Biomedical subjects

M A Shu

Publications and source records attributed to M A Shu.

8 recordsLinked to original sources

PubMed2001

DNA binding and modulation of gene expression by the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The latency-associated nuclear antigen (LANA) is highly expressed in these malignancies and has been shown to play an important role in episomal maintenance, presumably by binding to a putative oriP. In addition, LANA modulates cellular and viral gene expression and interacts with the cellular tumor suppressors p53 and retinoblastoma suppressor protein. Many of these features are reminiscent of Epstein-Barr virus nuclear antigens (EBNAs), a family of six proteins expressed during latency. EBNA-1 is required for episome maintenance, binds to oriP, and strongly activates transcription from two promoters, including its own. We have previously shown that LANA can transactivate its own promoter and therefore asked whether LANA, like EBNA-1, activates transcription by direct binding to DNA. By using recombinant LANA expressed from vaccinia virus vectors for electrophoretic mobility shift assays, we found that LANA does not bind to its own promoter. In contrast, LANA binds specifically to sequences containing an imperfect 20-bp palindrome in the terminal repeat (TR) of KSHV. We further show that the C-terminal domain of LANA is sufficient for site-specific DNA binding. Unlike EBNA-1, which activates transcription through binding of oriP, we found that LANA inhibits transcription from a single TR binding site. A multimerized TR as found in the viral genome results in strong transcriptional suppression when linked to a heterologous promoter. These data suggest that LANA, although fulfilling functions similar to those of EBNA-1, does so by very different mechanisms.

Animals↗

PubMed1998

Secretion of plasminogen activator by cultured rat endometrial stromal cells from uteri differentially sensitized for the decidual cell reaction.

Endometrial stromal cells from rat uteri differentially sensitized for the decidual cell reaction in vivo and which undergo differing degrees of decidualization in vitro were cultured and plasminogen activator (PA) in the medium determined. The cells were obtained by enzymatic dispersion from the uteri of ovariectomized, steroid-treated rats at the equivalent of day 4, 5, or 6 of pseudopregnancy or on day 5 from rats treated on day 4 with 0, 0.3, or 1.0 microgram estradiol (low, intermediate, or high dose of estradiol, respectively) and cultured for 24, 48, or 72 hr. For cells from day 4, 5, and 6 uteri cultured under control conditions, PA activity in the medium was greatest for day 5 cells, which were from uteri maximally sensitized for decidualization both in vivo and in vitro. By contrast, for cells from low-, intermediate-, and high-estradiol uteri, PA activity in the medium was greatest for the high-estradiol cells; these cells do not undergo decidualization in vivo or in vitro to the same extent as intermediate-estradiol cells. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, reduced PGE2 accumulation to nondetectable amounts and for most cultures decreased PA activity in the medium, suggesting that endogenous PG production regulated in part PA secretion under control conditions. The addition of PGE2 with indomethacin increased PA activities above those under control conditions, but activities were still lower for day 4 and 6 cells compared with day 5 cells, and for low- and intermediate-estradiol cells compared with high-estradiol cells. This indicates that the differences in PA secretion are not explainable by differences in PGE2 production. Northern blot analysis of RNA from cells cultured for 72 hr under control conditions did not reveal significant differences in steady-state concentrations of mRNA for urokinase-type PA or plasminogen activator inhibitor 1, but those for tissue-type PA were lower in day 6 cells compared with day 4 and 5 cells. It is concluded that PA activity secreted by the cultured endometrial stromal cells, although controlled in part by the endocrine milieu to which they were exposed prior to culture, does not simulate decidualization in vitro and, therefore, that PA activity is not a marker for decidualization in vitro.

Animals↗

PubMed1996

Regulation of plasminogen activator in rat endometrial stromal cells: the role of prostaglandin E2.

