Branched chain amino acids catabolism as a source of new drug targets in pathogenic protists.

Leucine, isoleucine, and valine, collectively termed Branched Chain Amino Acids (BCAA), are hydrophobic amino acids (AAs) and are essential for most eukaryotes since in these organisms they cannot be biosynthesized and must be supplied by the diet. These AAs are structurally relevant for muscle cells and, of course, important for the protein synthesis process. The metabolism of BCAA and its participation in different biological processes in mammals have been relatively well described. However, for other organisms as pathogenic parasites, the literature is really scarce. Here we review the BCAA catabolism, compile evidence on their relevance for pathogenic eukaryotes with special emphasis on kinetoplastids and highlight unique aspects of this underrated pathway.

Analysis of Biofilm Adhesion on CAD/CAM Lithium Disilicates Under Different Types of Intraoral Polishing.

PURPOSE: To evaluate the adhesion of Streptococcus mutans (S. mutans) on lithium disilicate ceramics, submitted to different intraoral polishing protocols, and the degree of surface smoothness obtained. MATERIALS AND METHODS: Fifty lithium disilicate specimens were divided into 5 groups (n=10): G1-Glaze Group (positive control); G2-Glaze Group + Wear + Glaze; G3-Wear Group (negative control); G4-Ceramisté Wear Group; G5-Optrafine Wear Group. Surface roughness (Ra - µm) was evaluated and the surface characteristics were assessed using a scanning electron microscope (SEM); to assess S. mutans biofilm, the number of cultured cells was evaluated by counting colony-forming units (CFU/mL). The data underwent one-way ANOVA followed by Tukey's test (P⟨.05). RESULTS: There was a significant difference in the surface roughness of all groups compared with G3. There was no significant difference between the G4 and G5 groups that received polishing. G1 group had the lowest mean roughness values. There was a difference in Log values (CFU/mL) between the G3 group and the groups that received glaze (G1 and G2). The G3 group had the highest adhesion of S. mutans (4.53 Log). CONCLUSION: The most effective polishing protocol after wear is glazing, presenting the lowest roughness and CFU/mL values.

Monensin associated or not with virginiamycin or functional oil for feedlot beef cattle.

The objective of this study was to evaluate diets containing monensin (MON) associated or not with virginiamycin (VM) or functional oil based on cashew nut shell and castor beans (FOcc) for beef cattle in feedlots on nutritional (intake and digestibility) and productive parameters. A total of 1410 non-castrated Nellore cattle were selected, with an average age of 18 months and with an initial mean body weight (BW) of 305 ± 41.52 kg. The diet showed a roughage to concentrate ratio of 23:77, with the supply of corn silage as a source of roughage. The following additive inclusions in the diet were evaluated: (1) MON: 27 mg MON/kg dry matter (DM); (2) MON + VM: 22 mg MON/kg DM + 19 mg VM/kg DM; and (3) MON + FOcc: 22 mg MON/kg DM + 500 mg FOcc/kg DM. Statistical analyses were obtained through a linear model using initial BW and days of feedlot as covariables and comparisons between treatments using mutually orthogonal linear contrasts with a 5% significance level. The association or not of MON with VM or FOcc does not affect any of the nutritional and productive parameters evaluated. Animals that receive diets with MON + VM have higher average daily gain and feed efficiency (FE) than those that receive MON + FOcc without showing differences in nutritional parameters. The supply of MON associated with VM or FOcc does not increase intake and productive performance and, consequently, efficiency of feedlot beef cattle. However, in the case of use associated with MON, the VM provides greater performance than FOcc without changing food intake.

Activation of TP receptors induces high release of PGI2 in coronary arteries of renal hypertensive rats.

