Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae)

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ß-ATP, and ADP failed to do so in a 1- to 200-µm con- centration. The EC50 obtained for the compounds tested was 41.77 µM for UTP, 48.11 µM for GTP, 53.11 µM for UDP, and 30.78 µM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

The action of red wine and purple grape juice on vascular reactivity is independent of plasma lipids in hypercholesterolemic patients

Although red wine (RW) reduces cardiovascular risk, the mechanisms underlying the effect have not been identified. Correction of endothelial dysfunction by RW flavonoids could be one mechanism. We measured brachial artery reactivity by high-resolution ultrasonography, plasma lipids, glucose, adhesion molecules (ICAM-1 and VCAM), and platelet function in 16 hypercholesterolemic individuals (8 men and 8 women; mean age 51.6 ± 8.1 years) without other risk factors. Twenty-four normal subjects were used as controls for vascular reactivity. Subjects randomly received RW, 250 ml/day, or purple grape juice (GJ), 500 ml/day, for 14 days with an equal wash-out period. At baseline, all 16 subjects were hypercholesterolemic (mean LDL = 181.0 ± 28.7 mg/dl) but HDL, triglycerides, glucose, adhesion molecules, and platelet function were within normal limits. Brachial artery flow-mediated dilation was significantly decreased compared to controls (9.0 ± 7.1 vs 12.1 ± 4.5%; P < 0.05) and increased with both GJ (10.1 ± 7.1 before vs 16.9 ± 6.7% after: P < 0.05) and RW (10.1 ± 6.4 before vs 15.6 ± 4.6% after; P < 0.05). RW, but not GJ, also significantly increased endothelium-independent vasodilation (17.0 ± 8.6 before vs 23.0 ± 12.0% after; P < 0.01). GJ reduced ICAM-1 but not VCAM and RW had no effect on either molecule. No significant alterations were observed in plasma lipids, glucose or platelet aggregability with RW or GJ. Both RW and GJ similarly improved flow-mediated dilation, but RW also enhanced endothelium-independent vasodilation in hypercholesterolemic patients despite the increased plasma cholesterol. Thus, we conclude that GJ may protect against coronary artery disease without the additional negative effects of alcohol despite the gender.

Prevalence of BRCA1 and BRCA2 gene mutations in families with medium and high risk of breast and ovarian cancer in Brazil

Of all malignant neoplasias affecting women, breast cancer has the highest incidence rate in Brazil. The objective of the present study was to determine the frequency of genetic modifications in families with medium and high risk for breast and ovarian cancer from different regions of Brazil. An exploratory, descriptive study was carried out on the prevalence of the BRCA1 and BRCA2 mutations in case series of high-risk families for breast and/or ovarian cancer. After heredogram construction, a blood sample was taken and DNA extraction was performed in all index cases. The protein truncation test was used to screen for truncated mutations in exon 11 of the BRCA1 gene and in exons 10 and 11 of the BRCA2 gene. Of the 612 individuals submitted to genetic testing, 21 (3.4%), 19 women and 2 men, had mutations in the BRCA1 or BRCA2 genes. Of the 19 BRCA1 mutations found in the 18 participants, 7 consisted of ins6kb mutations, 4 were 5382insC, 3 were 2156delGinsCC, 2 were 185delAG, 1 was C1201G, 1 was C3522T, and 1 was 3450del4. With respect to the BRCA2 gene, 3 mutations were found: 5878del10, 5036delA and 4232insA (one case each). The prevalence of germline mutations in the BRCA1 and BRCA2 genes found in the present study was lower than reported by other studies on high-risk Brazilian populations. The inclusion of individuals with medium risk may have contributed to the lower prevalence observed.

