Copy number determination of different derivatives of the streptomycete mini-plasmid pSLG33

Copy numbers of the streptomycete plasmid vector pRS410 and five other recombinant plasmid derivatives of the original cryptic streptomycete plasmid pSLG33 were determined using calibrated laser densitometry. DNA preparations, electrophoretically separated on agarose gels, were stained with ethidium bromide, photographed and the negatives were subsequently scanned in a laser densitometer. The pSLG33 replicon is very stable, as no effect of the selective pressure was observed. It is a multicopy plasmid with up to 220 detected copies per chromosome. The use of deletion and/or insertion mutants allowed us to define two regions of the pSLG33 molecule involved in the control of plasmid replication.     

Natriuretic Peptides Inhibit Nicotine-Induced Whole-Cell Currents and Catecholamine Secretion in Bovine Chromaffin Cells: Evidence for the Involvement of the Atrial Natriuretic Factor R2 Receptors

Abstract: There is increasing evidence that members of the natriuretic peptide family display sympathoinhibitory activity, but it remains uncertain which receptor pathway is implicated. We performed cyclic GMP production studies with chromaffin cells treated with either atrial natriuretic factor (ANF) or C-type natriuretic peptide (CNP) and found that these cells specifically express the ANF-R_1C but not the ANF-R_1A receptor subtype. Evidence for the existence of ANF-R₂ receptors was obtained from patch-clamp experiments where C-ANF, an ANF-R₂-specific agonist, inhibited nicotinic currents in single isolated chromaffin cells. Involvement of ANF-R₂ receptors in the modulation of nicotinic currents was further supported by the significant loss of this inhibitory activity after the cleavage of the disulfide-bridged structure of C-ANF. This linearized form of C-ANF also displayed a lower binding affinity for ANF-R₂ receptors. Like the patch-clamp studies, secretion experiments demonstrated that both CNP and C-ANF are equally effective in reducing nicotine-evoked catecholamine secretion by cultured chromaffin cells, raising the possibility that this effect of CNP is predominantly mediated by the ANF-R₂ and not the ANF-R_1C receptors. Finally, this response appears to be specific to nicotinic agonists because neither histamine- nor KCI-induced secretions were affected by natriuretic peptides. In the present study, we report (1) the presence of ANF-R_1C and ANF-R₂ receptor subtypes in bovine chromaffin cells, (2) the inhibition by natriuretic peptides of nicotinic whole-cell currents as well as nicotine-induced catecholamine secretion, (3) the possible mediation of these effects by the ANF-R₂ class of receptors, and (4) the specificity of this inhibition to nicotinic agonists. Because bovine chromaffin cells release ANF, BNP, and CNP together with catecholamines, all three peptides might exert negative feedback regulation of catecholamine secretion in an autocrine manner by interacting with the nondiscriminating ANF-R₂ receptor subtype.     

REPRODUCTION IN THE FEMALE SUBANTARCTIC FUR SEAL, ARCTOCEPHALUS TROPICALIS

The reproductive tracts of 89 female subantarctic fur seals, taken at Gough Island between November 1977 and October 1978, were examined. Females started ovulating at age 4 yr and all 6-yr-old females were sexually mature. They are mono-ovulatory, alternating between ovaries, and only single embryos were found. Females older than 13 yr ( n = 11) showed poor follicular development and some failed to ovulate. The gestation period (first recorded ovulation to first recorded birth) was 360 d, while delayed implantation (first recorded ovulation to first recorded implantation) lasted for 139 days. Follicle numbers in the functional ovary declined sharply after ovulation while the corpus luteum increased in size until at least 1 mo prior to parturition. The number of follicles in the contralateral ovary increased after midwinter (June/July), and the mean size of the largest follicles peaked prior to ovulation in December. The mean size of the largest follicles increased in both ovaries near implantation time, after reaching a low subsequent to, ovulation. The regressing corpus albicans, conspicuous for approximately 3 mo after parturition, could not be detected macroscopically within one year postpartum. Subantarctic fur seals at Gough Island have a distinct postreproductive class of older females. The pregnancy rate for all females ≥4 yr of age was 79%, and it was 84.5% for the sexually mature group of ≥6 yr of age, while the mean age at puberty was 4.80 yr.     

Phosphate regulation of phosphatases in submerged cultures of Claviceps purpurea 129 producing clavine alkaloids

Summary Claviceps purpurea strain 129 was cultivated under submerged conditions in a sucrose-citrate medium containing high (36.8 mM) or low (1.84 mM) KH₂PO₄ concentrations. The permeabilized cells and culture supernatants contained alkaline and acid phosphatases. In the medium containing a high phosphate concentration, the synthesis of extracellular phosphatases was repressed, but that of cellular phosphatases was not. Extracellular phosphatases, especially alkaline phosphatases, were derepressed by transferring the mycelium into a phosphate-free medium. This derepression was inhibited by cycloheximide. In the presence of cycloheximide, the activities of the cellular phosphatases decreased markedly, indicating turnover of these enzymes. The cellular acid phosphatase was inhibited by phosphate (0.025 M–0.1 M) and NaF (0.01 M) while the cellular alkaline phosphatase was only inhibited by phosphate. Both cellular and extracellular alkaline phosphatases were more sensitive to repression by phosphate than the acid phosphatases. The alkaloid synthesizing enzymes were: a) present in mycelia grown in high levels of phosphate and b) activated by decreasing the intracellular phosphate level.     

