Characterization of rat testicular peritubular myoid cells in culture: alpha-smooth muscle isoactin is a specific differentiation marker

In frozen sections of testes from 20-day-old rats, alpha-smooth muscle (SM) isoactin was prominently immunostained in the peritubular tissue and in vascular walls, but not in areas populated by germinal cells, interstitial cells, or Sertoli cells. Peritubular myoid cell (PMC)-enriched preparations were isolated by two different procedures involving our previously published sequential enzymatic treatment ("conventional peritubular cell [PC]-enriched preparation") and by density-gradient purification of PMC from these preparations. The properties of different populations of PMC in culture were compared with respect to plating efficiency, rates of proliferation, and presence of cytoskeletal proteins. PMC, maintained in culture under defined conditions, contained proteins immunoreactive with monoclonal antibodies against alpha-SM isoactin. This was detected by immunostaining and by Western blots of cell extracts subjected to gel electrophoresis. Neither Sertoli cells, skin fibroblasts, bovine endothelial cells, nor glial cells contained alpha-SM isoactin detectable by the above techniques. We report the ontogeny of alpha-SM isoactin in the peritubular tissue of testes at different stages of gonadal development, and show that it is detectable within 8 days after birth. In addition, we describe immunocytochemical changes that occur during culture in various media of PMC prepared from testes of 20-day-old rats. We compare the use of alpha-SM isoactin as a differentiation marker for PMC with the use of desmin in facilitating the identification of PMC, and in following alterations in phenotype during culture in various culture media. Data presented demonstrate that about 81% of cells in the "conventional PC-enriched preparation," and about 94% of cells in the more purified populations of PMC were positive for alpha-SM isoactin in cells maintained in culture for 18 h after plating. These same PMC also were shown to express vimentin and plasminogen activator inhibitor, type 1. We conclude that alpha-SM isoactin is an excellent specific marker for PMC in seminiferous tubules and in culture.     

Stress factors affecting expression of O6-methylguanine-DNA methyltransferase mRNA in rat hepatoma cells.

O6-Methylguanine-DNA methyltransferase (MGMT) is decisively involved in protecting mammalian cells against genotoxic effects of alkylating carcinogens. We analysed regulation of MGMT expression after exposing rat hepatoma H4IIE cells to various 'stress' factors. Treatments that damage DNA such as alkylation, hydrogen peroxide, ultraviolet or X-ray exposure, as well as restriction enzymes introduced into cells by electroporation or arrest of replication by hydroxyurea significantly induced MGMT mRNA (2.5 to 5-fold). Slight induction (up to 2.5-fold) was observed after heat shock or cadmium/zinc treatment. No or only a very weak induction (less than 1.5-fold) was observed after treatment with 6-thioguanine, 5-azacytidine, transfection of methylated DNA, depletion of MGMT by feeding with O6-methylguanine or O6-benzylguanine, serum starvation and feeding of starved cells, cAMP, TPA and dexamethasone treatment. Inhibitors of protein kinases, H8 and H9, induced MGMT mRNA. On the other hand, an inhibitor of phosphatases (sodium vanadate) prevented induction of MGMT by N-methyl-N'-nitro-N-nitrosoguanidine. The data indicate that DNA breaks are an ultimate signal for MGMT mRNA induction and that protein phosphorylation is involved in regulating MGMT expression.     

Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum

Immunoglobulin light and heavy chains are synthesized in mammalian cells as precursors containing a signal peptide. Processing and assembling result in formation of active antibodies. Chimeric genes have been made containing the coding sequence of the barley -amylase signal peptide which has been fused to cDNAs coding for either the mature light or the mature heavy chain of a monoclonal antibody. A plasmid was constructed linking both chimeric genes under the control of plant active promoters in an expression cassette. This DNA fragment was stably integrated into the genome of Nicotiana tabacum by Agrobacterium tumefaciens mediated gene transfer. Synthesis of light and heavy chains and assembly to antibodies was detected in transgenic tobacco tissue using specific secondary antibodies. By electron microscopic immunogold labeling, the presence of assembled antibody could be detected within the endoplasmic reticulum. Affinity chromatography indicated biological activity of the assembled immunoglobulin produced in plant cells. Unexpectedly, a significant amount of assembled antibodies was found within chloroplasts.     

The Status of Cassava Bacterial Blight Caused by Xanthomonas campestris pv. manihotis in Trinidad

Disease surveys conducted in Trinidad between 1985—1987 showed that Cassava Bacterial Blight (CBB) is present in all but one county of the country with disease severity ratings varying from 1—5 depending on day/night temperatures. Field and greenhouse screening identified varieties such as Point Fortin fine leaf and CMC 40 as being resistant whereas M col 22 was moderately resistant to susceptible. Using a combination of antiserum produced to whole cells of Xanthomonas campestris pv. manibotis and a broth enrichment technique, dissemination of the pathogen by flood water was confirmed. The pathogen was detected at distances of up to 300 meters from infected fields. The significance of this mode of pathogen dissemination in initiating primary infection in Trinidad is discussed.     

The lysis effect of bull spermatozoa on gelatin substrate film methodical investigations.

