDNAs coding for monkey cytochrome P-450 (P450) 2C, 2E and 3A (MKmp13, MKj1 and MKnf2, respectively) were isolated from a lambda gt11 cDNA library of a liver from a 3-methylcholanthrene (3MC)-treated crab-eating monkey, using cDNA fragments for human P450 2C, 2E and 3A as respective probes. MKmp13 and MKnf2 were 1901 and 2032 bp long, containing entire coding regions for polypeptides of 490 and 503 residues, respectively. The deduced N-terminal amino acid sequences of MKmp13 and MKnf2 were identical with those of P450-MK1 and P450-MK2, which had been purified from liver microsomes of untreated and polychlorinated biphenyl (PCB)-treated crab-eating monkeys, respectively. MKj1 was 1508 bp long, encoding a polypeptide of 449 residues, which is presumed to lack N-terminal 45 residues as compared with the sequence for human P450 2E1. Northern blot analysis indicated that monkey P450 2C, 2E and 3A mRNAs were expressed constitutively in monkey livers. P450 2E and 3A mRNAs were induced by both 3MC and PCB, while P450 2C mRNA was induced only by PCB. The deduced amino acid sequences of four monkey cytochrome P-450 cDNAs, including P450 1A1 (MKah1) which we isolated previously, were more than 92% identical with those of corresponding human cytochrome P-450 cDNAs.

Duodenal mucosal hemodynamics in patients with liver cirrhosis.

Clinical studies show that patients with liver cirrhosis associated with portal hypertension have a high incidence of duodenal ulcer and duodenitis. However, little information is available concerning pathophysiological process of such duodenal diseases in liver cirrhosis. Hemodynamics of the duodenal mucosa was studied in cirrhotics with esophageal varices (68 cases) and in noncirrhotics with non-ulcer dyspepsia (37 cases) as well. In each group, hemoglobin concentration in the peripheral venous blood was measured, and mucosal hemodynamics was examined in 4 regions of the duodenum by endoscopic reflectance spectrophotometer. No significant intergroup difference was noted in the mean age or sex ratio. Hemoglobin concentration in the peripheral venous blood was significantly lower (p less than 0.01) in the cirrhotics. There were no significant intergroup differences in duodenal mucosal blood volume. However, the cirrhotics showed significantly lower oxygen saturation of hemoglobin in all regions of the duodenum (p less than 0.01). These results show that the cirrhotics with esophageal varices had relative increase in blood volume and decrease in oxygen saturation of hemoglobin in the duodenal mucosa. Such microcirculatory disturbances seem to predispose liver cirrhosis patients to duodenal injury.

Growth of condylar, epiphyseal and spheno-occipital synchrondrosal cartilages cultured on medium BGJ.

The purpose of this experiment was to examine the growth of condylar, epiphyseal, and spheno-occipital synchrondrosal cartilages cultured on medium BGJ. Materials from 12 neonatal rabbits were cultured for 7 days on this medium with the addition of 0, 10, or 20% fetal calf serum. Epiphyseal cartilages cultured with the addition of 20% fetal calf serum showed slightly better maintenance of in situ state, though little morphological changes were observed during 7 days. Spheno-occipital synchondrosal cartilages cultured with the addition of 10% or 20% fetal calf serum were histologically relatively similar to in situ state than those cultured without fetal calf serum, but no growth in length and width of these cartilages was observed. On the other hand, cultured condylar cartilages showed the least maintenance of histological and histochemical features. It was assumed that medium BGJ was not a recommendable medium for the organ culture of condylar cartilage.