Studies on Proteinases from Gliocladium roseum

The alkaline proteinases of Gliocladium roseum (Link) Bainier were purified in crystalline forms by procedures of alcoholic precipitation, fuller’s earth- and acrinol-treatment, and isolated in two types. (Proteinases I and II). Both of these proteinases were homogeneous on zone electrophoresis with polyacrylamide gel (Gyanogum 41), and had the optimal pH values of 11 (Proteinase I) and 10 (Proteinase II), and the optimal temperature of 45°C.

The enzymatic reaction of proteinase I was remarkably promoted by Fe⁺⁺ and Co⁺⁺, and that of proteinase II was promoted by Fe⁺⁺, Go⁺⁺ and Ca⁺⁺, and both proteinases were protected from heat-inactivation by Ca⁺⁺ Proteinase II was activated remarkably by Cl^? under the existence of Fe⁺⁺, but proteinase I was unaffected by the anion.

The order of strength of proteolytic power of these proteinases and chymotrypsin on casein was as follows; proteinase I> proteinase II> chymotrypsin.     

Studies on Components of Mammalian Urine

Hexahydrohippuric acid was detected from the urine of cattles together with hippuric and phenaceturic acids as one of the conjugated compounds with glycine. Furthermore, cyclohexanecarboxylic acid was also detected. These experimental results suggest the interesting synthetic processes in vivo or in rumen of the cattle, though both acids have not determined whether they are metabolites of cattle or synthesized by miccroorganisms in rumen.     

Purification and Characterization of an Opioid Antagonist from a Peptic Digest of Bovine κ-Casein

The hepatotoxicity of orally administered secondary autoxidation products of linoleic acid was investigated, as compared with the administration of a saline solution and linoleic acid as controls. The de novo synthesis of fatty acids was strongly reduced in the secondary products group. The level of NADPH in the liver significantly decreased while that of NADH did not. The activities of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase apparently decreased. The activities of NAD⁺ kinase and NAD⁺ synthetase decreased and that of NAD ⁺ nucleosidase increased in the secondary products group. Therefore, the depletion of NADPH can be attributed to the inhibition of two metabolic systems (an NADPH-supplemental system, and a synthetic system of NADP and NAD), and resulted in the reduction of lipogenesis in the liver.     

Detailed Structure of an Acidic Polysaccharide in Soy Sauce,Confirmed by Use of Two Kinds of Purified Pectinases

The partially degraded polysaccharide obtained by means of mild acid hydrolysis of APS–I, an acidic polysaccharide in soy sauce, was incubated with an endo-polygalacturonase, and some acidic sugars liberated were fractionated and purified from the enzymatic hydrolyzate. They were identified as d-galacturonic acid (Gal A), its α-l,4-linked dimer and trimer, d-xylose (Xyl) β1→3 Gal A and Xyl β1→3 Gal A α1→4 Gal A, and an acidic polymer composed of the above-mentioned sugars through methylation analysis and other methods.

Methyl-esterized APS–I was subjected to β-elimination with pectin lyase or by heat treatment. The high molecular fraction which was highly resistant to β-elimination, was concluded to be β-1,4-linked d-galactan of which reducing-end group was attached to d-galacturonic acid. On the basis of these findings and previous knowledges, the construction of APS–I was also discussed.     

Euryspongins A–C,three new unique sesquiterpenes from a marine sponge Euryspongia sp.

Three new unique sesquiterpenes, euryspongins A–C (1–3), were isolated from a marine sponge Euryspongia sp. collected at Iriomote Island, Okinawa, Japan. Compound 1 possessed a bicyclic furanosesquiterpene structure with six- and eight-membered rings, whereas compounds 2 and 3 had an α,β-unsaturated-γ-lactone ring instead of the furan ring in 1. Only five natural products in this class have been reported, and compounds 1–3 are the sixth–eighth examples of natural products. Compounds 1–3 had no inhibition effect against PTP1B, an important target enzyme for the treatment of diabetes, while the dehydro derivative of 1 [dehydroeuryspongin A (4)] exhibited inhibitory activity (IC₅₀ = 3.6 μM).     

Paradoxical effects of chondroitin sulfate-E on Japanese encephalitis viral infection

Glycosaminoglycans (GAGs) have diverse functions in the body and are involved in viral infection. The purpose of this study was to evaluate the possible roles of the E-disaccharide units GlcAβ1–3GalNAc(4,6-O-disulfate) of chondroitin sulfate (CS), a GAG involved in neuritogenesis and neuronal migration, in Japanese encephalitis virus (JEV) infection. Soluble CS-E (sCS-E) derived from squid cartilage inhibited JEV infection in African green monkey kidney-derived Vero cells and baby hamster kidney-derived BHK cells by interfering with viral attachment. In contrast, sCS-E enhanced viral infection in the mouse neuroblastoma cell line Neuro-2a, despite the fact that viral attachment to Neuro-2a cells was inhibited by sCS-E. This enhancement effect in Neuro-2a cells seemed to be related to increased viral RNA replication and was also observed in a rat infection model in which intracerebral coadministration of sCS-E with JEV in 17-day-old rats resulted in higher brain viral loads than in rats infected without sCS-E administration. These results show the paradoxical effects of sCS-E on JEV infection in different cell types and indicate that potential use of sCS-E as an antiviral agent against JEV infection should be approached with caution considering its effects in the neuron, the major target of JEV.