Semen cryopreservation in golden‐headed lion tamarin,Leontopithecus chrysomelas

Wild animal genetic resource banking (GRB) represents a valuable tool in conservation breeding programs, particularly in cases involving endangered species such as the golden‐headed lion tamarin (Leontopithecus chrysomelas). Thus, we aimed to assess a sperm freezing protocol for golden‐headed lion tamarins using two different exenders: BotuBOV® (BB) and Test Yolk Buffer® (TYB). Ejaculates were collected by penile vibrostimulation from animals housed at São Paulo Zoological Park Foundation, São Paulo, Brazil, and after immediate analysis, two aliquots were diluted in BB and TYB. Postthawing samples were evaluated for total and progressive motility, plasma membrane and acrosome integrities, mitochondrial activity, susceptibility to oxidative stress, and sperm–egg‐binding. No differences between BB and TYB were found for most seminal parameters, except for acrosome integrity and susceptibility to oxidative stress (in both cases BB showed higher values). However, in spite of these differences and regardless of the extender used, postthaw sperm motility and viability with the described protocol were encouraging (on average >50% and >80%, respectively), indicating that sperm cryopreservation may be a short‐term measure for the conservation of golden‐headed lion tamarins.     

An improved purification procedure and properties of casein kinase II from brain

A simple and short purification procedure applicable to casein kinase II has been developed, for fully characterizing the enzyme from calf cerebral cortex cytosol. The procedure consists of four chromatographic steps: DEAE-cellulose, phosphocellulose, phosvitin-Sepharose and ATP-agarose which yields 87% pure casein kinase II. The purified enzyme shows three major bands with apparent molecular masses of 42, 38, and 27 kDa by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and is self-autophosphorylated on its 27 kDa polypeptide. The enzyme shows all the characteristics described for casein kinase II from other sources: it is independent of cyclic nucleotides, calcium/phospholipids, and double-stranded poly(I).poly(C); it can utilize both ATP and GTP as phosphoryl donors and can phosphorylate both casein and phosvitin but not histone. The kinetic studies establish that theK _m for ATP is 12.5 M and 25.1 M when using phosvitin and casein respectively as phosphoryl acceptors. TheK _m for phosvitin is 0.91 mg/ml and for casein 1.43 mg/ml, while theV _max is 315 nmol/min/per mg protein and 479 nmol/min/per mg protein for phosvitin and casein respectively. The activity of the kinase is highly stimulated by KCl or NaCl, and almost completely inhibited by heparin concentrations of 1 g/ml (92%). This inhibition is reduced to only 33% in the presence of optimal KCl concentrations (150 mM). Spermine stimulates enzyme activity, whilst hemin produces a slight inhibition.     

Effects of the administration of cadmium and zinc on the concentration of zinc in the thymus of rats

The zinc content of thymus glands of male Wistar rats has been determined during five weeks of treatment with ZnCl₂ and CdCl₂, and compared with a group of control rats. THymus gland extracts were chromatographed on columns of Sephadex G-75 and the zinc content of the one hundred fractions obtained were determined by atomic absorption spectrophotometry. The rats treated with ZnCl₂ showed an increase in the thymus concentration of zinc bound to high and low molecular weight proteins. The rats treated with CdCl₂ showed an increase in zinc concentration, as opposed to the control group, during the first three weeks of treatment, and thereafter show a toxic effect of cadmium on the gland, with ulterior regression of the latter, and a decrease in the concentration of zinc.     

Isolation and biochemical characterization of Streptomyces clavuligerus mutants in the biosynthesis of clavulanic acid and cephamycin C

Summary Seven mutants of Streptomyces clavuligerus blocked in the biosynthesis of clavulanic acid, cephamycin C, or both antibiotics, have been isolated and characterized. Mutants nca1 and nca2 were unable to synthesize clavulanic acid but produced cephamycin C. Mutants nce1 and nce2 were completely blocked in cephamycin C production but formed clavulanic acid. A third group (mutants ncc1, ncc4 and ncc5) failed to produce both antibiotics. Arginase activity (forming ornithine) was very low in mutants ncc1 and ncc5. All the mutants blocked in clavulanic acid biosynthesis showed a normal ornithine--aminotransferase activity. Mutant ncc1, blocked in cephamycin biosynthesis, lacked completely lysine--aminotransferase (forming -aminoadipic acid) and isopenicillin N synthase. Two other mutants (nce2 and nce5) lacked isopenicillin N synthase. There was a good correlation between the isopenicillin N synthase and the lysine--aminotransferase activities of the nca mutants and the ability of those strains to produce cephamycin C. The condensing enzyme involved in the formation of the clavulanic acid nucleus appears to be different from the isopenicillin N synthase.Dedicated to Professor H.-J. Rehm on the occasion of his 60th birthday     

