The synthetic peptide PnPP-19 induces peripheral antinociception via activation of NO/cGMP/KATP pathway: Role of eNOS and nNOS.

BACKGROUND: and purpose: The peptide PnPP-19, derived from the spider toxin PnTx2-6 (renamed as δ-CNTX-Pn1c), potentiates erectile function by activating the nitrergic system. Since NO has been studied as an antinociceptive molecule and PnPP-19 is known to induce peripheral antinociception, we intended to evaluate whether PnPP-19 could induce peripheral antinociception through activation of this pathway. EXPERIMENTAL APPROACH: Nociceptive thresholds were measured by paw pressure test. PGE2 (2 µg/paw) was administered intraplantarly together with PnPP-19 and inhibitors/blockers of NOS, guanylyl cyclase and KATP channels. The nitrite concentration was accessed by Griess test. The expression and phosphorylation of eNOS and nNOS were determined by western blot. KEY RESULTS: PnPP-19 (5, 10 and 20 µg/paw) induced peripheral antinociception in rats. Administration of NOS inhibitor (L-NOarg), selective nNOS inhibitor (L-NPA), guanylyl cyclase inhibitor (ODQ) and the blocker of KATP (glibenclamide) partially inhibited the antinociceptive effect of PnPP-19 (10 µg/paw). Tissue nitrite concentration increased after PnPP-19 (10 µg/paw) administration. Expression of eNOS and nNOS remained the same in all tested groups, however the phosphorylation of nNOS Ser852 (inactivation site) increased and phosphorylation of eNOS Ser1177 (activation site) decreased after PGE2 injection. Administration of PnPP-19 reverted this PGE2-induced effect. CONCLUSIONS AND IMPLICATIONS: The peripheral antinociceptive effect induced by PnPP-19 is resulting from activation of NO-cGMP-KATP pathway. Activation of eNOS and nNOS might be required for such effect. Our results suggest PnPP-19 as a new drug candidate to treat pain and reinforce the importance of nNOS and eNOS activation, as well as endogenous NO release, for induction of peripheral antinociception.

Synergic effect of oral contraceptives, GSTP1 polymorphisms, and high-risk HPV infection in development of cervical lesions.

Human papillomavirus (HPV) infection is considered a risk factor for cervical cancer. Even if the high-risk HPV (HR-HPV) infection is necessary, environmental co-factors and genetic susceptibility also play an important role in cervical cancer development. In this study, a possible association of rs1695 GSTP1 polymorphisms, HR-HPV infection, and oral contraceptive use with cancer lesion development in women was investigated. The study population comprised 441 Brazilian women from the Northeast region including 98 HPV-infected women with high-grade squamous intraepithelial lesions, 77 HPV-infected women with low-grade squamous intraepithelial lesions, and 266 HPV-negative women with no lesion, used as a control. Our data did not show a significant association between the GSTP1 polymorphism A/G (rs1695) and any HPV-related cervical abnormalities. However, considering the use of oral contraceptives, the GSTP1 rs1695 polymorphism was associated with higher susceptibility to the development of cervical lesions in HR-HPV-infected women. Our study suggests a synergic effect of oral contraceptive use, GSTP1 polymorphisms, and HR-HPV infection in the development of cervical lesions. Together, these risk factors may induce neoplastic transformation of the cervical squamous epithelium, setting conditions for secondary genetic events leading to cervical cancer.

Prevalence of human papillomavirus (HPV), distribution of HPV types, and risk factors for infection in HPV-positive women.

