Characterization of renin mRNA expression and enzyme activity in rat and mouse mesangial cells

Renin is an enzyme involved in the stepwise generation of angiotensin II. Juxtaglomerular cells are the main source of plasma renin, but renin activity has been detected in other cell types. In the present study we evaluated the presence of renin mRNA in adult male Wistar rat and mouse (C-57 Black/6) mesangial cells (MC) and their ability to process, store and release both the active and inactive forms of the enzyme. Active renin and total renin content obtained after trypsin treatment were estimated by angiotensinogen consumption analyzed by SDS-PAGE electrophoresis and quantified by angiotensin I generation by HPLC. Renin mRNA, detected by RT-PCR, was present in both rat and mouse MC under basal conditions. Active renin was significantly higher (P<0.05) in the cell lysate (43.5 ± 5.7 ng h-1 10(6) cells) than in the culture medium (12.5 ± 2.5 ng h-1 10(6) cells). Inactive prorenin content was similar for the intra- and extracellular compartments (9.7 ± 3.1 and 3.9 ± 0.9 ng h-1 10(6) cells). Free active renin was the predominant form found in both cell compartments. These results indicate that MC in culture are able to synthesize and translate renin mRNA probably as inactive prorenin which is mostly processed to active renin inside the cell. MC secrete both forms of the enzyme but at a lower level compared with intracellular content, suggesting that the main role of renin synthesized by MC may be the intracellular generation of angiotensin II.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Distribution of versican and hyaluronan in the mouse uterus during decidualization

Preparation for embryo implantation requires extensive adaptation of the uterine microenvironment. This process consists of cell proliferation and cell differentiation resulting in the transformation of endometrial fibroblasts into a new type of cell called decidual cell. In the present study, we followed the space-time distribution of versican and hyaluronan (HA) in different tissues of the uterus before and after embryo implantation. Fragments of mouse uteri obtained on the fourth, fifth, sixth and seventh days of pregnancy were fixed in Methacarn, embedded in Paraplast and cut into 5-µm thick sections. HA was detected using a biotinylated fragment of the proteoglycan aggrecan, which binds to this glycosaminoglycan with high affinity and specificity. Versican was detected by a polyclonal antibody. Both reactions were developed by peroxidase methods. Before embryo implantation, both HA and versican were present in the endometrial stroma. However, after embryo implantation, HA disappeared from the decidual region immediately surrounding the implantation chamber, whereas versican accumulated in the same region. The differences observed in the expression of HA and versican suggest that both molecules may participate in the process of endometrial decidualization and/or embryo implantation.

Normal HC11 and ras-transformed mouse mammary cells are resistant to the antiproliferative effects of retinoic acid

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR ß mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 µM 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of ß-casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR ß, as well as RXR a, ß and g expression was low in both HC11 and HC11ras cells. In addition, RAR ß mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse

Parkinson's disease, a major neurodegenerative disorder in humans whose etiology is unknown, may be associated with some environmental factors. Nocardia otitidiscaviarum (GAM-5) isolated from a patient with an actinomycetoma produced signs similar to Parkinson's disease following iv injection into NMRI mice. NMRI mice were infected intravenously with a non-lethal dose of 5 x 10(6) colony forming units of N. otitidiscaviarum (GAM-5). Fourteen days after bacterial infection, most of the 60 mice injected exhibited parkinsonian features characterized by vertical head tremor, akinesia/bradykinesia, flexed posture and postural instability. There was a peak of nocardial growth in the brain during the first 24 h followed by a decrease, so that by 14 days nocardiae could no longer be cultured. At 24 h after infection, Gram staining showed nocardiae in neurons in the substantia nigra and occasionally in the brain parenchyma in the frontal and parietal cortex. At 21 days post-infection, tyrosine hydroxylase immunolabeling showed a 58% reduction of tyrosine hydroxylase in the substantia nigra, and a 35% reduction of tyrosine hydroxylase in the ventral tegmental region. Dopamine levels were reduced from 110 ± 32.5 to 58 ± 16.5 ng/mg protein (47.2% reduction) in brain from infected mice exhibiting impaired movements, whereas serotonin levels were unchanged (191 ± 44 protein in control and 175 ± 39 ng/mg protein in injected mice). At later times, intraneuronal inclusion bodies were observed in the substantia nigra. Our observations emphasize the need for further studies of the potential association between Parkinson's disease or parkinsonism-like disease and exposure to various nocardial species.