AIM: To investigate the molecular mechanisms and cellular signaling pathways involved in the activation of TP receptors and the consequent induction of contractile responses in coronary arteries of renal hypertensive (2K-1C) rats. METHODS AND RESULTS: The coronary perfusion pressure (CPP) was lower in 2K-1C rats during increased coronary flow as measured by the Langendorff technique. The coronary contraction and relaxation were evaluated by vascular reactivity studies, and the molecular mechanisms were investigated on the basis of the protein expression of TP receptors, Cav-1, eNOS, COX-1, and COX-2, as measured by Western blot. The levels of eicosanoids were determined by ELISA immunoassay and analyzed by reverse-phase HPLC coupled to electrospray ionization mass spectrometry (HPLC-MS/MS). The metabolites from NO production were evaluated by the Griess reaction. The coronary arteries of 2K-1C rats expressed COX-2 to a larger extent and TP receptors to a lesser extent than the coronary arteries of normotensive (2K) rats. Selective COX-1 and non-selective COX inhibitors reversed the reduction in the contraction induced by TP receptors in the coronary arteries of 2K-1C rats. U46619, an agonist of TP receptors, induced a contractile response that was relaxed by acetylcholine (ACh). In the coronary arteries of 2K-1C rats, this ACh-induced relaxation depended on COX. The activation of TP receptors increased the production of PGI2 in the coronary arteries of 2K-1C rats. The results demonstrated that increased COX signaling in the coronary arteries of 2K-1C rats mediated the low levels of CPP, the contraction induced by the activation of TP receptors, and the endothelium-dependent relaxation. The vasodilator PGI2 seemed to be the major product. CONCLUSION: Activation of TP receptors increases production of PGI2 in coronary arteries of 2K-1C rats.

Cervical dilatation patterns of 'low-risk' women with spontaneous labour and normal perinatal outcomes: a systematic review.

BACKGROUND: The call for women-centred approaches to reduce labour interventions, particularly primary caesarean section, has renewed an interest in gaining a better understanding of natural labour progression. OBJECTIVE: To synthesise available data on the cervical dilatation patterns during spontaneous labour of 'low-risk' women with normal perinatal outcomes. SEARCH STRATEGY: PubMed, EMBASE, CINAHL, POPLINE, Global Health Library, and reference lists of eligible studies. SELECTION CRITERIA: Observational studies and other study designs. DATA COLLECTION AND ANALYSIS: Two authors extracted data on: maternal characteristics; labour interventions; the duration of labour centimetre by centimetre; and the duration of labour from dilatation at admission through to 10 cm. We pooled data across studies using weighted medians and employed the Bootstrap-t method to generate the corresponding confidence bounds. MAIN RESULTS: Seven observational studies describing labour patterns for 99 971 women met our inclusion criteria. The median time to advance by 1 cm in nulliparous women was longer than 1 hour until a dilatation of 5 cm was reached, with markedly rapid progress after 6 cm. Similar labour progression patterns were observed in parous women. The 95th percentiles for both parity groups suggest that it was not uncommon for some women to reach 10 cm, despite dilatation rates that were much slower than the 1-cm/hour threshold for most part of their first stage of labours. CONCLUSION: An expectation of a minimum cervical dilatation threshold of 1 cm/hour throughout the first stage of labour is unrealistic for most healthy nulliparous and parous women. Our findings call into question the universal application of clinical standards that are conceptually based on an expectation of linear labour progress in all women. FUNDING: UNDP/UNFPA/UNICEF/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP), Department of Reproductive Health and Research, World Health Organization, and the United States Agency for International Development (USAID). TWEETABLE ABSTRACT: Cervical dilatation threshold of 1 cm/hour throughout labour is unrealistic for most women, regardless of parity.

Socio-epidemiological characterisation of blood donors with asymptomatic Leishmania infantum infection from three Brazilian endemic regions and analysis of the transfusional transmission risk of visceral leishmaniasis.