The aim of this study was to describe the prevalence of human papillomavirus (HPV), the distribution of different HPV types, and the putative risk factors for infection among HPV-positive women from the State of Alagoas, Northeast Brazil. We analyzed data from 515 patients attending public and private health centers. HPV DNA from cervical samples was extracted and HPV genotyping was performed by polymerase chain reaction using MY09/11 consensus primers followed by direct sequencing. The chi-squared test for independence was used to assess statistical differences between the HPV groups. HPV DNA was found in 111 (21.55%) cervical samples. Twenty genotypes were detected: HPV6, 11, 16, 31, 33, 35, 39, 52, 53, 54, 58, 61, 62, 66, 70, 72, 81, 82, 83, and 84. In addition, multiple sexual partners (P = 0.002) and the use of oral contraceptives (P = 0.015) were associated with the presence of HPV. These findings may be relevant to the design of screening and vaccination strategies targeting specific groups of women in Northeast Brazil.

Polymorphism in the A2M gene associated with high-quality milk in Murrah buffaloes (Bubalus bubalis).

The study of genes associated with host defense mechanisms, such as the A2M gene, plays a critical role in preventing diseases that reduce milk yield and its constituents. The aim of this study was to identify polymorphisms in the A2M gene in Murrah buffaloes (Bubalus bubalis), and investigate their associations with milk yield, fat and protein production, fat and protein percentages, and somatic cell count. Hair follicle samples of 136 animals were collected for DNA extraction, and polymorphisms were identified by polymerase chain reactions and sequencing. Statistical analyses were performed to ascertain the allelic and genotypic frequencies, the Hardy-Weinberg equilibrium, and association analysis was conducted between the polymorphisms and the traits studied. Comparative analysis between buffalo and bovine sequences revealed seven nucleotide substitutions. Alignments among the buffalo sequences identified three single nucleotide polymorphisms (SNPs), including one in exon 29, g.241A>G, which was used in subsequent statistical analyses. A Hardy-Weinberg test indicated that this SNP was in equilibrium in this population, and was significantly associated (P < 0.05) with fat production and fat and protein percentages. Therefore, this SNP can be used as a molecular marker for these traits.

Short communication: Variable number of tandem repeat polymorphisms in DGAT1 gene of buffaloes (Bubalus bubalis) is associated with milk constituents.

The diacylglycerol-O-transferase 1 gene is a positional and functional candidate for milk composition traits. The objective of this study was to evaluate the segregation of the variable number of tandem repeat polymorphisms in the regulatory region of diacylglycerol-O-transferase 1 gene in a water buffalo herd, and to assess the association of this mutation with milk production traits. For this purpose, 196 Murrah buffalo cows were genotyped by PCR. The association of the marker with total milk, fat, and protein yields at 305 d of lactation, milk fat and protein percentage, and somatic cell scores were evaluated by single-trait analyses using a generalized mixed model. Two segregating alleles were identified in the population. The allele with 2 repeats affected fat percentage favorably. The present results suggest that this polymorphism is an interesting marker to include in the genetic evaluation of buffaloes.

Biological treatment with fungi of olive mill wastewater pre-treated by photocatalytic oxidation with nanomaterials.

Olive mill wastewater (OMW) still is a major environmental problem due to its high chemical oxygen demand (COD) and total phenolic content (TPC), contributing for the high toxicity and recalcitrant nature. Several attempts have been made for developing more efficient treatment processes, but no chemical or biological approaches were found to be totally effective, especially in terms of toxicity reduction. In this context, the main purpose of this study was to investigate the treatability of OMW by the combination of photocatalytic oxidation, using two nanomaterials as catalysts (TiO2 and Fe2O3), with biological degradation by fungi (Pleurotus sajor caju and Phanerochaete chrysosporium). Photocatalytic oxidation was carried out using different systems, nano-TiO2/UV, nano-Fe2O3/UV, nano-TiO2/H2O2/UV and nano-Fe2O3/H2O2/UV. The effectiveness of the treatment was assessed through color (465nm), aromatics (270nm), COD and TPC reductions, as well as by the decrease in toxicity using the bacterium Vibrio fischeri. The chemical treatment with the system nano-TiO2/H2O2/UV promoted 43%, 14%, 38% and 31% reductions in color, aromatics content, COD and TPC, respectively. However no toxicity reduction was observed. The combination with a biological treatment increased the reduction of COD and TPC as well as a reduction in toxicity. The treatment with P. chrysosporium promoted the highest reduction in toxicity, but P. sajor caju was responsible for the best reduction in COD and TPC. However, the biological treatment was more effective when no hydrogen peroxide was used in the pre-treatment.

