A simple method for the spectrophotometric determination of cephalosporins in pharmaceuticals using variamine blue

A simple spectrophotometric method for the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin with variamine blue is presented. The determination is based on the hydrolysis of β-lactam ring of cephalosporins with sodium hydroxide which subsequently reacts with iodate to liberate iodine in acidic medium. The liberated iodine oxidizes variamine blue to violet colored species of maximum absorption at 556 nm. The absorbance is measured within the pH range of 4.0-4.2. Beer's law is obeyed in the range of 0.5-5.8 µg mL-1, 0.2-7.0 µg mL-1, 0.2-5.0 µg mL-1 and 0.5-8.5 µg mL-1 for cefotaxime, ceftriaxone, cefadroxil and cephalexin respectively. The analytical parameters were optimized and the method is successfully applied for the determination of cefotaxime, ceftriaxone, cefadroxil and cephalexin in pharmaceuticals.

Sensitive spectrophotometric determination of lamotrigine in bulk drug and pharmaceutical formulations using bromocresol green

Two new, simple, rapid and reproducible spectrophotometric methods have been developed for the determination of lamotrigine (LMT) both in pure form and in its tablets. The first method (method A) is based on the formation of a colored ion-pair complex (1:1 drug/dye) of LMT with bromocresol green (BCG) at pH 5.02±0.01 and extraction of the complex into dichloromethane followed by the measurement of the yellow ion-pair complex at 410 nm. In the second (method B), the drug-dye ion-pair complex was dissolved in ethanolic potassium hydroxide and the resulting base form of the dye was measured at 620 nm. Beer's law was obeyed in the concentration range of 1.5-15 µg mL-1 and 0.5-5.0 µg mL-1 for method A and method B, respectively, and the corresponding molar absorptivity values are 1.6932 x 10(4) and 3.748 x 10(4) L mol-1cm-1. The Sandell sensitivity values are 0.0151 and 0.0068 µg cm-2 for method A and method B, respectively. The stoichiometry of the ion-pair complex formed between the dug and dye (1:1) was determined by Job's continuous variations method and the stability constant of the complex was also calculated. The proposed methods were applied successfully for the determination of drug in commercial tablets.

Development of a molecular method for detection and identification of Xanthomonas campestris pv. viticola

In order to develop a molecular method for detection and identification of Xanthomonas campestris pv. viticola (Xcv) the causal agent of grapevine bacterial canker, primers were designed based on the partial sequence of the hrpB gene. Primer pairs Xcv1F/Xcv3R and RST2/Xcv3R, which amplified 243- and 340-bp fragments, respectively, were tested for specificity and sensitivity in detecting DNA from Xcv. Amplification was positive with DNA from 44 Xcv strains and with DNA from four strains of X. campestris pv. mangiferaeindicae and five strains of X. axonopodis pv. passiflorae, with both primer pairs. However, the enzymatic digestion of PCR products could differentiate Xcv strains from the others. None of the primer pairs amplified DNA from grapevine, from 20 strains of nonpathogenic bacteria from grape leaves and 10 strains from six representative genera of plant pathogenic bacteria. Sensitivity of primers Xcv1F/Xcv3R and RST2/Xcv3R was 10 pg and 1 pg of purified Xcv DNA, respectively. Detection limit of primers RST2/Xcv3R was 10(4) CFU/ml, but this limit could be lowered to 10² CFU/ml with a second round of amplification using the internal primer Xcv1F. Presence of Xcv in tissues of grapevine petioles previously inoculated with Xcv could not be detected by PCR using macerated extract added directly in the reaction. However, amplification was positive with the introduction of an agar plating step prior to PCR. Xcv could be detected in 1 µl of the plate wash and from a cell suspension obtained from a single colony. Bacterium identity was confirmed by RFLP analysis of the RST2/Xcv3R amplification products digested with Hae III.

