Frozen bovine semen quality and bovine cervical mucus penetration test

Research has been carried out to test bovine cervical mucus penetration (penetration) as a means for evaluating frozen-thawed bovine semen. A commercially available cervical mucus penetration test kit (the kit) was used. A total of 158 previously frozen semen samples collected from 61 bulls were thawed in a 37 C water-bath for 2 minutes. Four ways to estimate penetration were compared using the distance traveled during 90 minutes 1) at 21 C, or 2) at 37 C, by 3) the first solitary mobile spermatozoon, or by 4) the front of the mass of the mobile spermatozoa. Penetration was measured using phase contrast microscopy and a millimeter grid. Spermatozoal quality parameters (concentration, total motility, progressive motility, acrosome integrity, total sperm integrity and cytoplasmic droplets) were measured and the correlation to penetration was calculated. The best way to assay penetration with the kit was by measuring the penetration of the first solitary mobile spermatozoon at 37 C. Semen quality variability was significant (P < 0.05) relative to penetration. Linear correlations between penetration and acrosome integrity r=0.42 as well as between penetration and total sperm integrity r=0.53 were highly significant (P < 0.001). There was significant linear multiple regression between penetration and acrosome integrity (expressed as percentage and number) and total sperm integrity (expressed as percentage and number) (r=0.62; F=23.5147; P<0.0001). There was a significant difference between the average progressive motility of samples with penetration > 20 mm and samples with penetration 20%), but it is not useful to define the fertility level of semen samples.     

A new human growth hormone production process using a recombinant Bacillus subtilis strain.

We constructed a series of hybrid plasmids which directed the synthesis of different human growth hormone (hGH) precursor sequences in Bacillus subtilis. In addition to the 191 amino acids of the hormone, the precursors had in common an amino-terminal extension characterized by the presence of a methionine at position 1 and of the tetrapeptide Ile-Glu-Gly-Arg preceding the first residue (Phe) of hGH. The sequence between the methionine and the tetrapeptide was specific for each precursor and, because of the presence of charged residues, conferred particular properties to the molecules. Long homopolymeric tail-containing precursors such as MRRRRRRIILM-IEGR appeared insoluble whereas shorter sequences of the type MRR-IEGR and MEELM-IEGR augmented the solubility of the precursors with respect to Met-hGH. The soluble precursors could be easily purified from the bulk proteins taking advantage of the charged residues present on the N-terminal tail. After purification, the natural hGH was obtained by treating the precursors with the protease Factor Xa which cleaves after the arginine residue of the tetrapeptide IEGR. A protocol for the production and purification of authentic hGH from a strain expressing one of these soluble precursors is reported.     

Prochymosin expression in Bacillus subtilis

Prochymosin (PC) sequence was cloned in Bacillus subtilis using two kinds of plasmid constructions. In plasmid pSM316 the cDNA was inserted to obtain the intracellular expression of the enzyme. The enzyme turned out to be expressed in an insoluble form which could be converted to native enzyme under proper denaturing and refolding conditions. The levels of intracellular expression of PC were further enhanced by modifying the 5' region of the gene in a way that a two-cistron expression system was created. For the PC secretion, the cDNA was fused to the subtilisin leader sequence and expressed under the control of the B. subtilis neutral protease promoter. A properly folded PC was secreted by the cells, although to low levels.     

Inducibility of gene conversion in Saccharomyces cerevisiae treated with MMS

At high survival levels (85%), point mutation and gene conversion frequencies were determined in strain D7 of Saccharomyces cerevisiae after treatment with methyl methanesulfonate (MMS) either after cells were incubated in complete medium before plating or following a split-dose protocol. It is shown that induction of gene conversion by MMS post-incubation leads to an additional enhancement in frequency. This increase is not observed for point mutation. By fractionation of the MMS dose (1 mM + 1 mM) with incubation in complete medium between the 2 doses the frequency of gene conversion is twice as high as with a single equal total dose (2 mM). This treatment does not modify the frequencies of point mutation. These data support the notion that an inducible recombinogenic function exists in wild-type yeast.     

