Effects of epidermal growth factor and progesterone on development, ultrastructure and gene expression of bovine secondary follicles cultured in vitro.

The aims of this study were to investigate the effects of epidermal growth factor (EGF) and progesterone on the development, viability and the gene expression of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (∼0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5 °C, with 5% CO2 in air, for 18 days, in TCM-199+ (n = 63) alone (control medium) or supplemented with 10 ng/mL progesterone (n = 64), 10 ng/mL EGF (n = 61) or both EGF and progesterone (n = 66). The effects of these treatments on growth, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and cyclin B1 were evaluated, significantly different (p < 0.05). The results showed that there was a progressive increase in follicular diameter in all treatments, but only follicles cultured in medium supplemented with EGF had increased significantly in diameter when compared to follicles cultured in the control medium at the end of the culture period, significantly different (p < 0.05). A positive interaction between EGF and progesterone was not observed. In addition, the presence of EGF, progesterone or both in culture medium did not influence the rate of follicle survival and antrum formation. However, the presence of only progesterone in cultured medium increased the expression of mRNAs for GDF9 and cyclin B1 in oocytes. EGF also significantly increased the levels of mRNAs for cMOS and GDF9 when compared to follicles cultured in control medium. Ultrastructural analyzes showed that cultured follicles in all treatments maintained the integrity of granulosa cells. In conclusion, the EGF promotes the development of secondary follicles cultured in vitro for 18 days and increases the expression of cMOS and GDF9, while progesterone alone or in association with EGF have not a positive effect on follicular growth. However, progesterone increases the expression of GDF9 and cyclin B1 in oocytes.

Helicobacter pylori antimicrobial resistance rates in the central region of Portugal.

Helicobacter pylori resistance to antimicrobial agents is steadily increasing. It is extremely important to be aware of the local prevalence of antibiotic resistance so as to adjust treatment strategies. During this single-centre, prospective study, we aimed to determine primary and secondary resistance rates of H. pylori to antibiotics as well as host and bacterial factors associated with this problem. Overall, 180 patients (131 female; mean age 43.4±13.5 years; primary resistance 103; secondary resistance 77) with positive (13) C-urea breath test were submitted to upper endoscopy with gastric biopsies. Helicobacter pylori was cultured and antimicrobial susceptibility was determined by Etest and molecular methods. Clinical and microbiological characteristics associated with resistance were evaluated by logistic regression analysis. Among the 180 isolates 50% were resistant to clarithromycin (primary 21.4%; secondary 88.3%), 34.4% to metronidazole (primary 29.1%; secondary 41.6%), 33.9% to levofloxacin (primary 26.2%; secondary 44.2%), 0.6% to tetracycline and 0.6% to amoxicillin. Being female was an independent predictor of resistance to clarithromycin and metronidazole. Previous, failed, eradication treatments were also associated with a decrease in susceptibility to clarithromycin. History of frequent infections, first-degree relatives with gastric carcinoma and low education levels determined increased resistance to levofloxacin. Mutations in the 23S rRNA and gyrA genes were frequently found in isolates with resistance to clarithromycin and levofloxacin, respectively. This study revealed that resistance rates to clarithromycin, metronidazole and levofloxacin are very high and may compromise H. pylori eradication with first-line and second-line empiric triple treatments in Portugal.

The effect of Helicobacter pylori infection on apoptosis and cell proliferation in gastric epithelium.

BACKGROUND/AIMS: Helicobacter pylori (Hp) may affect the normal balance between gastric epithelial cell proliferation and epithelial cell death, interfering thus with the maintenance of gastric mucosa integrity. The aim of this study was to investigate the effect of Hp infection on cell proliferation index (PI) and apoptotic index (AI) in gastric epithelium of the antrum and corpus. METHODOLOGY: Prospective study in forty-one patients with functional dyspepsia (14 males, 27 females, average age = 54+/-16.1 years). Day one: upper digestive endoscopy with biopsies of the antrum and corpus, and a cytological smear of the antral area for AgNOR (argyrophilic nucleolar organizer region) analysis. Biopsies for the programmed tests were sent in separate labeled containers: to study AI (antibody anti-M30), PI (antibody anti-Ki 67) and histology (Sydney criteria and the detection of Hp). Detection of the AgNORs through the Giménez-Mas et al. technique, using Visilog-Microptic Software. Day two: a blood sample was drawn from each patient for the serologic detection of the status CagA and VacA, and a breath test was carried out with 13C-Urea. STATISTICS: SPSS program with the application of Student's t, chi-square and Fisher tests. RESULTS: 24 patients were Hp(+) and 17 Hp(-). The PI (Ki 67 and AgNORs) in the antral area was significantly increased in the Hp(+) patients. AI showed no significant difference in the subgroups Hp(+) and Hp(-). Both subgroups showed increased PI in the antrum and increased AI in the gastric corpus. There was significantly higher PI in CagA(+), without an increase in the AI. The AI was significantly higher in CagA(-), when compared with CagA(+). The VacA protein had no influence on PI and AI. Acute and chronic gastritis was more frequent and more severe in Hp(+) patients. This group lacked any correlation between the histological findings and the PI, but the opposite was the case between AI and the degree of cellular infiltration. CONCLUSIONS: In patients with functional dyspepsia, Hp infection induces an increase of PI, with significant presence in the antrum area, without the corresponding increase in AI. Cag A(+) promotes the increase of PI, and Cag A(-) promotes the increase of AI. The Vac A status has no influence on the PI or AI. The degree of cellular infiltration interferes with AI.

