Exposure to maternal smoking during fetal life affects food preferences in adulthood independent of the effects of intrauterine growth restriction

Experimental animal studies have shown that nicotine exposure during gestation alters the expression of fetal hypothalamic neuropeptides involved in the control of appetite. We aimed to determine whether the exposure to maternal smoking during gestation in humans is associated with an altered feeding behavior of the adult offspring. A longitudinal prospective cohort study was conducted including all births from Ribeirao Preto (Sao Paulo, Brazil) between 1978 and 1979. At 24 years of age, a representative random sample was re-evaluated and divided into groups exposed (n = 424) or not (n = 1586) to maternal smoking during gestation. Feeding behavior was analyzed using a food frequency questionnaire. Covariance analysis was used for continuous data and the chi(2) test for categorical data. Results were adjusted for birth weight ratio, body mass index, gender, physical activity and smoking, as well as maternal and subjects` schooling. Individuals exposed to maternal smoking during gestation ate more carbohydrates than proteins (as per the carbohydrate-to-protein ratio) than non-exposed individuals. There were no differences in the consumption of the macronutrients themselves. We propose that this adverse fetal life event programs the individual`s physiology and metabolism persistently, leading to an altered feeding behavior that could contribute to the development of chronic diseases in the long term.

Transcriptional profiling reveals genes in the human pathogen Trichophyton rubrum that are expressed in response to pH signaling

Trichophyton rubrum is a dermatophyte that infects human skin and nails. Its growth on keratin as its carbon source shifts the ambient pH from acidic to alkaline, which may be an efficient strategy for its successful infection and maintenance in the host. In this study, we used suppression subtractive hybridization to identify genes preferentially expressed in T rubrum incubated at either pH 5.0 or pH 8.0. The functional grouping of the 341 overexpressed unigenes indicated proteins putatively involved in diverse cellular processes, such as membrane remodeling, cellular transport, metabolism, cellular protection, fungal pathogenesis, gene regulation, interaction with the environment, and iron uptake. Although the basic metabolic machinery identified under both growth conditions seems to be functionally similar, distinct genes are upregulated at acidic or alkaline pHs. We also isolated a large number of genes of unknown function, probably unique to T rubrum or dermatophytes. Interestingly, the transcriptional profiling of several genes in a pacC mutant suggests that, in T rubrum, the transcription factor PacC has a diversity of metabolic functions, in response to either acidic or alkaline ambient pH. (C) 2009 Elsevier Ltd. All rights reserved.

Insights on calcium-independent phospholipid membrane damage by Lys49-PLA(2) using tryptophan scanning mutagenesis of bothropstoxin-I from Bothrops jararacussu

Bothropstoxin-I (BthTx-I) is a homodimerie Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca2+- independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca2+-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA in a 9:1 molar ratio was unaffected by these substitutions. Binding studies revealed low protein affinity for these liposomes and no changes were observed in the ITFE properties. In contrast, the binding of all mutants to DPPC/DMPA liposomes in a 1:1 molar ratio was stronger, and was correlated with altered ITFE properties. The blue-shifted emission spectra and increased emission intensity of mutants at positions 31, 67 and 115-117 in the interface recognition surface of the protein suggest these regions are partially inserted into the membrane. These results are consistent with a model for the Ca2+-independent membrane damaging mechanism that involves a transient interaction of the protein with the outer phospholipid leaflet of the target membrane. (C) 2007 Elsevier Masson SAS. All rights reserved.

Compound 48/80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta

Compound 48/80 (C48/80) is a synthetic condensation product of N-methyl-p-methoxyphenethyl am me with formaldehyde and is an experimental drug used since the 1950s to induce anaphylactic shock through histamine release. This study was carried out to further elucidate the mechanism by which this drug induces nitric oxide (NO) release. Our specific goals were: (a) to verify if C48/80`s relaxation occurs through the stimulation of histamine receptors; (b) to evaluate the endothelium-dependent relaxation induced by C48/80; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NO synthase (NOS) responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent. Rabbit aorta segments, with and without endothelium, were suspended in organ chambers (25 ml) filled with Krebs solution maintained at 37 degrees C, bubbled with 95% O-2/5% CO2 (pH 7.4). Phenylephrine was used to contract the segments. Other protocol drugs included H-1- and H-2-receptor antagonists, cyclooxygenase, NOS, guanylyl cyclase and phospholipase C (PLC) inhibitors. Endothelium-dependent relaxation induced by C48/80 was also studied in calcium-free Krebs solution associated with a calcium chelator. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H-1 and H-2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the endothelial constitutive NOS (NOS-3) isoform activation; (d) is cGMP-dependent; and that NOS-3 activation by C48/80: (a) is independent of PLC up to 25 mu g/ml and (b) is partially dependent of this lipase in higher doses. (C) 2007 Elsevier Inc. All rights reserved.