Genomic alterations detected by comparative genomic hybridization in ovarian endometriomas

Endometriosis is a complex and multifactorial disease. Chromosomal imbalance screening in endometriotic tissue can be used to detect hot-spot regions in the search for a possible genetic marker for endometriosis. The objective of the present study was to detect chromosomal imbalances by comparative genomic hybridization (CGH) in ectopic tissue samples from ovarian endometriomas and eutopic tissue from the same patients. We evaluated 10 ovarian endometriotic tissues and 10 eutopic endometrial tissues by metaphase CGH. CGH was prepared with normal and test DNA enzymatically digested, ligated to adaptors and amplified by PCR. A second PCR was performed for DNA labeling. Equal amounts of both normal and test-labeled DNA were hybridized in human normal metaphases. The Isis FISH Imaging System V 5.0 software was used for chromosome analysis. In both eutopic and ectopic groups, 4/10 samples presented chromosomal alterations, mainly chromosomal gains. CGH identified 11q12.3-q13.1, 17p11.1-p12, 17q25.3-qter, and 19p as critical regions. Genomic imbalances in 11q, 17p, 17q, and 19p were detected in normal eutopic and/or ectopic endometrium from women with ovarian endometriosis. These regions contain genes such as POLR2G, MXRA7 and UBA52 involved in biological processes that may lead to the establishment and maintenance of endometriotic implants. This genomic imbalance may affect genes in which dysregulation impacts both eutopic and ectopic endometrium.

The efficacy of YKL-40 and CA125 as biomarkers for epithelial ovarian cancer

Our objective was to estimate the efficacy of the measurement of serum YKL-40 alone or with CA125 as biomarkers for the diagnosis of epithelial ovarian cancer (EOC) using the YKL-40 ELISA kit. An experimental group of 49 ovarian cancer patients included 42 patients with EOC (53 ± 15 years, range: 19-81 years) and 7 patients (48 ± 13 years, range: 29-36 years) with borderline epithelial ovarian tumor. A control group of 88 non-malignant cases included 42 patients (43 ± 10 years, range: 26-77 years) with benign gynecological disease and 46 healthy women (45 ± 14 years, range: 30-68 years) at a teaching hospital. Both YKL-40 (220.1 ± 94.1 vs 61.6 ± 48.4 and 50.1 ± 41.2 ng/mL) and CA125 (524.9 ± 972.5 vs 13.4 ± 7.6 and 28.5 ± 29.6 U/mL) levels were significantly higher (P < 0.05) in patients with ovarian cancer compared to the healthy and non-malignant groups. YKL-40 had 92.9% sensitivity and 94.4% specificity for the diagnosis of EOC. When YKL-40 and CA125 were tested in parallel, the sensitivity was increased to 98.2%, but the specificity was decreased to 81.3%. The correlations between serum YKL-40 and tumor stage, grade histology, performance status, patient age, and extension of debulking surgery were tested. With increasing stage and grade of EOC, preoperative serum YKL-40 levels were significantly increased (P = 0.029, P = 0.05, respectively). Serum YKL-40 alone or with serum CA125 levels are useful, although with some limitations, to diagnose ovarian cancer. Our study showed that YKL-40 may not be an independent prognostic factor for ovarian cancer. This prospective study may be a new trend in looking for biomarkers that optimize diagnosis of ovarian cancer.

Women with primary ovarian insufficiency have lower bone mineral density

The aim of the present study was to assess the prevalence of osteoporosis in a sample of 32 patients with spontaneous primary ovarian insufficiency (POI) in comparison to reference groups of 25 pre- and 55 postmenopausal women. Hip (lumbar) and spinal bone mineral density (BMD) measurements were performed by dual-energy X-ray absorptiometry in the three groups. The median age of POI patients at the time of diagnosis was 35 years (interquartile range: 27-37 years). The mean ± SD age of postmenopausal reference women (52.16 ± 3.65 years) was higher than that of POI (46.28 ± 10.38 years) and premenopausal women (43.96 ± 7.08; P = 0.001) at the time of BMD measurement. Twenty-seven (84.4%) POI women were receiving hormone replacement therapy (HRT) at the time of the study. In the postmenopausal reference group, 30.4% were current users of HRT. Lumbar BMD was significantly lower in the POI group (1.050 ± 0.17 g/cm²) compared to the age-matched premenopausal reference group (1.136 ± 0.12 g/cm²; P = 0.040). Moreover, 22 (68.7%) POI women had low bone density (osteopenia/osteoporosis by World Health Organization criteria) versus 47.3% of the postmenopausal reference group (P = 0.042). In conclusion, the present data indicate that BMD is significantly lower in patients with POI than in age-matched premenopausal women. Also, the prevalence of osteopenia/osteoporosis is higher in POI women than in women after natural menopause. Early medical interventions are necessary to ensure that women with POI will maintain their bonemass.