The rat endometrium undergoes decidualization, a tissue remodeling process, during embryo implantation. Plasminogen activator (PA), particularly the urokinase-type PA (uPA), has been implicated in tissue remodeling. The present study determined whether rat endometrial stromal cells secrete uPA during decidualization in vitro and, if so, whether the secretion is regulated by prostaglandins that are required in decidualization. Endometrial stromal cells were obtained from rats that had been treated with estrogen and progesterone to sensitize their uteri for decidualization, and the cells were cultured for up to 72 h in a serum-free medium. PA activity in the conditioned medium, as determined by a chromogenic assay, increased steadily during the 72-h culture period. PA secretion decreased when endogenous prostaglandin synthesis was inhibited by the addition of indomethacin to the cell cultures. The inhibitory effect of indomethacin on PA secretion was reversed by prostaglandin E2, and much less effectively by prostaglandin F2 alpha. PA activity in the medium was due primarily to uPA because 1) PA activity was inhibited by a uPA-specific inhibitor-amiloride-and by an anti-mouse uPA antibody, and 2) the predominant PA activity in the medium, as identified in zymography, had a molecular mass of approximately 40 kDa, similar to that reported for uPA. Northern blot analyses of RNA from the cultured cells demonstrated that the steady-state levels of mRNA for uPA, but not for tPA and plasminogen activator inhibitor-1, were decreased by indomethacin; this decrease was reversed by prostaglandin E2. Taken together, the data indicate that rat endometrial stromal cells secrete uPA during decidualization in vitro, and that prostaglandin E2 regulates uPA secretion by the decidualizing cells by directly increasing uPA gene transcription and/or stabilizing its transcripts. These findings may help to partially elucidate the mechanism of action of prostaglandin E2 in decidualization.

Animals↗

PubMed1996

Regulation of urokinase plasminogen activator production in implanting mouse embryo: effect of embryo interaction with extracellular matrix.

Embryo implantation in the mouse is an invasive process and requires the action of proteinases, including plasminogen activator (PA) and metalloproteinases. After the implanting embryo establishes close contact with the endometrium, the invasion process begins, at least in part, through interactions of the embryo with the extracellular matrix in the endometrium. This study determined whether embryo interaction with extracellular matrix components would affect the secretion of PA in vitro. PA in vitro. Mouse embryos were collected from the uterus on Day 3.5 of development, just before implantation, and were cultured dishes precoated with bovine serum, plasma fibronectin, or BSA (control). Embryos cultured on serum- or fibronectin-coated dishes secretes significantly more PA than those cultured on BSA. The effect of fibronectin was inhibited by hexapeptides that contained the integrin-recognizing Arg-Gly-Asp sequence. This indicates that the action of fibronectin in enhancing PA secretion is mediated through its receptor (integrins) in the embryo. Fibronectin fragments reproduced the effect of the whole fibronectin molecule, suggesting that the clustering of integrins by specific ligands is responsible, at least in part, for the increase PA secretion. The increase in PA secretion was a specific response to fibronectin rather than a reflection of increased total protein secretion, and was at least partially a result of the increased steady-state level of PA mRNA in the cultured embryos. Laminin was as effective as fibronectin in promoting PA secretion. Epidermal growth factor increased PA secretion, probably by promoting the interaction of the embryos with the extracellular matrix. In summary, our findings indicate that the interactions of the implanting embryos with their extracellular matrix may regulate trophoblast invasion by controlling PA secretion.

Amino Acid Sequence↗

PubMed1990

A simplified approach to sperm-cervical mucus interaction testing using a hyaluronate migration test.

Fifty-one comparisons were made of human sperm migration into capillary tubes containing either human cervical mucus ('Kremer test') or a synthetic mucus substitute consisting of a 5 mg/ml solution of sodium hyaluronate (average mol. wt 2 x 10(6)) in a phosphate-buffered medium. The results of these two tests were highly significantly correlated and dependent upon the same sperm characteristics reflecting sperm progressive ability (including the specific movement characteristic of lateral head displacement amplitude), morphological normality and cellular vitality as well as the concentration of these more functional cells in the semen. The result of the hyaluronate migration test, in conjunction with the mucus quality measures of Insler score and pH, allowed a 92.2% correct prediction of the Kremer test outcome (90.9% of normal tests and 93.1% of abnormal tests). In this data set, these values also corresponded to the sensitivity and specificity of the analysis, respectively. From these studies, we propose the hyaluronate migration test as a useful adjunct to routine semen analysis, sperm movement analysis and the more traditional in-vitro tests of sperm-cervical mucus interaction in the diagnostic investigation of infertile couples. It effectively assesses the mucus-penetrating potential of a semen sample without the need for relatively large quantities of midcycle cervical mucus; it will therefore augment (as an internal control), although not necessarily replace, the homologous Kremer test and reduce the quantity of both patient and donor mucus needed for comprehensive crossed-hostility format testing of sperm-mucus interaction.

