Cigarette Smoke Affects Apoptosis in Rat Tongue Mucosa: Role of bcl-2 Gene Family

While it has been clearly demonstrated that smoking is the most significant exogenous factor involved in oral carcinogenesis, little is known about the global molecular and cellular changes that occur prior to the appearance of clinically detectable symptoms. Thus, the aim of this study was to investigate the expressivity of bcl-2, bax and PCNA in the rat tongue mucosa exposed to cigarette smoke by means of immunohistochemistry. A total of twelve male Wistar rats were distributed into 2 groups: negative control and experimental group exposed to cigarette smoke during 75 days. After experimental period, no histopathological changes in the tongue mucosa were detected in the negative control and the experimental group. On the other hand, an overexpression of bcl-2 was detected (p < 0.01) throughout all layers of the epithelium, whereas bax did not show significant differences (p > 0.05). Also, the labeling index for bcl-2 and bax showed an increase 75 days after cigarette exposure (p < 0.01). PCNA-labeling index did not show remarkable changes between groups. Taken together, our results show that bcl-2 is overexpressed in the rat tongue keratinocytes after cigarette smoke exposure.     

Pseudomonas aeruginosa-induced production of free radicals by IFNgamma plus TNFalpha-activated human endothelial cells: mechanism of host defense or of bacterial pathogenesis?

We have previously shown that human umbilical vein endothelial cells (HUVEC) can be activated by IFNgamma plus TNFalpha to kill intracellular (IC) Pseudomonas aeruginosa through production of reactive oxygen intermediate, but the cumulative effects of cytokine activation and bacterial infection on host cells has not been extensively addressed. In this study we investigated the fate of IFNgamma plus TNFalpha-activated HUVEC that have harboured IC bacteria for up to 24 h. At 10 h, the endothelial cell killing of P. aeruginosa isolates exceeded 90%. IC bacteria enhanced the expression of inducible nitric oxide synthase (iNOS) and induced overproduction of NO and superoxide by infected HUVEC. P. aeruginosa IC infection also induced a slight decrease in the cellular level of reduced glutathione (GSH). Overproduction of NO correlated with a marked peroxidation of plasma membrane lipids and decline in HUVEC viability. Treatment of cells with the antioxidant alpha-lipoic acid significantly increased the survival of infected cells. Our data suggest that with the failure of adequate scavenger mechanisms, oxidant radicals overproduced in response to bacterial infection were highly toxic to host cells. Therefore, instead of contributing to defence against infectious agents, the upregulation of free radicals production by endothelial cells in response to cytokine activation would be detrimental to the host.     

Age determination in Pomatoschistus mi nut us (Pallas) and Pomatoschistus microps (Kreyer) (Pisces: Gobiidae) from the upper Tagus estuary, Portugal

The use of scale inspection as a means of age-determination in Pomatoschistus microps and Pomatoschistus minutus in the Tagus estuary is confirmed, but annulus formation pattern is different from that described for P. microps in the area of the British Isles (Miller, 1975). In Britain, two annuli are laid down on the scales each year, but in the Tagus, only one annulus is formed. One possible reason for this difference is the fact that in the Tagus the peak breeding season and the lowest water temperatures coincide. Therefore, the two causes of annulus formation coexist at the same time of year.
Population structure in both species is dominated by the 0-group, which appears earlier (April/May) than reported for other areas. Maximum age seems not to exceed 26 months in P. microps and 32 months in P. minutus .     

Intersubtype Differences in the Effect of a Rare p24 Gag Mutation on HIV-1 Replicative Fitness

Certain immune-driven mutations in HIV-1, such as those arising in p24^Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24^Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24^Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness.     

Postactivation potentiation: effect of various recovery intervals on bench press power performance

Postactivation potentiation (PAP) is a strategy used to improve performance in power activities. The aim of this study was to determine if power during bench press exercise was increased when preceded by 1 repetition maximum (1RM) in the same exercise and to determine which time interval could optimize PAP response. For this, 11 healthy male subjects (age, 25 ± 4 years; height, 178 ± 6 cm; body mass, 74 ± 8 kg; bench press 1RM, 76 ± 19 kg) underwent 6 sessions. Two control sessions were conducted to determine both bench press 1RM and power (6 repetitions at 50% 1RM). The 4 experimental sessions were composed of a 1RM exercise followed by power sets with different recovery intervals (1, 3, 5, and 7 minutes), performed on different days, and determined randomly. Power values were measured via Peak Power equipment (Cefise, Nova Odessa, S?o Paulo, Brazil). The conditions were compared using an analysis of variance with repeated measures, followed by a Tukey test. The significance level was set at p < 0.05. There was a significant increase in PAP in concentric contractions after 7 minutes of recovery compared with the control and 1-minute recovery conditions (p < 0.05). Our results indicated that 7 minutes of recovery has generated an increase in PAP in bench press and that such a strategy could be applied as an interesting alternative to enhance the performance in tasks aimed at increasing upper-body power performance.     

