A simple HPLC-fluorescence method for the measurement of R,S-sotalol in the plasma of patients with life-threatening cardiac arrhythmias

R,S-sotalol, a ß-blocker drug with class III antiarrhythmic properties, is prescribed to patients with ventricular, atrial and supraventricular arrhythmias. A simple and sensitive method based on HPLC-fluorescence is described for the quantification of R,S-sotalol racemate in 500 µl of plasma. R,S-sotalol and its internal standard (atenolol) were eluted after 5.9 and 8.5 min, respectively, from a 4-micron C18 reverse-phase column using a mobile phase consisting of 80 mM KH2PO4, pH 4.6, and acetonitrile (95:5, v/v) at a flow rate of 0.5 ml/min with detection at lex = 235 nm and lem = 310 nm, respectively. This method, validated on the basis of R,S-sotalol measurements in spiked blank plasma, presented 20 ng/ml sensitivity, 20-10,000 ng/ml linearity, and 2.9 and 4.8% intra- and interassay precision, respectively. Plasma sotalol concentrations were determined by applying this method to investigate five high-risk patients with atrial fibrillation admitted to the Emergency Service of the Medical School Hospital, who received sotalol, 160 mg po, as loading dose. Blood samples were collected from a peripheral vein at zero, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 and 24.0 h after drug administration. A two-compartment open model was applied. Data obtained, expressed as mean, were: CMAX = 1230 ng/ml, TMAX = 1.8 h, AUCT = 10645 ng h-1 ml-1, Kab = 1.23 h-1, a = 0.95 h-1, ß = 0.09 h-1, t(1/2)ß = 7.8 h, ClT/F = 3.94 ml min-1 kg-1, and Vd/F = 2.53 l/kg. A good systemic availability and a fast absorption were obtained. Drug distribution was reduced to the same extent in terms of total body clearance when patients and healthy volunteers were compared, and consequently elimination half-life remained unchanged. Thus, the method described in the present study is useful for therapeutic drug monitoring purposes, pharmacokinetic investigation and pharmacokinetic-pharmacodynamic sotalol studies in patients with tachyarrhythmias.

The use of wide-mesh gauze impregnated with lambda-cyhalothrin covering wall openings in huts as a vector control method in Suriname

An alternative vector control method, using lambda-cyhalothrin impregnated wide-mesh gauze covering openings in the walls of the houses was developed in an area in the Eastern part of the interior of Suriname. Experimental hut observations showed that Anopheles darlingi greatly reduced their biting activity (99-100%) during the first 5 months after impregnation. A model assay showed high mortality both of mosquitoes repelled by the gauze as well as of those that succeeded in getting through it. A field application test in 270 huts showed good acceptance by the population and good durability of the applied gauze. After introducing the method in the entire working area, replacing DDT residual housespraying, the malaria prevalence, of 25-37% before application dropped and stabilized at between 5 and 10% within one year. The operational costs were less than those of the previously used DDT housespraying program, due to a 50% reduction in the cost of materials used. The method using widemesh gauze impregnated with lambdacyhalothrin strongly affects the behavior of An. darlingi. It is important to examine the effect of the method on malaria transmission further, since data indirectly obtained suggest substantial positive results.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

Molecular and cellular basis of hepatic fibrogenesis in experimental schistosomiasis mansoni infection

Morbidity in schistosomiasis mansoni occurs primaryly as a result of the complications of hepatic fibrosis. Yet, the pathogenesis of schistosomal hepatic fibrosis is poorly understood. The fact that hepatic egg granuloma is the hallmark of this infection suggests a potential role for granulomatous inflamation in hepatic fibrogenesis. Our studies in a murine schistosomiasis model indicate that hepatic granuloma cells secrete a variety of fibrogenic cytokines that may initiate the scarring process. Among these cytokines, we identified a novel protein that we designated fibroplast stimulating factor-1 (FsF-1). FsF-1 is a lymphokine that can stimulate fibroplast growth and matrix synthesis. A notable feature of hepatic fibrosis in this model is that production of FsF-1 and other granuloma-derived fibrogenic cytokines is down-regulated in chronic infection, an event that may be under immunological control. The spontaneous reduction of FsF-1 secretion presumably accounts for reduced scar formation late in infection of mice. In the context of relevant clinical studies, our findings engender the hypothesis that Symmer's fibrosis may develop in a small suppopulation of individuals as a result of immunogenetically-determined dysregulation of fibrogenic cytokine production.