OBJECTIVES: This study assessed the prevalence of visceral leishmaniasis in blood donors from three endemic regions in Brazil and evaluated the risk of transmission by transfusion. BACKGROUND: Despite strong evidence of the transmission of visceral leishmaniasis through blood transfusion, the real risk, an essential condition for taking effective measures to control this serious disease, has not been determined. METHODS: A multicentre study was performed in highly endemic areas. Candidates eligible for their first blood donation underwent a socio-epidemiological interview, and blood samples were collected for enzyme-linked immunosorbent assay (ELISA) analysis, Western blot and polymerase chain reaction (PCR). Patients transfused with red blood cells or random platelet concentrates collected from these donors were also studied. The results were analysed using descriptive statistics and prevalence estimates, with significance defined as p-values <0·05. RESULTS: Of the 608 eligible donors, 37 (6·1%) were positive for visceral leishmaniasis as per ELISA. The socio-epidemiological analysis showed a significantly higher prevalence in non-Caucasians (p = 0·008). Among 296 patients who received blood components from these donors, the pre-transfusion seropositivity was 7·7%, and 13 patients received blood positive for Leishmania infantum. Six patients were followed up for 90 days, of which two (33·3%) had serological conversion at 60 days. CONCLUSIONS: The results confirm the high prevalence of L. infantum seropositivity among donors in the three regions; the seroconversion in a short period of time in two of six patients suggests the possibility of transmission of the infection by transfusion.

Effects of glycerol, equilibration time and antioxidants on post-thaw functional integrity of bovine spermatozoa directly obtained from epididymis.

This work aimed to evaluate the effect of stabilisation times, glycerol concentration, and the catalase and superoxide dismutase supplementation of diluent on parameters of frozen-thawed spermatozoa from epididymis of Nelore bulls: Experiment 1: spermatozoa diluted in Tris-egg yolk with glycerol (3%, 5% or 7%) and stabilisation times (0, 2 or 4 hr at 5°C); Experiment 2: Tris-egg yolk only, Tris-egg yolk with catalase (CAT, 50 or 100 U ml-1 ) or superoxide dismutase (SOD, 50 or 100 U ml-1 ). Frozen-thawed spermatozoa were evaluated for kinetic parameters, plasma membrane and acrosome integrity, mitochondrial activity and IVF capacity. ALH and BCF were affected (p < .05) by glycerol at 3% after 4-hr equilibration time and 7% after 2-hr equilibration time. Glycerol 3% had lower (p < .05) iPM and iAc after 4 hr. Glycerol 5% had greater (p < .05) hPMM after 4 hr and iAc after 2 hr than at 0 hr. SOD 100 U ml-1 had lower (p < .05) linearity and wobble compared to control group. No was observed differences to fertilisation rate (p < .05) among groups. In conclusion, glycerol 5% in Tris-egg yolk extender for 4 hr is suitable for the preservation of sperm kinetics and membrane integrity. CAT (50 and 100 U ml-1 ) or SOD (50-100 U ml-1 ) had no beneficial effects on sperm kinetics, plasma and acrosomal membrane integrity, mitochondrial activity or the capacity for IVF of frozen-thawed spermatozoa from epididymis of Nelore bulls.

Influence of polymer-coated slow-release urea on total tract apparent digestibility, ruminal fermentation and performance of Nellore steers.

OBJECTIVE: Two experiments were performed to evaluate the effects of coated slow-release urea on nutrient digestion, ruminal fermentation, nitrogen utilization, blood glucose and urea concentration (Exp 1), and average daily gain (ADG; Exp 2) of steers. METHODS: Exp 1: Eight ruminally fistulated steers [503±28.5 kg body weight (BW)] were distributed into a d 4×4 Latin square design and assigned to treatments: control (CON), feed grade urea (U2), polymer-coated slow-release urea A (SRA2), and polymer-coated slow-release urea B (SRB2). Dietary urea sources were set at 20 g/kg DM. Exp 2: 84 steers (350.5±26.5 kg initial BW) were distributed to treatments: CON, FGU at 10 or 20 g/kg diet DM (U1 and U2, respectively), coated SRA2 at 10 or 20 g/kg diet DM (SRA1 and SRA2, respectively), and coated SRB at 10 or 20 g/kg diet DM (SRB1 and SRB2, respectively). RESULTS: Exp 1: Urea treatments (U2+SRA2+SRB2) decreased (7.4%, p = 0.03) the DM intake and increased (11.4%, p<0.01) crude protein digestibility. Coated slow-release urea (SRA2+SRB2) showed similar nutrient digestibility compwared to feed grade urea (FGU). However, steers fed SRB2 had higher (p = 0.02) DM digestibility compared to those fed SRA2. Urea sources did not affect ruminal fermentation when compared to CON. Although, coated slow-release urea showed lower (p = 0.01) concentration of NH3-N (-10.4%) and acetate to propionate ratio than U2. Coated slow-release urea showed lower (p = 0.02) urinary N and blood urea concentration compared to FGU. Exp 2: Urea sources decreased (p = 0.01) the ADG in relation to CON. Animals fed urea sources at 10 g/kg DM showed higher (12.33%, p = 0.01) ADG compared to those fed urea at 20 g/kg DM. CONCLUSION: Feeding urea decreased the nutrient intake without largely affected the nutrient digestibility. In addition, polymer-coated slow-release urea sources decreased ruminal ammonia concentration and increased ruminal propionate production. Urea at 20 g/kg DM, regardless of source, decreased ADG compared both to CON and diets with urea at 10 g/kg DM.

