Search

Entry of human immunodeficiency type 1 virus (HIV-1) into target cells requires both CD4and one of the chemokine receptors. Viruses predominantly use one, or occasionally both, of the major co-receptors CCR5 and CXCR4, although other receptors, including CCR2B and CCR3, function as minor co-receptors. A 32-nucleotide deletion (delta32) within the beta-chemokine receptor 5 gene (CCR5) has been described in subjects who remain uninfected despite extensive exposition to HIV-1. The heterozygous genotype delays disease progression. This allele is common among Caucasians, but has not been found in people of African or Asian ancestry. A more common transition involving a valine to isoleucine switch in transmembrane domain I of CCR2B (64I), with unknown functional consequences, was found to delay disease progression but not to reduce infection risk. As the Brazilian population consists of a mixture of several ethnic groups, we decided to examine the genotype frequency of these polymorphisms in this country. There were 11.5% CCR5 heterozygotes among the HIV-1 infected population and 12.5% among uninfected individuals, similar to data from North America and Western Europe. The prevalence of CCR2-64I homozygotes and heterozygotes was 0.06 and 15.2%, respectively, also similar to what is known for North America and Western Europe.

The clinical significance of isolated anti-HBc is still a challenge. To elucidate the real importance of this finding in our blood donors, an investigation algorithm was tested. One hundred and twelve isolated anti-HBc seropositive blood donors underwent clinical evaluation and retesting of HBV markers. Those who presented repeatedly reactive isolated anti-HBc, received a single dose of hepatitis B recombinant vaccine to verify anti-HBs early response. A HBV-DNA determination by PCR was done for those who did not test positive to anti-HBs after vaccine. The level of anti-HBc was recorded as a ratio of the sample-to-cut-off values (S:C ratio) in 57 candidates at donation. Comparing true and false-positive anti-HBc results, the different S:C ratios of them were statistically significant and when less than 2, implying in a false-positive result probability over 80%. A high percent of false-positive results (16.07%) was verified after anti-HBc retesting. HBV immunity was characterized in 49.11%, either by anti-HBs detection in retesting (15.18%), or after a single dose HBV vaccination (33.93%). HBV-DNA was negative in all tested donors. In conclusion, this algorithm was useful to clarify the meaning of isolated anti-HBc in most of our blood donors.

Abstract Background Lamivudine inhibits replication of both human immunodeficiency virus (HIV) and hepatitis B virus (HBV) and is commonly used as part of antiretroviral therapy. The main limitation in the use of lamivudine is resistant mutation selection. Most of these mutations affect the YMDD motif of the HBV DNA polymerase. The resistance occurs through M550V or M550I aminoacid replacements. The M550V variation may be accompanied by L526M mutation, notably in HIV-HBV co-infected patients. The aim of this study was to investigate mutations associated with lamivudine resistance in a hemodialysis patient chronically co-infected with HIV-1 and HBV, who was submitted to several antiretroviral treatments. Methods HBV isolates derived from three blood samples collected at different times of antiretroviral therapies with and without lamivudine, were titred and submitted to nucleotide sequencing. Results HBV isolate derived from a sample collected in 1999 during an antiretroviral treatment with lamivudine showed the lamivudine resistant double mutation (L526M, M550V). However, no mutation associated with lamivudine resistance was observed in the HBV genome derived from the sample collected during a period of treatment without lamivudine (2001). After reinstitution of lamivudine (2002), the predominant HBV population exhibited a rare triple mutation (V519L, L526M, M550V), which has previously been associated with an in vitro reduction of virus antigenicity (escape mutant). HBV DNA was detected at high levels (108–109 copies/ml) in the three blood samples. Conclusions Reintroduction of lamivudine as part of antiretroviral treatment in a patient who had developed lamivudine resistant HBV strains favored the predominance of an HBV isolate with reduced antigenicity. The absence of hepatitis acute exacerbation in this patient may be correlated to the absence of significant variations of the viral load, which was independent of the presence of mutations in the HBV DNA polymerase.

