Caspase 3 activation and PARP cleavage in lymphocytes from newborn babies of diabetic mothers with unbalanced glycaemic control.

Several epidemiological studies showed that gestational diabetes mellitus is the most frequent metabolic disorder of pregnancy, the pathogenesis of which has yet to be completely clarified. The aim of this study was to investigate the presence and processing of caspase 3 (Casp3) and poly(ADP-ribose) polymerase 1 (PARP1) in cord blood lymphocytes as markers of apoptosis in relation to glycaemic control during intrauterine life. Our results showed a specific positive correlation between the levels of active Casp3 (17-19 kDa) and the inactive form of PARP1 (89 kDa) in lymphocytes isolated from newborn babies of diabetic women with unbalanced glycaemic control, with a direct correlation between the activation of casp3 and the inactivation of PARP1, that makes lymphocytes unresponsive towards lipopolysaccharide stimulation, highlighting an altered functional response. Besides more studies are required to fully correlate the activation of the apoptotic process during the intrauterine life with the foetal health later in life, our study indicates that a cord blood lymphocyte, an easily accessible source, is informative about the activation of apoptotic stimuli in circulating cells of newborn babies in relation to the glycaemic control reached by the mother during pregnancy.

Phytoplasmas Associated with Grapevine Yellows Disease in Chile.

An extensive survey was performed from 2002 to 2006 to detect and identify phytoplasmas associated with Chilean grapevines. Nested polymerase chain reaction assays using phytoplasma universal primer pairs P1/P7 and R16F2n/R2 detected phytoplasmas in 34 out of the 94 samples tested (36%). Restriction fragment length polymorphism (RFLP) analyses, cloning, and sequencing allowed identification of phytoplasmas belonging to ribosomal subgroups 16SrI-B, 16SrI-C, 16SrVII-A, and 16SrXII-A. The 16SrVII-A phytoplasma represents a new finding in grapevine; moreover, variability of the RFLP profile was observed in some of the 16SrXII-A phytoplasmas, indicating possible new ribosomal subgroups. Mixed phytoplasma infections and infections of phytoplasmas together with one or more viruses also occurred.

[Ticlopidine use in Spain: a pharmacy-based drug utilization study]. / Estudio de la utilización de ticlopidina en oficinas de farmacia de España.

BACKGROUND: In 1997 a program was set up to improve the use of ticlopidine. In the present study we assess whether this objective was achieved. PATIENTS AND METHODS: We carried out a pharmacy-based cross-sectional study. RESULTS: Out of 346 patients interviewed, 56% presented an off-label indication for ticlopidine. In 23% of patients the daily dose used was lower or higher than the recommended. Only 28% patients had the fortnightly blood monitoring performed at the time of interview. CONCLUSIONS: The use of ticlopidine in Spain is not consistent with the summary of product characteristics and the program set up to improve it did not achieve a satisfactory result.

Dietary isoflavones affect sex hormone-binding globulin levels in postmenopausal women.

The studies presented in this report were designed to further investigate the causal association between phytoestrogen action and increase in sex hormone-binding globulin (SHBG) levels. Phytoestrogens include isoflavones that bind to estrogen receptors and therefore exert estrogenic action. This study included 20 postmenopausal women that ingested 30 g soy milk daily for 10 weeks. Plasma concentrations of isoflavones and SHBG were measured. Total isoflavones significantly increased from 0.014 +/- 0.01 micromol/L (baseline) to 0.53 +/- 0.19 ,micromol/L, and paired responses showed that some subjects clearly increased their SHBG levels. The percent change in SHBG showed a positive correlation with phytoestrogen concentration; all women who had circulating phytoestrogen levels above 0.6 micromol/L increased by at least 30% their SHBG values. Results suggest that phytoestrogens may significantly increase SHBG in subjects whose SHBG concentrations are in the low end of the concentration range.

Abnormal osteogenesis in osteoporotic patients is reflected by altered mesenchymal stem cells dynamics.

Bone marrow contains a population of mesenchymal stem cells with the ability to differentiate into cells that form bone, cartilage, adipose, and other connective tissues. Stem cells can be isolated from bone marrow aspirates and expanded in vitro. Presently, most stem cells studies have been performed in cells obtained from "healthy" control subjects. The goal of this study was to compare the functional characteristics of mesenchymal stem cells derived from "healthy" control and osteoporotic postmenopausal women to better understand the mechanisms involved in the pathogenesis of this disease. Osteoporotic and control stem cells have similar morphology and size and express similar cell surface antigens as evidenced by their reactivity with cell specific monoclonal antibodies. Mesenchymal stem cells from osteoporotic women differ from controls in having a lower growth rate than control cells, being refractory to the mitogenic effect of IGF-1, and exhibiting a deficient ability to differentiate into the osteogenic linage as evidenced by the alkaline phosphatase activity and calcium phosphate deposition. We conclude that in osteoporosis stem cell growth, proliferative response and osteogenic differentiation are significantly affected. Also, the study of mesenchymal stem cells from osteoporotic postmenopausal women may provide a better understanding of the mechanisms involved in the pathogenesis of the osteoporosis. It may also serve to test in vitro in rapid manner novel new therapeutic strategies.