Long-term melatonin treatment reduces ovarian mass and enhances tissue antioxidant defenses during ovulation in the rat

Melatonin regulates the reproductive cycle, energy metabolism and may also act as a potential antioxidant indoleamine. The present study was undertaken to investigate whether long-term melatonin treatment can induce reproductive alterations and if it can protect ovarian tissue against lipid peroxidation during ovulation. Twenty-four adult female Wistar rats, 60 days old (± 250-260 g), were randomly divided into two equal groups. The control group received 0.3 mL 0.9% NaCl + 0.04 mL 95% ethanol as vehicle, and the melatonin-treated group received vehicle + melatonin (100 µg·100 g body weight-1·day-1) both intraperitoneally daily for 60 days. All animals were killed by decapitation during the morning estrus at 4:00 am. Body weight gain and body mass index were reduced by melatonin after 10 days of treatment (P < 0.05). Also, a marked loss of appetite was observed with a fall in food intake, energy intake (melatonin 51.41 ± 1.28 vs control 57.35 ± 1.34 kcal/day) and glucose levels (melatonin 80.3 ± 4.49 vs control 103.5 ± 5.47 mg/dL) towards the end of treatment. Melatonin itself and changes in energy balance promoted reductions in ovarian mass (20.2%) and estrous cycle remained extensive (26.7%), arresting at diestrus. Regarding the oxidative profile, lipid hydroperoxide levels decreased after melatonin treatment (6.9%) and total antioxidant substances were enhanced within the ovaries (23.9%). Additionally, melatonin increased superoxide dismutase (21.3%), catalase (23.6%) and glutathione-reductase (14.8%) activities and the reducing power (10.2% GSH/GSSG ratio). We suggest that melatonin alters ovarian mass and estrous cyclicity and protects the ovaries by increasing superoxide dismutase, catalase and glutathione-reductase activities.

Early responses of the STAT3 pathway to platinum drugs are associated with cisplatin resistance in epithelial ovarian cancer

Cisplatin resistance remains one of the major obstacles when treating epithelial ovarian cancer. Because oxaliplatin and nedaplatin are effective against cisplatin-resistant ovarian cancer in clinical trials and signal transducer and activator of transcription 3 (STAT3) is associated with cisplatin resistance, we investigated whether overcoming cisplatin resistance by oxaliplatin and nedaplatin was associated with the STAT3 pathway in ovarian cancer. Alamar blue, clonogenic, and wound healing assays, and Western blot analysis were used to compare the effects of platinum drugs in SKOV-3 cells. At an equitoxic dose, oxaliplatin and nedaplatin exhibited similar inhibitory effects on colony-forming ability and greater inhibition on cell motility than cisplatin in ovarian cancer. Early in the time course of drug administration, cisplatin increased the expression of pSTAT3 (Tyr705), STAT3α, VEGF, survivin, and Bcl-XL, while oxaliplatin and nedaplatin exhibited the opposite effects, and upregulated pSTAT3 (Ser727) and STAT3β. The STAT3 pathway responded early to platinum drugs associated with cisplatin resistance in epithelial ovarian cancer and provided a rationale for new therapeutic strategies to reverse cisplatin resistance.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.

