Relation of mode of administration of testosterone to evocation of male sex behavior in frogs

In Rana pipiens, mating behavior could be induced readily in intact males by several pituitary implantations, but never in castrates. Systemic testosterone injection (1 mg daily), with or without pituitary implantation, failed to restore mating behavior in castrated frogs. On the other hand, intracranial implantation of testosterone (approximately 60-μg pellets in which testosterone is mixed with cholesterol 1:1) in castrates evoked mating behavior, including mating calls and clasping. The most effective implantation site was the rostral part of the preoptic nucleus. Thus, the rostral part of the preoptic nucleus is the androgen-sensitive site which governs sexual behavior in this species. The relative ineffectiveness of systemic injection of testosterone is discussed.     

Biochemical studies on strain differences of mice in the susceptibility to nitrogen dioxide

Strain differences of mice in their susceptibility to nitrogen dioxide (NO₂) were examined by measuring the activities of antioxidative protective enzymes, and the amounts of antioxidants and lipid peroxides in lungs. Four strains of mice: ICR, BALB/c, ddy and C57BL/6 were used in this study and their LC₅₀ values after exposure to NO₂ for 16 hr were: 38, 49, 51 and 64 ppm, respectively (1).Genetic strain differences were observed in the enzyme activities, the antioxidant contents and lipid peroxide contents among these four different strains. The activities of glutathione peroxidase (GP_x), glutathione S-transferase, and superoxide dismutase (SOD), and the contents of non-protein sulfhydryls (NPSH), α-tocopherol (α-Toc) and total lipids in lungs of the four strains were related to their LC₅₀, while TBA reactants in lungs of the four strains were inversely related to their LC₅₀.After exposure to 20 ppm NO₂ for 16 hr, the activities of the protective enzymes and the contents of NPSH decreased, while the level of α-Toc increased markedly. The activities of GP_x, 6-phosphogluconate dehydrogenase, SOD and disulfide reductase, and the contents of NPSH, α-Toc and total lipids were also related to their LC₅₀. On the other hand, TBA reactants increased higher than those of the control groups and were inversely related to their LC₅₀.These results suggest that the protective enzymes and the antioxidants are important factors as defence mechanism in lungs to NO₂ and that the intensity of the protective systems in pigmented strains is generally greater than that in albino strains.     

One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell

Erythrocyte ghosts containing a known number of molecules of purified fragment A of diphtheria toxin with a constant amount of FITC-BSA as a fluorescence marker were prepared by dialyzing a mixture of erythrocytes and these substances against hypotonic solution. These substances were then introduced into diphtheria toxin-resistant mouse L cells by virus-mediated cell fusion of the cells with the ghosts, and mononuclear recipients that had fused with only one erythrocyte ghost were separated in a fluorescence-activated cell sorter (FACS) on the basis of their cell size and fluorescence intensity. After separation, the viability of cells containing known numbers of fragment A was examined by measuring colony-forming ability. The results demonstrated that a single molecule of fragment A was sufficient to kill a cell.This fact was confirmed by introduction into cells of fragment A from an immunologically related mutant toxin, CRM 176 (fragment A-176); this has a completely functional fragment B region, but in cell extracts, the enzymic activity of its fragment A is about 10 fold less than that of wild toxin. The cytotoxicity of CRM 176 is about two hundredths of that of the wild-type (Uchida, Pappenheimer and Greany, 1973). As expected, about 100–200 fold excess of fragment A-176 was needed to kill the cells.     

Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between Trp62 and Asp101.

A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the beta-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroacetone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH2COCH2SCH2CH2NH-bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3 degrees than that of native lysozyme at pH 3.     

Central effects of yohimbine on copulatory behavior in aged male rats

It is well known that yohimbine has a history of popular use because of its supposed aphrodisiac properties. The present study was done to determine whether yohimbine can modify the copulatory behavior of aged male rats. Adult male rats of the Wistar-Imamichi strain, 52 weeks of age and weighing 600-650g, were injected intracerebroventricularly with yohimbine hydrochloride (5, 10 micrograms/10 microliters/rat) or vehicle. Each male was then given the opportunity to mate with a receptive female for 30 min after administration of yohimbine or vehicle. Yohimbine produced significant decreases in the latency to initial mounting and significant increases in the number of mountings. However, there was no ejaculation in the yohimbine-and vehicle-treated males. This study is the first to clearly establish an important modulator of sexual arousal for yohimbine in aged male rats.     

A novel dihydrodiol dehydrogenase in bovine liver cytosol: purification and characterization of multiple forms of dihydrodiol dehydrogenase.

Three enzymes (DD1, DD2, and DD3) having dihydrodiol dehydrogenase activity were purified to homogeneity from bovine cytosol. DD1 and DD2 were identified as 3 alpha-hydroxysteroid dehydrogenase and high-Km aldehyde reductase, respectively, as judged from their molecular weights, substrate specificities and inhibitor sensitivities. DD3 was a unique enzyme which could specifically catalyze the dehydrogenation of trans-benzenedihydrodiol and trans-naphthalenedihydrodiol without any activity toward the other tested alcohols, aldehydes, ketones, and quinones. The Km value of DD3 (0.18 mM) for benzenedihydrodiol was lower than those of other dihydrodiol dehydrogenases so far reported. DD3 immunologically crossreacted with DD1, but showed no crossreactivity with DD2. Additionally, DD3 was inhibited in a competitive manner, with a low Ki value of 1 microM, by androsterone, which was a good substrate for DD1. It was assumed that DD3 is a novel enzyme which is specific to dihydrodiols, exhibiting similarity to DD1 in immunological and structural properties.     

Immunohistochemical and immunoelectron microscopic analyses of alpha-amylase isozymes in human intrahepatic biliary epithelium and hepatocytes.

The expression and localization of the pancreatic and salivary isozymes of alpha-amylase in the intrahepatic biliary epithelium and hepatocytes were examined by the immunohistochemical method with polyclonal and monoclonal antibodies in 45 normal autopsied human livers. Immunoelectron microscopic studies with the protein A-gold method were performed with the monoclonal antibodies (MAb) on seven of the livers. The intrahepatic biliary system was divided into large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands. Immunohistochemically, pancreatic isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands in almost all livers. Interlobular ducts expressed pancreatic isozyme in only four (9%) livers. Bile ductules and hepatocytes were negative for pancreatic isozyme in all cases. Expression of salivary isozyme was observed in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, interlobular ducts, bile ductules, and peribiliary glands in almost all livers, although the expression in interlobular ducts and bile ductules was weak. Hepatocytes were weakly positive for salivary isozyme. Immunoelectron microscopy revealed that both pancreatic and salivary isozymes were located in the supranuclear cytoplasm of the epithelium of large ducts, septal ducts, and peribiliary glands, and that hepatocytes had no pancreatic isozyme but contained salivary isozyme. These data suggest that pancreatic and salivary isozymes of alpha-amylase are produced by the intrahepatic biliary epithelium and secreted into intrahepatic biliary lumens, and that they may play an important role in the physiology of the intrahepatic biliary tree and hepatic bile. It is also suggested that hepatocytes produce a small amount of salivary alpha-amylase that may be secreted into the biliary tree.     

Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion

Summary Recombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon . The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C₁₈ reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses. Offprint requests to: J. Ishizaki