This study presented for the first time the development and validation of a sensitive method for quantification of dopamine, noradrenaline, and adrenaline in Krebs-Henseleit solution by LC-tandem mass spectrometry. Aliquots of 2.0 mL calibrators, quality controls, and samples of Krebs-Henseleit solution incubated with tortoise's aortic ring for 30 min were extracted by solid-phase extraction. Catecholamine separation was achieved on a 100 × 4.6 mm LiChrospher RP-8 column and the quantification was performed by a mass spectrometer equipped with an electrospray interface operating in positive ion mode. The run time was 4 min and the calibration curve was linear over the range of 0.1-20.0 ng/mL. The method was applied to the measurement of basal release of dopamine, noradrenaline, and adrenaline from the tortoise Chelonoidis carbonaria aortae in vitro. One aortic ring (30 mm) per tortoise (n = 5) was incubated for 30 min in a 5 mL organ bath filled with Krebs-Henseleit solution. The method demonstrated sensitivity, precision, and accuracy enough for its application in the measurement of basal release of these catecholamines from C. carbonaria aortic rings in vitro. The mean (standard deviation) concentrations of dopamine, noradrenaline, and adrenaline were 3.48 (2.55) ng/mL, 1.40 (0.57) ng/mL, and 1.87 (1.09) ng/mL, respectively., (© 2020 John Wiley & Sons, Ltd.)

Epigenetic modifications refer to a number of biological processes which alter the structure of chromatin and its transcriptional activity such as DNA methylation and histone post-translational processing. Studies have tried to elucidate how the viral genome and its products are affected by epigenetic modifications imposed by cell machinery and how it affects the ability of the virus to either, replicate and produce a viable progeny or be driven to latency. The purpose of this study was to evaluate epigenetic modifications in PBMCs and CD4+ cells after HIV-1 infection analyzing three approaches: (i) global DNA- methylation; (ii) qPCR array and (iii) western blot. HIV-1 infection led to methylation increases in the cellular DNA regardless the activation status of PBMCs. The analysis of H3K9me3 and H3K27me3 suggested a trend towards transcriptional repression in activated cells after HIV-1 infection. Using a qPCR array, we detected genes related to epigenetic processes highly modulated in activated HIV-1 infected cells. SETDB2 and RSK2 transcripts showed highest up-regulation levels. SETDB2 signaling is related to transcriptional silencing while RSK2 is related to either silencing or activation of gene expression depending on the signaling pathway triggered down-stream. In addition, activated cells infected by HIV-1 showed lower CD69 expression and a decrease of IL-2, IFN-γ and metabolism-related factors transcripts indicating a possible functional consequence towards global transcriptional repression found in HIV-1 infected cells. Conversely, based on epigenetic markers studied here, non-stimulated cells infected by HIV-1, showed signs of global transcriptional activation. Our results suggest that HIV-1 infection exerts epigenetic modulations in activated cells that may lead these cells to transcriptional repression with important functional consequences. Moreover, non-stimulated cells seem to increase gene transcription after HIV-1 infection. Based on these observations, it is possible to speculate that the outcome of viral infections may be influenced by the cellular activation status at the moment of infection.

Recombination is an important way to generate genetic diversity. Accumulation of HIV-1 full-length genomes in databases demonstrated that recombination is pervasive in viral strains collected globally. Recombinant forms achieving epidemiological relevance are termed circulating recombinant forms (CRFs). CRF12_BF was up to now the only CRF described in South America. The objective was to identify the first CRF in Brazil conducting full genome analysis of samples sharing the same partial genome recombinant structure. Ten samples obtained from individuals residing in Santos, Brazil, sharing the same recombination pattern based on partial genome sequence data, were selected from a larger group to undergo full length genome analysis. Near full length genomes were assembled from overlapping fragments. Mosaic genomes were evaluated by Bootscan, alignment inspection, and phylogenetic analysis using neighbor joining and maximum likelihood. Full genomes were also analyzed by split decomposition. We were able to identify five mosaic genomes. Two of these structures were represented by at least three samples derived from epidemiologically unlinked individuals. These structures were named CRF28_BF and CRF29_BF and are the second and third CRFs composed exclusively by subtypes B and F as well as the second and third CRFs encountered in South America. Other recombinant forms studied here resembled CRF28_BF and CRF29_BF. Our results suggest that a diverse population of related recombinants, including CRFs may play an important part in the Brazilian and South American epidemic.

