Mutations for the autosomal recessive and autosomal dominant forms of polycystic kidney disease are not allelic

Summary The autosomal dominant form of polycystic kidney disease (ADPKD) has been linked to the -globin gene locus on 16p. Linkage studies between the autosomal recessive type (ARPKD) and the 3 HVR of the -globin gene cluster showed that the ARPKD and ADPKD are not allelic.     

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells

Summary DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h ⁵¹Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants. Abbreviations used: NK, natural killer; NC, natural cytotoxic; NCMC, natural cell-mediated cytotoxicity; asGM1, asialo GM1; LL, large lymphocytes; LGL, large grnular lymphocytes; LAL, large agranular lymphocytes; PBMNC, peripheral blood mononuclear cells; E:T, effector to target cell ratio; C:H, cold to hot cell ratio; FBS, fetal bovine serum     

Structure and carcinogenicity of Dibenzo(c,g)carbazole derivatives

The carcinogenicity of several groups of carcinogens is evoked with particular reference to Dibenzo(c,g)carbazole derivatives. The activity of these derivatives is discussed with respect to their species and organ specificity. The enzymatic equipment is decisive as to whether the compounds formed can react with DNA or are simply detoxified and eliminated. All these carcinogens are complete carcinogens, i.e. they have the property of both initiation and promotion.     

Properties of the 12-O-Tetradecanoylphorbol-13-Acetate-Stimulated S6 Kinase from Rat Astroglial Cells

The S6 kinase activity of astroglial cells in primary culture stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied. This activity was eluted as a single peak at 0.15 M NaCl from a DEAE-Sephacel column. The chromatography of this peak on phosphocellulose revealed an activity eluted at 0.15 M NaCl. This partially purified enzyme had a sedimentation coefficient of 3.7S; Km values were 2 X 10(-5) M for ATP and 10(-6) M for 40S ribosomal subunits. The optimal Mg2+ concentration requirement was 2-3 mM. Mn2+ and Co2+ could substitute for Mg2+ (optimum concentrations 1.5 and 0.8 mM, respectively), but these cations were strong inhibitors in the presence of Mg2+. The enzyme was inhibited by N-ethylmaleimide, indicating that it contained thiol groups. This S6 kinase used ATP, but not GTP, as a phosphate donor, and exhibited great specificity for S6 as phosphate acceptor. Whole histones and protamine were slightly phosphorylated whereas phosvitin, histone H1, and surprisingly the peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala were not phosphorylated. The TPA-stimulated S6 kinase resembles the insulin-, fibroblast growth factor- and cyclic AMP-stimulated enzymes, suggesting that several pathways might activate the same entity.     

Formation of the Neurotransmitter Glycine from the Anticonvulsant Milacemide Is Mediated by Brain Monoamine Oxidase B

Milacemide (2-n-pentylaminoacetamide) is a secondary monoamine that in the brain is converted to glycinamide and glycine. This oxidative reaction was suspected to involve the reaction of monoamine oxidase (MAO). Using mitochondrial preparations from tissues that contain MAO-A and -B (rat brain and liver), MAO-A (human placenta), and MAO-B (human platelet and bovine adrenal chromaffin cell), it has been established that mitochondria containing MAO-B rather than MAO-A oxidize (H2O2 production and glycinamide formation) milacemide. The apparent Km (30-90 microM) for milacemide oxidation by mitochondrial MAO-B preparations is significantly lower than that for milacemide oxidation by mitochondrial MAO-A (approximately 1,300 microM). In vitro MAO-B (l-deprenyl and AGN 1135) rather than MAO-A (clorgyline) selectively inhibited the oxidation of milacemide. These in vitro data are matched by ex vivo experiments where milacemide oxidation was compared to oxidation of serotonin (MAO-A) and beta-phenylethylamine (MAO-B) by brain mitochondria prepared from rats pretreated with clorgyline (0.5-10 mg/kg) and l-deprenyl (0.5-10 mg/kg). Furthermore, in vivo experiment demonstrated that l-deprenyl selectively increased the urinary excretion of [14C]milacemide and the total radioactivity with a concomitant decrease of [14C]glycinamide. Such changes were not observed after clorgyline treatment, but were evident only at doses beyond clorgyline selectivity. The present data therefore demonstrate that milacemide is a substrate for brain MAO-B, and its conversion to glycinamide, further transformed to the inhibitory neurotransmitter, glycine, mediated by this enzyme may contribute to its pharmacological activities.     

