Genetic Transformation in Helicobacter pylori

Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells, with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.     

Characterization of antibody against human liver guanase by immunoblotting and immunohistochemical staining of human liver guanase by a direct labeling antibody-enzyme method

Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).     

Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog.

Capsiate is a nonpungent capsaicin analog, a recently identified principle of the nonpungent red pepper cultivar CH-19 Sweet. In the present study, we report that 2-wk treatment of capsiate increased metabolic rate and promoted fat oxidation at rest, suggesting that capsiate may prevent obesity. To explain these effects, at least in part, we examined uncoupling proteins (UCPs) and thyroid hormones. UCPs and thyroid hormones play important roles in energy expenditure, the maintenance of body weight, and thermoregulation. Two-week treatment of capsiate increased the levels of UCP1 protein and mRNA in brown adipose tissue and UCP2 mRNA in white adipose tissue. This dose of capsiate did not change serum triiodothyronine or thyroxine levels. A single dose of capsiate temporarily raised both UCP1 mRNA in brown adipose tissue and UCP3 mRNA in skeletal muscle. These results suggest that UCP1 and UCP2 may contribute to the promotion of energy metabolism by capsiate, but that thyroid hormones do not.     

Superovulation of Holstein heifers by a single subcutaneous injection of FSH dissolved in polyvinylpyrrolidone

This study was undertaken to determine whether a single injection of porcine FSH (pFSH) would induce a superovulatory response in cattle. Holstein heifers were given a single injection of pFSH (30 mg, s.c.) dissolved in saline (Group 1, n = 5); 50% polyvinylpyrrolidone (PVP; Group 2, n = 5); or 25% PVP (Group 3, n = 4). Group-4 heifers (n = 5) were given multiple intramuscular injections of pFSH every 12 h for 3 d at decreasing doses, for a total of 30 mg. All animals received a single injection of 750 microg PGF2 alpha 48 h after the initiation of pFSH treatment. Animals exhibiting estrus were artificially inseminated twice throughout estrus. Ova and embryos were recovered nonsurgically. Ovaries were examined by transrectal ultrasonography or by palpation per rectum on Day 7 or 8 of estrus. Plasma concentrations of pFSH, bovine FSH progesterone, estradiol-17 beta and inhibin were determined by specific radioimmunoassays. The number of corpora lutea (CL) and the numbers of total and transferable embryos which were detected and recovered in Groups 2 and 3 were equivalent to the numbers detected and recovered in Group 4. In Group 1, however, only 1 of 5 animals ovulated even a single oocyte. The present study demonstrated that only a single injection of pFSH dissolved in PVP was capable of inducing a superovulatory response by maintaining a high plasma FSH concentration to allow for the recovery of a sufficient number of embryos for transplantation.     

Podocytes in metanephric organ culture express characteristic in vivo phenotypes

 Podocytes outgrown from isolated glomeruli in vitro have failed to express fully differentiated in vivo phenotypes. In an attempt to determine whether podocytes in metanephric culture accomplish terminal differentiation, as observed in vivo, we investigated expression of their characteristic phenotypic features in rat metanephric organ cultures using immunohistochemistry and electron microscopy. Rat metanephroi were harvested on embryonic day 12.5 and cultured on transmembrane filters for 9 days. Morphological examination revealed two maturation stages when the podocytes resembled those of the S-shaped body stage and maturational stages of glomeruli in vivo. Electron microscopy revealed that, firstly podocytes lost their intercellular contacts and, simultaneously, the tight junctions shifted into close proximity to cell bases, followed by foot process development. Immunohistochemistry demonstrated that the tight junction protein, ZO-1, and specific podocytic markers, pp44, 5-1–6, podocalyxin and vimentin were expressed in a cell maturity-dependent manner, as observed in newborn rat kidneys. Furthermore, glomerular basement membrane components, collagen type IV and laminin, were expressed in the glomerular center. Our findings that cell maturity-dependent expression of structural and functional phenotypes in podocytes in metanephric culture was the same as that observed in developing kidneys in vivo indicate that podocyte differentiation during glomerulogenesis may be operated by an intrinsic property, such as programmed cell fate. Furthermore, these highly differentiated podocytes in vitro may provide clues that will help to establish a podocyte culture system. Accepted: 26 February 1997     

Isolation of Cytopathic Small Round Virus (Aichi Virus) from Pakistani Children and Japanese Travelers from Southeast Asia

Aichi virus was isolated in Vero cells from 5 (2.3%) of 222 Pakistani children with gastroenteritis but none was found in 91 healthy children. Aichi virus was also isolated from 5 (0.7%) of 722 Japanese travelers returned from tours to Southeast Asian countries and complained of gastrointestinal symptoms at the quarantine station of Nagoya International Airport in Japan. Of 5 Japanese travelers, 3 were returning from Indonesia, and 2 from Thailand or Malaysia. These results indicate that Aichi virus or a similar agent is endemic in Southeast Asian countries and is a cause of gastrointestinal symptoms in children in these areas or in Japanese travelers who visit there.     

Deactivation of macrophage oxidative burstin vitro by different strains ofHistoplasma capsulatum

It was previously reported thatHistoplasma capsulatum (Hc) yeast not only failed to stimulate a murine macrophage oxidative burst (OB), but they also blunted or abolished OB stimulation by a subsequent encounter with potent stimuli such as zymosan or phorbol 12-myristate 13-acetate (PMA). The present studies show that macrophage deactivation is proportional to the time of incubation and the dose of Hc yeast that induce the deactivated state. Hc yeast derived from a virulent strain (G217B) are more efficient inducers of macrophage deactivation than similar preparations derived from the avirulent Downs Hc strain. Yeast cells of two other pathogenic fungi,Candida albicans andCryptococcus neoformans are shown to stimulate rather than deactivate a macrophage OB.     

Localization of immuno-analogues of erythrocyte protein 4.1 and spectrin in epidermis of psoriasis vulgaris

The presence and localization of immuno-analogues of human erythrocyte protein 4.1 and spectrin were examined in the epidermis of psoriasis vulgaris. Immunoblot analysis with antibodies against human erythrocyte protein 4.1 revealed that psoriatic epidermis contains a 4.1-like protein of 80 kDa, and also minor immunoreactive polypeptides, including a 45-kDa polypeptide. The 45-kDa band was not detected in non-lesional epidermis. Lesional epidermis of psoriasis contains spectrin-like proteins of 240 kDa. Analysis with immunofluorescence microscopy revealed that 4.1-like proteins were detected mainly in the cytoplasm of the suprabasal cells in lesional epidermis and in the peripheral cytoplasm of the basal cells in non-lesional epidermis. On the other hand, spectrin-like proteins were localized to the peripheral cytoplasm of basal keratinocytes in both lesional and non-lesional psoriatic epidermis. The present results indicate that proteins related to protein 4.1 and spectrin are consistently detected within epidermal cells of psoriasis, a chronic skin disease characterized by epidermal hyperplasia; the expression and distribution of protein 4.1 in lesional epidermis of psoriasis differs from that in non-lesional epidermis. These membrane skeletal proteins may be of significance in the hyperproliferative epidermis of psoriasis.