Purified cytochrome b561 catalyzes transmembrane electron transfer for dopamine beta-hydroxylase and peptidyl glycine alpha-amidating monooxygenase activities in reconstituted systems

Cytochrome b561 from bovine adrenal medulla chromaffin granules has been purified by fast protein liquid chromatography chromatofocusing. The purified cytochrome was reconstituted into ascorbate-loaded phosphatidylcholine vesicles. With this reconstituted system transmembrane electron transfer for extravesicular soluble dopamine beta-hydroxylase activity was demonstrated. In accordance with the model proposed by Njus et al. (Njus, D., Knoth, J., Cook, C., and Kelley, P. M. (1983) J. Biol. Chem. 258, 27-30), catalytic amounts of a redox mediator were necessary to achieve electron transfer between cytochrome and soluble dopamine beta-hydroxylase. Our observations also showed that when membranous dopamine beta-hydroxylase was reconstituted on cytochrome containing vesicles, electron transfer occurred only in the presence of a redox mediator. Since cytochrome b561 has been found in secretory vesicles associated with peptidyl glycine alpha-amidating monooxygenase, electron transfer to this enzyme was also examined. Analogous to the results obtained for dopamine beta-hydroxylase, transmembrane electron transfer to peptidyl glycine alpha-amidating monooxygenase appears to require a redox mediator between cytochrome and this monooxygenase. These observations indicate that purified cytochrome b561 is capable of providing a transmembrane supply of electrons for both monooxygenases. Since no direct protein to protein electron transfer occurs, the results support the hypothesis that the ascorbate/semidehydroascorbate redox pair serves as a mediator for these enzymes in vivo.     

Behavioral changes of a juvenile gorilla after a transfer to a more naturalistic environment

Play behavior and stress-associated behavior of a captive juvenile gorilla were observed before and after his transfer to a larger and more natural environment. The gorilla was observed for 4 months after the transfer and at 1 and 4 years after the transfer. Throughout his first month in the new environment play decreased dramatically. Although play subsequently increased again 2 months and 1 year after the transfer, it never reached the levels of play in the old environment. Four years after the move his play had decreased again to the low level of his first month in the new environment. Two of his stress-associated behaviors, coprophagy and regurgitation/reingestion, decreased after the transfer. Self-clasping behavior increased initially in the new environment and remained at high levels 1 year after the move. Four years after the move his self-clasping behavior was significantly less than at 1 year after the move; however, it continued to be significantly greater than in the old environment. These findings suggest that larger and more natural environments do not necessarily result in more play activity or a reduction in all stress-associated behavior.     

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Upregulation ofE. coli 38kDa proteins induced by glutaraldehyde and formaldehyde

Exposure of the W3110 strain ofEscherichia coli K12 to low concentrations of glutaraldehyde or formaldehyde results in an unusual pattern of protein expression, as determined by high-resolution, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A decline in total protein synthesis is accompanied by the upregulation of three proteins of approximate molecular weight 38 kDa. In the presence of 0.1 mM glutaraldehyde this response occurs within the first 5 min of incubation, and with 0.1 mM formaldehyde, within the first 30 min of incubation. The 38 kDa proteins continue to be expressed at high levels until cell death. Comparison of our 2-D PAGE patterns withE. coli gene-protein and plasmid indexes indicates that one of the proteins may be the major gene product of thepyrC locus. This pattern of protein synthesis may indicate a novelE. coli stress response.     

Human papillomaviruses in anogenital warts in children: typing by in situ hybridisation.

OBJECTIVE--To identify the types of human papillomaviruses found in anogenital warts in children and to relate these to clinical and social information. DESIGN--In situ hybridisation using biotin labelled DNA probes to 11 types of human papillomavirus was performed on biopsy specimens from 17 children with anogenital warts. SETTING--Nuffield department of pathology and the department of dermatology, Oxford. PATIENTS--Children in one group were referred by general practitioners or paediatricians to the dermatology department, where biopsies were performed. The other children were seen in four different hospitals, and biopsy specimens were submitted to the laboratory at the physician''s or pathologist''s request. RESULTS--Of the 17 biopsy specimens, 10 contained cells positive with a probe to a genital human papillomavirus type (types 6 or 11), while six were positive with a skin virus type (types 2 or 3). One was negative. The virus type present bore no relation to the site or appearance of the warts. The virus type did, however, appear to correlate with groups of children. Skin types were commoner in older children (over 4 years), in those with a relative who had skin warts, and in children with warts elsewhere; there was no relation with the child''s sex and no suspicion of sexual abuse in these children. These circumstances suggested non-sexual transmission, such as autoinoculation. In contrast, genital types were commoner in girls, in children under 3 years, in children with relatives with genital warts, and in those with no warts elsewhere. Nevertheless, there was suspicion or evidence of sexual abuse in only half these children, suggesting that other routes of transmission--for example, perinatal--might have been implicated. CONCLUSION--Anogenital warts in children may contain either skin or genital wart virus type. Although the type of human papillomavirus present may give some indication of the likely mode of transmission, this can be interpreted only in conjunction with all available clinical and social information. The type of virus does not provide proof of the presence or absence of sexual transmission.     

Expectations of assisted conception for infertility.

OBJECTIVE--To provide reliable prognostic information for couples seeking assisted conception. DESIGN--Analysis of four years'' practice (1988-91). SETTING--Private university service linked with NHS reproductive medicine services. PATIENTS--804 couples with various causes of subfertility, median duration five years, median age of women 34 years. INTERVENTIONS--1280 completed cycles: 950 in vitro fertilisation, 144 gamete intrafallopian transfer, and 186 intrauterine insemination and superovulation. MAIN OUTCOME MEASURES--Pregnancy and birth rates per cycle and cumulative pregnancy and take home baby rates per couple. RESULTS--In women under 40 years and men with normal sperm, whatever the cause of infertility, results with in vitro fertilisation improved steadily reaching a pregnancy rate per cycle of 30% (95% confidence interval 26% to 35%) during 1990-1 and birth rate per cycle of 29% (23% to 35%) in 1990. Pregnancy and birth rates for gamete intrafallopian transfer were 36% (28% to 44%) and 26% (17% to 37%) and for intrauterine insemination 18% (12% to 24%) and 16% (10% to 22%). After six cycles cumulative probability of pregnancy was 82% and cumulative take home baby rate 70%. Considering only in vitro fertilisation and gamete intrafallopian transfer after four cycles the pregnancy rate was 78% (66% to 91%). CONCLUSIONS--Conception is less likely in women over 40 and men with sperm dysfunction. For other couples the prognosis for a live birth is at least as good as for fertile couples if they persist with treatment.     

Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells

In attempts to determine the mechanism of proliferation of arterial smooth muscle cells (SMC) in intimal atheromatous lesions, autocrine secretion of growth factors by SMC has recently received much attention. Here we report a new growth factor named smooth muscle cell derived growth factor (SDGF). Cultured rabbit medial SMC secreted SDGF for 1 week during their incubation in serum-free media only after at least 4 passages. SDGF differed from platelet derived growth factor (PDGF) physicochemically, immunologically, and biologically. The properties of SDGF also seemed different from those of other known growth factors that stimulate the proliferation of mesenchymal cells.