Development of a DNA-based vaccine strategy against bovine papillomavirus infection, involving the E5 or L2 gene.

Papillomaviruses are known to cause tumor lesions, generally benign, in epithelial tissues of diverse organisms; these lesions may progress to cancer under suitable conditions. Bovine papillomavirus (BPV) can cause urinary bladder cancer and cancer of the upper gastrointestinal tract. Furthermore, BPV1 and BPV2 are implicated in the development of tumors in equids. Many studies with animal models clearly demonstrate that DNA vaccines are very effective tools in controlling viral infections, providing strong humoral and cellular immune responses. In this study, we have described the development of two vaccine constructs for the control of diseases caused by BPV. The 1st strategy is prophylactic and is based on the L2 gene; the 2nd is therapeutic and is based on the E5 gene. Vaccine constructs were obtained and evaluated in vitro in mammalian cells. The results show the occurrence of E5 and L2 transcription and viral protein production. These results confirm the functionality of the vaccine constructs in mammalian cells. This is the 1st step in the development of a DNA-based vaccine strategy for the control and/or treatment of diseases caused by BPV.

Association between single-nucleotide polymorphisms and milk production traits in buffalo.

The aim of this study was to identify single-nucleotide polymorphisms (SNPs) in buffaloes associated with milk yield and content, in addition to somatic cell scores based on the cross-species transferability of SNPs from cattle to buffalo. A total of 15,745 SNPs were analyzed, of which 1562 showed 1% significance and 4742 with 5% significance, which were associated for all traits studied. After application of Bonferroni's correction for multiple tests of the traits analyzed, we found 2 significant SNPs placed on cattle chromosomes BTA15 and BTA20, which are homologous to buffalo chromosomes BBU16 and BBU19, respectively. In this genome association study, we found several significant SNPs affecting buffalo milk production and quality. Furthermore, the use of the high-density bovine BeadChip was suitable for genomic analysis in buffaloes. Although extensive chromosome arm homology was described between cattle and buffalo, the exact chromosomal position of SNP markers associated with these economically important traits in buffalo can be determined only through buffalo genome sequencing.

Human papillomavirus vertical transmission: review of current data.

Despite the increasing evidence of human papillomavirus (HPV) vertical transmission, this route is regarded as less clinically important because of the detections of transient HPV DNA. However, recent studies have provided clear evidence of papillomavirus productive infection in lymphocytes, placenta, and bovine fetal tissue. Furthermore, a model of papillomavirus latency has been recently proposed that could explain the failure or transience in HPV detection observed in some infected infants. This new evidence of hematogeneous and vertical spread of HPV suggests that these modes of transmission should be investigated in greater detail to obtain a better understanding of the infection and a fuller awareness of the preventive measures that can be taken against HPV-related diseases.

Entropy-based approach for selecting informative regions in the L1 gene of bovine papillomavirus for phylogenetic inference and primer design.

Bovine papillomaviruses (BPVs) cause many benign and malignant lesions in cattle and other animals. Twelve BPV types have been identified so far, and several putative novel BPV types have been detected based on the analysis of L1 gene fragments, generated by FAP59/64 and MY11/09 primers. Phylogenetic trees are important in studies that describe novel BPV types. However, topological mistakes could be a problem in such studies. Therefore, we made use of entropy to find phylogenetic informative regions in the BPV L1 gene sequences from all 12 BPVs. Six data sets were created and phylogenetically compared to each other using neighbor-joining and maximum likelihood methods of phylogenetic tree reconstruction. We found two major regions in the L1 gene, using an entropy-based approach, which selects regions with low information complexity. More robust phylogenetic trees were obtained with these regions, when compared to the ones obtained with FAP59/64 and MY11/09 primers. More robust phylogenetic trees are important to accurately position novel BPV types, subtypes and variants. We conclude that an entropy-based approach is a good methodology for selecting regions of the L1 gene of BPVs that could be used to design more specific and sensitive degenerate primers, for the development of improved diagnostic methods.