[Androgen receptor gene structure and protein function. Gene structural defects that promote androgen resistance in men]. / Estructura del gen del receptor de andrógeno y función de la proteína. Defectos estructurales del gen que determinan resistancia a andrógenos en el hombre.

The human androgen receptor is a member of the superfamily of steroid hormone receptors and contains three functional domains: an amino-terminal region involved in the expression of androgen regulated genes, a central cysteine-rich DNA binding region and a carboxy-terminal hormone binding region. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. Androgen action is currently studied in vitro, using fibroblasts culture from genital skin and complementary DNA of the androgen receptor gene has been recently cloned and sequenced. During recent years a substantial progress has been made elucidating the structure-function relationship of the androgen receptor and the characterization of the molecular defects associated with androgen insensitivity syndromes. There appears to be a broad correlation between the degree of receptor dysfunction caused by the mutation and the patient phenotype.

In vitro effect of hCG on steroidogenesis in the testicular tissue from a patient with complete androgen resistance.

The present study was performed to determine the in vitro steroidogenic capacity of a gonadal sample from a patient suffering from a complete androgen resistance syndrome. Testosterone and estradiol production by the testicular tissue from this patient as well as gonadotropin binding to a membrane fraction prepared from this tissue were measured. hCG bound with high affinity but with a very low capacity and the gonadotropin induced a clear dose response for both testosterone and estradiol production. The ED50 of hCG on testosterone and estradiol production were 2.5 and 5.0 nM, respectively. We conclude that estradiol originates from Leydig cell activity, since estradiol synthesis does not depend on testosterone availability and it shows a clear hCG dose response.

Detection of testosterone secretion from individual rat Leydig cells.

The purpose of the present study was to analyze testosterone secretion from individual purified Leydig cells, using a reverse hemolytic plaque assay (RHPA) as an approach for identifying and characterizing subtypes of Leydig cells. Leydig cells from adult rats and protein A-coated ovine erythrocytes were mixed and incubated for appropriate lengths of time in the presence or absence of antitestosterone antibody, hormones or an analog of cyclic AMP. The slides from RHPA were histochemically stained for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). Results show that testosterone secreting cells can be clearly identified by the formation of hemolytic plaques. The proportion of plaque-forming cells increases with incubation time, reaching a plateau at 60 min in the presence of gonadotropin. It was observed that not all 3 beta-HSD positive cells form plaques. It is concluded that the purified Leydig cell population has cells with differential steroidogenic and androgen-secretory activities.

Evidence for a Leydig cell progesterone receptor in the rat.

Tritiated promegestone [3H] R 5020 is bound with high affinity by charcoal-treated cytosol prepared from purified Leydig cells. The binding is characterized by high affinity (Kd = 2 x 10(-9) M) and specificity (R 5020 = progesterone greater than testosterone = dehydrotestosterone greater than hydroxyprogesterone greater than cortisol = dexamethasone greater than estradiol) appropriate for progesterone receptors. In vitro, progestin-bound cytosol was quantitatively translocated to nuclei fractions, only if cytosol samples were previously labeled at 25 degrees C. However no translocation of binding activity was observed when previous cytosol labeling was done in the presence of sodium molybdate. Effects of glucocorticoids, androgens and estrogens on the Leydig cell are well documented, the demonstration of a putative progesterone receptor raises the possibility of direct effect of progesterone on the Leydig cell.

RNA synthesis in the Leydig cells of the mature rat: effect of oestradiol-17 beta.

In view of the evidence that there may be an effect of high concentrations of oestradiol on testicular steroidogenic function, we have investigated the effect of this steroid on [3H]uridine incorporation into RNA by testicular cells. Our results have shown that oestradiol in vitro induced a marked dose-dependent inhibition of RNA synthesis by purified Leydig cells. The concentrations of oestradiol tested varied from 2 to 40 mumol/l; these concentrations also impaired net testosterone synthesis in vitro after human chorionic gonadotrophin (hCG) stimulation. Under the effect of oestradiol, the kinetics of [3H]nucleoside incorporation into RNA were impaired early and the inhibition of RNA synthesis was specific for oestrogenic compounds. It was concluded that, in Leydig cells, oestradiol, in addition to its known inhibitory action on the response of testosterone to hCG, triggers a more extensive response that also includes RNA synthesis in vitro.

RNA synthesis in Leydig cells during sexual maturation in the rat: effect of LH.

