Characterization of a 22 kDa protein with widespread tissue distribution but which is uniquely present in secretions of the testis and epididymis and on the surface of spermatozoa

The purification is reported of a 22 kDa protein which was first identified as one of the major components of the luminal secretion of the rat testis and epididymis. Antibodies against the 22 kDa protein cross-reacted with a protein of the same molecular weight in cytosolic extracts of other tissues from both male and female rats. However, since the protein could not be detected in blood, peritoneal fluid, saliva, milk, uterine fluid, seminal vesicle secretion, coagulating gland secretion or prostatic secretion, it would appear that the testis and epididymis may be unique in containing the protein in a soluble form within their luminal secretions. Proteins with slightly lower molecular weight were detected by the antibodies in cytosolic extracts of tissues from other animals (mice, rabbits, sheep, pigs, cattle), indicating that the protein may be conserved in a variety of species. However, in contrast to the rat, the protein was apparently not present in the testicular and epididymal secretions of these species. In addition to the occurrence of the 22 kDa protein as a soluble moiety in rat testicular and epididymal fluids, the protein was also located on sperm plasma membranes where its distribution was restricted to the surface of the flagellum. Amongst sperm surface proteins, the 22 kDa protein was the major protein containing sulphydryl groups and one of the major entities containing disulphide bonds. These properties may be of importance in the maintenance of sperm viability.     

Viability of Culex pipiens pipiens eggs affected by nutrition and aposymbiosis

Raisins were a better source of carbohydrate than sucrose for reproduction by autogenous Culex pipiens. A blood meal increased the number of eggs per raft from 49 autogenously to 114. Eggs of aposymbiotic females produced autogenously did not hatch, but 34% of the eggs were viable if the mosquitoes fed on chickens. With repeated blood meals the number of eggs per raft and the rate of embryonation and hatching declined in each successive gonotrophic cycle. In about $\text{1}\text{4}$ of the unhatched eggs of normal females there were no fully developed embryos, while many more of the unhatched eggs of aposymbiotic females contained no evidence of embryonic development. After the fifth blood meal, neither normal nor aposymbiotic insects oviposited. The ovaries of the nulliparous females contained approximately 10% of the potential number of mature terminal oocytes. Proximally in the ovarioles there were dilatations and coiled tracheoles indicating egg resorption. There were fewer parous follicles in aposymbiotic than in normal females. Larval rearing water, i.e., infusion in which larvae had been reared, was more attractive than fresh infusion for oviposition by normal, autogenous mosquitoes. The degree of embryonation of the eggs was lower and the hatching success rate was poorer in fresh infusion than in larval rearing water.     

Genotoxicity studies with mineral oils; effects of oils on the microbial mutagenicity of precursor mutagens and genotoxic metabolites

In vitro genotoxicity assays are extensively used to predict carcinogenic activity in vivo. The standard microbial mutagenicity assays however often fail to yield positive results with mineral oils which are carcinogenic to mice in long-term skin-cancer studies. A comprehensive programme of studies has therefore investigated the basis of this apparently anomalous behaviour. This investigation has addressed the possible effects of oils on the bioactivation of precursor mutagens and the disposition of mutagenic metabolites by studying the microbial mutagenicity of selected precursor mutagens (benzo[a]pyrene, benzo[a]anthracene, 2-aminoanthracene and 2-naphthylamine) and intrinsically reactive mutagens [+/- )-benzo[a]pyrene-4,5-oxide and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) in the presence and absence of mineral oils. Notably the mutagenicity associated with the deliberate additions of these mutagens or precursor mutagens to oils was readily detected by the microbial assays. The mutagenicity of only one of the precursor mutagens, benzo[a]pyrene, was significantly reduced by the oils, and then only in the standard plate-incorporation assay. Interestingly the degree of suppression appeared to be related to the polycyclic aromatic hydrocarbon content of the oils. In the case of 2-aminoanthracene large enhancements in its mutagenicity were observed in the presence of oils. These latter findings appear to be due to effects of oils on the bioactivation of precursor mutagens rather than on the disposition of their bioactivation products. The mutagenicity of intrinsically reactive mutagens, of a type generated by bioactivation of polycyclic aromatic hydrocarbons, was not significantly reduced in the presence of mineral oils. This indicates that it is unlikely that components in oils trap or facilitate the deactivation of ultimate mutagens whether these pre-exist in the oil or are formed from precursors by bioactivation in the in vitro test system. Viewed overall these results suggest that mineral oils judged to be carcinogenic on the basis of in vivo studies in mouse skin may possess only very weak genotoxic potential. While this potential is likely to be a prerequisite for carcinogenic action, the current results cause attention to be focussed on other factors, e.g. promotion, as potentially important determinants of the carcinogenic potencies of mineral oils in mouse skin.     

Transformation of Azotobacter vinelandii with plasmid DNA.

Azotobacter vinelandii cells can be transformed at high frequencies with the broad-host-range plasmids pRK2501, RSF1010, and pGSS15, using a modification of the procedure developed by Page and von Tigerstrom (J. Bacteriol. 139:1058-1061, 1979) for chromosomal DNA-mediated transformation. The frequency of transformation per microgram of plasmid DNA per viable cell with pRK2501 and pGSS15 was about 5 X 10(-2) and 2 X 10(-2), respectively. With RSF1010, transformation frequencies ranged from 3 X 10(-4) to 4 X 10(-2). With each plasmid, the frequency of transformation was independent of the phase of the growth cycle. When concentrations of pRK2501 ranging from 0.1 to 51 micrograms of DNA were tested, the frequency of transformation was directly proportional to the amount of DNA. This linear response indicated that, although the uptake of plasmid DNA with this procedure may be inefficient, there is a high probability that once inside a cell the plasmid will be stably maintained. Cells that have been transformed with pRK2501 did not grow well on transforming medium which lacks iron and contains fixed nitrogen. However, on growth medium which contains iron and lacks fixed nitrogen, transformants produced distinctive colonies larger than those of nontransformed cells. Resistance to kanamycin due to transformation by pRK2501 was stably maintained for at least 10 successive generations in the absence of selective pressure. The present protocol should facilitate the molecular cloning of genes in Azotobacter spp.     