Detection and expression of bovine papillomavirus in blood of healthy and papillomatosis-affected cattle.

Papillomaviruses (PV) are double-stranded DNA viruses that can cause benignant and malignant tumors in amniotes. There are 13 types of bovine papillomavirus (BPV-1 to -13); they have been found in reproductive tissues and body fluids. Normally these viruses are detected in epithelial tissue. We looked for BPV in the blood of healthy cattle and cattle with papillomatosis, using PCR and RT-PCR. BPV types 1 and 2 were detected in 8/12 blood samples of asymptomatic bovines and in 8/9 samples from cattle with papillomatosis. Six of 8 asymptomatic samples positive for BPV also showed expression for BPV. Five of 6 samples were positive for E2 expression, while 3/6 samples were positive for E5 expression. Five of 8 symptomatic samples positive for BPV also showed BPV expression. Five of 5 were positive for E2 expression, while 1/5 was positive for E5 expression. Two of 6 blood samples of asymptomatic cattle and 1/5 symptomatic blood samples scored positive for both E2 and E5 expression. This is the first study showing expression of BPV genes in the blood of asymptomatic and papillomatosis-affected animals.

Biological Activities of Libidibia (Caesalpinia) ferrea var. parvifolia (Mart. ex Tul.) L. P. Queiroz Pod Preparations.

Libidibia ferrea has been used in folk medicine throughout Brazil, and this study evaluated the biological activities of crude extract (CE) as well as a partially purified fraction (F80) obtained from its pods. Results from the MTT assay revealed that only F80 inhibited NCI-H292 cell growth; however, neither CE nor F80 reduced HEp-2 cell growth or sarcoma 180 tumor weight with the in vivo assay. Acute oral toxicity of the extract and fraction was evaluated following the steps of Guideline 423, using female mice; LD(50) for both preparations was determined as 2,500 mg/kg body weight. CE and F80 promoted a reduction of the leukocyte number and nitrite level in inflammatory exudates when the anti-inflammatory assay (carrageenan-induced peritonitis) was performed. CE and F80 inhibited writhing regarding antinociceptive activity (acetic acid-induced writhing response in mice). In conclusion, CE and F80 have no significant cytotoxic or antitumor activities in cell lines showing low toxicity and no action against tumors in vivo. Both preparations revealed anti-inflammatory and antinociceptive activities, corroborating the pharmacological basis of L. ferrea for ethnomedical use.

Influence of customized composite resin fibreglass posts on the mechanics of restored treated teeth.

AIM: To evaluate the mechanical behaviour of the dentine/cement/post interface of a maxillary central incisor using the finite element method and to compare the stresses exerted using conventional or customized post cementation techniques. METHODOLOGY: Four models of a maxillary central incisor were created using fibreglass posts cemented with several techniques: FGP1, a 1-mm-diameter conventionally cemented post; CFGP1, a 1-mm-diameter customized composite resin post; FGP2, a 2-mm-diameter conventionally cemented post; CFGP2, a 2-mm-diameter customized composite resin post. A distributed load of 1N was applied to the lingual aspect of the tooth at 45° to its long axis. Additionally, polymerization shrinkage of 1% was simulated for the resin cement. The surface of the periodontal ligament was fixed in the three axes (X =Y = Z = 0). The maximum principal stress (σ(max) ), minimum principal stress (σ(min)), equivalent von Mises stress (σ(vM) ) and shear stress (σ(shear)) were calculated for the dentine/cement/post interface using finite element software. RESULTS: The peak of σ(max) for the cement layer occurred first in CFGP1 (1.77 MPa), followed by CFGP2 (0.99), FGP2 (0.44) and FGP1 (0.2). The shrinkage stress (σ(vM) ) of the cement layer occurred as follows: FGP1 (35 MPa), FGP2 (34), CFGP1 (30.7) and CFGP2 (30.1). CONCLUSIONS: Under incisal loading, the cement layer of customized posts had higher stress concentrations. The conventional posts showed higher stress because of polymerization shrinkage.