The 5'' to 3'' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae

Three different mutations were introduced in the polA gene of Streptococcus pneumoniae by chromosomal transformation. One mutant gene encodes a truncated protein that possesses 5' to 3' exonuclease but has lost polymerase activity. This mutation does not affect cell viability. Other mutated forms of polA that encode proteins with only polymerase activity or with no enzymatic activity could not substitute for the wild-type polA gene in the chromosome unless the 5' to 3' exonuclease domain was encoded elsewhere in the chromosome. Thus, it appears that the 5' to 3' exonuclease activity of the DNA polymerase I is essential for cell viability in S. pneumoniae. Absence of the polymerase domain of DNA polymerase I slightly diminished the ability of S. pneumoniae to repair DNA lesions after ultraviolet irradiation. However, the polymerase domain of the pneumococcal DNA polymerase I gave almost complete complementation of the polA5 mutation in Escherichia coli with respect to resistance to ultraviolet irradiation.     

Chromosome-mediated iron uptake system in pathogenic strains of Vibrio anguillarum.

We describe in this work a new iron uptake system encoded by chromosomal genes in pathogenic strains of Vibrio anguillarum. This iron uptake system differs from the plasmid-encoded anguibactin-mediated system present in certain strains of V. anguillarum in several properties. The siderophore anguibactin is not utilized as an external siderophore, and although characteristic outer membrane proteins are synthesized under iron-limiting conditions, these are not related to the plasmid-mediated outer membrane protein OM2 associated with ferric anguibactin transport. Furthermore, the siderophore produced by the plasmidless strains may be functionally related to enterobactin as demonstrated by bioassays with enterobactin-deficient mutants, although its behavior under various chemical treatments suggested major differences from that siderophore. Hybridization experiments suggested that the V. anguillarum chromosome-mediated iron uptake system is unrelated genetically to either the anguibactin or enterobactin-associated iron assimilation systems.     

Functional heterogeneity of GEF-free initiation factor 2 purified from suckling and adult rat brain

The functional behavior of initiation factor 2 was studied in purified preparations from the brains of suckling (4-12-day-old) and adult (60-day-old) rats. Adult eIF2 has lower GDP and GTP affinity than suckling eIF2, even in the presence of a large excess of GTP, whereas suckling eIF2 has a lower capacity to bind GTP. Since these two factors are free of guanine nucleotide exchange factor (GEF), and ribosomal fractions show an age-dependent difference in GEF activity, the observed functional heterogeneity may be due to a different ratio in eIF2 species (eIF2-GDP, eIF2(P)).

Developing brain Protein synthesis Initiation factor 2 Guanine nucleotide exchange factor     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

Summary The 10-kb chromosomal fragment of Streptococus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome.A spontaneous deletion beginning 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (10^-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed.Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanil-amide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.     

Effect of rhodium (III) complex on experimental carcinogenesis in the livers of rats treated with thioacetamide

Enzyme activities and protein content were determined in the cytosolic and mitochondrial fractions of liver homogenates obtained from Rh(III) complex-, thioacetamide- and thioacetamide + Rh(III) complex-treated rats. The Rh(III) complex administered to nonthioacetamide-treated rats produced no significant changes either in the enzymatic activities assayed or in the protein concentration. The Rh(III) complex administered to thioacetamide-treated rats produced significant restoration of the following altered values: cytosolic and mitochondrial aspartate aminotransferase, glutamate dehydrogenase, NADP-isocitrate dehydrogenase, and protein concentration. However, a further increase was produced in the activities of glucose-6-phosphate dehydrogenase and malic enzyme. These increases can be interpreted in terms of an enhancement of the NADPH-dependent detoxifying processes and of nucleic acid synthesis and repair.