Application of synthetic peptides in development of a serologic method for laboratory diagnosis of schistosomiasis mansoni

The immunoreactivity of seven peptides synthesized from Schistosoma mansoni proteins, was evaluated by dot-blot and ELISA assays using two different sensitization methodologies. The best results were obtained on wells of the Costar 3590 microplates coated with peptides P1, P2, P3, P6, and P7 using conventional methodology. The signals increased considerably (p < 0.0003) on wells sensitized with P1 to P6 using alternative methodology. In contrast, the well coated with peptide P7 presented lower signal when compared with conventional methodology (p = 0.0019). These results, establish the basis for the application of synthetic peptides for laboratory diagnosis of schistosomiasis mansoni.

Method to estimate soil macroporosity and microporosity based on sand content and bulk density

Macroporosity is often used in the determination of soil compaction. Reduced macroporosity can lead to poor drainage, low root aeration and soil degradation. The aim of this study was to develop and test different models to estimate macro and microporosity efficiently, using multiple regression. Ten soils were selected within a large range of textures: sand (Sa) 0.07-0.84; silt 0.03-0.24; clay 0.13-0.78 kg kg-1 and subjected to three compaction levels (three bulk densities, BD). Two models with similar accuracy were selected, with a mean error of about 0.02 m³ m-3 (2 %). The model y = a + b.BD + c.Sa, named model 2, was selected for its simplicity to estimate Macro (Ma), Micro (Mi) or total porosity (TP): Ma = 0.693 - 0.465 BD + 0.212 Sa; Mi = 0.337 + 0.120 BD - 0.294 Sa; TP = 1.030 - 0.345 BD 0.082 Sa; porosity values were expressed in m³ m-3; BD in kg dm-3; and Sa in kg kg-1. The model was tested with 76 datum set of several other authors. An error of about 0.04 m³ m-3 (4 %) was observed. Simulations of variations in BD as a function of Sa are presented for Ma = 0 and Ma = 0.10 (10 %). The macroporosity equation was remodeled to obtain other compaction indexes: a) to simulate maximum bulk density (MBD) as a function of Sa (Equation 11), in agreement with literature data; b) to simulate relative bulk density (RBD) as a function of BD and Sa (Equation 13); c) another model to simulate RBD as a function of Ma and Sa (Equation 16), confirming the independence of this variable in relation to Sa for a fixed value of macroporosity and, also, proving the hypothesis of Hakansson & Lipiec that RBD = 0.87 corresponds approximately to 10 % macroporosity (Ma = 0.10 m³ m-3).

Genetic diversity between improved banana diploids using canonical variables and the Ward-MLM method

The objective of this work was to estimate the genetic diversity of improved banana diploids using data from quantitative analysis and from simple sequence repeats (SSR) marker, simultaneously. The experiment was carried out with 33 diploids, in an augmented block design with 30 regular treatments and three common ones. Eighteen agronomic characteristics and 20 SSR primers were used. The agronomic characteristics and the SSR were analyzed simultaneously by the Ward-MLM, cluster, and IML procedures. The Ward clustering method considered the combined matrix obtained by the Gower algorithm. The Ward-MLM procedure identified three ideal groups (G1, G2, and G3) based on pseudo-F and pseudo-t² statistics. The dendrogram showed relative similarity between the G1 genotypes, justified by genealogy. In G2, 'Calcutta 4' appears in 62% of the genealogies. Similar behavior was observed in G3, in which the 028003-01 diploid is the male parent of the 086079-10 and 042079-06 genotypes. The method with canonical variables had greater discriminatory power than Ward-MLM. Although reduced, the genetic variability available is sufficient to be used in the development of new hybrids.