Frequency of human papillomavirus types 16, 18, 31, and 33 and sites of cervical lesions in gynecological patients from Recife, Brazil.

Human papilloma virus (HPV) is a well-established cause of cervical cancer. While many studies have been performed so far on HPV viral biology, mode of infection and prevention measures, scanty information is available on lesion sites of infected women and the incidence of viral types at specific locations. We looked for a possible relationship between the most common viral types (HPVs 16, 18, 31, 33) found in Recife, PE, Brazil, and lesion sites. We examined 396 HPV-positive women at the Gynecological Unit of the IMIP at Recife; 288 women were positive for HPV 16, 18, 31, or 33, present as a single-virus type or as co-infection. HPV 16 was the most frequent virus type found in the vulva, vagina, uterine cervix-vagina, and uterine cervix. HPV 31 was the second prevalent virus type in vulva, vagina, uterine cervix-vagina, uterine cervix, and mole. HPVs 18 and 33 were present with similar frequencies in the mole-vulva region. Among the co-infections, HPV 16/18 and HPV16/31 were the most frequent in our study group, followed by HPV 16/33.

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.

Impact of a secondary treated bleached Kraft pulp mill effluent in both sexes of goldfish (Carassius auratus L.).

The aim of the present study was to assess the toxic effects of a secondary treated effluent from a modern pulp mill processing Eucalyptus globulus on both sexes of goldfish. The effects in the exposed fish were investigated by measuring biomarkers which have been used to assess the effects of BKME on aquatic biota, with particular emphasis in fish. However, few studies have been focused on effluents from pulp mill plants processing eucalyptus. The relative proportion of cytochrome P450 (CYP1A) and vitellogenin (VTG) induction were analyzed in the livers of males and females goldfish, exposed to different concentrations of a bleached Kraft pulp mill (BKPM) effluent. Somatic indices such as HSI (hepatosomatic index) and GSI (gonadosomatic index) were calculated and a significant reduction was found in males GSI. A significant increase of HSI was recorded for both sexes exposed to 50% BKPM effluent. Exposure to BKPM effluent induced CYP1A synthesis in both sexes and decreased VTG synthesis in females according to the different effluent concentrations suggesting that the secondary treated effluent contained compounds that affect fish negatively. The results suggest that BKPM effluent can cause sex-related biochemical changes in xenobiotic metabolism.

The effectiveness of a biological treatment with Rhizopus oryzae and of a photo-Fenton oxidation in the mitigation of toxicity of a bleached kraft pulp mill effluent.

Huge efforts have been made both in adopting more environmental-friendly bleaching processes, and in developing advanced oxidation processes and more effective biological treatments for the reduction of deleterious impacts of paper mill effluents. Even so, the success of such treatments is frequently reported in terms of chemical parameters without a proper evaluation of the effluent's toxicity mitigation. This is the first study reporting an exhaustive evaluation of the toxicity of a secondary bleached kraft pulp mill effluent, after either tertiary treatment with the soft-rot fungi Rhizopus oryzae or with a photo-Fenton oxidation, using a battery of freshwater species. As it has been reported the photo-Fenton/UV treatment has proved to be the most effective in reducing the colour and the COD (chemical oxygen demand) of the effluent. Nevertheless, extremely low EC(50) values were reported for almost all species, after this tertiary treatment. The treatment with R. oryzae was less effective in terms of colour removal and COD reduction, but proved to be the most promising in reducing toxicity.

Biological treatment of the effluent from a bleached kraft pulp mill using basidiomycete and zygomycete fungi.