A fluorescent antibody staining technique to detect bacterial adherence to urinary tract epithelial cells

A fluorescent antibody technique has been devised to assess specifically the adherence of Escherichia coli in vitro to uroepithelial cells from healthy women and bacterial adherence in vivo to cells from women with symptomatic urinary tract infection. Similar values can be obtained using methylene blue as the bacterial stain, but this depends on the experience of the observer. The results indicate that E. coli adherence to uroepithelial cells is a factor in the infection process. We suggest that uroepithelial cells from patients with symptoms of a urinary tract infection whose urine has a low bacterial count (less than 10(3) cells/ml) could be examined for the presence of adherent uropathogens, which may be indicative of an infection. Although the fluorescent staining technique possibly would be expensive, the results would be specific and reliable. Other diagnostic and research applications suggest themselves as in studies of bacterial colonization of mucosal tissues or plastic catheters, where conventional light microscopy and radiolabelling methods are not effective.     

Production of citric acid from sugars present in wood hemicellulose usingAspergillus niger andSaccharomycopsis lipolytica

Summary One strain each of the fungus,Aspergillus niger, and the yeast,Saccharomycopsis lipolytica, were investigated for their ability to produce citric acid from the sugars present in hemicellulose hydrolysates.S. lipolytica produced citric acid as efficiently from mannose as from glucose, but failed to assimilate xylose, arabinose or galactose.A. niger readily assimilated mannose, xylose and arabinose, and produced citric acid from these sugars although the yields were lower than from glucose. A possible inhibitory effect of arabinose on citric acid production from other sugars was observed usingA. niger.     

A B cell growth factor-like activity is secreted by cloned, neoplastic B cells

Supernatants from synchronized clones of neoplastic B cells (BCL1) were found to contain a B cell growth factor (BCGFI)-like activity. The BCGF activity in the BCL1 supernatants (SN) could synergize with EL-4 SN in the late phases of an IL 1-dependent BCGF I assay (days 5 through 8). These SN did not contain any detectable BCGF II, IL, 1, or IL 2 activity. In contrast to EL-4-derived BCGF I, which has a m.w. of approximately 18,000, the BCL1-BCGF activity has a m.w. of approximately 4500. Because the BCGF activity in BCL1 SN fluctuates with cell cycle in synchronized cultures, this BCL1-BCGF may play an auto-stimulatory role in B cell proliferation.     

Use of inversion spin transfer to monitor creatine kinase kinetics in rat skeletal muscle in vivo

A simple multipulse sequence has been used to monitor creatine kinase kinetics in rat skeletal muscle in vivo. Using these procedures, the forward (ATP synthesis) and reverse fluxes (phosphocreatine synthesis) have been calculated to be 8.98 +/- 0.6 and 10.7 +/- 0.8 mumoles/g wet wt/s (n = 5) respectively. These results suggest that in resting skeletal muscle most of the gamma ATP observed in 31P NMR spectra is cytosolic and rapidly exchanging with phosphocreatine. The high flux rates reflect the high catalytic capacity of creatine kinase in skeletal muscle.     

The effect of methanol on citric acid production from galactose by Aspergillus niger

Summary The effect of methanol on the ability of several strains of Aspergillus to produce citric acid from galactose has been investigated. In the absence of methanol, very little production (less than 1 g/l) was observed. In the presence of methanol (final concentration 1% v/v), however, citric acid production and yeilds were increased considerably. Strong relationships were observed between citric acid production and the activities of the enzymes 2-oxoglutarate dehydrogenase and pyruvate carboxylase in cell-free extracts. During citric acid production, in the presence of methanol, the activity of 2-oxoglutarate dehydrogenase was low and that of pyruvate carboxylase high. In the absence of methanol, where little citric acid was produced, the reverse was true. It is suggested that the presence of methanol may increase the permeability of the cell to citrate, and the cell responds to the diminished intracellular level by increasing production via repression of 2-oxoglutarate dehydrogenase.     

Selenium compounds in the fathead minnow (Pimephales promelas)--I. Uptake, distribution, and elimination of orally administered selenate, selenite and l-selenomethionine

Treatment of fathead minnows (Pimephales promelas) with either [75Se]selenate, -selenite or -l-selenomethionine by gavage at 20 ng Se/g resulted in organ uptake and early distribution patterns which differed significantly between compounds. The greatest differences in uptake between compounds was observed in liver tissue which accumulated much less [75Se]selenate than either selenite or l-selenomethionine. The 75Se burdens and relative distribution among the various organs were nearly identical during the elimination phase for [75Se]selenate and -selenite. This suggests that selenium derived from these compounds converge to a common metabolic pool. The whole body T1/2, rate of 75Se uptake and magnitude of 75Se accumulation were generally greater for [75Se]selenomethionine than the inorganic forms. Selenium-75 was present in the bile following the oral administration of each compound. The partitioning of selenate and selenite into the plasma and cellular fraction of blood differs with both the compound and time following exposure.