Age-related deregulation of Aire and peripheral tissue antigen genes in the thymic stroma of non-obese diabetic (NOD) mice is associated with autoimmune type 1 diabetes mellitus (DM-1)

Gene expression of peripheral tissue antigens (PTAs) in stromal medullary thymic epithelial cells (mTECs) is a key process to the negative selection of autoreactive thymocytes. This phenomenon was termed ""promiscuous gene expression"" (PGE), which is partially controlled by the Aire gene. Nevertheless, reasons for the correlation of Aire and PTAs with the emergence of autoimmune diseases are largely unknown, though it may be a result of a chronological effect. Although the effect of Aire mutations in pathogenic autoimmunity is well know, it could not be a unique cause for autoimmunity. Independently of mutations, temporal deregulation of Aire expression may imbalance Aire-dependent PTAs and/or wide PGE. This deregulation may be an early warning sign for autoimmune diseases as it guarantees autoantigen representation in the thymus. To assess this hypothesis, we studied the expression levels of Aire, Aire-dependent (Ins2) and Aire-independent (Gad67 and Col2a1) PTAs using real-time-PCR of the thymic stromal cells of NOD mice during the development of autoimmune type 1 diabetes mellitus (DM-1). Wide PGE was studied by microarrays in which the PTA genes were identified through parallel CD80(+) mTEC 3.10 cell line expression profiling. The results show that Aire gene was down-regulated in young pre-autoimmune (pre-diabetic) NOD mice. PGE and specific PTA genes were down-regulated in adult autoimmune diabetic animals. These findings represent evidence indicating that chronological deregulation of genes important to negative selection may be associated with the development of an autoimmune disease (DM-1) in mice.

Protective effects of carvedilol on systemic vascular damage induced by angiotensin II: Organ-specific effects independent of antihypertensive effects

Background: The protective effect of carvedilol on multiple organ damage induced by angiotensin II (Ang II) remains unclear. The aim of this study was to evaluate the protective effect of carvedilol on the heart, liver, and kidney in rats infused with Ang II. Material/Methods: Wistar rats were randomly distributed into three groups: control (no treatment), continuously infused with Ang II (150 eta g/min for 72 hr), and treated with Ang II + carvedilol (90 mg/kg/d). Histological sections of the myocardium, kidney, and liver were analyzed for the presence of necrosis. Results: Ang II induced arterial hypertension which was not affected by carvedilol treatment (tail-cuff blood pressures, control: 125 +/- 13.6, Ang II: 163 +/- 27.3, Ang II + CV: 178 +/- 39.8 mmHg, p<0.05). Also, there were perivascular inflammation and necrosis in the myocardium, kidney, and hepatocytes necrosis around the terminal vein. Carvedilol treatment fully prevented damage to the heart and kidney and attenuated liver lesions induced by the Ang II infusion. Conclusions: The protective effect of carvedilol on perivascular damage induced by Ang II infusion depended on the target organ. The prevention of heart damage occurred independently of the antihypertensive effects of carvedilol.

Correlation between mastitis occurrence and the count of microorganisms in bulk raw milk of bovine dairy herds in four selective culture media

Milk is the normal secretion of the mammary gland, practically free of colostrum and obtained by the complete milking of oil(? or more healthy animals. Mastitis is all inflammatory process of the mammary gland and it may cause alterations in the milk. The present work aimed to verify whether it is possible, by means of the counts of microorganism in the bulk raw milk ill four selective culture media, to establish a correlation with the occurrence of mastitis and therefore, to monitor this disease in bovine dairy herds. The following selective culture media were Used: KF Streptococcus Agar, Edwards Agar, Baird-Parker Agar, Blood Agar plus potassium tellurite. Spearman`s correlation coefficient was calculated in order to compare the Occurrence of mastitis (percentage) in each herd with respective selective culture media counts of microorganisms in bulk raw milk. Thirty-six possibilities were analysed (Tamis and CMT-positive rates were compared with the log-transformed count in four selective Culture media) and there was a negative correlation between Tamis 3 and the Baird-Parker Agar plate count. The total results of microbiological tests showed that there were three correlations of the counts in selective culture media. Fifty-two possibilities were analysed and there was a negative correlation between no-bacteria I-growth mastitis rates and log(10) of KF Streptoccocus Agar plate Count and there were two positive correlations between coagulase-positive staphylococci and log(10) of Baird-Parker Agar plate count and Blood Agar plus potassium tellurite plate count.