Indirect spectrophotometric method for determination of captopril using Cr(VI) and diphenylcarbazide

A spectrophotometric method for the indirect determination of captopril (CP) in pharmaceutical formulations is proposed. The proposed procedure is based on the oxidation of captopril by potassium dichromate and the determination excess oxidant on the basis of its reaction with diphenylcarbazide (DPC). Under the optimum conditions, a good linear relationship (r = 0.9997) was obtained in the range of 0.08-3.5 µg mL-1. The assay limits of detection and quantitation were 0.024 and 0.08 µg mL-1, respectively. The results obtained for captopril determination in pharmaceuticals using the proposed method and those obtained with the US Pharmacopoeia method were in good agreement at the 95% confidence level.

Genetic transformation of Eucalyptus camaldulensis by agrobalistic method

Eucalyptus stands in the setting of worldwide forestry due to its adaptability, rapid growth, production of high-quality and low cost of wood pulp fibers. The eucalyptus convetional breeding is impaired mainlly by the long life cycle making the genetic transformation systems an important tool for this purpose. However, this system requires in vitro eficient protocols for plant induction, regeneration and seletion, that allow to obtain transgenic plants from the transformed cell groups. The aim of this work was to evaluate the callus formation and to optimize the leaves and callus genetic transformation protocol by using the Agrobacterium tumefaciens system. Concerning callus formation, two different culture media were evaluated: MS medium supplemented with auxin, cytokinin (M1) and the MS medium with reduced nitrogen concentration and supplemented with auxin, cytokinin coconut water (M2). To establish the leave genetic transformation, those were exposed to agrobiolistics technique (gene gun), to tissue injury, and A. tumesfasciens EHA 105 contening the vetor pCambia 3301 (35S::GUS::NOS), for gene transference and to establish the callus transformation thoses were exposed only to A. tumefasciens. For both experiments, the influence of different infection periods was evaluated. The M2 medium provided the best values for callus sizea and fresh and dry weight. The leaves genetic transformation using the agrobiolistics technique was effective, the gus gene transient expression could be observed. No significant differences were obtained in the infection periods (4, 6 and 8 minutes). The callus genetic transformation with A. tumefaciens also promotend the gus gene transient expression on the callus co-cultiveted for 15 e 30 minutes. The transformed callus was transfered to a regeneration and selection medium and transformed plants were obtained.

Changes in motor behavior during pregnancy in rats: the basis for a possible animal model of restless legs syndrome

PURPOSE:Pregnant women have a 2-3 fold higher probability of developing restless legs syndrome (RLS – sleep-related movement disorders) than general population. This study aims to evaluate the behavior and locomotion of rats during pregnancy in order to verify if part of these animals exhibit some RLS-like features.METHODS:We used 14 female 80-day-old Wistar rats that weighed between 200 and 250 g. The rats were distributed into control (CTRL) and pregnant (PN) groups. After a baseline evaluation of their behavior and locomotor activity in an open-field environment, the PN group was inducted into pregnancy, and their behavior and locomotor activity were evaluated on days 3, 10 and 19 of pregnancy and in the post-lactation period in parallel with the CTRL group. The serum iron and transferrin levels in the CTRL and PN groups were analyzed in blood collected after euthanasia by decapitation.RESULTS:There were no significant differences in the total ambulation, grooming events, fecal boli or urine pools between the CTRL and PN groups. However, the PN group exhibited fewer rearing events, increased grooming time and reduced immobilization time than the CTRL group (ANOVA, p<0.05).CONCLUSION:These results suggest that pregnant rats show behavioral and locomotor alterations similar to those observed in animal models of RLS, demonstrating to be a possible animal model of this sleep disorder.

Algorithm to determine the intersection curves between bezier surfaces by the solution of multivariable polynomial system and the differential marching method

The determination of the intersection curve between Bézier Surfaces may be seen as the composition of two separated problems: determining initial points and tracing the intersection curve from these points. The Bézier Surface is represented by a parametric function (polynomial with two variables) that maps a point in the tridimensional space from the bidimensional parametric space. In this article, it is proposed an algorithm to determine the initial points of the intersection curve of Bézier Surfaces, based on the solution of polynomial systems with the Projected Polyhedral Method, followed by a method for tracing the intersection curves (Marching Method with differential equations). In order to allow the use of the Projected Polyhedral Method, the equations of the system must be represented in terms of the Bernstein basis, and towards this goal it is proposed a robust and reliable algorithm to exactly transform a multivariable polynomial in terms of power basis to a polynomial written in terms of Bernstein basis .

Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin

In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 ± 0.19 and Dex = 0.35 ± 0.13 vs saline (S) = 2.85 ± 0.59; fMLP: Ptx = 0.43 ± 0.09 and Dex 0.01 ± 0.01 vs S = 1.08 ± 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 ± 1.4 and Dex = 3.06 ± 0.76 vs S = 15.94 ± 3.97; fMLP: Ptx = 3.85 ± 0.56 and Dex = 0.40 ± 0.16 vs S = 7.15 ± 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 µm), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 ± 0.38 vs S = 4.20 ± 1.01; fMLP: Dex = 0.25 ± 0.11 vs S = 2.20 ± 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 ± 2.25 vs S = 4.20 ± 1.01; fMLP: Ptx = 4.66 ± 1.24 vs S = 2.20 ± 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.

Determination of serum aluminum, platelet aggregation and lipid peroxidation in hemodialyzed patients

Aluminum (Al3+) overload is frequently associated with lipid peroxidation and neurological disorders. Aluminum accumulation is also reported to be related to renal impairment, anemia and other clinical complications in hemodialysis patients. The aim of the present study was to determine the degree of lipid peroxidation, platelet aggregation and serum aluminum in patients receiving regular hemodialytic treatment. The level of plasma lipid peroxidation was evaluated on the basis of thiobarbituric acid reactive substances (TBARS). Mean platelet peroxidation in patients undergoing hemodialysis was significantly higher than in normal controls (2.7 ± 0.03 vs 1.8 ± 0.06 nmol/l, P<0.05). Platelet aggregation and serum aluminum levels were determined by a turbidimetric method and atomic absorption spectrophotometry, respectively. Serum aluminum was significantly higher in patients than in normal controls (44.5 ± 29 vs 10.8 ± 2.5 µg/l, P<0.05). Human blood platelets were stimulated with collagen (2.2 µg/ml), adenosine diphosphate (6 µM) and epinephrine (6 µM) and showed reduced function with the three agonists utilized. No correlation between aluminum levels and platelet aggregation or between aluminum and peroxidation was observed in hemodialyzed patients.

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

A low-protein diet leads to functional and structural pancreatic islet alterations, including islet hypotrophy. Insulin-signaling pathways are involved in several adaptive responses by pancreatic islets. We determined the levels of some insulin-signaling proteins related to pancreatic islet function and growth in malnourished rats. Adult male Wistar rats (N = 20 per group) were fed a 17% protein (normal-protein diet; NP) or 6% protein (low-protein diet; LP), for 8 weeks. At the end of this period, blood glucose and serum insulin and albumin levels were measured. The morphometric parameters of the endocrine pancreas and the content of some proteins in islet lysates were determined. The β-cell mass was significantly reduced (≅65%) in normoglycemic but hypoinsulinemic LP rats compared to NP rats. Associated with these alterations, a significant 30% reduction in insulin receptor substrate-1 and a 70% increase in insulin receptor substrate-2 protein content were observed in LP islets compared to NP islets. The phosphorylated serine-threonine protein kinase (pAkt)/Akt protein ratio was similar in LP and NP islets. The phosphorylated forkhead-O1 (pFoxO1)/FoxO1 protein ratio was decreased by 43% in LP islets compared to NP islets (P < 0.05). Finally, the ratio of phosphorylated-extracellular signal-related kinase 1/2 (pErk1/2) to total Erk1/2 protein levels was decreased by 71% in LP islets compared to NP islets (P < 0.05). Therefore, the reduced β-cell mass observed in LP rats is associated with the reduction of phosphorylation in mitogenic-related signals, FoxO1 and Erk proteins. The cause/effect basis of this association remains to be determined.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.