Antithrombotic effect of topically applied 3-Oxa-Methano-prostaglandin I1 in the microcirculation and antiplatelet functions of its active form

The antithrombotic effect of topical application of the 3-oxamethano-prostaglandin (PG) I₁ analog, SM-10902 in the microcirculation and in vitro antiplatelet functions of its active form SM-10906 were estimated in comparison with PGI₂ and PGE₁. In rat platelets, SM-10906 evoked accumulation of intracellular cyclic adenosine 3′,5′-monophosphate, and exhibited antiaggregatory and disaggregatory activities, which were all enhanced by the phosphodiesterase inhibitor theophylline. Additionally, SM-10906 was shown to inhibit platelet adhesion to collagen in human platelet-rich plasma. PGI₂ and PGE₁ also showed in vitro antiplatelet effects in the order of PGI₂ > SM-10906 ≥ PGE₁. SM-10902 exhibited a dose-dependent antithrombotic effect in the guinea pig mesenteric arteriole by a topical application, and this activity might be exerted by the antiplatelet functions of SM-10906. Although SM-10906, PGI₂ and PGE₁ also showed the antithrombotic effects, SM-10902 was the most potent. In conclusion, the present studies indicate that an external topical preparation of SM-10902 may be useful for the therapy of peripheral circulatory insufficiency.     

Hormonal effects on the development changes of mouse small intestinal glycolipids

The composition of intestinal glycosphingolipids during normal and hormone-perturbed development was investigated. The concentrations of glycosphingolipids of mouse small intestine were affected by the injection of thyroxine or cortisone during suckling and weaning periods. GDla was reduced by the hormonal treatment among major gangliosides, GM3, GM1 and GD1a, of mouse small intestine during the suckling period. In contrast, asialo GM1 was precociously produced by the treatment, which scarcely found in control suckling mouse small intestine. The results showed that these hormones were related to developmental alteration of small-intestinal glycolipids.     

Purification and some properties of a soluble cytochrome (cytochrome c-551) from Erythrobacter species strain OCh 114

A soluble cytochrome, cytochrome c-551 was purified from an aerobic photosynthetic bacterium Erythrobacter species strain OCh 114 (ATCC No. 33942) by ammonium sulfate fractionation, ion-exchange chromatography and gel-filtration. The cytochrome had absorption maxima at 277, 410, and 524–525 nm in the oxidized form, and at 415, 522, and 550.5 nm in the reduced form. At 77 K, the -band of the absorption spectrum of the reduced form split in two at 547 and 549 nm. The millimolar absorption coefficient at 550.5 nm was 26.8 mM^-1 cm^-1 in the reduced form. This cytochrome was an acidic protein with an isoelectric point of 4.9. Its molecular weight was determined to be 15,000 by gel-filtration on Sephadex G-100 and 14,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The midpoint potential of this cytochrome was +250 mV at pH 7.0. This cytochrome did not bind CO.     

Studies on organ specificity in experimental murine cryptococcosis

The chief histopathological features found in patients with cryptococcosis are both a cystic (gelatinous) lesion and a granulomatous reaction. These two tissue reactions are definitely different from each other, because a cyst is not accompanied with a significant cellular response, while a granuloma is formed as a result of various cell reactions. Therefore, it is very interesting that these two types of lesion can be observed in the same patient or in the same animal infected with Cryptococcus neoformans. From our previous paper (II) the authors reach such a thought that two steps may be required for the granuloma formation against C. neoformans infection: first, of phagocytosis by sessile macrophages of C. neoformans and second is related to T-cell function. This experiment was done to verify that the granulomatous response against C. neoformans infection might occur easily in the organs rich in sessile macrophages as compared with those poor in them and a polysaccharide capsule surrounding cryptococci may have effects to inhibit a migration of polymorphonuclear leucocytes or monocytes toward C. neoformans. C. neoformans strain RIB 12 (serological type A, mating type α) was used in this experiment. After a culture of a brain heart infusion glucose agar slant at 37 C for 3 days, yeast cells of the strain were harvested, and suspended in 1/15 M(p_H7.4) sterile phosphate buffered saline solution. Infective inoculum was prepared by adjusting the number of the yeast cells to 10⁵, 10⁶ or 5×10⁶/0.2 ml in a hemacytometer. Fourty-two male mice strain ddY were divided into 3 groups consisting of 14 each and one group was allotted to one of the cell suspensions. Each mouse was inoculated with 0.2 ml of the cell suspension into a tail vein and one mouse from each group was sacrificed at adequate intervals. At necropsies the brain, thymus, lungs, heart, liver, kidneys, spleen, pancreas, mesenteric lymph nodes, a part of the small intestine, testes and fat tissue were removed. From these organs histopathological sections, stained with HE or by PAS, were prepared. To investigate effects of a polysaccharide capsule to a migration of polymorphonuclear leucocytes or monocytes, double infections with C. neoformans and Aspergillus fumigatus, and an observation by the ‘Agar-Implantation method’ were done. As results, granulomata were formed easily in the organs rich in macrophages or lymphocytes such as the liver, spleen, lymph nodes, thymus, lungs, small intestine and fat tissue. On the contrary, in organs poor in the macrophages such as the brain, heart, pancreas, kidneys, adrenal glands and testes, the chief histopathological feature was a cyst formation containing numerous yeast cells. In the double infection, two types of lesions such as cysts and abscesses were observed in the sections of the brain. The former occurred against C. neoformans infection and the latter, against A. fumigatus infection. Even though a cyst was very close to an abscess, polymorphonuclear leucocytes or monocytes were never induced to C. neoformans. In the observation using the ‘Agar-Implantation method’, a severe cellular infiltration occurred to a perfect (teleomorphic) state of C. neoformans and very weak response, to yeast cells with a polysaccharide capsule. The difference may be due to the existence of the capsule, because a perfect state of C. neoformans is not surrounded by it.     

Biosynthesis of ribulose-1,5-bisphosphate carboxylase in spinach leaf protoplasts

Spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts sustain protein-synthesizing activity as measured by the incorporation of [¹⁴C]-leucine into the protein fraction both in the light and in the dark. By the immunoprecipitation of ribulose-1,5-bisphosphate (RuP₂) carboxylase with rabbit antibody raised against the purified spinach enzyme preparation, it was found that approximately 7% of the total radiocarbon incorporated into the protein fraction in the light was in the carboxylase molecules. However, there was no measurable net increase observed in the content of the enzyme protein in the experimental conditions employed. It was found that both chloramphenicol and cycloheximide inhibited the incorporation of [¹⁴C]leucine into RuP₂ carboxylase and its constituent subunits, as measured by the immunoprecipitation of the enzyme molecule and its subunits, A and B.     

Rat cathepsin H-catalyzed transacylation: comparisons of the mechanism and the specificity with papain-superfamily proteases.

We found that rat cathepsin H showed strong transacylation activity under physiological conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl-acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (pKapp = 7.5) could be regarded as the pKa of the alpha-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKapp = 5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the N alpha-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH5), the transacylation was detectable even at the acidic pH-range because of the high S1'-site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the S1 and S1' site appeared to prefer hydrophobic residues without and regardless of a branch at beta-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the S1' site, presumably reflecting the good conservation of Trp177 in papain-superfamily proteases.     

A novel cDNA clone for acid invertase in tomato fruit.

The sequence of a novel cDNA clone, Aiv-1, for tomato acid invertase was similar to that of TIV1 (Klann et al., 1992) for the enzyme except for a unique intron-like insertion. It is considered that Aiv-1 is derived from either an alternatively spliced mRNA for an isozyme or a pre-mRNA of TIV1.