Three white-rot fungi (Pleurotus sajor caju, Trametes versicolor and Phanerochaete chrysosporium) and one soft-rot fungi (Rhizopus oryzae) species confirmed their potential for future applications in the biological treatment of effluents derived from the secondary treatment of a bleached kraft pulp mill processing Eucalyptus globulus. Among the four species P. sajor caju and R. oryzae were the most effective in the biodegradation of organic compounds present in the effluent, being responsible for the reduction of relative absorbance (25-46% at 250 nm and 72-74% at 465 nm) and of chemical oxygen demand levels (74 to 81%) after 10 days of incubation. Laccase (Lac), lignin (Lip) and manganese peroxidases (MnP) expression varied among fungal species, where Lac and LiP activities were correlated with the degradation of organic compounds in the effluent treated with P. sajor caju. The first two axes of a principal component analysis explained 88.9% of the total variation among sub-samples treated with the four fungus species, after different incubation periods. All the variables measured contributed positively to the first component except for the MnP enzyme activity which was the only variable contributing negatively to the first component. Absorbances at 465 nm, LiP and Lac enzyme activities were the variables with more weight on the second component. P. sajor caju revealed to be the only species able to perform the biological treatment without promoting an increment in the toxicity of the effluent to the Vibrio fischeri, as it was assessed by the Microtox assay. The opposite was recorded for the treatments with the other three species of fungus. EC(50-5 min) values ranging between 28 and 57% (effluent concentrations) were recorded even after 10 to 13 days of treatment with P. chrysosporium, R. oryzae or with T. versicolor.

Effects of ECF-Kraft pulp mill effluent treated with fungi (Rhizopus oryzae) on reproductive steroids and liver CYP1A of exposed goldfish (Carassius auratus).

The toxicity of bleached Kraft pulp mill effluents (BKME) is usually attributed to chemical compounds which are produced and released throughout various stages of pulp and paper production. The main objective of the present work was to detect sub-lethal responses of goldfish (Carassius auratus) to secondary treated BKME which was treated with Rhizopus oryzae. A total of 96 carps (C. auratus; 11 +/- 3 g) were exposed to different concentrations of the post-treated effluent (0, 1, 10, 25, 50, and 100%), in 28 days semi-static tests. Several biomarkers were then evaluated to assess the toxicological effects: induction of CYP1A (metabolic processes of organic compounds in liver), change in steroid profiles (11-Ketotestosterone, 17beta-estradiol), histopathology of liver and gonads and somatic indices (GSI, HSI) for endocrine disruption and other physiological disturbances. The most significant results show an induction of CYP1A in both sexes and a decrease of 17beta-estradiol concentrations in females. Histopathological changes such as liver tissue degeneration were observed in fish exposed to 50 and 100% of the BKME. Although the BKME was biologically treated there are some chemical compounds in the effluent that are capable to affect fish physiology, however, a clear evidence for endocrine disruption was not found.

Simultaneous presence of bovine papillomavirus in blood and in short-term lymphocyte cultures from dairy cattle in Pernambuco, Brazil.

Bovine papillomaviruses (BPV) are the causal agents of benign and malignant lesions; they can cause dramatic economic losses in cattle. Although 10 virus types have been described, three types are most common in tumors, namely BPV-1, -2 and -4. Previous studies have reported BPV in blood cells and the possibility of blood acting as a latent virus site and/or transmission agent of virus dissemination. We studied a Holstein dairy herd in Pernambuco, Brazil, in which several animals showed severe cutaneous papillomatosis, without previous determination of BPV types. Blood samples and short-term lymphocyte cultures were collected from 54 cows. We compared the BPV types detected in peripheral blood to those identified in the respective lymphocyte cultures: BPV-1 was detected in 74% and BPV-2 in 87% of the whole blood samples. Simultaneous virus presence (BPV-1 and BPV-2) was found in 65% of the blood samples. BPV-1 or BPV-2 were detected in the lymphocyte cultures in 93% of the samples, and both in 89%. The detection of viral DNA in whole blood and in lymphocyte cultures is evidence that this virus is carried by lymphocytes.