Low-level red laser therapy alters effects of ultraviolet C radiation on Escherichia coli cells

Low-level lasers are used at low power densities and doses according to clinical protocols supplied with laser devices or based on professional practice. Although use of these lasers is increasing in many countries, the molecular mechanisms involved in effects of low-level lasers, mainly on DNA, are controversial. In this study, we evaluated the effects of low-level red lasers on survival, filamentation, and morphology of Escherichia colicells that were exposed to ultraviolet C (UVC) radiation. Exponential and stationary wild-type and uvrA-deficientE. coli cells were exposed to a low-level red laser and in sequence to UVC radiation. Bacterial survival was evaluated to determine the laser protection factor (ratio between the number of viable cells after exposure to the red laser and UVC and the number of viable cells after exposure to UVC). Bacterial filaments were counted to obtain the percentage of filamentation. Area-perimeter ratios were calculated for evaluation of cellular morphology. Experiments were carried out in duplicate and the results are reported as the means of three independent assays. Pre-exposure to a red laser protected wild-type and uvrA-deficient E. coli cells against the lethal effect of UVC radiation, and increased the percentage of filamentation and the area-perimeter ratio, depending on UVC fluence and physiological conditions in the cells. Therapeutic, low-level red laser radiation can induce DNA lesions at a sub-lethal level. Consequences to cells and tissues should be considered when clinical protocols based on this laser are carried out.

Low-intensity red and infrared laser effects at high fluences on Escherichia coli cultures

Semiconductor laser devices are readily available and practical radiation sources providing wavelength tenability and high monochromaticity. Low-intensity red and near-infrared lasers are considered safe for use in clinical applications. However, adverse effects can occur via free radical generation, and the biological effects of these lasers from unusually high fluences or high doses have not yet been evaluated. Here, we evaluated the survival, filamentation induction and morphology of Escherichia coli cells deficient in repair of oxidative DNA lesions when exposed to low-intensity red and infrared lasers at unusually high fluences. Cultures of wild-type (AB1157), endonuclease III-deficient (JW1625-1), and endonuclease IV-deficient (JW2146-1) E. coli, in exponential and stationary growth phases, were exposed to red and infrared lasers (0, 250, 500, and 1000 J/cm2) to evaluate their survival rates, filamentation phenotype induction and cell morphologies. The results showed that low-intensity red and infrared lasers at high fluences are lethal, induce a filamentation phenotype, and alter the morphology of the E. coli cells. Low-intensity red and infrared lasers have potential to induce adverse effects on cells, whether used at unusually high fluences, or at high doses. Hence, there is a need to reinforce the importance of accurate dosimetry in therapeutic protocols.

Ghrelin attenuates the growth of HO-8910 ovarian cancer cells through the ERK pathway

Ovarian cancer is one of the most common causes of death from gynecologic tumors and is an important public health issue. Ghrelin is a recently discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR). Several studies have identified the protective effects of ghrelin on the mammalian reproductive system. However, little research has been done on the effects of ghrelin on ovarian cancer cells, and the underlying mechanisms of these effects. We sought to understand the potential involvement of mitogen-activated protein kinases (MAPKs) in ghrelin-mediated inhibition of growth of the ovarian line HO-8910. We applied different concentrations of ghrelin and an inhibitor of the ghrelin receptor (D-Lys3-GHRP-6) to HO-8910 cells and observed the growth rate of cells and changes in phosphorylation of the MAPKs ERK1/2, JNK and p38. We discovered that ghrelin-induced apoptosis of HO-8910 cells was though phosphorylated ERK1/2, and that this phosphorylation (as well as p90rsk phosphorylation) was mediated by the GHSR. The ERK1/2 pathway is known to play an essential part in the ghrelin-mediated apoptosis of HO-8910 cells. Hence, our study suggests that ghrelin inhibits the growth of HO-8910 cells primarily through the GHSR/ERK pathway.