The effects of methyltestosteone on reproductive function in male greyhounds

Oral administration of methyltestosterone (MT) at 50 mg/dog/day to intact adult male greyhounds for 90 d resulted in decreased (P < 0.05) mean daily sperm output and mean testicular length. Additionally, the mean diameter of seminiferous tubules in MT-treated dogs tended to decrease (P = 0.08). Mean concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and concentrations of testosterone in serum were also decreased or tended to decrease (P = 0.0003 to 0.059) at various sampling periods during MT treatment, suggesting alterations in spermatogenesis resulted from decreased serum concentrations of gonadotropins and steroids. Mean daily sperm output, mean testicular length, mean seminiferous tubule diameter and mean concentrations of FSH in serum were not decreased (P > 0.05) at the end of a 90-d recovery period. However, mean concentrations of serum LH and concentrations of testosterone were still lower (P < 0.05) during five of six and one of six sampling times, respectively, during the recovery period than the pretreatment levels, suggesting a prolonged effect of MT treatment on the pituitary/gonadal axis.     

Protein Synthesis and Breakdown during Heat Shock of Cultured Pear (Pyrus communis L.) Cells

Cultured pear (Pyrus communis L. cv Passe Crassane) cells were subjected to temperatures of 39, 42, and 45[deg]C. Heat-shock protein (hsp) synthesis was greater at 30[deg]C than at temperatures above 40[deg]C and continued for up to 8 h. Both cellular uptake of radiolabeled methionine and total protein synthesis were progressively lower as the temperature was increased. Polysome levels decreased immediately when cells were placed at 39 or 42[deg]C, although at 39[deg]C the levels began to recover after 1 h. In cells from both temperatures, reassembly occurred after transfer of cells to 25[deg]C Four heat-shock-related mRNAs[mdash]hsp17, hsp70, and those of two ubiquitin genes[mdash]all showed greatest abundance at 39[deg]C and decreased at higher temperatures. Protein degradation increased with time at 42 and 45[deg]C, but at 39[deg]C it increased for the first 2 h and then decreased. In the presence of cycloheximide, which prevented hsp synthesis, protein degradation at 39[deg]C was as great as that at 45[deg]C in the absence of cycloheximide. The data suggest that hsps may have a role in protecting proteins from degradation at the permissive temperature of 39[deg]C. At temperatures high enough to inhibit hsp synthesis, protein degradation was enhanced. Although ubiquitin may play a role in specific protein degradation, it does not appear to be involved in increased protein degradation occurring above 40[deg]C.     

GLOBAL POPULATION STRUCTURE AND NATURAL HISTORY OF THE GREEN TURTLE (CHELONIA MYDAS) IN TERMS OF MATRIARCHAL PHYLOGENY

To address aspects of the evolution and natural history of green turtles, we assayed mitochondrial (mt) DNA genotypes from 226 specimens representing 15 major rookeries around the world. Phylogenetic analyses of these data revealed (1) a comparatively low level of mtDNA variability and a slow mtDNA evolutionary rate (relative to estimates for many other vertebrates); (2) a fundamental phylogenetic split distinguishing all green turtles in the Atlantic-Mediterranean from those in the Indian-Pacific Oceans; (3) no evidence for matrilineal distinctiveness of a commonly recognized taxonomic form in the East Pacific (the black turtle C.m. agassizi or C. agassizi); (4) in opposition to published hypotheses, a recent origin for the Ascension Island rookery, and its close genetic relationship to a geographically proximate rookery in Brazil; and (5) a geographic population substructure within each ocean basin (typically involving fixed or nearly fixed genotypic differences between nesting populations) that suggests a strong propensity for natal homing by females. Overall, the global matriarchal phylogeny of Chelonia mydas appears to have been shaped by both geography (ocean basin separations) and behavior (natal homing on regional or rookery-specific scales). The shallow evolutionary population structure within ocean basins likely results from demographic turnover (extinction and colonization) of rookeries over time frames that are short by evolutionary standards but long by ecological standards.     

The interaction of the steroidal antagonist faslodex and methotrexate.

Faslodex (FAS, ICI 182, 780), a novel steroidal estrogen antagonist decreased high-dose methotrexate (MTX) cytotoxicity in MCF-7 breast cancer cells. When FAS is given at least 24 hr prior to MTX, the resultant interaction is antagonistic. However, when breast cancer cells are exposed to FAS 24 hr after MTX, the interaction between FAS and MTX is not antagonistic. The proliferation of cells exposed to 0.1 microM FAS and 10 microM MTX alone or in combination with FAS 24 hr prior to MTX was in the following order: FAS>FAS 24 hr prior to MTX>MTX. MTX administration 24 hr prior to FAS had the following inhibitory effects on the growth of cells: MTX 24 hr prior to FAS >MTX>FAS 24 hr prior to MTX>FAS>control (no drug exposure). To determine if the antagonistic interaction between FAS and MTX was a function of sequence and time, cells were exposed to FAS 24 hr and 36 hr prior to MTX exposure. The percentages of control rates were 42.70 +/- 4.60% and 57.89 +/- 0.55%, respectively, from a 24 hr and 36 hr exposure of FAS prior to MTX. The growth rates after 24 and 36 hr exposures to MTX alone were 30.30 +/- 0.61% and 33.11 +/- 2.57% of control rates, respectively. These studies suggest that: a) the interactions between FAS and MTX are sequence-dependent; b) FAS antagonizes the effect of MTX when FAS administration precedes MTX, and c) FAS antagonism to MTX is a function of time.     

