Polymorphisms in the melatonin receptor gene promoter and their associations with fertility characteristics in buffalo herd in Eastern Amazon.

Buffalo production is spreading globally because of its economic advantage. Then, it has become necessary to improve the reproductive and productive efficiency of these animals, as well as to look for genetic factors that increase this efficiency. The objectives of this study were to characterize the promoter region of the melatonin 1A receptor gene (MTRN1A), to detect possible SNPs and associate them with fertility characteristics, and identify binding sites of transcription factors involved in the regulation of genetic expression in buffaloes in the Amazon. The conventional PCR method was carried out using the two primers designed from the reference sequence deposited in the GenBank AY52466.1. The products of the PCRs were purified, sequenced, and subsequently edited and aligned. Twenty-six SNPs were found, where 73% presented allele frequencies of wild nucleotides above 0.5, and 73% presented deviations from the Hardy-Weinberg equilibrium (P < 0.05) and FIS varying between 0.06 and 1.00, characterizing high degrees of inbreeding within the population. A block of ACAA deletion (position -1483) was observed in 25% of samples. The associations between these SNPs and reproductive characteristics were observed for calving interval and 5 SNPs: -1289, -1139, -911, -724, and -656 (P < 0.05), and three other SNPs: -1395, -724, and -94 (P < 0.05) were associated significantly with age at first calving, and were not associated with calving concentration. The promoter region was characterized by the different types of binding factors, where only 11 sites are significantly strong enough for transcription factor bindings. The ACAA deletion also exhibited a strong association with transcription factors. As a result, it would be necessary to test the SNPs above with other reproductive characteristics of economic relevance to approve the gene as a strong candidate for the selection of buffaloes in the Amazon.

Polymorphism in the melatonin receptor gene in buffalo populations of the Brazilian Amazon.

Buffalo farming in Brazil is increasing, as is the challenge of identifying molecular markers that will improve productivity. Therefore, the aim of this study was to analyze single nucleotide polymorphisms of the receptor gene for the hormone melatonin in buffaloes from northern Brazil by polymerase chain reactions (PCRs) and restriction fragment length polymorphism assays. The PCR products exhibited a cutting point for HpaI at the 318th position of the gene, indicating a transition substitution (T↔C). This substitution was synonymic, and did not alter the stability of the mRNA structure. Allelic and genotypic frequencies differed between the populations studied, and all of the populations demonstrated endogamy and were in Hardy-Weinberg equilibrium. Therefore, the HpaI restriction marker in the melatonin receptor gene cannot be used for genetic improvement, but is an excellent marker for population genetic studies.

Corrigendum Polymorphism in the melatonin receptor gene in buffalo populations of the Brazilian Amazon - Genet. Mol. Res. 15 (2): gmr.15027960.

The correction is only in the name of the first author and should be: E.M. Barbosa(1), B.B. Souza(2), R.C. Guimarães(2), J.S.N. Azevedo(3), E.C. Gonçalves(4), H.F.L. Ribeiro(2), S.T. Rolim Filho(2), E. Silva Filho(2).

Novel polymorphism in exon 1 of the melatonin receptor gene unassociated with reproductive characteristics of buffaloes in the Amazon Region.

The objective of this study was to sequence part of the exon 1 in the melatonin receptor 1A gene (MTRN1A) in buffaloes to detect a novel polymorphism with which to associate reproductive characteristics, such as age at first birth and the interval between births, in buffaloes from the northeastern region of the State of Pará (Brazil). Buffalo hair samples (77) were collected from the Terra Firme region of Pará. DNA was extracted and polymerase chain reactions (PCRs) were carried out with a primer that was designed using the GenBank accession No. AY524665 reference sequence. PCR products were purified and sequenced. After editing and analysis of the sequences, a mutation was observed at the 62nd position in exon 1 of MTRN1A (T↔C), which corresponded with a change in the 21st amino acid from leucine to proline. All possible genotypes were observed, with the most common being genotype CC (0.481). The allele frequencies were T = 0.377 and C = 0.623. Statistical analysis of FIS showed inbreeding within the sample group (FIS = 0.397) and deviations from the Hardy- Weinberg equilibrium were observed (P < 0.05). Associations between genotypes and reproductive characteristics were not significant (P > 0.05). Although the related SNP was not synonymous, there were no observable effects on the reproductive characteristics under investigation. As such, it would be ideal to detect other SNPs in exon 1 of the MTRN1A gene that can be associated with reproductive characteristics in Amazonian buffaloes.