PGC-1ß regulates HER2-overexpressing breast cancer cells proliferation by metabolic and redox pathways.

Breast cancer is a prevalent neoplastic disease among women worldwide which treatments still present several side effects and resistance. Considering that cancer cells present derangements in their energetic homeostasis, and that peroxisome proliferator-activated receptor- gamma coactivator 1 (PGC-1) is crucial for cellular metabolism and redox signaling, the main objective of this study was to investigate whether there is a relationship between PGC-1 expression, the proliferation of breast cancer cells and the mechanisms involved. We initially assessed PGC-1ß expression in complementary DNA (cDNA) from breast tumor of patients bearing luminal A, luminal B, and HER2-overexpressed and triple negative tumors. Our data showed that PGC-1ß expression is increased in patients bearing HER2-overexpressing tumors as compared to others subtypes. Using quantitative PCR and immunoblotting, we showed that breast cancer cells with HER2-amplification (SKBR-3) have greater expression of PGC-1ß as compared to a non-tumorous breast cell (MCF-10A) and higher proliferation rate. PGC-1ß expression was knocked down with short interfering RNA in HER2-overexpressing cells, and cells decreased proliferation. In these PGC-1ß-inhibited cells, we found increased citrate synthase activity and no marked changes in mitochondrial respiration. Glycolytic pathway was decreased, characterized by lower intracellular lactate levels. In addition, after PGC-1ß knockdown, SKBR-3 cells showed increased reactive oxygen species production, no changes in antioxidant activity, and decreased expression of ERRα, a modulator of metabolism. In conclusion, we show an association of HER2-overexpression and PGC-1ß. PGC-1ß knockdown impairs HER2-overexpressing cells proliferation acting on ERRα signaling, metabolism, and redox balance.

Analysis of the mitochondrial COI gene and its informative potential for evolutionary inferences in the families Coreidae and Pentatomidae (Heteroptera).

The mitochondrial cytochrome oxidase subunit 1 (COI) gene is one of the most popular markers used for molecular systematics. Fragments of this gene are often used to infer phylogenies, particularly the region near the 5'-end, which is used by the DNA Barcoding Consortium. With a growing number of sequences being deposited in the DNA barcoding database, there is an urgent need to understand the evolution of this gene and its evolutionary relationship among species; it is also important to analyze the informative potential of the gene for phylogenetic inferences for each group used. In this study, the COI gene was divided into three distinct regions: a 5'-region, a central region, and a 3'-region. The nucleotide composition of these regions was analyzed, and their potential for making informative phylogenetic inferences using species in the families Coreidae and Pentatomidae (Heteroptera) was assessed. It was found that the same region in the COI gene may present different behaviors for each family analyzed, and that using additional regions from the same gene may even prejudice the analysis.

Frequency of human platelet antigens (HPA)-1, -2, -5 and -15 in Brazilian blood donors and establishment of a panel of HPA-typed donors.