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mm 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mm 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1–3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mm 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mm octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3–5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 m monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mm 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.We acknowledge the technical assistance of Richard Lewis and Meghan Abella. We thank Dr. Hugh Dookwah for contributions to the myometrial cell isolation protocol and Drs. Stephen H. Safe, Timothy D. Phillips, and Evelyn Tiffany-Castiglioni for helpful discussions. This work was funded by NIH (HD-26182, P42-ES04917, ES05871-01A1), the March of Dimes Birth Defects Foundation Basic Research grant #1-0796, and USDA 92-37203-7952.     

Properties of microfiltration membranes: Mechanisms of flux loss in the recovery of an enzyme

The transmission and rate of filtration of the enzyme yeast alcohol dehydrogenase (YADH) has been studied at capillary pore microfiltration membranes. Photon correlation spectroscopy (PCS) with nanometer resolution showed that the enzyme existed as discreate molecules only for a narrow range of pH and ionic strength. Under such conditions, the transmission of the enzyme was high. However, the rate of filtration still decreased continuously with time. Analyssis of the time dependence of the rate of filtration indicated that this decrease was due to in-pore enzyme deposition at low concentration ("standard blocking model") and suface depositon at high concentration ("cake filtration model"). Use of atomic force microscopy (AFM) gave unequivocal and quantitative confirmation of these inferences. The work shows the great advantage of using advanced physical characterization techniques, both for the identification of the optimum conditions for filtration (PCS) and for the elucidation of mechanisms giving rise to inefficiencies in the filtration process (AFM). (c) 1995 John Wiley & Sons, Inc.     

Expression of Ty-lacZ fusions in Saccharomyces cerevisiae

We have determined the nucleotide sequence of about 520 bp spanning the 5' delta regions (Figure 1) of two Tyl elements. There is an open reading frame running out of the deltas for at least 180 nucleotides into the internal region of each element. The functional significance of these open reading frames has been tested by fusing them to a defective E.coli lacZ gene. Expression of B-galactosidase in yeast transformants containing these fusions shows that Tyl elements contain functional translation signals.     

Metabolism of [14C]diethylstilboestrol epoxide by rat liver in vitro.

1. The trans-epoxide of diethylstilboestrol and its pinacolone were synthesized chemically and the pinacolone shown to be formed from the epoxide by a non-enzymic process. 2. [14C]Diethylstiboestrol epoxide was converted by rat liver microsomal fraction into 4'-hydroxypropiophenone by a new type of cleavage reaction involving mono-oxygenase. Conditions for the formation of this metabolite and also water-soluble products were investigated together with the effect of inhibitors. A sex-difference in the conversion of diethylstilboestrol epoxide into 4'-hydroxypropiophenone and to polar and water-soluble products was observed. 3. Diethylstilboestrol epoxide was found to be a relatively stable compound that did not form a glutathione conjugate readily without further microsomal activation. A purified preparation of epoxide hydratase did not enhance its rate of conversion into the pinacolone. 4. Diethylstilboestrol epoxide was found to have about one-tenth the oestrogenic activity of diethylstilboestrol as measured by the increase in uterine weight or the induction of peroxidase in immature rat uteri. It was inactive as a mutagen when tested for its ability to inhibit bacteriophage phi X174 DNA viral replication. 5. The possible role of diethylstilboestrol epoxide as an intermediate in the metabolism of diethylstilboestrol and in mediating the harmful effects of this synthetic estrogen is discussed.     

DNA fragments associated with chromosome scaffolds

Following extensive digestion of HeLa metaphase chromosomes with either Hae III endonuclease or micrococcal nuclease, nonhistone protein scaffolds may be isolated. Scaffolds isolated after Hae III digestion have about 1.5% of the chromosomal DNA attached to them. This DNA is heterogeneous in size, ranging from about 0.2 to 20 kbp. It can be cleaved with either Eco RI or Hae III - Eco RI, producing a series of repeated fragments, of which the most abundant is 1.7 kbp in length. The 1.7-kdp fragment is tandemly repeated and is enriched (about 50-fold) in the scaffold-associated DNA. It is located primarily on human chromosome 1 and is probably a component of human satellites II and III. Scaffolds isolated after micrococcal nuclease digestion have about 0.1% of chromosomal DNA attached. This DNA is present in two size classes - fragments larger than 10 kbp and fragments approximately 0.2 kbp long. Restriction enzyme digestion of this DNA gives no prominent repeated fragments. Its reassociation kinetics are similar to those of total DNA, indicating that it is not enriched in either highly repetitive or middle repetitive sequences.