Differential effects of various vasoactive drugs on basal and stimulated levels of TXB2 and 6-keto-PGF1α in rat brain

Thromboxane B₂ and 6-keto-PGF_1α (6KPGF_1α), the major stable metabolites of thromboxane and prostacyclin, are present in the CNS, where they appear to be mainly produced within and/or acting upon the vascular district. Their concentrations are of few pg/mg protein in rat brain cortex of animals sacrificed by microwave (MW) radiation, procedure which inactivates tissue enzymes and allows the determination of endogenous “basal” levels of eicosanoids. Levels of 6KPGF_1α and especially those of TxB₂ increase several fold over the basal values in brain cortex of animals sacrificed by decapitation followed by a few minute interval before analysis (post-decapitation ischemia, PDI). Pretreatment of animals with the vasoactive drug papaverine, resulted in elevation of brain basal levels of 6KPGF_1α and with the carbochromene derivartive AD₆ in reduction of basal levels of TxB₂, whereas the calcium antagonist nifedipine and dipyridamole did not modify basal levels of the two eicosanoids. Treatments with papaverine and AD₆ reduced the accumulation of TxB₂ and enhanced that of 6KPGF_1α occurring after PDI, to different extents, both resulting, however, in reduction of the TxB₂/6KPGF_1α ratio. Nifedipine instead, decreased the release of both eicosanoids and resulted in elevation of the TxB₂/6KPGF_1α ratio, whereas dipyridamole had no effect. In conclusion, the evaluation of the overall effects of drug treatments on the TxB₂/6KPGF_1α ratio in cerebral tissue, provided useful informations on the pharmacological modulation of vascular eicosanoids in this district.     

BRAIN RECOVERY FROM ESSENTIAL FATTY ACID DEFICIENCY IN DEVELOPING RATS 1

Abstract— Rats were supplied from before birth with an essential fatty acid (EFA) deficient, a control, or an EFA deficient-control combination diet for various periods up to 6 months. It was found that EFA deficiency resulted in brain weights decreased in comparison with control values throughout development. The brain weight/body weight relationship, however, expressed by Donaldson's equation was generally maintained in animals fed either totally deficient or control diets. Animals deficient even during the brain's most actively growing period were able to recover completely on restoration of the control diet for a sufficiently long period. Fatty acid alterations in brain ethanolamine phosphoglyceride (EPG) during EFA deficiency were extensive. Acids of the ω6 family (18:2, 20:2, 20:3, 20:4, 22:3, 22:4 but not 22:5) were reduced from control figures. In the w9 family 20:3 and 22:3 were especially elevated whereas 22:6 ω3 levels were similar to those of the controls, finally decreasing only after a lengthy period of EFA deprivation. Mean unsaturation contents, as expressed by the proposed unsaturation index notation (Ul_mol) agreed closely in EPG fatty acids of deficient and control rats at a particular age. On substitution of the control for the deficient diet the ω6 family rebounded in a manner such that values for 20:4, 22:4, and 22:5 exceeded comparable figures in control animals. Concomitantly the ω9 family receded below control levels, and ω3 acids remained or returned to normal. This overadjustment in ω6 and ω9 families continued even after a prolonged period on the control diet.     

LIPID COMPOSITION OF THE CENTRAL NERVOUS SYSTEM OF MARINE VERTEBRATES

—The lipid composition of the central nervous system of some marine vertebrates and two mammalian species (rat and man) was analysed by one- and two-dimensional quantitative thin-layer chromatography, and the cerebroside fatty acids were analysed by gas chromatography. The concentrations of sphingomyelin and cerebrosides are higher in mammals than in fishes, while no significant differences are observed for other lipid classes. Furthermore, in mammals the ratio between hydroxy and normal fatty acids in the cerebrosides is much higher than in fishes. The cerebrosides of mammals contain more very long chain fatty acids than those of marine vertebrates.     

A new member of a secretory protein gene family in the dipteranChironomus tentans has a variant repeat structure

Summary We describe the structure of a gene expressed in the salivary gland cells of the dipteranChironomus tentans and show that it encodes 1 of the approximately 15 secretory proteins exported by the gland cells. This sp115,140 gene consists of approximately 65 copies of a 42-bp sequence in a central uninterrupted core block, surrounded by short nonrepetitive regions. The repeats within the gene are highly similar to each other, but divergent repeats are present in a pattern which suggests that the repeat structure has been remodeled during evolution. The 42-bp repeat in the gene is a simple variant of the more complex repeat unit present in the Balbiani ring genes, encoding four of the other secretory proteins. The structure of the sp115,140 gene suggests that related repeat structures have evolved from a common origin and resulted in the set of genes whose secretory proteins interact in the assembly of the secreted protein fibers.