Positive correlation of serum leptin with estradiol levels in patients with polycystic ovary syndrome

Patients with polycystic ovary syndrome (PCOS) usually are obese, insulin resistant and hyperinsulinemic. The known association between leptin, obesity andinsulin action suggests that leptin may have a role in PCOS but this has only been addressed peripherally. This study was designed to assess the relationship between serum leptin and the anthropometric, metabolic and endocrine variables of obese (body mass index, BMI ³30 kg/m²) and non-obese (BMI <30 kg/m²) PCOS patients. Twenty-eight PCOS patients and 24 control women subdivided into obese and non-obese groups were evaluated. Leptin, androgens, lipids, gonadotrophins and insulin-glucose response to the oral glucose tolerance test were measured by radioimmunoassay in all participants. The assays were done all in one time. The areas under the insulin curve (AUC-I) and the glycemia curve were calculated to identify patients with insulin resistance. Mean leptin levels were not significantly higher in patients with PCOS compared to the control group (21.2 ± 10.2 vs 27.3 ± 12.4 ng/ml). Leptin levels were found to be significantly higher in the obese subgroups both in patients with PCOS (26.9 ± 9.3 vs 14.1 ± 7.0 ng/ml) and in the control group (37.3 ± 15.5 vs 12.9 ± 5.8 ng/ml). The leptin of the PCOS group was correlated with BMI (r = 0.74; P < 0.0001) and estradiol (r = 0.48; P < 0.008) and tended to be correlated with the AUC-I (r = 0.36; P = 0.05). Of the parameters which showed a correlation with leptin in PCOS, only estradiol and probably insulinemia (AUC-I) did not show a significant correlation with BMI, suggesting that the other parameters were correlated with leptin due to their correlation with BMI. Estradiol correlated with leptin in PCOS patients regardless of their weight.

Fuzzy expert system in the prediction of neonatal resuscitation

In view of the importance of anticipating the occurrence of critical situations in medicine, we propose the use of a fuzzy expert system to predict the need for advanced neonatal resuscitation efforts in the delivery room. This system relates the maternal medical, obstetric and neonatal characteristics to the clinical conditions of the newborn, providing a risk measurement of need of advanced neonatal resuscitation measures. It is structured as a fuzzy composition developed on the basis of the subjective perception of danger of nine neonatologists facing 61 antenatal and intrapartum clinical situations which provide a degree of association with the risk of occurrence of perinatal asphyxia. The resulting relational matrix describes the association between clinical factors and risk of perinatal asphyxia. Analyzing the inputs of the presence or absence of all 61 clinical factors, the system returns the rate of risk of perinatal asphyxia as output. A prospectively collected series of 304 cases of perinatal care was analyzed to ascertain system performance. The fuzzy expert system presented a sensitivity of 76.5% and specificity of 94.8% in the identification of the need for advanced neonatal resuscitation measures, considering a cut-off value of 5 on a scale ranging from 0 to 10. The area under the receiver operating characteristic curve was 0.93. The identification of risk situations plays an important role in the planning of health care. These preliminary results encourage us to develop further studies and to refine this model, which is intended to implement an auxiliary system able to help health care staff to make decisions in perinatal care.