OBJECTIVES: The aim of this study is to keep a record of regular human platelet antigens (HPA)-typed blood donors and to compare their allele frequencies with those reported in other populations. BACKGROUND: HPA are polymorphisms expressed on platelet membrane glycoproteins. They can generate an immune response leading to platelet alloimmunisation that may show clinical manifestations, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura and platelet refractoriness. Platelet alloimmunisation is not uncommon, therefore, for an optimum management, it is advantageous to establish a panel of typed platelets to help matched platelets selection in HPA-alloimmunised patients transfusion. METHODS: The polymerase chain reaction (PCR)-allele-specific primers and PCR-restriction fragment length polymorphism methods were used to determine the genotypes of HPA-1, -2, -5 and -15 systems of 337 blood donors. RESULTS: The three genotypes (AA, AB and BB) were found in all HPA systems analysed, and the most frequent genotypes were AA for HPA-1, -2 and -5 systems (mean: 0·732) and AB for HPA-15 system (mean: 0·523). Allele frequencies were 0·148, 0·155, 0·140 and 0·430 for HPA-1b, -2b, -5b and -15b, respectively, and they were similar to those found in Caucasian populations, especially for HPA-1. However, the B allele was more frequent in all HPA systems when compared with Amazon Indians, and the frequency of the B allele in our study was higher in HPA-1 and -15 systems and lower in HPA-2 and -5 systems in comparison with sub-Saharan African populations. CONCLUSIONS: A record of HPA-typed donors would enable rapid identification and selection of donors when HPA-compatible platelets are required for transfusion.

Novel polymorphisms in the promoter region of the perforin gene among distinct Brazilian populations and their functional impact.

Cytotoxic T lymphocytes and natural killer cells play a crucial role in eliminating tumour and virus-infected cells. The perforin is a key part of the arsenal that these cells use to destroy their targets. In this study, we characterized single-nucleotide polymorphisms (SNPs) located in the promoter region of the perforin gene among distinct Brazilian ethnic groups. The study was carried out by sequencing this region in three groups: European, African and Asian descents. We demonstrated for the first time the occurrence of three new polymorphisms in the promoter region of gene PRF1: 494A/G (rs78058707), 720G/A (rs75925789) and 1176C/T (rs75183511). Three other SNPs already described in the literature 63A/G (rs35401316), 112A/G (rs10999428) and 1012C/T (rs35069510) were also detected. The SNPs are distributed differently in the ethnic groups studied. The 112G allele was observed at high frequency, especially among Asian descents (48.1%). The 1012T allele was detected only among European descents, the 494G allele only among Asian descents and 1176T allele only in African descents. Based on the association between the polymorphisms described, ten new haplotypes were originated. In functional analysis, we noticed that SNPs present in most common haplotypes cannot induce significant differences in expression levels of perforin alone. In conclusion, this study demonstrates for the first time the existence of three new polymorphisms in perforin promoter and, contrary to what was stated, the presence of these SNPs does not alter the levels of protein expression.

Effects of training periodization on cardiac autonomic modulation and endogenous stress markers in volleyball players.

We investigated the effects of selective loads of periodization model (SLPM) on autonomic modulation of heart rate variability (HRV) and endogenous stress markers before and after a competition period in volleyball players (N=32). The experimental protocol for the evaluation of HRV consisted of using spectral analysis of time series composed of the R-R intervals derived from electrocardiogram obtained in the supine position and during the tilt test. Stress marker levels were determined by quantifying the plasma concentration of endogenous catecholamines, cortisol and free testosterone. The results showed no changes between the levels of HRV before and after a competition period. In contrast, the quantification of the plasma concentration of endogenous stress markers revealed reductions in the levels of total catecholamines, noradrenaline and cortisol. These changes were accompanied by increases in the concentration of free testosterone and in the testosterone/cortisol ratio. In conclusion, our results demonstrate that the SLPM did not change the cardiac autonomic modulation of HRV, but promoted beneficial adaptations in athletes, including positive changes in the plasma concentration of the endogenous stress markers. The absence of changes in HRV indicates that there is no direct relationship between cardiac autonomic modulation and endogenous stress markers in the present study.

Combined effect of physical exercise and hormone replacement therapy on cardiovascular and metabolic health in postmenopausal women: A systematic review and meta-analysis.

