Fertility after bilateral cryptorchidism. Evaluation by paternity, hormone, and semen data.

PURPOSE: Evaluation of the fertility of a cohort of formerly bilaterally cryptorchid men in comparison with a group of formerly unilaterally cryptorchid men, and a group of control men. MATERIALS AND METHODS: Using a detailed questionnaire concerning paternity and factors related to paternity, a cohort of formerly bilateral cryptorchid men were studied and compared with men who had undergone orchiopexy for unilateral cryptorchidism, and a group of control men. All study subjects had had surgery at the Children's Hospital of Pittsburgh, Pittsburgh, Pa., between 1955 and 1975. A subset of the full cohort underwent clinical evaluation that included a physical examination, serum hormonal determination and semen analyses. RESULTS: Paternity rates are significantly lower among the formerly bilaterally cryptorchid men who have attempted to father a child (65.3%) as compared to the formerly unilaterally cryptorchid (89.7%; p < 0.001) and control men (93.2%; p < 0.001). Differences in the ability to father children are also apparent when semen and hormone levels are compared between the three groups. The bilateral group has significantly lower sperm density and inhibin B levels, and higher FSH and LH levels, than the unilateral and control groups. CONCLUSIONS: Men born with bilateral cryptorchidism have severely compromised fertility in adulthood. This reduction in fertility is clearly shown in comparisons of both paternity rates, and in semen and hormone analyses, between the formerly bilateral, formerly unilateral, and control groups.     

Autophosphorylation of brain microtubule protein: evidence for endogenous protein kinase/phosphoprotein phosphatase cycling and multiple phosphorylation of a microtubule associated protein

Brain microtubule protein, prepared by two types of recycling methods, undergoes “flash” phosphorylation in the presence of [γ-³²p]ATP through sequential action of protein kinase and phosphoprotein phosphatase present in microtubule protein. SDS electrophoretic analysis indicates that MAP₁, tau protein, and tubulin are poorly phosphorylated, and MAP₂ is the major site of phosphorylation. To improve [³²P]phosphoprotein stability in the presence of the kinase/phosphatase cycle, 3′,5′-cyclicAMP, orthophosphate, or fluoride ion may be added. After separation from tubulin by phosphocellulose chromatography, the MAP fraction exhibits autophosphorylation. Finally, the maximal extent of autophosphorylation is observed with an ATP regenerating system using ADP, [³²P]acetyl-P, and bacterial acetate kinase; this results in the incorporation of 3–4 phosphoryl groups per MAP₂ subunit.     

Thrombin-induced events in non-platelet cells are mediated by the unique proteolytic mechanism established for the cloned platelet thrombin receptor

We recently isolated a cDNA clone encoding a functional platelet thrombin receptor that defined a unique mechanism of receptor activation. Thrombin cleaves its receptor''s extracellular amino terminal extension, unmasking a new amino terminus that functions as a tethered peptide ligand and activates the receptor. A novel peptide mimicking this new amino terminus was a full agonist for platelet secretion and aggregation, suggesting that this unusual mechanism accounts for platelet activation by thrombin. Does this mechanism also mediate thrombin''s assorted actions on non-platelet cells? We now report that the novel thrombin receptor agonist peptide reproduces thrombin-induced events (specifically, phosphoinositide hydrolysis and mitogenesis) in CCL-39 hamster lung fibroblasts, a naturally thrombin- responsive cell line. Moreover, these thrombin-induced events could be recapitulated in CV-1 cells, normally poorly responsive to thrombin, after transfection with human platelet thrombin receptor cDNA. Our data show that important thrombin-induced cellular events are mediated by the same unusual mechanism of receptor activation in both platelets and fibroblasts, very likely via the same or very similar receptors.     

Production of L-phenylacetyl carbinol by immobilized yeast cells: II. Semicontinuous fermentation

The cyclic, semicontinuous production of L-phenylacetyl carbinol (L-PAC) from a benzaldehyde substrate by Saccharomyces cerevisiae ATCC 834 immobilized in calcium alginate beads was substantially enhanced to about 4.5 g/L in a second cycle by reactivation in fresh medium for 24 h, following an earlier 24-h period of production from substrate. Intermittent feeding of benzaldehyde was employed (four doses in 3 h). In subsequent similar cycles, however, the production returned to that produced in the first cycle, viz. L-PAC concentration of 2-3 g/L in the medium. Production of L-PAC was also increased by adaptation of the cells over 200 h of exposure to the benzaldehyde substrate (compared to wild-type cells) and by continuous (as compared to intermittent) feeding of the substrate. A liter as great as 10 g/L was obtained with wild-type cells by continuous feeding of benzaldehyde over 6 h. Immobilization not only protected the cells from toxic effects of substrate but also permitted them to be used during 7 cycles of semicontinuous operation over more than 200 h.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Enzymatic hydrolysis of cellulose to glucose lung immobilized β-glucosidase

The production of sugars by enzymatic hydrolysis of cellulose is a multistep process which includes conversion of the intermediate cellobiose to glucose by β-glucosidase. Aside from its role as an intermediate, cellobiose inhibits the endoglucanase components of typical cellulase enzyme systems. Because these enzyme systems often contain insufficient concentrations of β-glucosidase to prevent accumulation of inhibitory cellobiose, this research investigated the use of supplemental immobilized β-glucosidase to increase yield of glucose. Immobilized β-glucosidase from Aspergillus phoenicis was produced by sorption at controlled-pore alumina with about 90% activity retention. The product lost only about 10% of the original activity during an on-stream reaction period of 500 hr with cellobiose as substrate; maximum activity occurred near pH 3.5 and the apparent activation energy was about 11 kcal/mol. The immobilized β-glucosidase was used together with Trichoderma reesei cellulase to hydrolyze cellulosic materials, such as Solka Floc, corn stove and exploded wood. Increased yields of glucose and greater conversions of cellobiose of glucose were observed when the reaction systems contained supplemental immobilized β-glucosidase.