The kinetics of the first wave of spermatogenesis in the newborn male mouse

A combination of autoradiography and air-dried techniques was used to calculate the duration of the major meiotic stages in the first wave of spermatogenesis in the newborn mouse. The data indicated that the entry into meiosis occurred asynchronously over 2 days, and the time required for each stage and the total cycle was constant. These time intervals were nearly identical with those estimated for adult animals in the present study and by other authors.     

Role of 2-deoxy-D-glucose in the inhibition of phagocytosis by mouse peritoneal macrophage

2-Deoxy-D-glucose inhibits Fc and complement receptor-mediated phagocytosis of mouse peritoneal macrophages. To understand the mechanism of this inhibition, we analyzed the 2-deoxy-D-glucose metabolites in macrophages under phagocytosis inhibition conditions and conditions of phagocytosis reversal caused by glucose, mannose and 5-thio-D-glucose, and compared their accumulations under these conditions. Macrophages metabolized 2-deoxy-D-glucose to form 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucose 1-phosphate, UDP-2-deoxy-D-glucose, 2-deoxy-D-glucose 1, 6-diphosphate, 2-deoxy-D-gluconic acid and 2-deoxy-6-phospho-D-gluconic acid. The level of bulk accumulation as well as the accumulation of any of these 2-deoxy-D-glucose metabolites did not correlate with changes in macrophage phagocytosis capacities caused by the reversing sugars. 2-Deoxy-D-glucose inhibited glycosylation of thioglycolate-elicited macrophage by 70-80%. This inhibition did not cause phagocytosis inhibition, since (1) the reversal of phagocytosis by 5-thio-D-glucose was not followed by increases in the incorporation of radiolabelled galactose, glucosamine, N-acetylgalactosamine or fucose; (2) cycloheximide at a concentration that inhibited glycosylation by 70-80% did not affect macrophage phagocytosis. The inhibition of protein synthesis by 2-deoxy-D-glucose similarly could not account for phagocytosis inhibition, since cycloheximide, when used at a concentration that inhibited protein synthesis by 95%, did not affect phagocytosis. 2-Deoxy-D-glucose lowered cellular nucleoside triphosphates by 70-99%, but their intracellular levels in the presence of different reversing sugars did not correlate with the magnitude of phagocytosis reversal caused by these sugars. The results show that 2-deoxy-D-glucose inhibits phagocytosis by a mechanism distinct from its usual action of inhibiting glycosylation, protein synthesis and depleting energy supplies, mechanisms by which 2-deoxy-D-glucose inhibits other cellular processes.     

Immunohistochemical studies on the localization of cellular retinol-binding protein in rat testis and epididymis

The immunohistochemical localization of cellular retinol-binding protein (CRBP) was studied in rat testis and epididymis. Parallel studies were also carried out on the localization of plasma retinol-binding protein (RBP) and transthyretin (TTR) in testis. The studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. For RBP and TTR, specific immune staining was found in the interstitial spaces between the seminiferous tubules, and not in the tubules themselves. In contrast, strong specific immune staining for CRBP was found in the seminiferous tubules, with a striking localization within Sertoli cells. Moreover, a distinct cyclic variation of specific staining for CRBP within Sertoli cells was observed during the spermatogenic cycle. This cyclic variation was seen with regard to both the intensity of staining and to the anatomic distribution of CRBP within the Sertoli cells. Within the epididymis CRBP was selectively localized to the proximal portion of the caput epididymidis, with variations in intensity of the staining of the epithelium of the ducts in different histological zones. Specific immune staining for CRBP was very weak or absent in the other portions of the epididymis. These results were confirmed by radioimmunoassay. Vitamin A-deficient rats showed markedly reduced specific immune staining for CRBP in both testes and epididymides, and greatly reduced levels of CRBP in these tissues on radioimmunoassay. These studies on the localization of CRBP provide information concerning the specific cells and anatomic loci within the testis and epididymis where retinol may be playing an important role in sperm formation and maturation.     

Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons

We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.     