Effect of culture media on porcine embryos produced by in vitro fertilization or parthenogenetic activation after oocyte maturation with cycloheximide

This study evaluated the effects of reversible meiotic inhibition and different culture media (PZM3 or NCSU23) on production of porcine embryos by either in vitro fertilization (IVF) or parthenogenetic activation (PA). Oocytes from abattoir-derived ovaries were allocated into two groups for maturation: CHX (5 mu g/ml cycloheximide for 10 h) or Control (no CHX). The percentage of metaphase II (MII) oocytes was determined at 36, 40 or 44 h of in vitro maturation. For IVF and PA, denuded oocytes were fertilized with purified sperm for 6 h or activated by electric stimuli. Zygotes were then subdivided into two culture groups: NCSU23 or PZM3. No effect of treatment with CHX and culture media was observed on cleavage (D3) and blastocyst (D7) rates in IVF and PA groups. There are no differences of quality or development rates between IVF-derived embryos cultured in NCSU23 or PZM3. However, we observed high quality PA embryos in PZM3 compared with NCSU23. Maturation arrest with CHX decreased the average blastocyst cell number in IVF while it was increased in PA embryos. As older oocytes are more effectively activated, CHX-blocked oocytes reached the mature stage faster than the control group. In conclusion, the CHX treatment for 10 h, followed by oocyte maturation for 40 h, is an efficient protocol to produce high quality parthenote embryos, especially when they are cultured in PZM3. However, this protocol is not satisfactory for IVF embryos production. In this case, a shorter maturation period could provide better embryo quality.

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17 beta on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17 beta and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 mu g ml(-1)) of estradiol-17 beta and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17 beta), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17 beta supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.

Squamous Cell Carcinoma of the Tongue and Floor of the Mouth: Analysis of Survival Rate and Independent Prognostic Factors in the Amazon Region

Oral squamous cell carcinoma (OSCC) accounts for more than 95% of all malignant neoplasms in the oral cavity. Although several studies have shown the epidemiology of this cancer in Brazil, there do not seem to be any studies that describe the prognostic factors related to OSCC in the Amazon region. Therefore, the aim of this study was to determine the survival rate and prognostic significance of different factors in patients from this region affected by OSCC. Data from 85 patients with histologically confirmed squamous cell carcinoma of the tongue and floor of the mouth identified from the Ofir Loyola Hospital archives were collected and analyzed using univariate (log-rank test) and multivariate (Cox proportional hazard model) tests. The overall 5-year survival rate was found to be 27%. Univariate analysis showed that the 5-year survival rate was significantly higher for younger (<= 45 y) female patients, patients with T1-2 tumors and clinically clear neck nodes (N0), patients with early stage cancers (AJCC stage I-II), and patients treated with surgical procedures. However, multivariate analysis showed that the 5-year survival rate was significantly higher only in the younger patients and those who underwent surgical treatment. The age of the patient at the moment of diagnosis and treatment with surgical procedures were the only independent prognostic factors that affected the 5-year survival rate of the patients in this region.

Comparison of two cellular harvesting methods for primary human oral culture of keratinocytes

The possibility of obtaining transplantable oral epithelia opens new perspectives for oral treatments. Most of them are surgical, resulting in mucosal failures. As reconstructive material this in vitro epithelia would be also useful for other parts of the human body. Many researchers still use controversial methods; therefore it was evaluated and compared the efficiency of the enzymatic and direct explant methods to obtain oral keratinocytes. To this project oral epithelia fragments were used. This work compared: time needed for cell obtainment, best cell amount, life-span and epithelia forming cell capacity. The results showed the possibility to obtain keratinocytes from a small oral fragment and we could verify the advantages and peculiar restrictions. We concluded that under our conditions the enzymatic method showed the best results: in the cells obtaining time needed, cell amount and life-span. Both methods showed the same capacity to form in vitro epithelia.

Human Alveolar Bone-Derived Cell-Culture Behaviour on Biodegradable Poly(L-lactic Acid)

Poly(L-lactic acid) (PLA) is a polymer of great technological interest, whose excellent mechanical properties, thermal plasticity and bioresorbability render it potentially useful for environmental applications, as a biodegradable plastic and as a biocompatible material in biomedicine. The interactions between an implant material surface and host cells play central roles in the integration, biological performance and clinical success of implanted biomedical devices. Osteoblasts from human alveolar bone were chosen to investigate the cell behaviour when in contact with PLA discs. Cell morphology and adhesion through osteopontin (OPN) and fibronectin (FN) expression were evaluated in the initial osteogenesis, as well as cell proliferation, alkaline phosphatase activity and bone nodule formation. It was shown that the polymer favoured cell attachment. Cell proliferation increased until 21 days but in a smaller rate when compared to the control group. On the other hand, ALP activity and bone mineralization were not enhanced by the polymer. It is suggested that this polymer favours cell adhesion in the early osteogenesis in vitro, but it does not enhance differentiation and mineralization. (C) Koninklijke Brill NV, Leiden, 2009

Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.