Assessment of nitrite oxide and maternal–fetal Doppler parameters during pregnancy

Objective: The objective was to evaluate and compare the whole blood nitrite concentration in the three trimesters of pregnancy. Additionally, we investigate whether there is any relation between nitrite concentrations and Doppler ultrasound analysis of some maternal and fetal vessels.

Methods: Thirty-three healthy pregnant women were examined at the first (11–14 weeks), second (20–24 weeks) and third trimester (34–36 weeks) of pregnancy. In the three exams, we determined the maternal whole blood nitrite concentration and uterine arteries Doppler analysis to determine pulsatility index (PI), and resistance index (RI). In the second and third trimester we also performed fetal umbilical and middle cerebral arteries PI and RI. We compared the concentrations of nitrite in three trimesters and correlated with Doppler parameters.

Results: No difference was observed in the whole blood nitrite concentrations across trimesters: 151.70?±?77.90?nmol/ml, 142.10?±?73.50?nmol/ml and 147.10?±?87.30?nmol/ml; first, second and third trimesters, respectively. We found no difference in correlation between whole blood nitrite concentration and Doppler parameters from the evaluated vessels.

Conclusions: In healthy pregnant women, the nitrite concentrations did not change across gestational trimesters and there was also no strong correlation with Doppler impedance indices from maternal uterine arteries and fetal umbilical and middle cerebral arteries.     

Familial glucocorticoid deficiency due to compound heterozygosity of two novel MC2R mutations

Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder characterized by isolated glucocorticoid deficiency. Mutations in the ACTH receptor (melanocortin 2 receptor, MC2R) or the MC2R accessory protein (MRAP) cause FGD types 1 and 2, respectively. A 2-year-old adopted Chinese girl presented with hypertonic seizures associated with hypoglycemia, skin hyperpigmentation, muscle weakness and mild jaundice. Hormonal analyses revealed high ACTH, low serum cortisol along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered, although further episodes occurred with infection. To date, her physical and neurocognitive development progress is normal. A clinical diagnosis of FGD was given. We undertook MC2R and MRAP mutation screening. Two novel MC2R mutations were identified: p.D107G localized in the transmembrane region, predicted to be trafficking-competent but is unable to bind to ACTH, and p.R145C, situated in the second intracellular loop, predicted to be trafficking-defective.     

13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy

Chronic allograft nephropathy is characterized by chronic inflammation and fibrosis. Because retinoids exhibit anti-proliferative, anti-inflammatory, and anti-fibrotic functions, the effects of low and high doses of 13-cis-retinoic acid (13cRA) were studied in a chronic Fisher344-->Lewis transplantation model. In 13cRA animals, independent of dose (2 or 20 mg/kg body weight/day) and start (0 or 14 days after transplantation) of 13cRA administration, serum creatinine was significantly lower and chronic rejection damage was dramatically reduced, including subendothelial fibrosis of preglomerular vessels and chronic tubulointerstitial damage. The number of infiltrating mononuclear cells and their proliferative activity were significantly diminished. The mRNA expression of chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, IP-10/CXCL10, RANTES/CCL5) and proteins associated with fibrosis (plasminogen activator inhibitor-1, transforming growth factor-beta1, and collagens I and III) were strikingly lower in treated allografts. In vitro, activated peritoneal macrophages of 13cRA-treated rats showed a pronounced decrease in protein secretion of inflammatory cytokines (eg, tumor necrosis factor-alpha, interleukin-6). The suppression of the proinflammatory chemokine RANTES/CCL5 x 13cRA in fibroblasts could be mapped to a promoter module comprising IRF-1 and nuclear factor-kappaB binding elements, but direct binding of retinoid receptors to promoter elements could be excluded. In summary, 13cRA acted as a potent immunosuppressive and anti-fibrotic agent able to prevent and inhibit progression of chronic allograft nephropathy.     