Glutamate Dehydrogenase Activity Is Markedly Higher in a "Golgi-Bergmann"-Like Glial Clone than in Other Astroglial Cell Lines

Glutamate appears to be the neurotransmitter of granule cells, the major neuronal population of the cerebellar cortex. To determine the role of astroglial cells in the synthesis of glutamate, we have measured the specific activity of glutamate dehydrogenase (GDH) in clonal cell lines that might be the in vitro equivalents of the different cerebellum astroglial cell types. In conditions where GDH operates in the direction of glutamate synthesis, the specific activity of GDH measured in the "Golgi-Bergmann"-like clone was 4-6 times higher than in the "velate protoplasmic"- or "fibrous-like" astrocytic clones. These data correlate well with the intense immunoreactivity to GDH in Golgi-Bergmann astrocytes in vivo that has been recently reported.     

Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus

Summary The structural genes (hup) of the H₂ uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization with the structural genes of the H₂ uptake hydrogenase of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup^-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68108 dalton) and by alignment with the NH₂ amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34 256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology, but to a lesser extent, with the hydrogenase of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues, (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe–4S] ferredoxins.     

Early quantitative method for measuring germination in non-green spores of Dryopteris paleacea using an epifluorescence-microscope technique

A method is described to determine germination by blue-light excited red fluorescence in the positively photoblastic spores of Dryopteris paleacea Sw. This fluorescence is due to chlorophyll as evidenced from 1) a fluorescence-emission spectrum in vivo, where a bright fluorescence around 675 nm is obtained only in red light (R)-irradiated spores and 2) in vitro measurements with acetone extracts prepared from homogenized spores. Significant amounts of chlorophyll can be found only in R-treated spores; this chlorophyll exhibits an emission band around 668 nm, when irradiated with 430 nm light at 21°C.
Compared to other criteria for germination, such as swelling of the cell, coat splitting, greening, and rhizoid formation, which require longer periods after induction for their expression, chlorophyll fluorescence can be used to quantify germination after two days. This result is confirmed by fluence-response curves for R-induced spore germination; the same relationship between applied R and germination is obtained by the evaluation with the epifluorescence method 2 days after the light treatment as compared with the evaluation with bright-field microscopy 5 days after the inducing R.
Using this technique we show for the first time that Ca²⁺ contributes to the signaltransduction chain in phytochrome-mediated chlorophyll synthesis in spores of Dryopteris paleacea .     

Production and Characterization of Monoclonal Antibodies to Wall-Localized Peroxidases from Corn Seedlings

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a M_r 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a M_r 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.     

Nuclear protein transitions in cuttle-fish spermiogenesis: Immunocytochemical localization of a protein specific for the spermatid stage

The changes in basic nuclear proteins throughout cuttle-fish spermiogenesis were investigated both by immunocytochemical procedures and by isolation of late spermatid nuclei (by virtue of their resistance to sonication). Antibodies were raised in rabbits to a protein, named protein T, isolated from testis chromatin. The anti-protein T immune serum was found to recognize protein T and not histones from the testis. Immunoperoxidase staining of sections or of smears of testis with anti-protein T antibodies showed that protein T appears in the nuclei of round spermatids, is abundant in elongating spermatid nuclei, but cannot be detected in elongated spermatids. Nuclei from these elongated spermatids were isolated by sonication treatment of testis cells. A protein, named protein Sp, with the characteristic mobility of a protamine, was isolated from elongated spermatid nuclei. This protein has the same mobility as the protamine present in mature spermatozoa. Taken together, the results indicate that in cuttle-fish, nuclear protein transitions involve the replacement of histones by a spermatid-specific protein (protein T), which is replaced at the end of elongation of the nucleus by a protamine (protein Sp). Thus, spermiogenesis of the cuttle-fish (and perhaps of other cephalopods), shows two basic nuclear protein transitions, which are similar to the transitions observed in higher vertebrates such as mammals.