The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

Homologous and heterologous β-adrenergic desensitization in hepatocytes. Additivity and effect of pertussis toxin

In hepatocytes obtained from hypothyroid rats, phorbol myristate acetate (PMA) and vasopressin diminished the accumulation of cyclic AMP and the stimulation of ureagenesis induced by isoprenaline or glucagon without altering significantly the accumulation of cyclic AMP induced by forskolin. Pretreatment with PMA markedly reduced the stimulation of ureagenesis and the accumulation of cyclic AMP induced by isoprenaline or glucagon. In membranes from cells pretreated with PMA, the stimulation of adenylate cyclase induced by isoprenaline + GTP, glucagon + GTP or by Gpp[NH]p were clearly diminished as compared to the control, whereas forskolin-stimulated activity was not affected. The data indicate heterologous desensitization of adenylate cyclase. It was also observed that the homologous (García-Sáinz J.A. and Michel, B. (1987) Biochem. J. 246, 331–336) and this heterologous β-adrenergic desensitizations were additive. Pertussis toxin treatment markedly reduced the heterologous desensitization of adenylate cyclase but not the homologous β-adrenergic desensitization. It is concluded that the homologous and heterologous desensitizations involve different mechanisms. The homologous desensitization seems to occur at the receptor level, whereas the heterologous probably involves the guanine nucleotide-binding regulatory protein, Ns.     

Mutation analysis of phenylketonuria in Spain: prevalence of two Mediterranean mutations

Summary We have screened seven Spanish phenylketonuric (PKU) families for the most prevalent Mediterranean and Caucasian mutations, and have subsequently found mutations P281L and IVS10. We have analyzed these two mutations in 23 of our patients. The frequencies found correspond to those of Mediterranean countries, such as Italy. This is the first report of a mutation analysis in the Spanish PKU population.     

Phosphorylation of the presynaptic protein B-50 (GAP-43) is increased during electrically induced long-term potentiation.

The protein B-50 (F1, GAP-43) is a presynaptic-specific substrate of protein kinase C, functionally related to neurotransmitter release. An increase in phosphorylation of this protein has been proposed as a molecular mechanism underlying long-term potentiation (LTP). B-50 phosphorylation measured by quantitative immunoprecipitation in rat hippocampal slices incubated in the presence of radiolabeled inorganic phosphate was increased for at least 1 hr after the induction of LTP in the CA1 region. No significant changes in B-50 phosphorylation were observed in untetanized slices stimulated at low frequency. The direct demonstration of an increased phosphorylation of the protein B-50 during LTP is consistent with the hypothesis that presynaptic mechanisms contribute to maintenance of LTP.     

Early human disturbance of the natural environment recorded in annually laminated sediments of Lake Gosciaz,central Poland

Palynological records from the annually laminated sediments of Lake Gosciaz are presented for the period ca. 7600–3900 conventional B.P. Indications in percentage and influx diagrams of Mesolithic and Early Neolithic human impact on the natural environment around the lake are discussed and compared with the archaeological evidence. The first indications of human disturbance start at ca. 6700 B.P. and in the following period four disturbance phases with interruptions are distinguished. A relationship between the Elm Decline and an increasing thickness of annual sediment layers as a consequence of increased soil erosion is suggested. Some hitherto unrecognized or incompletely known microfossils are identified and figured, and their palaeoecological significance is discussed. The records of akinetes of Aphanizomenon probably indicate increasing eutrophication of the lake. Charred fragments of grass epidermis are possibly indicative of openings in the forest.     

Monoclonal antibody-defined antigens of human prostate cancer cell line PC3

Summary Over 600 hybridomas were derived from the immunization of mice with live cells and aqueous extracts of the human prostatic carcinoma cell line PC3. A total of 26 hybridomas with restricted reactivities were selected, subcloned and antibodies tested on a variety of tumor and normal cells. Seven monoclonal antibodies showed reactivity for prostate cancer and other tumor cell lines, including breast carcinomas. Three of the antibodies obtained after immunization with live cells reacted with live cells only and three of the four antibodies obtained after immunization with cell extract reacted with cell extracts and spent culture media. The fourth antibody in the latter group was reactive only in the immunoperoxidase staining assay. Antibody PrS5 recognized a 90,000 molecular weight molecule from ¹²⁵I-surface-labeled cells in immunoprecipitation analysis. Antibodies PrE3 and PrD8 detected a nonacid glycolipid pentasaccharide from PC3 cells and meconium, and a glycoprotein of 115,000 molecular weight from ¹²⁵I-surface-labeled red blood cells. The similar patterns of reactivity in RIAs and antigen analysis suggest that antibodies PrE3 and PrD8 recognize the same molecule. The results emphasize the usefulness of immunohistochemistry in the testing of monoclonal antibodies and the impact of the form in which the antigen is presented on the resultant antibody specificity     

Characterization of inositolpolyphosphate binding to myocardial membranes

Although it is well-accepted that the phosphatidylinositol signalling transduction pathway, producing inositol-1,4,5-P3 (InsP₃) and inositol-1,3,4,5-P₄ (InsP4) as second messengers, functions in heart muscle, virtually nothing is known about the roles of the higher inositol polyphosphates such as inositolhexakisphosphate (InsP₆). This study demonstrates that InSP₆ has the ability to bind intracellularly, with different binding characteristics, to different myocardial membranes. Binding to purified sarcoplasmic reticulum (SR) membranes, purified sarcolemmal (SL) membranes as well as to viable mitochondria were characterized. Binding to all these membranes display high as well as low affinity binding sites, with differing affinities. Kd values of binding to SR were 32 and 383 nM, to SL 61 and 1312 nM, while those of mitochondrial binding were 230 and 2200 nM respectively.InsP₄ binding was also investigated and displayed the following characteristics: to SR, one low affinity binding site (Kd = 203 nM) and to SL, a high as well as a low affinity binding site with Kd values of 41 and 2075 nM respectively. Presence of InsP₃, the second messenger for SR calcium release, at concentrations of 1 nM, elevated the binding of InsP₄ to SR and SL by a mean of 30% and 20% respectively.Fractionation of SR and SL membranes on sucrose density gradients, after solubilization with CHAPS, indicated that InsP₆ bound to two separate protein peaks in both these membranes, while InsP₄ bound to only one. In SR membranes, InsP₄ bound preferentially to a protein separating at high sucrose density while it bound to a protein separating at low sucrose density in SL membranes.     

Non-motile mutants of Helicobacter pylori and Helicobacter mustelae defective in flagellar hook production

Flagellar hooks were purified from Helicobacter pylori and Helicobacter mustelae. The 70 × 16nm H. pylori hook was composed of FIgE subunits of 78kDa, while the 72 × 16nm H. mustelae hook was composed of 87kDa subunits. N-terminal sequence was obtained for the FIgH proteins of both species, and for an internal H. mustelae FlgE peptide. Degenerate oligonucleotide primers allowed amplification of a 1.2 kb fragment from the H. mustelae chromosome, which carried part of the flgE gene. The corresponding H. pylori gene was cloned by immunoscreening of a genomic library constructed in λZAP Express, The translated H. pylori flgE sequence indicated a protein with limited homology with the hook proteins from Salmonella typhimurium and Treponema phagedenis. Mutants of H. pylori and H. mustelae defective in hook production generated by allele replacement were non-motile and devoid of flagellar filaments but produced both flagellin subunits, which were localized in the soluble fraction of the cell. The level of flagellin production was unchanged in the mutants, indicating that the regulation of flagellin expression in Helicobacter differs from that in the Enterobacteriaceae.