Evidence for the involvement of the opioid system in the agmatine antidepressant-like effect in the forced swimming test

This study investigated the involvement of the opioid system in the antidepressant-like effect of agmatine in the mouse forced swimming test (FST). The antidepressant-like effects of agmatine (10 mg/kg, i.p.), as well as those of fluoxetine (32 mg/kg, i.p, a selective serotonin reuptake inhibitor, SSRI) or morphine (5 mg/kg, s.c., a nonselective opioid receptor agonist) in the FST was completely blocked by pretreatment of mice with naloxone (1 mg/kg, i.p., a nonselective opioid receptor antagonist). Pretreatment of mice with naltrindole (3 mg/kg, i.p., a selective delta-opioid receptor antagonist), clocinnamox (1 mg/kg, i.p., an irreversible mu-opioid receptor antagonist), but not with 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(3-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (DIPPA; 1 mg/kg, i.p., a selective kappa-opioid receptor antagonist) completely blocked the anti-immobility effect of agmatine (10 mg/kg, i.p.) in the FST. These results firstly demonstrate that the antidepressant-like effects of agmatine in the FST seem to be mediated, at least in part, by an interaction with the opioid system, that involves an activation of delta- and mu-opioid receptors.     

Hox genes specify vertebral types in the presomitic mesoderm

We show here that expression of Hoxa10 in the presomitic mesoderm is sufficient to confer a Hox group 10 patterning program to the somite, producing vertebrae without ribs, an effect not achieved when Hoxa10 is expressed in the somites. In addition, Hox group 11-dependent vertebral sacralization requires Hoxa11 expression in the presomitic mesoderm, while their caudal differentiation requires that Hoxa11 is expressed in the somites. Therefore, Hox gene patterning activity is different in the somites and presomitic mesoderm, the latter being very prominent for Hox gene-mediated patterning of the axial skeleton. This is further supported by our finding that inactivation of Gbx2, a homeobox-containing gene expressed in the presomitic mesoderm but not in the somites, produced Hox-like phenotypes in the axial skeleton without affecting Hox gene expression.     

Haplotype diversity and somatic instability in normal and expanded SCA8 alleles.

Spinocerebellar ataxia type 8 (SCA8) is an autosomal dominant late-onset neurodegenerative disorder, belonging to the group of diseases caused by trinucleotide repeat expansions. SCA8 remains one of the most intriguing SCAs, regarding the reduced disease penetrance, and the high instability and poorly understood functional meaning of the (CTA)(n)(CTG)(n) expansion. We performed haplotype and sequencing analysis in a large region, encompassing the repeat, in four SCA8 and 20 control Portuguese families. The results from the haplotype study including the combined repeat and six SNP markers showed two different haplotypes, AG-Exp-GTTG and AG-Exp-CTTG, in the SCA8 families. Among the control population, these were also the most frequent, in a total of five haplotypes found unequally distributed throughout repeat sizes. From cloning fragments of control, unstable normal and expanded chromosomes, eleven different base substitutions were identified in exon A of the SCA8 gene. In some instances, somatic variability in repeat size or base composition was found for a same chromosome, regardless of its normal or expanded nature. In conclusion, our results in Portuguese families with ataxia show that SCA8 expansions arose in common backgrounds; in addition, this region seems to be unstable beyond the repeat.     

Evaluation of stromal metalloproteinases and vascular endothelial growth factors in a spontaneous metastasis model

This study aims to investigate MMP2 and MT1-MMP protein as well as VEGF-C and VEGF-D mRNA expression in tumor cells and distant organs considered to be targets for metastasis in a tumor spontaneous metastasis model previously described. Cultured tumor cells, able to express pro-MMP2, MMP2, pro-MMP9, and MT1-MMP, develop tumor growth and metastasis, mainly in the liver and spleen, when they are injected in the mammary pad gland of Wistar rats. Immunohistochemical studies of tumor masses showed small groups of tumor cells staining for MT1-MMP but not for MMP2. In the liver, tumor metastatic foci and a stromal positive staining for both MMP2 and MT1-MMP were shown. The spleen and lymph nodes, with only scattered metastatic cells, did not show MMPs immunostaining. Using RT-PCR, a significantly higher VEGF-C and VEGF-D gene expression was shown in the liver of tumor-bearing rats respect to normal rats, whereas spleen and lymph nodes did not show significant differences in mRNA VEGF-C/D levels. Taken together, our results suggest that the stroma microenvironment of target organs for metastasis has the ability to produce MMPs and VEGFs that facilitate the anchorage of tumor cells and promote tumor cell growth and angiogenesis.     