Membrane lipid composition of Bacillus stearothermophilus as affected by lipophilic environmental pollutants: an approach to membrane toxicity assessment.

The thermophilic eubacterium Bacillus stearothermophilus is used as a model to identify membrane perturbing effects of lipophilic compounds. A parallelism has been established between the toxicity of the organochlorine insecticide DDT and its metabolite, DDE, in bacterial growth and the effects on cell functions and physical perturbations induced at the membrane (Donato et al. 1997a, Arch Environ Contam Toxicol 33:109-116; Donato et al. 1997b, Appl Environ Microbiol 63:4948-495). In the present work, the use of B. stearothermophilus as a model of screening for chemical toxicity has been implemented. Because the regulation of the lipid composition of the membrane is a common strategy in response to adverse growth conditions, we studied the effects of DDE on the lipid composition and the consequent alterations of membrane physical properties in comparison to the parental compound DDT. As expected, different adaptation responses were induced by the compounds, being DDT more effective as compared with DDE. Collected data are consistent with the stronger perturbations induced by DDT on growth and membrane functions. It is concluded that the membrane lipid composition of the bacterium is a very sensitive criterium to detect membrane-mediated toxic effects at low concentrations of lipophilic xenobiotics.

Bacillus stearothermophilus as a model to evaluate membrane toxicity of a lipophilic environmental pollutant (DDT).

The thermophilic eubacterium Bacillus stearothermophilus has been used as a model system to identify DDT-promoted events in biological membranes putatively related with the insecticide toxicity. Two strategies have been approached: a) bacterial growth and viability were followed and the effects of DDT (2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane) determined; b) biophysical studies with fluorescent probes were performed to elucidate the effects of DDT on the organization of the membrane lipid bilayer. The effects of DDT on growth and physical properties of the membrane were also determined in the presence of Ca2+ to further identify the interference of the insecticide at the membrane level and its putative contribution to cell toxicity. Growth inhibition by DDT is concentration-dependent, being attenuated or removed by the addition of 2.5-mM Ca2+ to bacterial cultures. Consistently, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its propionic acid derivative (DPH-PA) exhibited opposite effects of Ca2+ and DDT on the physical state of bacterial polar lipid dispersions. Growth and viability of bacterial cells are affected by DDT concentrations lower than those able to induce detectable bulk fluidity alterations, indicating high sensitivity of the intact bacterial system to alterations in limited membrane domains not directly probed by fluorescent probes that only report the average behavior of membrane lipid population.

Effects of a lipophilic environmental pollutant (DDT) on the phospholipid and fatty acid contents of Bacillus stearothermophilus.

Cultures of Bacillus stearothermophilus grown in a complex medium containing 1 microM DDT, exhibited longer lag adapting periods, decreased specific growth rates, and lower growth yield as compared to control cultures. The membrane lipid composition from cells grown in the presence of the insecticide was significantly different from that of control cells. The effects of DDT (2,2-bis(p-chlorophenyl)-1, 1,1-trichloroethane) on growth and lipid composition of bacterial cells were also determined in cultures grown in a medium supplemented with Ca2+ (membrane stabilizer) to further clarify the influence of growth conditions on bacterial responses to the toxicant. The main membrane-lipid changes induced by DDT relate to a very significant increase (74%) of the relative concentration of a phosphoglycolipid, an increase of the phosphatidylethanolamine content, with a parallel decrease of phosphatidylglycerol and an unidentified phospholipid X0. The changes of the phospholipid acyl chains relate to an increase of straight chains and a parallel decrease of branched chains. The effects of DDT-induced lipid composition alterations on membrane physical properties were monitored by fluorescence polarization studies with bacterial polar lipid dispersions. Changes in the membrane lipids upon growing the bacteria in a DDT-containing medium promoted, as expected, more ordered membranes with a shift of the phase transition temperature to higher values. Data are interpreted in the frame of an adaptation mechanism to counteract the membrane perturbation resulting from the accumulation of the insecticide molecules in the lipid bilayer.

Comparative study of the toxic actions of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane and 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene on the growth and respiratory activity of a microorganism used as a model.

A strain of Bacillus stearothermophilus was used as a model for a comparative study of the toxic effect of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane and 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene. Bacterial growth, the O2 consumption rate, and respiration-related enzymatic activities provided quantitative data in agreement with results reported for other systems. The use of this bacterium for screening for chemical toxicity is discussed.