A method based on LC-MS-MS is described for the determination of methyldopa in human plasma using dopa-phenyl-D3 as the internal standard. The method has a chromatographic run time of 5.5 min and was linear in the range of 20-5000 ng/ml. The limit of quantitation was 20 ng/ml, the intra-day precisions were 7.3, 5.4 and 4.3% and the intra-day accuracies were -8.0, -1.3 and -2.0% for 30, 600 and 3000 ng/ml, respectively. The inter-day precisions were 7.7, 0.5 and 0.7% and the inter-day accuracies were 0.2, -1.1 and -2.3%, respectively, for the above concentrations. This method was employed in a bioequivalence study of two tablet formulations of methyldopa.

A method based on liquid chromatography with negative ion electrospray ionization and tandem mass spectrometry is described for the determination of nimesulide in human plasma. Liquid-liquid extraction using a mixture of diethyl ether and dichloromethane was employed and celecoxib was used as an internal standard. The chromatographic run time was 4.5 min and the weighted (1/x) calibration curve was linear in the range 10.0-2000 ng x ml(-1). The limit of quantification was 10 ng x ml(-1), the intra-batch precision was 6.3, 2.1 and 2.1% and the intra-batch accuracy was 3.2, 0.3 and 0.1% for 30, 300 and 1200 ng x ml(-1) respectively. The inter-batch precision was 2.3, 2.8 and 2.7% and the accuracy was 3.3, 0.3 and 0.1% for 30, 300 and 1200 ng x ml(-1) respectively. This method was employed in a bioequivalence study of one nimesulide drop formulation (nimesulide 50 mg x ml(-1) drop, Medley S/A Indústria Farmacêutica, Brazil) against one standard nimesulide drop formulation (Nisulid, 50 mg x ml(-1) drop, Astra Médica, Brazil). Twenty-four healthy volunteers (both sexes) took part in the study and received a single oral dose of nimesulide (100 mg, equivalent to 2 ml of either formulation) in an open, randomized, two-period crossover way, with a 2-week washout interval between periods. The 90% confidence interval (CI) for geometric mean ratios between nimesulide and Nisulid were 93.1-109.6% for C(max), 87.7-99.8% for AUC(last) and 88.1-99.7% for AUC(0-infinity). Since the 90% CI for the above-mentioned parameters were included in the 80-125% interval proposed by the US Food and Drug Administration, the two formulations were considered bioequivalent in terms of both rate and extent of absorption., (Copyright 2001 John Wiley & Sons, Ltd.)

Fifty 3-4-mo-old piglets died of accumulative sodium poisoning, but none of the 60 adult pigs with the same feeding and management did. The average ambient temperature throughout the period was 32 C. The herd had been regulary fed whey, ground corn and vegetables, but for at least 2 d the pigs were deprived of water and then water was offered ad libitum. Twenty hours later 20 piglets had died and the remaining exhibited classical nervous signs and died within 14 h. Only the piglets had a high degree of dehydration caused by water deprivation and exacerbated by the high average ambient temperature. Pulmonary edema was evident in most piglets. Acute cerebral edema and meningoencephalitis were present in all animals, but there was no polioencephalomalacia. The sodium accumulation was caused by the continuing intake of whey which contained 10,810 mg sodium/L. To produce the whey, 2 kg of sodium chloride had been added to every 100 kg of milk.