Air pollution and neonatal deaths in São Paulo, Brazil

Air pollution has been associated with health effects on different age groups. The present study was designed to assess the impact of daily changes in air pollutants (NO2, SO2, CO, O3, and particle matter (PM10)) on total number of daily neonatal deaths (those that occur between the first and the 28th days of life) in São Paulo, from January 1998 to December 2000, since adverse outcomes such as neonatal deaths associated with air pollution in Brazil have not been evaluated before. Generalized additive Poisson regression models were used and nonparametric smooth functions (loess) were adopted to control long-term trend, temperature, humidity, and short-term trends. A linear term was used for holidays. The association between air pollutants and neonatal deaths showed a short time lag. Interquartile range increases in PM10 (23.3 µg/m³) and SO2 (9.2 µg/m³) were associated with increases of 4% (95% CI, 2-6) and 6% (95% CI, 4-8), respectively. Instead of adopting a two-pollutant model we created an index to represent PM10 and SO2 effects. For an interquartile range increase in the index an increase of 6.3% (95% CI, 6.1-6.5) in neonatal deaths was observed. These results agree with previous studies performed by our group showing the deleterious effects of air pollutants during the perinatal period. The method reported here represents an alternative approach to analyze the relationship between highly correlated pollutants and public health problems, reinforcing the idea of the synergic effects of air pollutants in public health.

Effects of melatonin on the ovarian response to pinealectomy or continuous light in female rats: similarity with polycystic ovary syndrome

The current study was conducted to investigate the relationship between melatonin and chronic anovulation. Adult (3-4 months old) female Wistar rats were submitted to pinealectomy: group I: pinealectomized ovariectomized melatonin-treated (N = 10); group II: pinealectomized ovariectomized placebo-treated (N = 12); group III: pinealectomized light-treated placebo-treated(N = 10) or maintained under continuous light; group IV: maintained under continuous light, ovariectomized melatonin-treated (N = 22); group V: maintained under continuous light, ovariectomized placebo-treated (N = 10); group VI: maintained under continuous light placebo-treated (N = 10). In order to assess ovarian modifications, unilateral ovariectomy was performed during the fourth month in groups I, II, IV, V and the other ovary was removed after 8 months. Ovariectomy was performed in groups III and VI only after eight months. Melatonin (200 µg/100 g body weight) dissolved in 0.02 ml absolute ethanol was injected intramuscularly daily during the last 4 months into groups I and IV. The other groups were treated with placebo (NaCl). The ovarian cysts were analyzed and their area, perimeter and maximum diameter, as well as the thickness of the ovarian capsule were measured. Daily colpocytological smears were performed throughout the study. Persistent estrous condition and ovarian cysts were observed in all groups. In pinealectomized rats the ovarian and vaginal alterations disappeared at the end of the study and in rats maintained under continuous light the vaginal and ovarian polycystic aspect was reversed only in those treated with melatonin. We conclude that melatonin may act on the ovarian response reverting chronic anovulation induced by pinealectomy or continuous light.

Toxicity of a cyanobacterial extract containing microcystins to mouse lungs

Toxic cyanobacteria in drinking water supplies can cause serious public health problems. In the present study we analyzed the time course of changes in lung histology in young and adult male Swiss mice injected intraperitoneally (ip) with a cyanobacterial extract containing the hepatotoxic microcystins. Microcystins are cyclical heptapeptides quantified by ELISA method. Ninety mice were divided into two groups. Group C received an injection of saline (300 µl, ip) and group Ci received a sublethal dose of microcystins (48.2 µg/kg, ip). Mice of the Ci group were further divided into young (4 weeks old) and adult (12 weeks old) animals. At 2 and 8 h and at 1, 2, 3, and 4 days after the injection of the toxic cyanobacterial extract, the mice were anesthetized and the trachea was occluded at end-expiration. The lungs were removed en bloc, fixed, sectioned, and stained with hematoxylin-eosin. The percentage of the area of alveolar collapse and the number of polymorphonuclear (PMN) and mononuclear cell infiltrations were determined by point counting. Alveolar collapse increased from C to all Ci groups (123 to 262%) independently of time, reaching a maximum value earlier in young than in adult animals. The amount of PMN cells increased with time of the lesion (52 to 161%). The inflammatory response also reached the highest level earlier in young than in adult mice. After 2 days, PMN levels remained unchanged in adult mice, while in young mice the maximum number was observed at day 1 and was similar at days 2, 3, and 4. We conclude that the toxins and/or other cyanobacterial compounds probably exert these effects by reaching the lung through the blood stream after ip injection.