Polymorphisms in the leptin gene promoter in Brazilian beef herds.

Brazil is the world's largest producer of beef cattle; however, the quality of its herds needs to be improved. The use of molecular markers as auxiliary tools in selecting animals for reproduction with high pattern for beef production would significantly improve the quality of the final beef product in Brazil. The leptin gene has been demonstrated to be an excellent candidate gene for bovine breeding. The objective of this study was to sequence and compare the leptin gene promoter of Brazil's important cattle breeds in order to identify polymorphisms in it. Blood samples of the Nellore, Guzerat, Tabapuã, and Senepol breeds were collected for genomic DNA extraction. The genomic DNA was used as a template for polymerase chain reaction (PCR) to amplify a 1575-bp fragment, which in turn was sequenced, aligned, and compared between animals of different breeds. Twenty-three single nucleotide polymorphic sites, including transitions and transversions, were detected at positions -1457, -1452, -1446, -1397, -1392, -1361, -1238, -963,-901, -578, -516, -483, -478, -470, -432, -430, -292, -282, -272, -211, -202, -170, and -147. Additionally, two insertion sites at positions -680 and -416 and two deletion sites at positions -1255 and -1059 were detected. As the promoter region of the leptin gene has been demonstrated to vary among breeds, these variations must be tested for their use as potential molecular markers for artificial selection of animals for enhanced beef production in different systems of bovine production in Brazil.

High-efficiency microwave-assisted digestion combined to in situ ultraviolet radiation for the determination of rare earth elements by ultrasonic nebulization ICPMS in crude oils.

A method for heavy and extraheavy crude oil digestion based on microwave-assisted wet digestion (MW-AD) and ultraviolet (UV) radiation using diluted HNO3 was applied for the determination of rare earth elements (REE) by inductively coupled plasma mass spectrometry (ICPMS) with an ultrasonic nebulizer (USN). Even using pressurized systems conventional acid digestion is not feasible for efficient crude oil digestion, especially for heavy and extraheavy crude oils that generally present high amounts of asphaltenes and resins. In the proposed system, UV radiation is generated in situ by immersed electrodeless Cd discharge lamps positioned inside quartz vessels. The use of diluted solutions (1-14.4 mol L(-1) HNO3 and 1-4 mol L(-1) H2O2) were evaluated for heavy and extraheavy crude oil digestion (API density of 11.1-19.0). With the proposed method the residual carbon content was lower than 13 mg C/100 mg of sample, and it was possible to digest sample masses up to 500 mg using 4 mol L(-1) HNO3 and 4 mol L(-1) H2O2. Interferences caused by excessive acid concentration and carbon content in digests were minimized allowing limits of quantification for REEs as low as 0.3 ng g(-1). Samples were also digested using MW-AD in pressurized systems with concentrated HNO3, but even using 280 °C, 80 bar, and concentrated HNO3, MW-AD method was not suitable for REE determination due to interferences in ICPMS determination. The combination of microwave heating with UV was considered a suitable and effective way to digest crude oil allowing further determination of low concentrations of REE by ICPMS.

Biotechnological and socio-environmental potential of Campomanesia adamantium (Myrtaceae): an interdisciplinary review.

