A method to test large numbers of bovine semen samples for viral contamination and results of a study using this method

A procedure to test large numbers of semen samples for viral contamination is described. The procedure has the advantages of being simple to perform, sensitive and a relatively inexpensive method to test pooled semen samples, but it has the disadvantage of requiring serologically negative calves and sheep, isolation facilities to prevent environmental contamination during the testing procedure and relatively long times to obtain results. Using this in vivo procedure called the "Cornell Semen Test", it was found that of 40,000 ejaculates tested during a four year period none contained Infectious Bovine Rhinotracheitis Virus, Bovine Herpes Mammalitis Virus, Bovine Leukemia Virus nor Bluetongue Virus at infectious levels. The only virus that was found in a limited number of pools of semen was Bovine Virus Diarrhea Virus. It is recommended that if large volumes of pooled semen are to be tested for viral contamination that this procedure be used.     

Potassium transport and intracellular potassium activities in rabbit gallbladder

Summary Intracellular K activities, (K) _c , in rabbit gallbladder were determined using conventional and ion-selective microelectrodes. (K) _c averaged 73mm and was 1.5 times that predicted for an equilibrium distribution of the ion across both apical and basolateral membranes. Thus, K must be actively transported into the cell, and the responsible mechanism is almost certainly the Na–K exchange pump in the basolateral membrane.Measurements of the bidirectional transepithelial fluxes of⁴²K indicate that K is secreted into the mucosal solution at a rate of 0.8 eq/cm² hr; this value is only 6% of the rate of transcellular Na absorption by this epithelium.Calculation of the conductance of the basolateral membrane,G ^s, reveals that it is too low to account for the maintenance of the steady-state (K) _c by a 3 Na2 K pump mechanism at the basolateral membrane if K exit across that barrier is entirely electrodiffusional.Our results together with those of others strongly suggest that a significant fraction of downhill K exit from the cell across the basolateral membrane is nonconductive and coupled to the movement of some other ion, perhaps Cl.     

Differential distribution of voltage-dependent calcium channels and guanylate cyclase in the excitable ciliary membrane from Paramecium tetraurelia

A novel method for isolation of cilia and ciliary membrane vesicles from Paramecium tetraurelia has been developed. Using a continuous Percoll gradient of low osmolarity after fragmentation of purified cilia by French Press treatment two membrane fractions with different buoyant densities were obtained. These fractions were further purified by conventional discontinuous sucrose density gradients and characterized biochemically and by electron microscopy. Guanylate cyclase, a membrane bound enzyme, was found almost exclusively in membrane vesicles of high buoyant density while the voltage-sensitive calcium-channel of the ciliary membrane was predominantly localized in low density vesicles. Examination of both fractions by SDS polyacrylamide gel electrophoresis revealed only minor differences in protein pattern in the 34 and 64 kilodaltons range. Morphologically both membrane vesicle fractions had a diameter of about 300 nm, however, the high density vesicle fraction contained a considerably larger amount of multilamellar structures with a multishell, onion-like appearance. Freeze-fracture analysis failed to detect differences in intramembrane particle content between low and high density vesicles. The possible biological relevance of the spatial separation of the calcium-sensor enzyme guanylate cyclase and the voltage-sensitive calcium-channels in the ciliary membrane is discussed in terms of a diffusion controlled mechanism for graded signal transmission.     

Inhibitory effect of ethylenediaminetetraacetate (EDTA) on the porcine lymphocyte response to mitogens and reversal of the effect with metal ions

The effect of ethylenediaminetetraacetate (EDTA) on the mitogen response of porcine lymphocytes and the role of metal ions in reversal of the inhibitory effect of EDTA were determined. Porcine lymphocyte responses to mitogens were totally suppressed when serum used to supplement Ca²⁺, Mg²⁺-free minimum essential medium (MEM) was dialyzed against saline or saline with 0.2 or 0.60 mM EDTA, but the responses were only partially reduced when the same serum was added to RPMI-1640 medium. The inhibition observed in MEM could be reversed by adding 1×10⁻³ M Ca²⁺ and 1×10⁻³ M Mg²⁺ to the dialyzed serum. Serum treated directly with 0.60 mM EDTA completely suppressed blastogenesis in lymphocyte cultures maintained in RPMI-1640 or Ca²⁺, Mg²⁺ free MEM. The inhibitory effect of EDTA-treated serum could be completely reversed by adding Zn²⁺ or a combination of Zn²⁺ with other cationic ions, or partially reversed by adding Ni²⁺ or Fe³⁺. Zn²⁺ was the most effective ion, in that it was the only ion that, when alone added to the serum, could completely restore lymphocyte responses to phytohemagglutinin (PHA) or pokeweed mitogen (PWM).     

In vitro RNA synthesis in oocyte nuclei of the newt Notophthalmus

An incubation medium is described which supports RNA synthesis in isolated oocyte nuclei of the newt Notophthalmus, and which permits subsequent autoradiographic examination of the lampbrush chromosomes and nucleoli. By using different concentrations of α-amanitin we distinguish RNA synthesis due to RNA polymerases I, II and III. All RNA synthesis on loops is inhibited by 0.5 μ/ml of α-amanitin and is therefore due to polymerase II. Polymerase III is responsible for RNA synthesis at a small number of discrete sites in condensed chromatm. These include the centromere bars of three of four chromosomes, which probably represent 5S RNA synthesis, as well as 15–20 lesser sites scattered elsewhere. Polymerase I activity is confined to the nucleoli. Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Laser Raman characterization of conformational changes in sarcoplasmic reticulum induced by temperature, Ca2+, and Mg2+