The trophic action of LH on Leydig cells involves the triggering of a number of cellular events including changes in protein synthesis. This latter change has led a number of workers to postulate an effect of LH on RNA synthesis. A direct action of LH on RNA synthesis, however, has been difficult to assess. The aim of the present work was to analyse the effect of LH on RNA synthesis in vitro during sexual development. Studies were performed using purified Leydig cells from rats of 20, 30, 40, 50, 60 and 90 days of age. The results obtained show that basal uridine incorporation into RNA increases in an age-dependent manner in rats from 20 to 60 days of age and then remains unchanged until 90 days of age. A stimulatory effect of LH on RNA synthesis was clearly demonstrated only in the youngest rats (20 and 30 days old). In order to differentiate the effect of LH on different RNA populations, the RNA synthesized by immature and mature rats was analysed using a poly(U)-Sepharose column. In 20-day-old rats, LH stimulated both unbound and poly(A) RNA, although a more marked effect was clearly demonstrated on the latter. On the other hand, LH had an identical effect on both unbound and poly(A) RNA obtained from Leydig cells of 60-day-old rats. This stimulatory effect of LH on RNA synthesis in Leydig cells from immature rats seemed specific, since effectors which act on interstitial cells, such as LH-releasing hormone, [Arg8]-vasopressin and FSH (which may act on macrophages) did not modify RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The transmethylations inhibitor 3-deazaadenosine, inhibits in vitro testosterone production by rat testis interstitial cells stimulated with HCG.

3-Deazaadenosine (3-DZA), an inhibitor of somatic cell transmethylations, inhibited in vitro HCG-stimulated testosterone synthesis by rat testis interstitial cells. A maximal inhibition of 50% was observed with 100 microM 3-DZA; in addition homocysteine-thiolactone (Hcy) enhanced the inhibitory effect of 3-DZA. On the other hand, when cells were stimulated with dibutyryl cyclic AMP (Bt)2-cAMP, 3-DZA did not exert any effect on the stimulation. The presence of 3-DZA in the incubation medium neither modified HCG Kd values nor the number of its binding sites to Leydig cells. These results demonstrate that inhibitors of transmethylation reactions interfere with hormone-stimulated testosterone synthesis, suggesting that those reactions (presumably phospholipid methylation) at the plasma membrane level are involved in hormone-stimulated testosterone synthesis by rat Leydig cells.

Estradiol and progesterone nuclear and cytosol receptors of hydrosalpinx.

Hydrosalpinx is usually associated with a low intrauterine pregnancy rate following restoration of tubal patency by microsurgery. The biochemical evaluation of pathologic tubes from 11 infertile patients showed the presence of estradiol (E2) and progesterone (P) receptors at the cytosol and nuclear levels. The binding constants (Kd) for these specific proteins for E2 and Promegestone (R5020) were of the same magnitude as observed in normal tissue. Mean levels of E2 cytosolic and nuclear receptors and cytosolic P receptors of hydrosalpinx were significantly lower than those of a normal fallopian tube (P less than 0.05). No correlation between the severity of the histologic appearance of the tissue and the subcellular distribution of receptors was observed. We conclude that the decrease in the steroid receptor population of these damaged tubes could be counted as another factor to be considered in the poor intrauterine rate of the salpingoneostomy.

Changes during the menstrual cycle in cytosolic and nuclear concentrations of progestagen receptor in the human Fallopian tube.

[3H]R5020 was bound to cytosolic and nuclear samples of human Fallopian tube with high affinity and specificity. The cytoplasmic and nuclear concentrations of progestagen receptor varied, throughout the menstrual cycle, in the ampulla, isthmus and fimbria. Concentrations were higher at the late proliferative stage of the cycle than at the early proliferative and late secretory stages. A positive linear regression was observed between cytosolic and nuclear progestagen receptor concentrations and plasma oestradiol levels. A negative linear relationship was observed between cytosolic progestagen receptor concentration and plasma progesterone levels during the secretory stages of the menstrual cycle.

[Pathomorphological changes in the organs of cattle dying in so-called sudden death]. / Patomorfologichni izmeneniia v organite na goveda, umreli ot t. nar. "vnezapna smurt".

Studied were morphologically the organs of 10 cattle originating from two provinces of Cuba that suddenly succumbed ( muerte subita ). There were hemorrahagic diathesis, and histologically--general activation of the reticulo-endothelial system, nonsuppurative encephalomyocarditis, interstitial nonsuppurative hepatitis, nephritis, and pneumonia as well as catarrhal hemorrhagic gastroenteritis. In all cases there were among the lymphoid proliferations diffusely disseminated eosinophile leukocytes ( hyperergia ). This finding showed that the disease had run a subacute or chronic course which was made acute by the action of some stress factors (continuous running, intoxications oligoelement disturbances, etc.). The finding was also characteristic of reactive processes taking place under the action of some specific virus that probably took part in the etiology of the disease and required an intermediary host that remained unknown at the time.

Changes in cytosolic and nuclear estradiol receptors of normal Fallopian tube throughout the menstrual cycle.

During the normal menstrual cycle, the changes in estradiol binding to cytosol and nuclear receptors of ampulla, isthmus and fimbria were analyzed. Cytosol estradiol receptor concentration showed little variation throughout the cycle, but were higher at the periovulatory phases. The concentration of nuclear estradiol receptor markedly varied in isthmus and ampulla; being significantly higher at the late proliferative phase than at the secretory phases. A positive linear correlation between nuclear receptor content and plasma estradiol concentration was observed, while a negative one was found with plasma progesterone.