Guavira (Campomanesia adamantium, Myrtaceae) is a native fruit from the Brazilian Cerrado savanna and is socio-economically important for the indigenous and traditional people living in the Central-West. This is a bibliographic review of the biological properties of guavira and its derivatives, and, after discussing experimental studies, an interdisciplinary approach is conducted highlighting the im-portance of Agroforestry Systems as an ecological restoration tool to leverage the production chain of guavira while providing ecosystem services. Many research groups studied effects of polyphenols and other bioactive compounds and biological properties of this fruit and other plant parts such as antibiotic, antioxidant, anti-inflammatory, anti-hyperlipidemic, anti-diarrheic and antitumoral activities, cardiovascular and hepatic protection and action against neuropathic pain. Besides, guavira by-products benefit poultry intestinal health, similarly to antibiotics added to their feed. Furthermore, several biotechnological products were found, like pulp flour, seasoning from the peel, sunscreen, and seed oil similar to olive oil with pharmaceutical and industrial potential. We conclude by emphasizing the importance of guavira for restoration and preservation of the threatened Brazilian Cerrado, and for the socio-environmental development of family agriculture. The same approach and study are welcome and necessary in other regions and domains worldwide having their native flora as means for a restorative end.

Trypanosomatids in Phlebotomine Sand Flies (Diptera: Phlebotominae) From Anthropic and Sinantropic Landscapes in a Rural Settlement in the Brazilian Amazon.

Trypanosomatids (Kinetoplastida:Trypanosomatidae) protozoa are a diverse group of obligate parasites. The genera Trypanosoma and Leishmania are the most studied because of their medical importance. This work aims to evaluate the effects of anthropization processes on the composition of the phlebotomine sand fly fauna and the natural infection by Trypanosomatids, with emphasis on Leishmania. At all 3,186 sand flies were collected, distributed in 13 genera and 52 species, being Ny. umbratilis the most abundant species. There was no difference in the diversity between canopy and soil environments. The species abundance and richness were higher in the forest environment while species diversity and evenness were highest in the forest edge. The ITS1 region was used by PCR-RFLP to identify the fragment profiles of Leishmania species, followed by genetic sequencing. Here were analyzed 100 pools of female sand flies, being six positive for DNA parasite. PCR-RFLP fragment patterns similar to Endotrypanum sp. were observed in Nyssomyia anduzei, Psychodopygus amazonensis and Lutzomyia gomezi, and those fragments similar to Leishmania (Leishmania) amazonensis were observed in Bichromomyia flaviscutellata. ITS1 sequencing confirmed the presence of Leishmania sp. in Bi. flaviscutellata, and Leishmania (Viannia) naiffi in Ny. anduzei, Psychodopygus amazonensis, and Lu. gomezi. This is the first record of Lu. gomezi and Ps. amazonensis infection by L. naiffi in the State of Amazonas. These results show the trypanosomatid infection in sandflies from different landscapes in a rural settlement, and the finding of species infected with L.(V.) naiffi suggest that they can develop a role in the transmission cycle of leishmaniasis.

Analysis of magnetic resonance imaging characteristics and pain in temporomandibular joints with and without degenerative changes of the condyle.

The aim of this study was to investigate temporomandibular joint (TMJ) pain and magnetic resonance imaging characteristics in 104 TMJs with and 58 without degenerative changes of the condyle, such as osteophytes, erosion, avascular necrosis, subcondral cyst and intra-articular loose bodies. TMJ images were also assessed for flattening, retropositioning and hypomobility of condyle and disc displacement. Comparison of the TMJ side-related data showed a significant relationship between disc displacement without reduction (DDwoR) and the presence of degenerative bony changes (p=0.00). Flattening, retropositioning and hypomobility of condyle showed no significant difference in relation to the presence or absence of degenerative bony changes. Retropositioning of the condyle was significantly associated to disc displacement with reduction (DDwR) (p=0.00), while condylar hypomobility was significantly more frequent in TMJ with DDwoR (p<0.05). Independent of the presence or type of DD, TMJ pain was more frequent in the presence of degenerative bony changes. When considering only DDwR, TMJ pain was significantly associated to a degenerative condition (p=0.03). When there were no degenerative bony changes, TMJ pain was significantly more frequent in DDwoR (p=0.04). Despite the present findings, the absence of symptoms in some patients with condylar bony changes suggests that the diagnosis of osteoarthritis should be established by evaluation of magnetic resonance images in association with clinical examination.

