Isolation of anti-endothelin receptor monoclonal antibodies for use in receptor characterization

Monoclonal antibodies reactive with endothelin (ET) receptors have been prepared by immunization of mice with rat lung membranes. Of four clones isolated, three clones preferentially recognized 32,000-dalton ET receptor and the other has a higher affinity for the 45,000-dalton receptor. The binding of 125I-ET-1 to detergent-solubilized ET receptors which were adsorbed to the antibodies was displaced by increasing concentrations of unlabeled ET isopeptides. These results demonstrate that the four clones specific for the receptor have the potential to be a useful tool in the characterization of ET receptors.     

Use of thionitrobenzoic acid to characterize the stability of nitric oxide in aqueous solutions and in porcine aortic endothelial cell suspensions

Nitric oxide is an important vasodilator which can be biologically produced from leukocytes and endothelial cells. However, it is highly unstable, which is an obstacle to detection and quantitation. We have exploited the reactivity of nitric oxide with thiols to establish an assay based on oxidation of thionitrobenzoic acid (TNB). The oxidation of thionitrobenzoic acid and the reaction with oxygen, which was measured by employing an oxygen electrode, were examined after the addition of nitric oxide solutions. The inhibition of aggregation of human platelets after challenge with 2.5 microM adenosine diphosphate was also investigated. These studies show the following properties of nitric oxide in aqueous solutions. (i) Nitric oxide is highly reactive to oxygen. (ii) Thiols react with a labile, highly reactive nitric oxide-oxygen product. (iii) Medium with very low oxygen content increases the life span of nitric oxide in aqueous solution. We also used the nitric oxide quantitation using TNB to study the metabolism of nitric oxide by porcine aortic endothelial cells and the results show that nitric oxide added to these cells in low oxygen content solution is stable. From these studies, we conclude that deoxygenated solutions stabilize nitric oxide. An important consequence of low oxygen content at localized tissue sites may be to augment biological effects mediated by nitric oxide.     

Changes in the levels of prostaglandins and thromboxane and their roles in the accumulation of exudate in rat carrageenin-induced pleurisy — a profile analysis using gas chromatography-mass spectrometry —

Injection of γ-carrageenin into t he pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B₂ were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethyllisopropylsilyl (DMiPS) ether or ME-methoxine-DMiPS ether derivatives, 6-keto-PGF_1α reached the maximum at 1 hr after carrageenin, then PGE₂ and TXB₂ showed peaks at 3 hr and waned off before 9 hr. he PGF_2α level was kept low, but PGD₂, PGE₁ and PGF_1α were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB₂ levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE₂, definitely play an important role in the exudation during the first 5 hr.     

Adductor muscles of the thigh of crab-eating monkeys (Macaca fascicularis)

On the basis of 44 hindlimbs of 14 male and 14 female crab-eating monkeys (Macaca fascicularis), the morphology of the adductor muscles of the thigh was described and some functional indices were calculated. The results obtained from this study agreed generally with those of otherMacaca species reported by various authors. For the classification and nomenclature of the adductors, the criteria proposed byUhlmann (1967, 1968) was well adapted to the crab-eating monkey. The adductors comprise the m. gracilis, m. pectineus, m. adductor longus, pars longa and pars brevis of m. adductor brevis, pars lata and pars minima of m. adductor magnus and m. obturatorius externus. In males, the adductors are generally inserted further down the femur, and the insertions of pars brevis of the m. adductor brevis and pars minima of the m. adductor magnus have longer attachments to the femur than in females. The arrangement of each adductor muscle and of each fasciculus of a thigh muscle may invoke a principle of organization.     

The nucleotide sequence of 5S ribosomal RNA from Schizosaccharomyces pombe

The nucleotide sequence of 5S rRNA from the fission yeast, S. pombe, has been established by post labeling procedures combined with cataloging RNase T1- and A-oligonucleotides derived from unlabeled 5S rRNA. The sequence consists of 119 nucleotides without a modified base and shows more dissimilarities (at 38 positions) from that of S. cerevisiae than from that of humans (at 33 positions).     