Inhibition of tumor growth in mice treated with synthetic muramyl dipeptide

Summary Treatment with synthetic MDP inhibited growth of transplantable, chemically induced tumors in syngeneic mice. The tumor-inhibitory effect was dependent on the schedule of MDP administration.Growth of SC transplants of a nonmetastasizing, MC-induced fibrosarcoma, MC11, was inhibited by local treatment with 200 g and 1,000 g MDP given SC 5–7 weeks before challenge. Treatment with lower (10 g and 100 g) doses of MDP and shorter (1–4 weeks) time intervals was not effective. Single doses of MDP (10–1,000 g) 1–3 weeks after challenge had no effect.Growth of IV-inoculated, metastasizing AAT-induced hepatoma A was inhibited by IV injections of 20 g MDP given 1 and 2 days prior to the challenge. Significant increases in the survival of hepatoma-bearing mice were observed only after injections of MDP incorporated in multilamellar liposomes.Abbreviations MDP n-acetylmuramyl-l-alanyl-d-isoglutamine - B10 C57BL/10ScSnPh mice - MC 3-methylcholanthrene - ATT o-amino-azotoluene - PBS phosphate-buffered saline     

Cytotaxonomic study ofTragopogon L. in czechoslovakia

Karyotypes ofTragopogon orientalis L. subsp.orientalis, T orientalis L. subsp.leiocarpos (Sauter)Trnka,T. pratensis L.,T. minor Miller,T. dubius Scop. subsp.dubius andT. dubius Scop. subsp.major (Jacq.)Vollmann were studied. The occurrence in Slovakia ofT. pratensis was karyologically proved.     

Growth of Streptomyces bambergiensis and glucose isomerase biosynthesis

Summary Growth and glucose isomerase biosynthesis in Streptomyces bambergiensis ATCC 13879 have been studied under different conditions. Some data concerning correlation between cultivation conditions and elemental analysis of the cells are also presented.     

Translocation of cadmium and mercury in straw columns colonized by the fungus Pleurotus cornucopiae Paul ex Fr.

Summary Using straw columns colonized by the lignocellulytic fungus Pleurotus cornucopiae, translocation of ¹⁰⁹Cd and ²⁰³Hg in the substrate-mycelium complex and via the substrate-mycelium complex into the fruiting bodies was studied. The translocation patterns generated were metal specific and were influenced by the temperature and the physiological conditions of the mycelium (growing mycelium, established mycelium, reproductive stage).Under all conditions, generally more mercury than cadmium was translocated. In growing mycelia, for instance, an average of about seven times more mercury than cadmium was translocated. Translocation was greatly enhanced, when fruiting bodies were present. Up to 7% and 20% (average: 3.5% and 12%) of the applied cadmium and mercury, respectively, were found in the fruiting bodies. In old columns bearing fruiting bodies (colonized for more than 50 days by the fungus) considerably more heavy metal (up to 45% of the applied radioactivity) was released from the point of application than in younger columns.With one exception, no substantial differences in the translocation patterns of the label in relation to the direction of mycelial growth could be detected.     

Length of human constitutive heterochromatin in relation to chromosomal contraction

Summary Linear measurement of blocks of constitutive heterochromatin and the euchromatin portion 1q-h in three members of a family was used to study the dependence of the size of C blocks on the degree of chromosomal contraction. The results demonstrate that the size of heterochromatin portions decrease regularly with an increases of the degree of euchromatin contraction. The dependence was found to be linear, except for mitoses with an extremely high or low degree of contraction. The finding was used for the development of a new method of evaluation of constitutive heterochromatin.     

Inactivation of uridine nucleosidase in yeast. Purification and properties of an inactivating protein.

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is subjected in yeast to inactivation. An inactivating fraction has been isolated and purified to homogeneity with a procedure which includes gel filtration, adsorption chromatography, and electrofocusing techniques. The molecular weight of the enzyme, estimated either by sodium dodecyl sulfate disc gel electrophoresis or by gel filtration is approximately 44,000. No quaternary structure was evidenced. The inactivating activity possesses proteolytic activity against casein and hemoglobin with pH optima of 2.5 and 3.2, respectively. The optimal pH for uridine nucleosidase inactivation is around 4.7. The inactivating activity as well as the proteolytic activity of the preparation can be inhibited by IA but not by IB2 and IC, yeast macromolecular inhibitors for proteinase A (EC 3.4.23.8), B (EC 3.4.22.9), and C (EC 3.4.12.8), respectively. The apparent isoelectric point is pH 4.03. The carbohydrate content is 8.5%. A comparison of the properties of the inactivating protein with those of known yeast proteinases leads to the conclusion that it is identical with the enzyme previously designated as proteinase A, which for the first time has been obtained homogeneous and characterized. It has been shown that proteinase A could play a physiological role in the uridine nucleosidase inactivation process when it is associated, as a complex, with proteinase B.