Gemcitabine and paclitaxel as salvage therapy in metastatic breast cancer.

In a phase II trial, 29 patients with anthracycline-pretreated or anthracycline-resistant metastatic breast cancer in whom anthracycline-containing first- or second-line chemotherapy failed received combination paclitaxel (Taxol)/gemcitabine (Gemzar). The initial regimen of paclitaxel at 175 mg/m2 on day 1 and gemcitabine at 1,000 mg/m2 on days 1, 8, and 15 of a 28-day cycle was given to five patients for a total of 27 cycles. The regimen resulted in excessive thrombocytopenia and was subsequently changed to gemcitabine at the same dose on days 1 and 8 of a 21-day cycle, with study treatment being given for a maximum of eight cycles. This regimen was well tolerated. Further evaluation of this regimen in minimally and heavily pretreated patients with advanced breast cancer is warranted.

Phase II trial of the use of paclitaxel and gemcitabine as a salvage treatment in metastatic breast cancer.

The purpose of this study was to evaluate gemcitabine plus paclitaxel in heavily pretreated patients with metastatic breast cancer (MBC). Patients with MBC with second or third relapse to anthracycline-containing regimens received a 3-hour infusion of paclitaxel 175 mg/m2 on day 1, and gemcitabine 1.0 g/m2 on days 1, 8, and 15, every 28 days. Because of unacceptable thrombocytopenia seen in the first 5 patients, the gemcitabine schedule was changed to days 1 and 8 (G-1,8) for the remainder of the study, every 21 days. Twenty-nine patients (median age, 46 years; range, 32-68 years) received 137 cycles (median: 4 per patient). The regimen was well tolerated. World Health Organization grades III and IV thrombocytopenia were observed in 5 (18.5%) of the first 27 cycles (G-1,8,15), and in 6 (5.4%) of the 110 subsequent cycles (G-1,8)--p = 0.04 for the difference between schedules. Five patients had grade I and two had grade III neuropathy. Eight patients had grade III neutropenia, two had grade IV neutropenia associated with fever (G-1,8,15), and eight had grades I and II myalgia and fatigue. There were 16 (55%) objective responses (95% CI 36-73%); 5 (17%) complete responses, 11 (38%) partial responses (95% CI 3-30% and 19-56%, respectively), and 6 (20.5%) patients with stable disease. Median response duration was 8 months (range, 4-26 months). Median overall survival was 12 months (range, 4-28+ months), and 1-year and 2-year survival rates were 45% and 30%, respectively. This phase II study demonstrated a manageable toxicity profile with the gemcitabine day 1, 8 schedule in combination with paclitaxel and significant and promising activity in heavily pretreated patients with MBC. A confirmatory phase III trial is warranted.

Phase II trial of the combination of paclitaxel and 5-fluorouracil in the treatment of advanced gastric cancer: a novel, safe, and effective regimen.

This prospective phase II clinical trial was performed to explore the activity and efficacy of the combination of paclitaxel and 5-fluorouracil in the treatment of advanced gastric adenocarcinoma. Thirty-one patients ages 18 to 70 years, with Karnofsky performance status (KPS) >50, adequate cardiac, renal, and hepatic functions, measurable metastatic or locally unresectable disease, life expectancy > or =3 months, signed written informed consent, and without any previous chemotherapy were assigned to receive on an outpatient basis: paclitaxel--175 mg/m2, in a 3-hour infusion on day 1 and 5-fluorouracil--1.5 g/m2, also in a 3-hour infusion on day 2 every 21 days, for a maximum of seven cycles. A system to assess clinical benefit based on KPS, analgesic consumption, and weight gain was also used in this trial. Median age was 61 years (range, 31-70 years). The 29 patients eligible for response and toxicity evaluation underwent 147 cycles of chemotherapy. There were 19 (65.5%) objective responses (95% confidence interval: 48%-83%), including 7 (24.1%) complete responses and 12 (41.4%) partial responses. Three patients had the complete response pathologically confirmed. In three of six patients who went to second-look laparotomy, a potentially curative esophagogastrectomy was possible. The toxicity of this combination was considered low, predictable, and manageable and was characterized mainly by reversible alopecia, peripheral neuropathy, myalgia, and mild neutropenia. Fifteen (51.7%) patients attained a clinical benefit response. The median overall survival was 12 months (range, 2-30+ months) and the 30-month overall survival was 20%. This novel regimen appears to be very effective in advanced gastric cancer. The projected 2-year survival of 20% is higher than that achieved with other first-line regimens. These encouraging results indicate the need for further studies to confirm the merit of this regimen.