NADPH oxidase-mediated oxidative stress: genetic studies of the p22(phox) gene in hypertension

Increased vascular production of reactive oxygen species, especially superoxide anion, significantly contributes to the oxidative stress associated with hypertension. An enhanced superoxide production causes an increased inactivation of nitric oxide that diminishes nitric oxide bioavailability, thus contributing to endothelial dysfunction and hypertrophy of vascular cells. It has been shown that NADPH oxidases play a major role as the most important sources of superoxide anion in phagocytic and vascular cells. Several experimental observations have described an enhanced superoxide generation as a result of NADPH oxidase activation in hypertension. Although these enzymes respond to stimuli such as vasoactive factors, growth factors, and cytokines, recent data suggest a significant role of the genetic background in the modulation of the expression of its different components. Several polymorphisms have been identified in the promoter and in the coding region of CYBA, the gene that encodes the essential subunit of the NADPH oxidase p22phox, some of which seem to influence significantly the activity of these enzymes in the context of cardiovascular diseases. Among CYBA polymorphisms, genetic investigations have provided a novel marker, the -930(A/G) polymorphism, which determines the genetic susceptibility of hypertensive patients to oxidative stress.     

Cyclooxygenase-2 protein reduces tamoxifen and N-(4-hydroxyphenyl)retinamide inhibitory effects in breast cancer cells

Approximately 30-40% of estrogen receptor alpha (ERalpha)-positive breast tumors express high levels of the cyclooxygenase-2 (COX-2) protein, and these high levels have been associated with a poorer prognosis in breast cancer patients. We speculate that high levels of COX-2 induce drug resistance in ERalpha-positive breast tumors, thus reducing the survival rate of patients with such tumors. Human breast cancer cell lines that express high levels of COX-2 are generally ERalpha negative. To determine whether COX-2 induces drug resistance, plasmids encoding the COX-2 gene were stably transfected into ERalpha-positive MCF-7 human breast cancer cells (MCF-7/COX-2). MCF-7/COX-2 cells were resistant to the selective estrogen receptor modulator tamoxifen but not to its analog, raloxifene. MCF-7/COX-2 cells were also resistant to the retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) but not to its analog, all-trans retinoic acid. In contrast, the sensitivities of MCF-7/COX-2 cells to doxorubicin and paclitaxel were similar to those of the parental MCF-7 cells. We then determined which COX-2 product, prostaglandin E2 (PGE2) or prostaglandin F2alpha is involved in the COX-2-mediated drug resistance. PGE2, but not PGF2alpha, blocked the antiproliferative effects of tamoxifen and 4-HPR. Agonists that activate PGE2 receptors and their downstream kinase effectors, protein kinases A and C, also blocked the growth inhibitory effects of these drugs. Increased levels of Bcl-2 and Bcl-XL proteins have been reported in mammary tumors of COX-2 transgenic mice and in human colon cancer cell lines that have high levels of COX-2. However, we did not observe any changes in Bcl-2, Bcl-XL, or Bax expression induced by COX-2 or PGE2. Here we report the novel findings that COX-2 uses PGE2 to stimulate the activities of protein kinases A and C to induce selectively tamoxifen and 4-HPR resistance in ERalpha-positive breast cancer cells.     

Involvement of intercellular adhesion molecule-1 and beta1 integrin in the internalization process to human endothelial cells of group B Streptococcus clinical isolates

The mechanism by which group B Streptococcus (GBS) interacts with human cells and disrupts physiological processes is an intriguing area of investigation and continues to unfold. The aim of this study was to investigate the adherence and intracellular viability within endothelial ECV304 cells of GBS serotypes Ia, III and V isolates from patients and asymptomatic carriers. The GBS isolates from patients (GBS-Ia 90222-urine, GBS-III 90356-liquor and GBS-V 90186-blood strains) exhibited a more efficient adherence and survival mechanisms to endothelial cells than those from asymptomatic carriers (GBS-Ia 85147-oropharynx, GBS-III 80340 and GBS-V 88641-vagina strains), independent of bacterial serotypes. Treatment of endothelial ECV304 cells with EDTA demonstrated that Ca2+-dependent molecules modulated the adherence and internalization process of GBS-Ia and III to ECV304 cells. SDS-PAGE analysis of samples of biotinylated ECV304 extracts treated with GBS clinical isolates (urine 90222-Ia, liquor 90356-III and blood 90186-V strains) revealed fragments ranging from approximately 61 to approximately 179 kDa. Results of immunoassays with ECV304 membrane proteins showed that ICAM-1 molecules interacted only with GBS-III liquor 90356 strain while beta1 integrin interacted with GBS-III liquor 90356 and GBS-V blood 90186 invasive strains. Thus, the interaction between ICAM-1 and beta1-integrin seems an additional means by which GBS exploits host endothelial cells during infection. These findings add to the current understanding of the roles played by multiple receptor-ligand systems in the uptake and pathogenesis of GBS infection.