Oligopeptidases are tissue endopeptidases that do not attack proteins and are likely to be involved in the maturation and degradation of peptide hormones and neuropeptides. The rabbit brain endooligopeptidase A and the rat testes soluble metallopeptidase (EC 3.4.24.15) are thiol-activated oligopeptidases which are able to generate enkephalin from a number of opioid peptides and to inactivate bradykinin and neurotensin by hydrolyzing the same peptide bonds. A monospecific antibody raised against the purified rabbit brain endooligopeptidase A allowed the identification of a 2. 3 kb cDNA coding for a truncated enzyme of 512 amino acids, displaying the same enzymatic features as endooligopeptidase A. In spite of all efforts, employing several strategies, the full-length cDNA could not be cloned until now. The analysis of the deduced amino acid sequence showed no similarity to the rat testes metalloendopeptidase sequence, except for the presence of the typical metalloprotease consensus sequence [HEXXH]. The antibody raised against recombinant endooligopeptidase A specifically inhibited its own activity and reduced the thiol-activated oligopeptidase activity of rabbit brain cytosol to less than 30%. Analysis of the endooligopeptidase A tissue distribution indicated that this enzyme is mainly expressed in the CNS, whereas the soluble metallo EC 3.4.24.15 is mainly expressed in peripheral tissues., (Copyright 2000 Academic Press.)

The effects of the Ca2+ channel blockers diltiazem, nifedipine and amlodipine were investigated on both arterial hypertension and myocardial changes induced by chronic blockade of nitric oxide synthesis. Control male Wistar rats received Nomega-nitro-L-arginine methyl ester (L-NAME; 20 mg rat(-1) day(-1)) in the drinking water for 8 weeks; blood pressure and body weight were monitored weekly. The Ca2+ channel blockers were given concomitantly to L-NAME, as follows: diltiazem (13.5 mg rat(-1) day(-1)) and amlodipine (6.25 mg rat(-1) day(-1)) were administered in the drinking water whereas nifedipine (6.25 mg rat(-1) day(-1)) was given in the chow. Nomega-nitro-L-arginine methyl ester induced a time-dependent increase in blood pressure which was significantly attenuated by diltiazem (154+/-1.6 vs. 139+/-1.6 mm Hg, p < 0.05), nifedipine (166+/-2.7 vs. 150+/-2.1 mm Hg, p < 0.05) and amlodipine (208+/-5.8 vs. 158+/-1.8 mm Hg, p < 0.05) at the last week of the treatment. Rats treated with the L-NAME also developed myocardial ischaemia, as indicated by the increased percentage of fibrous tissue found in the left ventricles of these animals (10.9+/-0.1%, p < 0.01) when compared to control ones (6.3+/-0.1%). Neither diltiazem (14.9+/-1.2%) nor nifedipine (11.1+/-1.5%) prevented this effect whereas amlodipine (6.9+/-1.1%, p < 0.01) virtually abolished the increase in fibrous tissue induced by L-NAME. The plasma concentration of the Ca2+ channel blockers was measured by liquid chromatography coupled to mass spectrometry at two different time points (morning and afternoon). Only amlodipine treatment was able to maintain constant levels (186+/-46 ng ml(-1) in the morning and 110+/-19 ng ml(-1) in the evening) compared to nifedipine (3003+/-578 ng ml(-1) in the morning and 436+/-100 ng ml(-1) in the evening) and diltiazem (77+/-51 ng ml(-1) in the morning and not detectable in the evening). In conclusion, our results indicate that amlodipine (but not diltiazem and nifedipine) can efficiently control myocardial ischaemia in nitric oxide deficient rats, probably due to its intrinsically long half-life.