Uptake of purine bases by Trypanosoma cruzi culture epimastigotes.

The uptake of adenine, guanine and hypoxanthine by Trypanosoma cruzi culture epimastigotes was studied over short time periods at 22 degrees C. The uptake process is concentrative, driven by the purine phosphoribosyltransferases and saturable at about 10 microM hypoxanthine and 1 microM adenine or guanine. Each purine base is apparently taken up by a separate route and the oxypurines are regulated in parallel. Adenine inhibits oxypurine uptake. When the material was incubated with guanine or hypoxanthine for more than 2 min, a decrease in the nucleotide/free base ratio was observed, indicating inhibition of the guanine and hypoxanthine phosphoribosyltransferases. The uptake rates during the ascending phase of uptake and of culture growth rates were of the order of 50 for hypoxanthine and 20 for guanine, relative to that of adenine. These rates were within the physiological range for cell growth in these cultures. During the phase of descending growth and decreased purine uptake activity rates, uptake was depressed to 20% of the rates required for the growth observed in the cultures. It is proposed that the decline in growth rate leads to an increase of the nucleotide pools in the cells which inhibit uptake. This depression may be a cause of the mitotic blockade which occurs during the stationary phase.

Noninvasive classification of liver disease in asymptomatic and oligosymptomatic male alcoholics.

The predominant type of liver alteration in asymptomatic or oligosymptomatic chronic male alcoholics (N = 169) admitted to a psychiatric hospital for detoxification was classified by two independent methods: liver palpation and multiple quadratic discriminant analysis (QDA), the latter applied to two parameters reported by the patient (duration of alcoholism and daily amount ingested) and to the data obtained from eight biochemical blood determinations (total bilirubin, alkaline phosphatase, glycemia, potassium, aspartate aminotransferase, albumin, globulin, and sodium). All 11 soft and sensitive, and 13 firm and sensitive livers formed fully concordant groups as determined by QDA. Among the 22 soft and not sensitive livers, 95% were concordant by QDA grouping. Concordance rates were low (55%) in the 73 firm and not sensitive livers, and intermediate (76%) in the 50 not palpable livers. Prediction of the liver palpation characteristics by QDA was 95% correct for the firm and not sensitive livers and moderate for the other groups. On a preliminary basis, the variables considered to be most informative by QDA were the two anamnestic data and bilirubin levels, followed by alkaline phosphatase, glycemia and potassium, and then by aspartate aminotransferase and albumin. We conclude that, when biopsies would be too costly or potentially injurious to the patients to varying extents, clinical data could be considered valid to guide patient care, at least in the three groups (soft, not sensitive; soft, sensitive; firm, sensitive livers) in which the two noninvasive procedures were highly concordant in the present study.

Purine base uptake in Trypanosoma cruzi: adaptations and effects of inhibitors.

1. The pattern of purine base uptake in culture epimastigotes of Trypanosoma cruzi can be predicted from cell growth rate and cell concentration, with the late log phase showing the greatest variability. 2. Uptake rates are unexpectedly low in the reproductive tissue amastigotes and high in the non-reproductive blood trypomastigotes. It is suggested that blood trypomastigotes metabolize and accumulate reserves of purine metabolites, whereas amastigotes depend on degradation of host cell RNA and nucleotides as purine sources. 3. All parasite forms salvage hypoxanthine and guanine in preference to adenine. Nifurtimox, dipyridamole and cytochalasin have no effect on uptake, whereas amphotericin B, allopurinol, xanthine and urate inhibit it. The alterations caused by urate are complex, apparently involving inhibition of the monooxypurine phosphoribosyltransferase and induction of permeation of purines into the cells.

tRNA-rRNA sequence matches from inter- and intraspecies comparisons suggest common origins for the two RNAs.