A strong deletion bias in nonallelic gene conversion

Gene conversion is the unidirectional transfer of genetic information between orthologous (allelic) or paralogous (nonallelic) genomic segments. Though a number of studies have examined nucleotide replacements, little is known about length difference mutations produced by gene conversion. Here, we investigate insertions and deletions produced by nonallelic gene conversion in 338 Drosophila and 10,149 primate paralogs. Using a direct phylogenetic approach, we identify 179 insertions and 614 deletions in Drosophila paralogs, and 132 insertions and 455 deletions in primate paralogs. Thus, nonallelic gene conversion is strongly deletion-biased in both lineages, with almost 3.5 times as many conversion-induced deletions as insertions. In primates, the deletion bias is considerably stronger for long indels and, in both lineages, the per-site rate of gene conversion is orders of magnitudes higher than that of ordinary mutation. Due to this high rate, deletion-biased nonallelic gene conversion plays a key role in genome size evolution, leading to the cooperative shrinkage and eventual disappearance of selectively neutral paralogs.     

Leukotrienes Are Upregulated and Associated with Human T-Lymphotropic Virus Type 1 (HTLV-1)-Associated Neuroinflammatory Disease

Leukotrienes (LTs) are lipid mediators involved in several inflammatory disorders. We investigated the LT pathway in human T-lymphotropic virus type 1 (HTLV-1) infection by evaluating LT levels in HTLV-1-infected patients classified according to the clinical status as asymptomatic carriers (HACs) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Bioactive LTB₄ and CysLTs were both increased in the plasma and in the supernatant of peripheral blood mononuclear cell cultures of HTLV-1-infected when compared to non-infected. Interestingly, CysLT concentrations were increased in HAM/TSP patients. Also, the concentration of plasma LTB₄ and LTC₄ positively correlated with the HTLV-1 proviral load in HTLV-1-infected individuals. The gene expression levels of LT receptors were differentially modulated in CD4⁺ and CD8⁺ T cells of HTLV-1-infected patients. Analysis of the overall plasma signature of immune mediators demonstrated that LT and chemokine amounts were elevated during HTLV-1 infection. Importantly, in addition to CysLTs, IP-10 was also identified as a biomarker for HAM/TSP activity. These data suggest that LTs are likely to be associated with HTLV-1 infection and HAM/TSP development, suggesting their putative use for clinical monitoring.     

Chitooligosaccharides antagonize the cytotoxic effect of glucosamine

Chitooligosaccharides (COS) are partially hydrolyzed compounds derived from chitosan that exhibit a number of biological activities, including antitumor, antibacterial and antifungal properties. In this work, we examined the cytotoxicity of pure COS and oligomers A, B and C (solutions composed of different amounts of COS) produced by enzymatic hydrolysis using a crude enzyme extract produced by the fungus Metarhrizium anisopliae. The antiproliferative effect of these molecules was analyzed using tumor cell lines (HepG2 and HeLa cells) and in a normal cell line (3T3). The antioxidant activity was analyzed in several in vitro experiments. Glucosamine showed higher toxicity (approximately 92%) to all cell lines studied. However, the oligomers obtained after hydrolysis demonstrated no toxic effects on the normal cells (3T3). Furthermore, we showed that a small amount of other COS can decrease the cytotoxic effect of glucosamine against 3T3 cells, indicating that glucosamine could be used as an antitumor drug in the presence of other COS. In addition, different effects were found in antiproliferative assays, which depended on the COS composition in the oligomers (A, B and C), showing that a combination of them may be essential for developing antineoplastic drugs. Superoxide anion scavenging was the main antioxidant activity demonstrated by the COS and oligomers. This activity was also dependent on the oligomer composition of the chitosan hydrolysates. Further work will identify the ideal proportions of COS and glucosamine for maximizing the effects of these biological activities.     

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.