Objective: To compare the bioavailability of 2 sertraline tablets formulations (Tolrest from Laboratórios Biosintética, and Zoloft from Laboratórios Pfizer, Brazil) in 24 healthy volunteers of both sexes (12 male and 12 female) who received a single 50 mg dose of each sertraline formulation., Material and Methods: The study was conducted open with randomized two-period crossover design and a 14-day washout period. Plasma samples were obtained over a 96-hour interval and sertraline concentrations were analyzed by combined reversed phase liquid chromatography and tandem mass spectrometry (LC-MS-MS) with positive ion electrospray ionization using selected ion monitoring method. From the plasma sertraline concentration vs. time curves the following pharmacokinetic parameters were obtained: AUC(0-96h), AUC(0-infinity), Cmax, Cmax/AUC(0-96h), Tmax, ke, and t(1/2)., Results: Pharmacokinetic parameters presented normal distribution according to Probit' s plot and Kolmogorov Smirnov's test, and the variance of AUC(0-96h), AUC(0-infinity) or Cmax were homoscedastic. Geometric mean Tolrest/Zoloft individual percent ratio was 95.22% for AUC(0-96h), 99.87% for Cmax, 100.4% for AUC(0-infinity), 103.6% for Ke, 96.0% for t(1/2) and 93.7% for Tmax., Conclusion: Since the 90% CI for both Cmax and AUC(0-96h) mean ratio were within the 80-125% interval proposed by the Food and Drug Administration, it was concluded that Tolrest was bioequivalent to Zolof for both extent and rate of absorption in a single dose administration.

The endopeptidase 22.19 (EC 3.4.22.19) has been associated with the metabolism of neuropeptides by its ability to convert small enkephalin-containing peptides (8 to 13 amino acids) into enkephalins. In addition, this enzyme cleaves the Arg8-Arg9 bond of neurotensin and the Phe5-Ser6 bond of bradykinin. We analyzed the circadian variation of endopeptidase 22.19 in the whole and individual areas of the rat brain. Endopeptidase 22.19 activity was analyzed by high-performance liquid chromatography (HPLC) using bradykinin as an operative substrate. Enzymatic specific activities were analyzed by rhythmometric methods and indicate a circadian fluctuation of endopeptidase 22.19 specific activity (mU of enzyme/mg of protein) in the whole brain [p less than 0.001, mesor (M) = 7.62, amplitude (A) = 2.89, and acrophase (phi) = 23:08 h], striatum (p less than 0.001, M = 2.92, A = 0.62, phi = 23:03 h), hypothalamus (p less than 0.001, M = 3.15, A = 0.86, phi = 01:12 h), periaqueductal gray matter (p less than 0.005, M = 2.62, A = 0.34, phi = 22:35 h), and cerebellum (p less than 0.014, M = 4.27, A = 0.88, phi = 17:12 h). The circadian rhythmicity in endopeptidase 22.19 specific activity suggests that light may have an effect on the peptidase activity in whole brain and in areas of the central nervous system and may be essential for the mechanisms of circadian fluctuations of neuropeptides in the brain.

A novel quenched fluorescence substrate, QF-ERP7 (Abz-G-G-F-L-R-R-V-EDDn), structurally related to enkephalins, proved to be suitable for assaying the endooligopeptidase A (E.C.3.4.22.19) activity. The enzyme only splits the L-R bond (Km 1.75 microM, Kcat 8.25 s-1), a reaction efficiently blocked by anti-endooligopeptidase A antibodies and by inhibitor and alternative substrates of the enzyme. Evidences based on the action of inhibitors and on the analysis of QF-ERP7 fragments demonstrated that endooligopeptidase A contributes with 100% of the QF-ERP7 cleaving activity found in the cytosol of rabbit brain homogenates and with 85% of that recovered in the membrane fraction. Homologous substrates, Abz-G-G-F-L-R-R-EDDn and Abz-G-G-F-L-R-EDDn, were resistant to hydrolysis. The convenience and sensitivity of the fluorimetric assay based on the QF-ERP7 moiety offers several advantages compared with previously described painstaking procedures for endooligopeptidase A activity measurements, what will certainly contribute to further our understanding of the role of this enzyme on the peptide hormone metabolism.