The cardioprotective effect of postmenopausal hormone replacement therapy (HRT) has been demonstrated in several studies. Similarly, physical exercise has yielded positive results. However, the effects of their combination remain inconclusive. This review describes the combined effects of physical exercise and hormone therapy on cardiovascular and metabolic health in postmenopausal women. We searched the Scopus, Web of Science, PubMed, and Embase databases and included randomized controlled trials published up to December 2021 on the combined effects of physical exercise and hormone therapy on cardiovascular and metabolic health in postmenopausal women. We identified 148 articles, of which only seven met the inclusion criteria (386 participants; 91 [23%] HRT + exercise; 104 [27%] HRT; 103 [27%] exercise; 88 [23%] placebo). The combined treatment further decreased systolic blood pressure (SBP) compared to the isolated effect of aerobic training (AT) (mean difference [MD]=-1.69; 95% confidence interval [CI]=-2.65 to -0.72, n=73). Nevertheless, it attenuated the decrease in diastolic blood pressure (DBP) (MD=0.78; 95%CI: 0.22-1.35, n=73), and the increase in peak oxygen consumption (VO2 peak) promoted by exercise (AT + HRT=2.8±1.4 vs AT + placebo=5.8±3.4, P=0.02). The combination of AT and oral HRT improved SBP. However, AT alone seemed to have a better effect on physical fitness and DBP in postmenopausal women.

Association study between ceftriaxone and a synthetic amide against strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a non-lactose fermenting Gram-negative bacteria responsible for causing numerous nosocomial infections. The present research aimed to analyze the anti-Pseudomonas aeruginosa potential of 2-Chloro-N-(4-fluoro-3-nitrophenyl)acetamide (A8). The antibacterial potential of A8 was evaluated from the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Association using the checkerboard method. MIC and MBC values were 512 µg/mL for all P. aeruginosa strains evaluated, demonstrating predominantly bactericidal activity. Furthermore, when A8 was associated with the drug ceftriaxone, pharmacological additivity and indifference were evidenced. In this sense, the synthetic amide was interesting, since it demonstrates the potential to become a possible candidate for an antimicrobial drug.

Chronic exercise promotes alterations in the neuroendocrine profile of elderly people.

Aging and physical inactivity are 2 factors that favour the development of cardiovascular disease, metabolic syndrome, obesity, and diabetes. In contrast, adopting a habitual moderate exercise routine may be a nonpharmacological treatment alternative for neuroendocrine aging disorders. We aimed to assess the effects of moderate exercise training on the metabolic profiles of elderly people with sedentary lifestyles. Fourteen sedentary, healthy, elderly male volunteers participated in a moderate training regimen for 60 min/day, 3 days/week for 24 weeks at a work rate equivalent to their ventilatory aerobic threshold. The environment was maintained at a temperature of 23±2°C, with a humidity of 60±5%. Blood samples for analysis were collected at 3 intervals: at baseline (1 week before training began), and 3 and 6 months after training. The training promoted increased aerobic capacity (relative VO(2), and time and velocity to VO(2)max; (p<0.05)) and reduced serum α-MSH (p<0.05) after 3 months of training when compared with the baseline data. In addition, serum thyroid hormone (T3 and T4) was reduced after 6 months of training compared with baseline levels. Our results demonstrate that a moderate exercise training protocol improves the metabolic profile of older people, and metabolic adaptation is dependent on time.

Isolation of high quality and polysaccharide-free DNA from leaves of Dimorphandra mollis (Leguminosae), a tree from the Brazilian Cerrado.

Dimorphandra mollis (Leguminosae), known as faveiro and fava d'anta, is a tree that is widely distributed throughout the Brazilian Cerrado (a savanna-like biome). This species is economically valuable and has been extensively exploited because its fruits contain the flavonoid rutin, which is used to produce medications for human circulatory diseases. Knowledge about its genetic diversity is needed to guide decisions about the conservation and rational use of this species in order to maintain its diversity. DNA extraction is an essential step for obtaining good results in a molecular analysis. However, DNA isolation from plants is usually compromised by excessive contamination by secondary metabolites. DNA extraction of D. mollis, mainly from mature leaves, results in a highly viscous mass that is difficult to handle and use in techniques that require pure DNA. We tested four protocols for plant DNA extraction that can be used to minimize problems such as contamination by polysaccharides, which is more pronounced in material from mature leaves. The protocol that produced the best DNA quality initially utilizes a sorbitol buffer to remove mucilaginous polysaccharides. The macerated leaf material is washed with this buffer until there is no visible mucilage in the sample. This protocol is adequate for DNA extraction both from young and mature leaves, and could be useful not only for D. mollis but also for other species that have high levels of polysaccharide contamination during the extraction process.