Effect of bullfrog (Rana catesbeiana) oil administered by gavage on the fatty acid composition and oxidative stress of mouse liver

The aim of the present study was to investigate the effects of daily intragastric administration of bullfrog oil (oleic, linoleic and palmitoleic acid-rich oil), corresponding to 0.4% of body weight for four weeks, on fatty acid composition and oxidative stress (lipid peroxidation and catalase activity) in mouse liver. The activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT), biomarkers of tissue injury, were determined in liver homogenates and serum. The proportions of 18:2n-6, 20:4n-6, 20:5n-3, and 22:6n-3 (polyunsaturated fatty acids, from 37 to 60%) in the total fatty acid content were increased in the liver of the bullfrog oil-treated group (P < 0.05) compared to control. At the same time, a significant decrease in the relative abundance of 14:0, 16:0, and 18:0 (saturated fatty acids, from 49 to 25%) was observed. The hepatic content of thiobarbituric acid reactive substances (TBARS) was increased from 2.3 ± 0.2 to 12.3 ± 0.3 nmol TBA-MDA/mg protein and catalase activity was increased from 840 ± 32 to 1110 ± 45 µmol reduced H2O2 min-1 mg protein-1 in the treated group. Bullfrog oil administration increased AST and ALP activities in the liver (from 234.10 ± 0.12 to 342.84 ± 0.13 and 9.38 ± 0.60 to 20.06 ± 0.27 U/g, respectively) and in serum (from 95.41 ± 6.13 to 120.32 ± 3.15 and 234.75 ± 11.5 to 254.41 ± 2.73 U/l, respectively), suggesting that this treatment induced tissue damage. ALT activity was increased from 287.28 ± 0.29 to 315.98 ± 0.34 U/g in the liver but remained unchanged in serum, whereas the GGT activity was not affected by bullfrog oil treatment. Therefore, despite the interesting modulation of fatty acids by bullfrog oil, a possible therapeutic use requires care since some adverse effects were observed in liver.

Neonatal administration of citalopram delays somatic maturation in rats

We investigated the somatic maturation of neonate rats treated during the suckling period with citalopram, a selective serotonin reuptake inhibitor. Groups with 6 male neonates were randomly assigned to different treatments 24 h after birth. Each litter was suckled by one of the dams until the 21st postnatal day. Body weight, head axis and tail length were measured daily from the 1st to the 21st postnatal day. Time of ear unfolding, auditory conduit opening, incisor eruption, and eye opening was determined. Pups received 5 mg (Cit5), 10 mg (Cit10) or 20 mg/kg (Cit20) citalopram sc, or saline (0.9% NaCl, w/v, sc). Compared to saline, body weight was lower (24.04%, P < 0.01) for Cit10 from the 10th to the 21st day and for Cit20 from the 6th to the 21st day (38.19%, P < 0.01). Tail length was reduced in the Cit20 group (15.48%, P < 0.001) from the 8th to the 21st day. A reduction in mediolateral head axis (10.53%, P < 0.05) was observed from the 11th to the 21st day in Cit10 and from the 6th to the 21st day in Cit20 (13.16%, P < 0.001). A reduction in anteroposterior head axis was also observed in the Cit20 group (5.28%, P < 0.05) from the 13th to the 21stday. Conversely, this axis showed accelerated growth from the 12th to the 21stday in the Cit5 group (13.05%, P < 0.05). Auditory conduit opening was delayed in the Cit5 and Cit20 groups and incisor eruption was delayed in all citalopram groups. These findings show that citalopram injected during suckling to rats induces body alterations and suggest that the activity of the serotoninergic system participates in growth mechanisms.

