Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of semen fractionation and dilution ratio on motility parameters of stallion spermatozoa. In Experiment 1, three ejaculates from each of three stallions were divided into sperm-rich (SR) and sperm-poor (SP) fractions to determine the difference in sperm concentration. Mean sperm concentration in SR fractions (349.5 x 10(6)/ml) was greater (P < 0.001) than that of SP fractions (96.9 x 10(6)/ml). In Experiment 2, three ejaculates from each of two stallions were divided into SR and SP fractions. Fifty percent of the original volume of SR fractions was combined with 50% of the original volume of SP fractions for each ejaculate to represent total ejaculates. SR and total ejaculates were diluted with skim milk-glucose semen extender as follows: 1) no dilution, or dilution to 2) 100 x 10(6)sperm/ml, 3) 50 x 10(6)sperm/ml, or 4) 25 x 10(6)sperm/ml. Semen samples were evaluated at 0.5, 3, 6, 12, and 24 h postejaculation (25 degrees C storage temperature) for percentages of total spermatozoal motility (TSM) and progressive spermatozoal motility (PSM). Mean TSM was greater (P < 0.05) in SR ejaculates than total ejaculates at 12 and 24 h postejaculation. Mean TSM of undiluted semen was lower (P < 0.05) than other dilution ratios over all periods. Mean TSM was greater (P < 0.05) at a 25 x 10(6)sperm/ml dilution ratio than a 50 x 10(6)sperm/ml dilution ratio at 12 and 24 h postejaculation, and greater (P < 0.05) than a 100 x 10(6)sperm/ml dilution ratio from 3 to 24 h postejaculation. Similar patterns were found for PSM. Collection of SR ejaculates and dilution to 25 x 10(6)sperm/ml improved longevity of spermatozoal motility.     

Chick oviduct progesterone receptor phosphorylation: characterization of a copurified kinase and phosphorylation in primary cultures

A protein kinase activity was copurified with the chick oviduct progesterone receptor. The enzyme is magnesium dependent and can use the B subunit of progesterone receptor or histones as substrates. The physiochemical parameters of the kinase were determined [pI approximately 5.3; Stokes radius approximately 7.2 nm; sedimentation coefficient (S 20,w) approximately 5.6] and compared to those of the purified B subunit. The results were consistent with the presence of an unique enzyme distinct from the receptor itself. The physiological significance of receptor phosphorylation was investigated in oviduct cells grown in primary culture. Cells were labeled with [32P]orthophosphate in presence or absence of progesterone and the receptor components were immunoprecipitated with a specific polyclonal antibody. Although progesterone treatment lead to the attachment of most of the receptor (approximately 80%) to nuclear structures, the 32P-labeled B subunit was only recovered in the cytosol fraction. Different procedures to extract the nuclear receptor did not allow detection of any 32P-labeled form in the nuclear-soluble fractions, suggesting that the B subunit was not further phosphorylated upon the exposure of cells to progesterone.     

Bactericidal activity of peripheral blood neutrophils during the oestrous cycle and early pregnancy in the mare

The oestrous cycles of 20 mixed-breed mares were synchronized with daily injections of 10 mg oestradiol-17 beta and 150 mg progesterone given i.m. for 10 days. On the 10th day, 10-15 mg prostaglandin F-2 alpha was administered i.m. to induce oestrus. Neutrophils were isolated from jugular blood on the 2nd or 3rd day of oestrus, Days 5 and 7 after ovulation or during early pregnancy (Days 18-34 of pregnancy). Neutrophils were challenged with Staphylococcus aureus and their bactericidal activity examined after 30 and 120 min of incubation for a reduction of colony forming units. Bactericidal activity increased with the time of incubation (P less than 0.01) but did not differ for the oestrous cycle or pregnancy (P greater than 0.05).     

Genetic and biological analyses of a herpes simplex virus intertypic recombinant reduced specifically for neurovirulence.

RS6 is a herpes simplex virus intertypic recombinant derived from type 1 strain 17 syn+ and type 2 strain HG52. With a 50% lethal dose of about 10(5) PFU after intracerebral inoculation of mice, RS6 was approximately 100,000 times less neurovirulent than either of its wild-type parental viruses were. When compared with strains 17 syn+ and HG52, RS6 replicated intermediately in primary mouse embryo fibroblasts in vitro at 38.5 degrees C (mouse temperature) and to wild-type peak titers in mouse feet in vivo. In contrast, following intracranial inoculation of mice, RS6 replicated significantly less well than did either of its parental viruses in brains. The genetic defect(s) responsible for the reduced neurovirulence of RS6 was stable after in vitro and in vivo serial passage, was not manifested as temperature-sensitive plaquing in vitro, and did not affect thymidine kinase expression. These data indicate that RS6 has a genetic defect(s) specifically affecting its ability to replicate in the mouse brain. Using marker rescue technologies, we increased the neurovirulence of RS6 and localized one genetic determinant(s) involved with the reduced neurovirulence of this agent to 0.72 to 0.87 map units (and, tentatively, to 0.79 to 0.83 map units) of the herpes simplex virus genome. When coupled with the work suggesting that thymidine kinase expression is essential for efficient replication in nerve tissues and earlier reports from this laboratory and others, the results presented in this study indicate that more than one herpes simplex virus gene is involved with neurovirulence.     