Laser Raman spectroscopy has been used to examine the conformations of the protein and phospholipid components of sarcoplasmic reticulum from rabbit white skeletal muscle. The phospholipid component is shown to have the conformation of fluid, liquid-crystalline lipids, even at 10 degrees C, and no breaks in the lipid conformation are observed in the range of 10-37 degrees C. Protein (predominantly the Ca2+-dependent ATPase) conformation is shown to contain very little beta-sheet structure under all conditions. Absolute content of alpha-helix and random coil or beta-turn could not be determined because of interference in the amide I and III regions. However, the Ca2+-ATPase in sarcoplasmic reticulum appears to undergo a conformational change at 15-18 degrees C which involves removal of a portion of the tryptophan residues from an aqueous environment and an increase in alpha-helical content. This conformation change coincides with a change in slope of Arrhenius plots of ATP hydrolysis activity. Increasing concentrations of Ca2+ and Mg2+ appear to slightly decrease the alpha-helical content of sarcoplasmic reticulum protein.     

Efficient extraction and quantitative determination of nanogram amounts of cellular RNA

An assay for quantitating nanogram amounts of cellular RNA is described. RNA is efficiently extracted from cells, using RNA-free DNA as carrier, by conventional chloroform: phenol procedures and the nucleic acids are precipitated with ethanol. Isolated RNA is hydrolyzed by RNase T2 to ribonucleoside 3′-monophosphates which in turn are converted to 5′-³²P-labeled ribonucleoside 3′,5′-diphosphates in the presence of T4 polynucleotide kinase and [γ-³²P]ATP. Radiolabeled products are separated from remaining [γ-³²P]ATP by chromatography on polyethyleneimine-cellulose, located by autoradiography, excised from the chromatogram, and subjected to liquid scintillation counting to quantitate the amount of RNA. Using mouse liver ribosomal RNA as a standard, the assay is linear over a range of 0 to 64 ng of RNA. The assay has been used to determine the amount of RNA in fully grown mouse oocytes arrested at the dietyate stage of first meiotic prophase. Each oocyte contains 0.61 ± 0.05 ng of RNA and only 25 oocytes have been used for such assays.     

Factors which may affect removal of protamine from sperm DNA during fertilization in the rabbit

In order to derive information about possible mechanisms by which the sperm head is converted into the male pronucleus during fertilization in the rabbit, unfertilized egg homogenate was assayed for two enzyme activities. Protamine was extracted from rabbit sperm, purified, and labelled with [¹⁴C] in an in vitro reaction and used as a probe to assay for a protein kinase which could transfer [³²P]PO₄ from [γ-³²P]ATP onto the substrate. A kinase with a pH optimum of approximately 8.0 to 8.5 is described. Assays for the enzyme glutathione reductase were performed using homogenates from eggs or embryos at three early stages of development. Results suggest that oocytes can oxidize 2.58 × 10^?6 μmol NADPH per minute per oocyte, unfertilized eggs 5.16 × 10^?7 μmol NADPH per minute per ovum, and 20- to 24-hour postcoitus fertilized eggs 2.30 × 10^?6 μmol NADPH per minute per ovum. The relevance of these observations to male pronuclear formation is discussed.     

Fermentation and anaerobic respiration by Rhodospirillum rubrum and Rhodopseudomonas capsulata

Rhodospirillum rubrum and Rhodopseudomonas capsulata were able to grow anaerobically in the dark either by a strict mixed-acid fermentation of sugars or, in the presence of an appropriate electron acceptor, by an energy-linked anaerobic respiration. Both species fermented fructose without the addition of accessory oxidants, but required the initial presence of bicarbonate before fermentative growth could begin. Major products of R. rubrum fermentation were succinate, acetate, propionate, formate, hydrogen, and carbon dioxide; R. capsulata produced major amounts of lactate, acetate, succinate, hydrogen, and carbon dioxide. R. rubrum and R. capsulata were also capable of growing strictly through anaerobic, respiratory mechanisms. Nonfermentable substrates, such as succinate, malate, or acetate, supported growth only in the presence of an electron acceptor such as dimethyl sulfoxide or trimethylamine oxide. Carbon dioxide and dimethyl sulfide were produced during growth of R. rubrum and R. capsulata on succinate plus dimethyl sulfoxide. Molar growth yields from cultures grown anaerobically in the dark on fructose plus dimethyl sulfoxide were 3.8 to 4.6 times higher than values obtained from growth on fructose alone and were 56 to 60% of the values obtained from aerobic, respiratory growth with fructose. Likewise, molar growth yields from anaerobic, respiratory growth conditions with succinate plus dimethyl sulfoxide were 51 to 54% of the values obtained from aerobic, respiratory growth with succinate. The data indicate that dimethyl sulfoxide or trimethylamine oxide as a terminal oxidant is approximately 33 to 41% as efficient as O₂ in conserving energy through electron transport-linked respiration.     

Summer distribution of Red-legged Partridges Alectoris rufa in relation to water availability on Mediterranean farmland

The suggestion that the summer distribution of Red-legged Partridges Alectoris rufa in the Mediterranean region is determined by the availability of surface water was examined on the agricultural farm, Alto Alentejo, southern Portugal. Partridge coveys were surveyed between 15 July and 15 August in 1993 and 1994. Using a vector-based Geographic Information System, we assessed, for each covey location and for the locations of a double number of random points, the distance to the nearest water point, distance to field boundaries, distance to water lines and land use classes. Univariate comparisons were made between the two groups of locations, and three multivariate logistic models were fitted through forward stepwise selection to the 1993, 1994 and pooled data sets to estimate the probability of sighting partridge coveys in the study area. Distance to water was significantly lower for partridge locations than for random points in both years and was the only variable selected for all logistic models. Apart from water availability, Red-legged Partridge locations were also affected by land use and distance to field boundaries.