Proteolytic activity in the acrosomes of ejaculated bull spermatozoa was demonstrated using an autoradiographic film as a gelatin substrate. Incubation of the spermgelatin adducts at +37 degrees C and 94% humidity, which was kept constant by ventilating an incubator with water-saturated compressed air, yielded reproducible results. Gelatin depolymerisation started adjacent to the posterior segment of the acrosome within 30 to 60 s after application of individual spermatozoa to the substrate membrane and, finally, increased to a white circular digestion area enveloping the entire sperm head. The observed gelatinolysis seems to be mainly caused by acrosin, the trypsin-like acrosomal proteinase. This conclusion is supported by the positive correlation (r = +0.83, P is less than or equal to 0.01) found between the mean values of the lysis areas of individual spermatozoa on gelatin films and the acrosin activity of the sperm population measured with Bz-Arg-OEt as substrate after acidic extraction of the spermatozoa. In addition, prior saturation of the substrate layers with acrosin inhibitor (SSPI-I, II) from boar seminal plasma prevented the lysis reaction. Extraction of acrosin from the spermatozoa before application to the gelatin membranes resulted in a complete loss of any proteolytic activity. If spermatozoa were stored for 4 to 6 days at +4 degrees C or -20 degrees C in Tris buffer and afterwards applied to the substrate layer, lysis areas of individual spermatozoa differed markedly. Spermatozoa from undiluted ejaculated frozen at -20 degrees C showed no proteolytic effect on gelatin films. In general, there was a high correlation (r = +0.83, P is less than or equal 0.01) between the number of "living cells" characterized by live-dead staining and the percentage of spermatozoa active on the substrate membranes.     

Extrachromosomal inheritance of paromomycin resistance in Saccharomyces cerevisiae. Genetic and biochemical characterization of mutants

Summary In Saccharomyces cerevisiae, mutants were isolated which show high resistance to the aminoglycoside paromomycin. Amino acid incorporation of mitochondria isolated from such mutant strains proved also to be paromomycin resistant. All of them are cross-resistant to the structurally related antibiotic neomycin. Three independent methods revealed the resistance to be extrachromosomally, presumably mitochondrially inherited.     

Relation between the nuclei and the nucleoli during cell differentiation in roots of Vicia faba volumetric and cytochemical analysis

Summary The behaviour of the nuclei and the nucleoli of roots of Vicia faba during cell differentiation was studied quantitatively. The relations between these cell constituents and the polyploidy was analysed. The study was made on isolated nuclei and nucleoli and on plastic sections. A method for the isolation of nuclei and nucleoli of secondary roots fixed in formol was modified and another developed for material fixed in ethanol/acetic acid mixture. The volumetric investigation showed that the nuclear volume increases while the nucleolar decreases during cell differentiation. The mean number of nucleoli decreases. In Vicia faba there is no relation between the ploidy and the volume of nuclei and nucleoli; the protein synthesis rate has an influence on the size of these organelles. Quantitative investigation has shown the proportionality of dry weight, DNA, total protein, histone, protein-bound lysine and arginine content of the nuclei and their ploidy. The same experiments made on nucleoli showed linear relation between their content and volume. The concentration of analysed substances in constant in nucleoli.     

Beobachtungen einer Familie mit Galaktosämie und “Duarte-Variante”

Zusammenfassung Die Familienuntersuchungen einer galaktosämischen Patientin ergaben bei 5 Verwandten das gleichzeitige Vorkommen eines galaktosämischen Merkmals und eines varianten Enzyms (Duarte-Variante). Aktivität der Gal-1-Phosphat-Uridylyltransferase wurde mit Hilfe eines modifizierten UDPG-Verbrauchstests nach Beutler u. Baluda bestimmt.

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Summary 5 relatives of a patient with galactosemia showed a simultaneous occurence of the characteristic sign of galactosemia and of a variant enzyme (Duarte variant). The activity of Gal-1-Phosphat-Uridylyltransferase was determined by a modification of the UDPG-consumptiontest of Beutler and Baluda.

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With the aid of: Landesamt für Forschung des Landes Nordrhein-Westfalen und Landschaftsverband Westfalen Lippe.     

Biosynthesis of the carbohydrate portion of immunoglobulins. Incorporation of radioactive fucose into immunoglobulin G1 synthesized and secreted by mouse plasma-cell tumour MOPC 21

Incorporation of radioactive fucose into the immunoglobulin G1 myeloma protein secreted by mouse plasma-cell tumour MOPC 21 is stereospecific for the l-isomer. Heavy chains of the secreted form of the myeloma protein carry 90% of the label in fucose residues of their carbohydrate moieties. A small but significant amount of the intracellular immunoglobulin G1 of the mouse plasma-cell tumour MOPC 21 appears to be labelled. Serum in the incubation medium supplies low-molecular-weight diffusible substances necessary to maintain continuous secretion of fucose-labelled myeloma protein beyond 2-3h, and of leucine-labelled myeloma protein beyond 6-8h. In medium containing extensively dialysed serum the secretion of leucine- and fucose-labelled myeloma protein can be restored by the addition of 250mum-d-mannose, 250mum-d-galactose and 250mum-glucosamine. Synthesis and secretion appear to be facilitated in the presence of these sugars, although secretion of myeloma protein devoid of terminal fucose residues is possible for a limited time-period.