1. Comparisons were made of the results of searches within and among different species of organisms for sequence matches between transfer RNAs and ribosomal RNAs. The purpose was to determine whether the matching sequences might result from selection acting on the two RNAs within a common cellular environment. 2. The results indicate that most matches do not reflect such selection. The matches described were more frequent than those found in searches among randomized sequences and the frequency of intraspecific matches was not significantly higher than that of interspecific matches. 3. The matches are thought to identify conserved vestiges of a molecule or molecules ancestral to both classes of RNAs (Bloch, D.P., McArthur, B. and Mirrop, S. (1985). BioSystems, 17: 209-225). The matching sequences are interpreted as homologies.

Microwave-induced combustion of crude oil for further rare earth elements determination by USN-ICP-MS.

A procedure for light and heavy crude oils digestion by microwave-induced combustion (MIC) is proposed for the first time for further rare earth elements (REE) determination by inductively coupled plasma mass spectrometry (ICP-MS) equipped with an ultrasonic nebulizer (USN). Samples of crude oil (API density of 10.8-23.5, up to 250 mg) were inserted in polycarbonate capsules and combusted using 20 bar of oxygen and 50 µL of 6 mol L(-1) ammonium nitrate as igniter. Nitric acid solutions (1-14.4 mol L(-1)) were evaluated for analyte absorption and a reflux step was applied after combustion (5 min of microwave irradiation at 1400 W) in order to achieve better analyte recoveries. Accuracy was evaluated using a spiked sample and also by comparison of results obtained by microwave-assisted digestion combined to ultraviolet radiation (MW-UV) and by neutron activation analysis (NAA). Using 3 mol L(-1) HNO3, quantitative recoveries (better than 97%) were obtained for all analytes. Blank values were always negligible. Agreement was higher than 96% for La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and Y by comparison of results with those obtained by MW-UV and by NAA (only for La, Ce, Nd, Sm, and Yb). Residual carbon content in digests using MIC was always below 1%. As an advantage over conventional procedures for crude oil digestion, using MIC, it was possible to use diluted acid as absorbing solution, obtaining better limits of detection and avoiding interferences in REE determination by USN-ICP-MS.

Towards functional repertoire of the earliest proteins.

The conserved protein sequence motifs present in all prokaryotic proteomes, "omnipresent motifs," presumably, correspond to the earliest proteins of the Last Universal Cellular Ancestor, from which all the proteomes have descended. Fifteen proteomes, each representing one of the total 15 diverse phyla of 131 Eubacteria and Archea, from which the omnipresent elements have been originally derived, are exhaustively screened. All those proteins which harbor the omnipresent motifs are identified. Six "omnipresent" protein types are revealed which are located in all 15 proteomes: ABC cassettes, FtsH proteases, translation initiation factors, translation elongation factors, isoleucyl-tRNA synthases, and RNA polymerases ß'. In addition to the omnipresent motifs, these proteins also contain other highly conserved motifs, standing for additional modules of the proteins. Remarkably, the identified tentative earliest proteins are responsible for only three basic functions: supply of monomers (ABC transporters and proteases), protein synthesis (initiation and elongation factors, aminoacyl-tRNA synthases), and RNA synthesis (polymerases). No enzymes involved in metabolic activities are present in the list of the earliest proteins derived by this approach. Some of the omnipresent sequence motifs are found, indeed, in the metabolic enzymes (e.g. NTP binding motifs), but these enzymes do not make a sequence matching collection of 15 sequences, i.e. they are not omnipresent. Future analysis of less conserved sequence motifs may reveal at what degree of conservation (stage of evolution) the metabolic enzymes could have entered the scene.