Phylogeography of the leaf beetle Chrysolina virgata in wetlands of Japan inferred from the distribution of mitochondrial haplotypes

The genetic differentiation among populations of the leaf beetle Chrysolina virgata living in wetlands of Japan was studied based on the sequence data of the mitochondrial cytochrome oxidase subunit I gene region (750 bp). Two distinct lineages of mitochondrial haplotypes were found: one (clade A) consisted of 26 haplotypes distributed over the distribution range of C. virgata between north‐east Honshu and Kyushu, whereas the other (clade B) was monotypic and confined to a small region in north‐east Honshu where it coexisted with clade A. Nested clade analysis for these haplotypes suggested that range expansion and following differentiation due to isolation by distance might have resulted in the present distribution pattern of the haplotypes in clade A. We discuss the evolutionary process leading to the occurrence of two distinct haplotype clades in Japan in terms of repeated colonization from the continent and range expansion and contraction during climatic changes.     

Glutamine Synthetase of the Human Brain: Purification and Characterization

Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver.     

Incidence of herpes infection of the uterine cervix observed in cytologic specimens in the Tokyo metropolitan area

In a high-volume cytology laboratory in the metropolitan Tokyo area, the incidence of cytologically diagnosed herpes infection in cervical scraping smears of the female genital tract was studied according to the year-by-year changes, age distribution, seasonal variation and types of cytologic alteration. The overall incidence over the 12 years studied was 0.007% (87 cases among 1,230,773 examined). The incidence varied from 0.003% to 0.005% in the early 1970s (except for 1973) and increased to 0.011% in the last three years (1980 to 1982). A large increase was noted in younger age groups in comparison with middle and older age groups. There was a tendency for the infection rate to be higher in the spring (0.011%) and lower in the fall (0.005%). Multinucleation and a ground-glass appearance were observed in the infected cells in almost every case while eosinophilic inclusion bodies were found in 20.7% of the cases.     

Cytolytic activity of cytotoxin isolated from Indian cobra venom against experimental tumor cells

Cytolytic activity of cytotoxin isolated from the venom of the Indian cobra (Naja naja) on experimental tumor cells was far stronger than that on normal cells such as peritoneal exudate cells, spleen cells, and erythrocytes of the rat. The effect on Yoshida sarcoma cells was temperature-dependent, being stronger at 37 degrees C than at 0 degrees C. Intramolecular disulfide linkages and free amino groups in the cytotoxin molecule were shown to be essential for the lytic action on the cell membrane. Yoshida sarcoma cells treated with 0.1 mM N-ethylmaleimide reduced the cytolytic action of the toxin. Antitumor activity of the cytotoxin toward a Yoshida sarcoma inoculated intraperitoneally into a rat was not observed.     

A case of pituitary adenoma producing both growth hormone (GH) and adrenocorticotropic hormone (ACTH).

The authors report a very rare case of pituitary adenoma producing both GH and ACTH. A 29-year-old female was admitted with obesity, amenorrhea, acromegaly, hirsutism, excessive pigmentation, acne, and diabetes mellitus. Computed tomography revealed an intrasellar tumor 16 mm in height, with a destroyed sellar floor. The blood concentrations of GH, ACTH and cortisol were increased (GH: 92 ng/ml, ACTH: 94 pg/ml, cortisol: 18.3 micrograms/dl). No diurnal variation in the amount of cortisol was observed. The urinary 17-OHCS was suppressed by 8 mg but not by 2 mg of dexamethasone. A subtotal adenomectomy was then performed through the transsphenoidal approach, which led to a sufficient reduction of both blood GH and ACTH (cortisol). Histologically the tumor was an acidophilic pituitary adenoma. Immunoperoxidase staining showed diffuse GH and sporadic ACTH producing cells, but failed to show any cells producing both hormones. The electron micrograms of neoplastic cells showed the ultrastructural characteristics of respective GH and ACTH cells. Another increase in both GH and cortisol, which occurred 19 months after the operation, has been controlled by bromocriptine administration. This case may be the first reported case of a pituitary adenoma producing both GH and ACTH, not accompanied by prolactin (PRL) hypersecretion, which has been fully confirmed endocrinologically and histopathologically.     

Delineation of the functional site of a snake venom cardiotoxin: preparation, structure, and function of monoacetylated derivatives

Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.     

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio‐lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1‐1 and 1‐2). Both PpAN1 genes complemented the A. thaliana an‐1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1‐promoter– uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1‐1 and PpAN1‐2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip‐growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α‐tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1‐1/1‐2 double‐knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.     

Anesthetic Effects of a Mixture of Medetomidine,Midazolam and Butorphanol in Two Strains of Mice

The combination of ketamine and xylazine is a widely used anesthetic for laboratory animals. However, due to an abuse problem in Japan, ketamine has been specified as a narcotic since 2007. Instead of using ketamine, Kawai et al. reported an injectable formula with an equivalent effect to the mixture of ketamine and xylazine [11]. The mixture of 0.3 mg/kg body weight (b.w.) medetomidine (Med.), 4.0 mg/kg b.w. midazoram (Mid.), and 5.0 mg/kg b.w. butorphanol (But.) produced an anesthetic duration of around 40 min in outbred ICR mice. However, the anesthetic effect of the mixture for inbred mice strains remains unknown. Therefore, we examined anesthetic effects of the mixture of Med., Mid., and But. in the BALB/c and C57BL/6J strains. After intraperitoneal injection into mice, right front paw, left hind paw, and tail pinch reflexes as well as corneal and righting reflexes were observed. Every 5 min, we scored each reflex category as 0 for reaction or 1 for no reaction. As long as the total score was at least 4 out of 5, we considered the mixture as putting a mouse in a surgical anesthetic state. The mixture produced an anesthetic duration of more than 45 min in both strains of mice. These results indicate that the mixture of Med., Mid., and But. can be a useful and effective anesthesia for the BALB/c and C57BL/6J strains of inbred mice as well as outbred ICR mice.