Rapid Ca2+ efflux from intracellular stores during cardiac muscle excitation-contraction coupling is mediated by the ryanodine-sensitive calcium-release channel, a large homotetrameric complex present in the sarcoplasmic reticulum. We report here the identification, primary structure and topological analysis of the ryanodine receptor-calcium release channel from human cardiac muscle (hRyR-2). Consistent with sedimentation and immunoblotting studies on the hRyR-2 protein, sequence analysis of ten overlapping cDNA clones reveals an open reading frame of 14901 nucleotides encoding a protein of 4967 amino acid residues with a predicted molecular mass of 564 569 Da for hRyR-2. In-frame insertions corresponding to eight and ten amino acid residues were found in two of the ten cDNAs isolated, suggesting that novel, alternatively spliced transcripts of the hRyR-2 gene might exist. Six hydrophobic stretches, which are present within the hRyR-2 C-terminal 500 amino acids and are conserved in all RyR sequences, may be involved in forming the transmembrane domain that constitutes the Ca(2+)-conducting pathway, in agreement with competitive ELISA studies with a RyR-2-specific antibody. Sequence alignment of hRyR-2 with other RyR isoforms indicates a high level of overall identity within the RyR family, with the exception of two important regions that exhibit substantial variability. Phylogenetic analysis suggests that the RyR-2 isoform diverged from a single ancestral gene before the RyR-1 and RyR-3 isoforms to form a distinct branch of the RyR family tree.
Mammalian brain possesses ryanodine-sensitive Ca2+ channels, which in muscle cells mediate rapid Ca2+ release from intracellular stores during excitation-contraction coupling. Analysis of bovine brain ryanodine receptor (RyR) channels suggests specific expression of the cardiac-muscle RyR isoform in mammalian brain. Localization using cardiac-muscle RyR-specific antibodies and antisense RNA revealed that brain RyRs were present in dendrites, cell bodies and terminals of rat forebrain, and highly enriched in the hippocampus. Activity of skeletal-muscle RyR channels is coupled to sarcolemmal voltage sensors, in contrast with cardiac-muscle RyR channels, which are known to be Ca(2+)-induced Ca(2+)-release channels. Thus Ca(2+)-induced Ca2+ release from intracellular stores mediated by brain RyR channels may be a major Ca(2+)-signalling pathway in specific regions of mammalian brain, and hence may play a fundamental role in neuronal Ca2+ homoeostasis.
and the §Department ofBiochemistry, Charing Cross and Westminster Medical School, London SUMMARY The prevalence of human papillomavirus (HPV) 6, 11, 16, and 18 in 36 heterosexual couples and seven male homosexual couples with genital warts was investigated for evidence ofsexual transmission of genital HPV. The prevalence of virus type and number of copies of viral genome equivalents/cell in the lesions were assessed, and the factors influencing transmission analysed. Our results show that HPV 6 and, to a lesser extent, HPV 11 were the types most readily transmitted, and that transmission appears to depend on the copy number and the duration and frequency ofexposure.The results of clinical studies of patients with genital warts have shown that these lesions are sexually transmissible.' To date, there have been no extensive studies of the transmission of different viral types present in genital warts using deoxyribonucleic acid (DNA) hybridisation techniques. We have developed a technique for assessing the type and quantity of human papillomavirus (HPV) DNA in cell samples and biopsy specimens,2 and have applied it to a study of the sexual transmissibility of HPV 6,11, 16, and 18,j which commonly cause genital warts.Campion et al reported that the female partners of men with genital warts have an increased risk of developing cervical intraepithelial neoplasia (CIN) and cited this evidence to support the hypothesis that HPV plays a part in cervical carcinogenesis.7 In this study we have also investigated that aspect of infection.
PATIENTS, MATERIALS AND METHODS
PATIENTSPatients presenting with genital warts to the Praed Street clinic for sexually transmitted diseases were Address for reprints:
Cervical scrapes were obtained from 215 women with cytologically normal cervices and 74 women with cervical intraepithelial neoplasia (CIN) and probed for HPV 6, 11, 16, and 18 by dot-blot hybridization. Viral copy numbers were determined by densitometric scanning of autoradiographs. The prevalence of HPV DNA in women with normal smears was as follows: 23 per cent of women attending a Family Planning Clinic (FPC), 16 per cent of women attending a Sexually Transmitted Diseases (STD) clinic, and 48 per cent of women attending a laser follow-up clinic after treatment of CIN. Ten per cent of women with normal cervices harboured HPV 16. Viral copy numbers ranged from 1300 to 52,800 genome equivalents per cell. Twenty-seven HPV-positive women with normal smears (including four patients infected with HPV 16) were followed for up to 3 years. None developed CIN irrespective of copy number. Our results show that HPV is present in normal cervices in the absence of CIN. Copy number has no relation to the development of CIN and factors other than the amount of HPV DNA may trigger the neoplastic process.
Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide a general mechanism for the fusion of lipid bilayers in biomembrane fusion reactions.
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