Isolation from urine of two Serratia marcescens strains excreting a diffusible yellow pigment

Two bacterial strains excreting a yellow pigment were isolated from human urine and identified as Serratia marcescens. The pigment was produced in the late exponential and early stationary phases of growth. Minimal media supplemented with tyrosine, phenylalanine, 3,4-dihydroxyphenylacetate or tryptophan, as well as complex media, induced pigment production. UV-visible spectra of the extracted pigment had peaks characteristic of 2-hydroxy-5-carboxymethylmuconate semialdehyde, produced from meta-cleavage of 3,4-dihydroxyphenylacetate by the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15). This enzyme was active when the bacteria were grown under conditions promoting pigment production. The kinetics and factors affecting pigment production are also reported.     

d-Aspartate in Human Brain

The presence of the biologically uncommon D-aspartic acid (D-aspartate) in human brain white matter has been previously reported. The earlier study has now been expanded to include D/L-aspartate ratios from 67 normal brains. The data show that the D-aspartate content increases rapidly from 1 year to approximately 35 years of age, levels off in middle age, and then appears to decrease somewhat. The D-aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L-aspartate ratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D-aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.     

A relationship between the inhibition of heparan sulfate and chondroitin sulfate synthesis and the inhibition of molting by selenate in the hemipteran Rhodnius prolixus

The insect Rhodnius prolixus synthesizes heparan sulfate and chondroitin sulfate after a blood meal containing [35S]-inorganic sulfate. A 40 to 80% inhibition of heparan sulfate synthesis was obtained when the meal was supplemented with 10(-5) and 10(-4) M sodium selenate respectively. Likewise an inhibition of the molting in the order of 30 to 60% was observed when the insects were fed with blood containing 10(-5) and 10(-4) M selenate respectively. The insects after a subsequent meal without selenate molted normally. Except for the inhibition of the ecdysis no gross physiological or morphological changes could be observed in the insects. Based on these and other findings the possible role of sulfated glycosaminoglycans in the control of cell growth is discussed.     

Specific binding of atrial natriuretic factor to renal glomeruli in Doca- and Doca-salt-treated rats correlation with atrial and plasma levels

Since volume expansion and high blood pressure (BP) are known stimuli of atrial natriuretic factor (ANF) release, and since this peptide may be involved in mineralocorticoid escape, we investigated the effects of chronic deoxycorticosterone (DOCA) and DOCA-NaC1 treatment on renal glomerular ANF receptor density and affinity in relation to atrial and plasma ANF levels. An increase in plasma immunoreactive ANF (IR-ANF) was observed both after two and four weeks of treatment. IR-ANF concentrations were elevated in the left atrium only in four-week DOCA treated rats. Administration of the mineralocorticoid alone resulted in a decreased density of glomerular ANF receptors in both time periods investigated. DOCA-NaC1-treated animals presented an increased receptor density during the pre-hypertensive stage (2 weeks) and a reduced density in the later hypertensive period (4 weeks). Receptor affinity in both groups was identical to that in the controls after 2 weeks and was augmented after 4 weeks of treatment. Our data suggest that the down-regulation of renal glomerular ANF receptors during chronic DOCA-NaC1 administration may play a role in the maintenance of high BP in this model of volume-expanded hypertension.     

Growth curves,morphology and ultrastructure of ten Paracoccidioides brasiliensis isolates

The yeast phase of ten P. brasiliensis isolates were studied to characterize their growth pattern, morphology and ultrastructure. Growth curves were determined after counts of total and viable fungi units (FU) during 20 days. Three growth patterns were observed: slow, reaching approximately 10–30× 10⁶ FU/tube (Pb 18, Pb 265 and PB 2); intermediate, reaching 60–150×10⁶ FU/tube (IVIC Pb 9, IVIC Pb 267, Pb SN, Pb Vitor and Pb Campo Grande) and fast, reaching 180–370×10⁶ FU/tube (Pb 2052 and Pb 192). The highest percentage of viable cells occurred on the 6th day of culture for Pb 192, Pb Campo Grande, Pb 2052 and IVIC Pb 9; on the 8th day for Pb Vitor, Pb SN, Pb 18 and IVIC Pb 267; on the 10th day for Pb 265 and on the 12th day of culture for Pb 2. Mean generation times varied from approximately 21.2 (Pb 2052) to 102.6 hours (Pb 265). The isolates showed similar morphology, except IVIC Pb 267 which did not present a typical yeast-phase at 35°C and the two fast-growing isolates (Pb 2052 and Pb 192) that presented smaller cell sizes and less tendency to clump. The ultrastructure of the isolates was similar: the cell walls presented a width of 0.1 to 0.2 °; the mitochondria presented few cristae and had equivalent patterns of distribution and morphology; the endoplasmic reticulum was scanty, presenting narrow cisternae; the vacuoles, empty or filled with electrondense material, were numerous and two to five nuclei with pores were constantly observed.     

Reconstructive surgery for immunosuppressed organ-transplant recipients

Prolonged vascularized organ allograft survival and an improved quality of life are now possible for many transplant recipients. These advances are due largely to greater understanding of the immune response, the development of potent immunosuppressive agents (cyclosporin A), and improved surgical techniques. Thus more of these patients may require surgical procedures related or unrelated to their original operation, and the plastic surgeon, among other specialists, should be aware of the special problems of the immunocompromised transplant recipient who needs to undergo reconstructive surgery. We report our experience with 15 kidney, heart, and liver transplant recipients who required reconstructive surgery for a variety of conditions. The combined team approach by reconstructive and transplant surgeons is described, as well as the perioperative drug protocol and the special problems that immunosuppressed transplant recipients present. We conclude that these patients can successfully undergo major reconstructive procedures as long as the plastic surgeon not only performs technically flawless surgery, but also familiarizes himself or herself with the special problems of the immunosuppressed host, including the ever-present risk of sepsis and delayed and impaired wound healing, the potential for acute Addisonian crisis, and the possibility of multiple complicating comorbid conditions.