Cervix Mucus↗

PubMed1989

A technical note on diluting semen for the haemocytometric determination of sperm concentration.

A comparison was made of the use of either an SMI positive displacement pipette or an Eppendorf Varipette as the method of sampling 63 liquefied semen samples for volumetric dilution and haemocytometry to determine sperm concentration. The 95% range of the differences between the values obtained using the SMI and the Varipette with whole tips was from -46.9 to 63.8 x 10(6)/ml. With the Varipette tips cut off 12.5 mm from the end the 95% range was from -52.4 to 55.8 x 10(6)/ml. Previous work had shown that the 95% ranges of differences between duplicate determinations using the SMI pipette were from -7.2 to 6.9 x 10(6)/ml for two 1 + 19 dilutions, or from -16.0 to 12.1 x 10(6)/ml for 1 + 19 and 1 + 49 parallel dilutions. Therefore, since the Varipette had a far greater potential for error, a positive displacement pipette should be used when taking precise volume aliquots of human semen.

Humans↗

PubMed1988

Evaluation of the CellSoft automated semen analysis system in a routine laboratory setting.

The sperm concentration and percentage motility values generated by version 3.2 of the CellSoft (Cryo Resources Ltd., New York, NY) automated semen analyzer on 200 ejaculates were compared with those obtained by standardized traditional methods. Overall, CellSoft gave mean concentrations that were 20.9 x 10(6)/ml lower (95% range of differences = -112.6 to +154.4 x 10(6)/ml). However, the difference between methods was not systematic. Below 50 x 10(6)/ml, CellSoft more often gave higher values, and above 100 x 10(6)/ml, it usually gave lower values. In the middle range, differences were randomly distributed. For motility, the CellSoft values were usually higher than those obtained by visual counting (mean difference = -17.5%, 95% range = -56.0% to +21.0%). Multiple regression analyses revealed a strong concentration dependency such that reliable values will probably be obtained only if all samples are diluted (with homologous seminal plasma) before CellSoft analysis. This upper concentration limit is of the order of 30 to 50 x 10(6)/ml. Without such dilution, this version of CellSoft will not provide sufficiently accurate values for basic semen characteristics and cannot be accepted as a routine diagnostic method.

Humans↗

PubMed1986

Standardization and quality control of sperm concentration and sperm motility counts in semen analysis.

The paper reports a study of standardization and quality control of sperm concentration counts and visual motility assessments in human semen analyses performed for infertility investigations and from internal quality control procedures. Sperm concentration determinations were performed in Improved Neubauer haemocytometers on volumetric dilutions made using a positive displacement pipettor for sampling the liquefied semen. In addition to a standard 1 + 19 dilution a second dilution of either 1 + 9, 1 + 19 or 1 + 49 was made according to whether the estimated sperm concentration was less than 20, 20-100 or greater than 100 X 10(6)/ml respectively. The duplicate determinations of sperm concentration were highly significantly correlated (P much less than 0.001) with less than 5% variability. Parallel visual sperm motility assessments were made by two pairs of technicians and showed highly significant correlations (P much less than 0.001) between technicians in the determination of the percentages of motile and progressive spermatozoa as well as the subjective rating of sperm progressivity. When these values were incorporated into a calculated motility index which gave added weight to the progressive spermatozoa and to their quality of progression the correlations between technicians remained highly significant (P much less than 0.001) with average differences of the order of 1.0%. Therefore, provided that sufficient attention is paid to technician training, regular standardization checks and the use of only proven reliable procedures, quantitatively accurate values for sperm concentration and motility can be obtained in routine semen analyses.

Humans↗