Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus

Deletions of polyoma virus DNA around the region that codes for the C-terminus of the viral middle T-antigen were created using a transforming fragment (BamH I/EcoR I) of viral DNA cloned in the plasmid vector pAT153. These species were recloned and assayed for their ability to transform Rat-1 cells in culture. Our results showed that whereas the DNA sequence between the presumed translational termination codon for the viral middle T-antigen and the single viral EcoR I site could be removed with no apparent effect on transformation, the removal of the termination codon itself or any amino acid coding sequences of this protein caused a drastic decrease in the transforming ability of the DNA. Transfection of Rat-1 cells with plasmids that contained viral DNA with deletions which corresponded to the last fourteen or more amino acids of the middle T-antigen never gave rise to cellular transformation.     

Role of gonadal hormones in development of the sexual phenotypes

Summary Male and female embryos develop in an identical fashion during the initial portion of gestation. If the indifferent gonad differentiates into an ovary (or if no gonad is present), a female phenotype is formed. Male phenotypic differentiation, however, requires the presence of an endocrinologically active testis. Two secretion of the fetal testis, Müllerian inhibiting substance and testosterone, are responsible for male development. Studies of single gene mutations that interfere with androgen action indicate that testosterone itself is responsible for virilization of the Wolffian duct system into the epididymis, vas deferens, and seminal vesicle, whereas the testosterone metabolite dihydrotestosterone induces development of the prostate and male external genitalia. Thus, impairment of dihydrotestosterone formation results in a characteristic phenotype consisting of predominantly female external genitalia but normally virilized Wolffian ducts. The molecular mechanisms by which testosterone and dihydrotestosterone act during fetal development appear to involve the same high affinity receptor, a protein that transports both testosterone and dihydrotestosterone to the nucleus of target cells. When this receptor is either absent, deficient, or structurally abnormal, the actions of both testosterone and dihydrotestosterone are impaired, and the resulting developmental anomalies involve both internal and external genital structures.The original work described in this review was supported by grant AM 03892 from the National Institutes of Health     

Reproductive studies of Brahman cattle II. Luteinizing hormone patterns in ovariectomized Brahman and Hereford cows before and after injection of gonadotropin releasing hormone

Six Brahman and six Hereford long-term ovariectomized cows were bled via tail vessel at 15 minute intervals for a period of 4 hours. Serum was collected and analyzed via radioimmunoassay (RIA) for luteinizing hormone (LH) to determine if ovariectomized Brahman and Hereford cows have pulsatile LH patterns and if breed of animal influenced LH patterns. Brahman and Hereford ovariectomized cattle did have pulsatile LH patterns. Although the trend was for higher LH levels in Hereford than Brahman cows there were no significant differences in mean serum LH levels, number or magnitude of LH peaks or serum LH pulse height.Six Brahman and five Hereford long-term ovariectomized cows were injected (IM) with a single dose of 500μg of gonadotropin releasing hormone (GnRH). Animals were bled via tail vessel at 15 minute intervals for a period of 6 hours. Serum was assayed for LH via RIA to determine if ovariectomized Brahman and Hereford cows differ in GnRH induced LH response. All animals showed increased serum LH in response to GnRH injection within the first 15 minute collection interval. There were no significant differences in duration of response between ovariectomized Brahman or Hereford cows. Ovariectomized Brahman cows had significantly lower (P<.005) Lh values per period than ovariectomized Hereford cows. It was therefore concluded that ovariectomized Brahman cows were significantly less responsive to GnRH induced LH release than were ovariectomized Hereford cows, although duration and shape of the response curves were not different.     

Orientation of Euglena gracilis by electromagnetic fields: theory and experiment.

Computer data derived from theoretical treatments of the orientation of ellipsoidal particles in alternating fields is compared with experimental data from microscopic studies on Euglena gracilis, and elongated free-living flagellate. Computed data based upon a theoretical treatment by SAITO, SCHWAN and SCHWARZ showed good agreement with experimental results, predicting the relationship between cellular orientation, the frequency of the impressed field, and the conductivity of the suspending medium.     

Toxic effect of water-soluble fractions of crude, refined, and weathered oils on the growth of a marine bacterium.

The water-soluble fractions of three crude and two refined oils reduced the growth rate and maximum cell density of the marine bacterium Serratia marinorubra grown in batch culture. The weathering of a crude and a refined oil was simulated in the laboratory. The water-soluble fractions remaining from this process were more toxic to S. marinorubra than were the parent unweathered oils. Increases in the magnitude of toxic effect of 3 to 30 times were observed as a function of decreasing the concentration of yeast extract in the cultures from 0.1 to 0.05 and 0.01%. The toxicity did not correlate with the concentration of total water-soluble fraction or of aromatic hydrocarbons in the water-soluble fraction. Affected cultures did not exhibit a residual toxicity after being back-inoculated into control media.     

Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.     

Cell cycle kinetics, tissue transglutaminase and programmed cell death (apoptosis).

Studies were undertaken on a highly metastatic hamster fibrosarcoma cell line with a view to assessing whether cells entering into apoptosis, measured by counting the number of transglutaminase mediated detergent insoluble envelopes, has any synchrony with a particular phase of the cell cycle. A double exposure of thymidine was used to block cells in early S-phase. Flow cytometry in combination with [3H]thymidine incorporation into DNA was used to assess the degree of synchrony and progression through the different phases of cell cycle. The apoptotic index was found to be at its maximum in mid-S-phase. Measurement of transglutaminase activity in each phase of the cell cycle indicated that the specific activity was also at its greatest during mid S-phase. The level of enzyme was relatively unchanged throughout the cell cycle indicating that the regulation of transglutaminase activity occurs primarily through effects on catalytic activity rather than enzyme synthesis.