Spermatogenesis of riffle bugs, Rhagovelia whitei and Rhagovelia sp (Veliidae), and backswimmers Martarega sp (Notonectidae).

We examined the course of spermatogenesis and the meiotic chromosome complements in aquatic species of true bugs, Heteroptera. The chromosome complement of the Veliidae species was 2n = 39 (38A + X0) and 23 (22A + X0) in Rhagovelia whitei and Rhagovelia sp, respectively, and in the species of the Notonectidae (Martarega sp) it was 26 (22A + 2m + XY); all collected from the region of São José do Rio Preto, SP, Brazil. An impressive characteristic of the first analysis was the size of the cells belonging to Martarega sp, which were six times larger than the same cells in Pentatomidae and twice as large as the cells in aquatic Heteroptera (Gerridae). Regarding spermatogenesis, all the species analyzed showed the same pattern: holocentric chromosomes and elongated spermatids with the chromatin distributed evenly along the head. The family Veliidae showed several bodies impregnated with silver nitrate at prophase, while the family Notonectidae displayed only one. The cells of Notonectidae also showed an evident and round body until the end of prophase I and in the family Veliidae the silver-impregnated bodies were disorganized, where the only region visualized was possibly that of the NOR. In metaphase, silver-stained regions were found at the periphery of all chromosomes in Veliidae and at the periphery of some chromosomes in Notonectidae. The spermatids of Veliidae showed a less silver-impregnated vesicle, while Notonectidae showed silver staining only in part of the nuclear membrane. Therefore, families of Heteroptera have some differences and features that can help identify and classify these species.

Expression of the bovine papillomavirus type 1, 2 and 4 L1 genes in the yeast Pichia pastoris.

Papillomaviruses are known to cause benign or malignant lesions in various animals. In cattle, bovine papillomavirus (BPV) is the etiologic agent of papillomatosis and neoplasia of the upper gastrointestinal tract and urinary bladder. Currently, there are no standard diagnostic tests or prophylactic vaccines. Protection against papillomavirus infection is conferred by neutralizing antibodies directed towards the major structural protein L1. These antibodies can be efficiently induced by immunization with virus-like particles that are formed spontaneously after L1 gene expression in recombinant systems. The yeast Pichia pastoris is known to provide an efficient system for expression of proteins due to reduced cost and high levels of protein production. We evaluated P. pastoris for expression of the L1 gene from BPV1, BPV2 and BPV4. After methanol induction, the recombinants were able to produce L1 proteins of the three different BPV types. To increase heterologous L1 protein levels, a codon optimization strategy was used for production under bioreactor conditions. The BPV1 L1 protein was identified by monoclonal antibody anti-6xHis. This is the first report of BPV L1 expression in yeast.

Acute and subchronic effects of petroleum on the freshwater fish Hoplias aff. malabaricus.

Petroleum water soluble fraction (WSF) impairs organisms, but damages may vary among cell and tissue levels. The aim of the present study was to evaluate the acute (24 h, 48 h, 72 h) and subchronic effects (36 days) of WSF (0%, 25% and 100%) in juveniles of the Neotropical top predator fish Hoplias aff. malabaricus. The effects of WSF were evaluated at a molecular level using the comet assay and micronucleus test for genome damage; and at a morphological level through histological identification of liver pathologic lesions. In both acute and subchronic exposure we found low levels of DNA damage (< 10% of comet tail) and non-significant frequency of micronucleus in WSF exposed fish. The most significant liver lesions in WSF exposed fish were fatty vacuolization, hypertrophy and focal necrosis. Since these tissue injuries were progressive and persistent, their irreversibility may negatively affect fish recruitment, even in a such resistant top predator.