Identification of metabolic model: Citrate production from glucose by Candida lipolytica

The concept of mass balance was used to analyze the metabolic pathways of citrate production by Candida lipolytica from glucose. Specific rates of glucose consumption, citrate and isocitrate productions, carbon dioxide evolution, and cellular syntheses of protein and carbohydrate were observed in an NH₄⁺-limited chemostat culture. These data permitted one to assess the carbon flux in vivo by solving simultaneous carbon balance equations with respect to intermediary metabolite pools in the steady State. Among the three models considered here, model I (which coordinates the pyruvate carboxylation with the tricarboxylic acid cycle, but disregards the glyoxylate cycle) was considered plausible because the carbon flux calculated so far was acceptable. On the other hand, models II and III (which overlook the pyruvate carboxylation and the 2-oxoglutarate dehydrogenation, respectively) were found to be most unlikely because of the unusual flux assessed from these models.     

On the phosphate linkages and the structure of a disaccharide unit of the type-specific polysaccharide of pneumococcus type XIX

The structure of the capsular polysaccharide (S-XIX) of Pneumococcus Type XIX, which contains residues of d-glucose, l-rhamnose, 2-acetamido-2-deoxy- d-mannose, and phosphate, has been investigated by acid hydrolysis, treatment with acid phosphatase, mass spectrometry, and ¹³C-n.m.r. spectroscopy. Phosphoric esters in S-XIX were largely resistant to hydrolysis (4M HCl, 100°, 3 h). With M or 2M HCl at 100° for 3 h, 4-O-(2-amino-2-deoxy-β-d-mannopyranosyl)-d-glucose 4′-phosphate was liberated. More-drastic hydrolysis of S-XIX gave 2-amino-2-deoxy-d-mannose 3-, 4-, and 6-phosphates, and 4-O-(2-amino-2-deoxy-d-mannopyranosyl)-d-glucose and its 4′-phosphate.     

Fast polyspermy block and activation potential : Correlated changes during oocyte maturation of a starfish

Sperm entry into the oocyte of the starfish, Asterina pectinifera, was prevented when the membrane potential of the oocyte was held more positive than −10 to −5 mV, and multiple sperm entries were induced when the potential was held more negative. Based on this potential-dependent fertilization block mechanism, it was demonstrated that an activation potential (AVP) which is induced immediately after the attachment of the first sperm to the egg surface plays the role of a fast polyspermy block. The AVP-mediated polyspermy block mechanism develops as the oocyte matures and deteriorates as it ages. AVPs of mature oocytes exceeded −5 mV (the critical potential level for fertilization block) within 1 sec, and the potential stayed at +12 mV even after the initiation of fertilization membrane elevation. Consequently, the entry of a second sperm is prevented. In contrast, AVPs of overripe oocytes took about 15 sec to attain −5 mV, or they did not attain −5 mV at all. In overripe oocytes multiple sperm entries were associated with “step depolarization(s)” in the rising phase of the AVPs before membrane elevation took place. Immature oocytes generated AVPs associated with sperm entries, but without membrane elevation. AVPs in immature oocytes were characterized by the step depolarization(s) in the rising phase, and an AVP could be evoked again by a second insemination 20 min after the first insemination. These findings indicate that immature oocytes lack both fast and slow polyspermy block mechanisms.     

A sperm factor as the counterpart to the sperm-binding factor of the homologous eggs

A substance was isolated from sperm of the sea urchin, $\text{Hemicentrotus pulcherrimus}$. When unfertilized eggs of homologous species were pre-treated with this substance, they rapidly lost the fertilizability due to loss of the sperm-binding capacity. Such an effect was not exerted upon eggs either in CaMg free sea water or of heterologous species. This substance caused neither iso-agglutination of eggs nor precipitation of jelly coat. When it was pre-incubated with the sperm-binding factor purified from eggs of homologous species, it lost the fertilization-inhibiting effect on eggs. It seems very likely that a complementary relationship exists between the present substance and the sperm-binding factor.