A protease inhibitor produced by Streptomyces lividans 66 exhibits inhibitory activities toward both subtilisin BPN' and trypsin.

A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.     

Anti-eye muscle antibodies and hypothyroid Graves' disease: a case report.

We report the case of a 70-year-old man who developed hypothyroidism associated with TSH receptor antibodies and severe ophthalmopathy during lithium therapy. He had received lithium therapy for more than 20 years for manic depression, when ophthalmopathy (class VI of the American Thyroid Association classification) and mild hypothyroidism developed. Orbital magnetic resonance imaging indicated marked enlargement of the superior, medial and inferior rectus muscles in the left eye. He had anti-eye muscle antibodies in his serum, detected by Western blotting and quantified by chromatoscanning, as well as anti-TSH receptor antibodies. He was treated with supplementation of levothyroxine and four cycles of methylprednisolone pulse therapy. After the pulse therapy, both anti-eye muscle antibodies and anti-TSH receptor antibodies decreased and disappeared in parallel with the improvement in eye symptoms and signs. These observations suggest the importance of anti-eye muscle antibodies as clinical markers in the development of thyroid-associated ophthalmopathy.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.     

Parturition in a free-ranging Japanese monkey (Macaca fuscata)

Parturition behavior of a multiparous female and her interactions with group members throughout the birth process were recorded for a free-ranging Japanese monkey (Macaca fuscata). The female showed evidence of 18 contractions during the 35 min prior to delivery, with a mean duration and a mean intercontraction interval of 30 sec and 96 sec, respectively. These values were similar to those in individually caged Japanese monkeys. Some adult females remained in proximity to the female who was giving birth during the prepartum phase, and her 2-year-old daughter watched the delivery of the infant. Even during the prepartum phase the female moved in order to keep up with the group which traveled from the feeding site to a sleeping site in the forest.     

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [³H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [³H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.     

Spectrophotometrical and immunochemical studies on the conformational changes in poly(dG-dC).poly(dG-dC) after modification by 4-hydroxyaminoquinoline 1-oxide

Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.     

AMP synthesis in aqueous solution of adenosine and phosphorus pentoxide

Possible formation of a P₄O₁₀ molecule in magma, the stability of the molecule in hydrous volcanic gas at high temperatures and a possible prebiotic phosphate cycle were discussed in relation to chemical evolution. To demonstrate the utility of phosphorus pentoxide as a phosphorylating agent, aqueous solutions of adenosine (0.02M) and phosphorus pentoxide (0.2M) were incubated at 37°C for 5 months. The pH of the solutions was adjusted every day or every few days to each fixed value (9.0, 10.5, 11.5, 12.5) with 10 N NaOH. The HPLC analysis showed the formation of 2-AMP, 3-AMP, 5-AMP, cyclic (2–3)-AMP and cyclic (3–5)-AMP. The main components of the products were 2- and 3-AMP, though cyclic (2–3)-AMP was the main component in the early period of the incubation at pH 9.0. The yields (conversion rate of adenosine to AMPs) were increased almost linearly with the incubation time for 5 months in the case of pH 9.0. The final yields were about 3% (pH 9.0), 6% (pH 9.0, 1 M NaCl), 5% (pH 9.0, 0.01 M CaCl₂, 0.01 M MgCl₂), 7% (pH 9.0, 0.5 M NaCl, 0.01 M CaCl₂, 0.01 M MgCl₂), 9% (pH 9.0, 1 M NaCl, 0.01 M CaCl₂, 0.01 M MgCl₂), 32% (pH 10.5), 43% (pH 11.5), 35% (pH 12.5).     

Thymidine triphosphate dephosphorylating activity in regenerating rat liver

The enzyme activity of dephosphorylation of thymidine triphosphate was found in microsomal fraction of rat liver. The enzyme activity decreased at the time when [³H]thymidine incorporation into DNA of regenerating liver increased. When the [³H]thymidine incorporation was suppressed by 1,3-diaminopropane, the enzyme activity remained elevated. These results suggest that the enzyme activity appears to be closely linked to DNA synthesis.     

Biochemical and Histochemical Studies on Catecholamines in Bullfrog Spinal Ganglia

Abstract: Bullfrog spinal ganglia were subjected to two types of examination to determine whether catecholamines were present. A biochemical microassay developed by Kissinger and co-workers and a histofluorescence technique for cellular demonstration of biogenic monoamines developed earlier by Falck and co-workers were used. Bullfrog spinal ganglia and dorsal roots were found to contain catecholamines, primarily adrenaline.     

Effect of short — chain fatty acids on electrical activity of the small intestinal mucosa of rat

Intravenous administration of short-chain fatty acid (SCFA), such as propionate, butyrate, valerate and caproate, caused a transient increase in transmural potential difference (p.d.) across the small intestine of rat $\text{in vivo}$. There was a sigmoid relationship between the change in the p.d. and the logarithm of the dose of SCFA. The median effective dose of propionate, n-butyrate, n-valerate and n-caproate, which was calculated from the each dose-response curve obtained from the terminal ileum, 1.31, 1.43, 0.83 and 0.81 μmole, respectively. Repeated administrations of the same dose of propionate evoked progressively smaller response. The dose-response curve of propionate was shifted to the left by neostigmine and to the right by atropine, suggesting that the action of SCFA may be mediated by acetylcholine, which was released from a nerve ending.