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.     

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.     

Spatiotemporal relationships among early events of fertilization in sea urchin eggs revealed by multiview microscopy.

Four early events of egg fertilization, changes in intracellular calcium concentration and intracellular pH, reorientation of the surface membrane, and the elevation of the fertilization envelope, were imaged in real time and in pairs in single sea urchin eggs. The paired imaging allowed the correlation of the four events spatially and temporally. Three of them propagated as waves starting at the sperm entry site. The earliest was the calcium wave, visualized with fluorescent indicator dyes. After a delay of 10 s there followed a large decrease in the fluorescence polarization of membrane-bound dyes, which we interpret as arising from membrane reorientation as a result of cortical granule exocytosis and microvillar elongation. With a further delay of 15 s the fertilization envelope was seen to rise in transmitted light. All three waves propagated with similar velocities of approximately 10 microns/s, supporting the view that calcium triggers the latter two events. The fluorescence polarization changed in two steps with a clear pause of 10-20 s in between. The second step, which also propagated as wave, reflects either further elongation of microvilli or straightening of irregular microvilli. This second step was abolished by cytochalasin B and was coincident with an increase in cytoplasmic pH, suggesting that pH-induced actin reorganization may play a role. The cytoplasmic alkalinization, imaged with a fluorescent probe, was quite different from the other events in that it took place homogeneously throughout the egg and slowly (over 100 s). Apparently, the alkalinization is not on a direct downstream pathway of calcium origin. An opposing possibility, that the alkalinization may in fact be triggered by the traveling calcium wave, is also discussed.     

Kinetics and collagenolytic role of eosinophils in chronic gastric ulcer in the rat

 An association between eosinophils and tissue damage has been observed in numerous disorders. However, few reports have addressed the role of infiltrating eosinophils in gastric ulcer healing. The aim of this study was to investigate the kinetics and role of eosinophils infiltrating experimental chronic gastric ulcers in the rat. We developed a monoclonal antibody against human matrix metalloproteinase 1 (MMP1) purified from conditioned culture medium of human skin fibroblasts. Acetic acid-induced gastric ulcers were resected from rats on days 1, 3, 5, 10, 20, 40, and 180 after the days of induction (day 0). Tissue specimens were immunostained with this antibody and examined with an electron microscope. Few eosinophils were observed in the granulation tissue until day 20. By days 40 and 180, MMP1-positive eosinophils had increased in the granulation tissue of open ulcers. Azan staining revealed dispersed collagen fibers around infiltrating eosinophils. In contrast, scars demonstrated few eosinophils in fibrous tissue on days 40 and 180. Eosinophils which express MMP1 infiltrate granulation tissue at the chronic stage of gastric ulceration. The results suggest that eosinophils may play a role in tissue remodeling and deterioration of ulceration. Accepted: 18 March 1997     

Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected miniF segments

Summary Various DNA subfragments were derived from miniF DNA by complete or partial PstI cleavage, and cloned in the plasmid vectors pBR322 or dv1. The recombinant plasmids obtained were introduced into an Escherichia coli minicell-producing strain, and the plasmid-coded proteins were radiolabeled and analyzed by gel electrophoresis. Six miniF-encoded proteins, larger than 11 000 daltons, were detected and their coding regions were mapped on the F plasmid genome. Three of them were assigned by taking into account the known nucleotide sequences (Murotsu et al. 1981; K. Yoshioka, personal communication). The coding directions of some proteins were determined by inserting the lac promotor into one of the recombinant plasmids and analyzing the increase in production of the proteins. The coding direction of the five proteins analyzed so far was uniform. Comparison of these results with a functional map of miniF suggested possible roles of the proteins.     

A neurochemical study of developmental impairment of the brain caused by the administration of cytosine arabinoside during the fetal or neonatal period of rats

Injection of pregnant rats with cytosine arabinoside (ara-C) (280 mg/kg) on day 15 of gestation caused a significant rise (about two times the control value) in monoamine concentrations (norepinephrine, dopamine, and serotonin) accompanied by a decrease (about 60% of the control) in the brain weight and DNA content in the cerebrum of the offspring at 60 days of age. When neonatal rats were injected with ara-C (30 mg/kg/day) for four consecutive days from the fourth to seventh days after birth, a decrease of DNA content per cerebellum and an elevation of monoamine concentrations in the cerebellum were found. However, the total content of each monoamine per cerebrum or cerebellum showed no difference from the control. These results suggest that monoaminergic neurons may remain intact, with normal monoaminergic synapses compressed into a small brain volume. The neonatal administration of ara-C caused an elevation of 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase) (EC 3.1.4.37) activity and myelin protein content in the cerebellum, suggesting a relative increase in myelin concentration as a result of hypoplasia of granule cells.     

Specific and abundant secretion of a novel hydroxyproline-rich glycoprotein from salt-adapted winged bean cells

Winged bean callus was adapted to increasing concentrations of NaCl by sequential transfer to medium with 0, 0.5, 1.0, 1.5, and 2.0% (w/v) NaCl. When the culture media, after cell suspension cultures of callus adapted to 0.5 (SA-0.5), 1.0 (SA-1.0), 1.5 (SA-1.5), or 2.0% (w/v) NaCl (SA-2.0), were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, six specific or enhanced polypeptide bands (SAP1, -2, -3, -4, -5, and -6) were observed. SAP1, with a molecular weight of 84,000, was abundantly secreted in suspension cultures of SA-1.0 and SA-1.5, and was observed as the most striking polypeptide band. The SAP1 yield was about 4 mg/g cells fresh weight. SAP1 was abundantly secreted after the suspension culture of SA-1.0 in the presence of AlCl₃, but little was secreted in the presence of KCl, LiCl, CaCl₂, MgCl₂, mannitol, sucrose, or abscisic acid. SAP1 was purified from the culture medium after suspension culture of SA-1.0 in the presence of 1.0% (w/v) NaCl. Two steps, ammonium sulfate fractionation and CM-cellulose chromatography, were sufficient for purification to homogeneity. Finally, about 5 mg of SAP1 could be isolated from 7 g of fresh callus cells. Of the amino-terminal 32 amino acid residues of SAP1, 10 and 5 were found to be hydroxyproline and proline, respectively. SAP1 on an acrylamide gel was stained by the periodic acid-Schiff method. It is interesting that SAP1 has pentahydroxyproline blocks (Hyp₅) instead of tetrahydroxyproline blocks (Hyp₄) common to many hydroxyproline-rich glycoproteins in dicotyledons. Thus, this novel hydroxyproline-rich glycoprotein was shown to be abundantly secreted from NaCl-adapted winged bean cells.     

Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.

Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.