In-vitro pulsatile flow visualization studies in a pulmonary artery model

In-vitro pulsatile flow visualization studies were conducted in an adult-sized pulmonary artery model to observe the effects of valvular pulmonic stenosis on the flow fields of the main, left and right pulmonary arteries. The flow patterns revealed that as the degree of stenosis increased, the jet-type flow created by the valve became narrower, and it impinged on the far (distal) wall of the left pulmonary artery further downstream from the junction of the bifurcation. This in turn led to larger regions of disturbed turbulent flow, as well as helical-type secondary flow motions in the left pulmonary artery, compared to the right pulmonary artery. The flow field in the main pulmonary artery also became more disturbed and turbulent, especially during peak systole and the deceleration phase. The flow visualization observations have been valuable in helping to conduct further quantitative studies such as pressure and velocity field mapping. Such studies are important to understanding the fluid mechanics characteristics of the main pulmonary artery and its two major branches.     

Influence of cholesterol content on red cell membrane viscoelasticity and fluidity.

The purpose of this investigation was to correlate the viscoelastic properties and lipid fluidity of the red blood cell membrane to its lipid composition. The viscoelastic properties of human red cells that had been enriched or depleted in cholesterol were determined by the micropipette technique. The lipid fluidity of the outer and inner leaflets of the erythrocyte membrane was concurrently assessed by steady state fluorescence depolarization. The elastic modulus and the viscosity moduli of the erythrocyte membrane showed no significant differences between the cholesterol-modified and the control cells. Cholesterol enrichment decreased the lipid fluidity of the outer membrane leaflet alone, and cholesterol depletion increased the fluidity mainly of the inner leaflet.     

Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages

We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 μg/ml and 10 μg/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles.     

A monoclonal antibody from infected mice to a Schistosoma mansoni egg proteinase

A number of monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells and spleen lymphocytes from mice infected with Schistosoma mansoni. These antibodies were tested for their ability to inhibit acidic, thiol-dependent proteinases previously isolated from Schistosoma mansoni eggs and adult worms. One of the monoclonal antibodies isolated inhibits egg proteinase activity measured in vitro with the use of a low m.w. synthetic substrate. This antibody, which is an IgG1 isotype, does not appreciably inhibit an acidic, thiol-dependent proteinase obtained from the adult stage of Schistosoma mansoni. Immunocytochemical methods with the monoclonal antibody have been used to localize the egg proteinase within a set of "penetration" glands in the unhatched miracidium.     

Comparison of Rumen Microbial Inhibition Resulting from Various Essential Oils Isolated from Relatively Unpalatable Plant Species

Essential oils were isolated from eight plant species which were relatively unpalatable to sheep and deer. The inhibitory potency of these essential oils upon sheep and deer rumen microorganisms was compared, in terms of total gas and volatile fatty acid (VFA) production, by use of an anaerobic manometric technique. Inhibitory effects of oils from the eight plant species may be placed in four groups: (i) essential oils from vinegar weed (Trichostema lanceoletum) and California bay (Umbellularia californica) inhibited rumen microbial activity most; (ii) lesser inhibition was exhibited by rosemary (Rosmarinus officinalis) and California mugwort (Artemisia douglasiana) oils, followed by (iii) blue-gum eucalyptus (Eucalyptus globulus) and sagebrush (Artemisia tridentata) oils; and (iv) oils from Douglas fir (Psuedotsuga menziesii) and Jerusalem oak (chenopodium botrys) resulted in the least inhibition, when 0.3 ml of each oil was used. A highly significant correlation coefficient (r = 0.98(**)) between total gas and VFA production indicated the validity of either method to measure the activity of rumen microorganisms. Our results are discussed in relation to the hypothesis that the selectivity and voluntary consumption of ruminants are related to the characteristic odor and antibacterial action of essential oils isolated from relatively unpalatable plant species.     

Phosphorylation of choline and ethanolamine in Ehrlich ascites-carcinoma cells

1. Ehrlich ascites-cell extracts convert choline and ethanolamine approximately equally well into their respective phosphoryl derivatives. 2. Choline is a potent inhibitor of ethanolamine phosphorylation, but ethanolamine has little effect on choline phosphorylation. 3. 2,3-Dimercaptopropanol, cysteine and Ca(2+) inhibit ethanolamine phosphorylation, but have no detectable effect on choline phosphorylation. 4. Choline-phosphorylating activity in Ehrlich ascites-cell extracts is more stable during storage than ethanolamine-phosphorylating activity. 5. Choline phosphorylation is stimulated in the presence of benzoylcholine, succinylcholine, butyrylcholine and propionylcholine, whereas ethanolamine phosphorylation is inhibited. This relationship is reciprocal: the compounds causing the greatest stimulation of choline phosphorylation bring about the greatest inhibition of ethanolamine phosphorylation.