Effects of metformin treatment on luteal phase progesterone concentration in polycystic ovary syndrome

The causes of luteal phase progesterone deficiency in polycystic ovary syndrome (PCOS) are not known. To determine the possible involvement of hyperinsulinemia in luteal phase progesterone deficiency in women with PCOS, we examined the relationship between progesterone, luteinizing hormone (LH) and insulin during the luteal phase and studied the effect of metformin on luteal progesterone levels in PCOS. Patients with PCOS (19 women aged 18-35 years) were treated with metformin (500 mg three times daily) for 4 weeks prior to the test cycle and throughout the study period, and submitted to ovulation induction with clomiphene citrate. Blood samples were collected from control (N = 5, same age range as PCOS women) and PCOS women during the late follicular (one sample) and luteal (3 samples) phases and LH, insulin and progesterone concentrations were determined. Results were analyzed by one-way analysis of variance (ANOVA), Duncan's test and Karl Pearson's coefficient of correlation (r). The endocrine study showed low progesterone level (4.9 ng/ml) during luteal phase in the PCOS women as compared with control (21.6 ng/ml). A significant negative correlation was observed between insulin and progesterone (r = -0.60; P < 0.01) and between progesterone and LH (r = -0.56; P < 0.05) concentrations, and a positive correlation (r = 0.83; P < 0.001) was observed between LH and insulin. The study further demonstrated a significant enhancement in luteal progesterone concentration (16.97 ng/ml) in PCOS women treated with metformin. The results suggest that hyperinsulinemia/insulin resistance may be responsible for low progesterone levels during the luteal phase in PCOS. The luteal progesterone level may be enhanced in PCOS by decreasing insulin secretion with metformin.

Photocytotoxicity of a 5-nitrofuran-ethenyl-quinoline antiseptic (Quinifuryl) to P388 mouse leukemia cells

Quinifuryl (MW 449.52), 2-(5'-nitro-2'-furanyl)ethenyl-4-{N-[4'-(N,N-diethylamino)-1'-methylbutyl]carbamoyl} quinoline, is a water soluble representative of a family of 5-nitrofuran-ethenyl-quinoline drugs which has been shown to be highly toxic to various lines of transformed cells in the dark. In the present study, the toxicity of Quinifuryl to P388 mouse leukemia cells was compared in the dark and under illumination with visible light (390-500 nm). Illumination of water solutions of Quinifuryl (at concentrations ranging from 0.09 to 9.0 µg/ml) in the presence of P388 cells resulted in its photodecomposition and was accompanied by elevated cytotoxicity. A significant capacity to kill P388 cells was detected at a drug concentration as low as 0.09 µg/ml. The toxic effect detected at this drug concentration under illumination exceeded the effect observed in the dark by more than three times. Moreover, the general toxic effect of Quinifuryl, which included cell proliferation arrest, was nearly 100%. Both dose- and time-dependent toxic effects were measured under illumination. The LC50 value of Quinifuryl during incubation with P388 cells was ~0.45 µg/ml under illumination for 60 min and >12 µg/ml in the dark. We have demonstrated that the final products of the Quinifuryl photolysis are not toxic, which means that the short-lived intermediates of Quinifuryl photodecomposition are responsible for the phototoxicity of this compound. The data obtained in the present study are the first to indicate photocytotoxicity of a nitroheterocyclic compound and demonstrate the possibility of its application as a photosensitizer drug for photochemotherapy.

Skin morphology of the mutant hairless USP mouse

The morphology of the skin of the mutant hairless USP mouse was studied by histological, histochemical and immunohistochemical methods and compared to the skin of BALB/c mice. Representative sections of the dorsal skin from mice of both strains aged 18 days, and 1, 3, 6, and 8 months were studied. Sections stained with hematoxylin and eosin showed cystic formations called utricles and dermal cysts in the dermis that increased in size and number during growth. Skin thickness increased significantly at 8 months. Sections stained with picrosirius and examined with polarized light, displayed different colors, suggesting different thicknesses of dermal collagen fibers (probably types I and III). Weigert, Verhoeff and resorcin-fuchsin stains revealed fibers of the elastic system. The PAS and Alcian blue methods revealed neutral and acid glycosaminoglycans in the skin ground substance of both mouse strains. Immunohistochemical staining for fibronectin and laminin did not show differences between the mutant and BALB/c mice. Mast cells stained by the Gomori method and macrophages positive for HAM 56 antibodies were observed in both mouse strains. Except for the presence of enlarged cysts in the hairless strain, no qualitative differences were found during development of the skin of BALB/c and the mutant hairless mice.