Learning through role-playing games: an approach for active learning and teaching

This study evaluates the use of role-playing games (RPGs) as a methodological approach for teaching cellular biology, assessing student satisfaction, learning outcomes, and retention of acquired knowledge. First-year undergraduate medical students at two Brazilian public universities attended either an RPG-based class (RPG group) or a lecture (lecture-based group) on topics related to cellular biology. Pre- and post-RPG-based class questionnaires were compared to scores in regular exams and in an unannounced test one year later to assess students' attitudes and learning. From the 230 students that attended the RPG classes, 78.4% responded that the RPG-based classes were an effective tool for learning; 55.4% thought that such classes were better than lectures but did not replace them; and 81% responded that they would use this method. The lecture-based group achieved a higher grade in 1 of 14 regular exam questions. In the medium-term evaluation (one year later), the RPG group scored higher in 2 of 12 questions. RPG classes are thus quantitatively as effective as formal lectures, are well accepted by students, and may serve as educational tools, giving students the chance to learn actively and potentially retain the acquired knowledge more efficiently.

Quali/quantitative characterization of biogas produced in batch digesters supplied with six distinct substrates

The present research was carried out at the DER/FCAV, São Paulo State University - Brazil, with the purpose of analyzing the quantity and quality of biogas produced by six types of substrates. The substrates used in the anaerobic digestion were characterized as: 1- Litter of broiler (LB); 2- Grinded broiler litter (GBL); 3- Broiler litter with wood shavings (BLWS); 4- Grinded broiler litter with wood shavings (GBLWS); 5- Broiler litter with peanut hulls (BLPH); 6- Grinded broiler litter with peanut hull (GBLPH). It was concluded from the collected data that: the (GBL) substrate showed a superior biogas accumulated production in relation to the other substrates, while the BLWS presented an inferior accumulated production; the grinded substrates showed higher quantities of accumulated biogas in relation to the non-grinded substrates, except for GBLPH, with 20.9 m³ inferior than BLPH; the period of maximum biogas production started from 45 to 60 days, declining after 120 days; at 57 days after filling up the digesters the biogas produced had levels of CH4 superior than 53%, and from 99 days all of them produced biogas with levels of CH4 superior than 70%; LB and GBL presented higher concentrations of CH4 in the biogas.

Performance of UASB reactors in two stages under different HRT and OLR treating residual waters of swine farming

In this study it was evaluated the effects of hydraulic retention time (HRT) and Organic Loading Rate (OLR) on the performance of UASB (Upflow Anaerobic Sludge Blanket) reactors in two stages treating residual waters of swine farming. The system consisted of two UASB reactors in pilot scale, installed in series, with volumes of 908 and 188 L, for the first and second stages (R1 and R2), respectively. The HRT applied in the system of anaerobic treatment in two stages (R1 + R2) was of 19.3, 29.0 and 57.9 h. The OLR applied in the R1 ranged from 5.5 to 40.1 kg CODtotal (m³ d)-1. The average removal efficiencies of chemical oxygen demand (COD) and total suspended solids (TSS) ranged, respectively, from 66.3 to 88.2% and 62.5 to 89.3% in the R1, and from 85.5 to 95.5% and 76.4 to 96.1% in the system (R1 + R2). The volumetric production of methane in the system (R1 + R2) ranged from 0.295 to 0.721 m³CH4 (m³ reactor d)-1. It was found that the OLR applied were not limiting to obtain high efficiencies of CODtotal and TSS removal and methane production. The inclusion of the UASB reactor in the second stage contributed to increase the efficiencies of CODtotal and TSS removal, especially, when the treatment system was submitted to the lowest HRT and the highest OLR.

Performance of UASB reactors in two stages followed by post-treatment with activated sludge in wastewater batch of wet-processed coffee

In this study it was evaluated the efficiency of the treatment of wet-processed coffee wastewater in upflow anaerobic sludge blanket (UASB) reactors in two stages, in bench scale, followed by post-treatment with activated sludge in batch. The first UASB reactor was submitted to an hydraulic retention time (HRT) of 6.2 d and organic loading rates (OLR) of 2.3 and 4.5g CODtotal (L d)-1, and the second UASB reactor to HRT of 3.1 d with OLR of 0.4 and 1.4g CODtotal (L d)-1. The average values of the affluent CODtotal increased from 13,891 to 27,926mg L-1 and the average efficiencies of removal of the CODtotal decreased from 95 to 91%, respectively, in the UASB reactors in two stages. The volumetric methane production increased from 0.274 to 0.323L CH4 (L reactor d)-1 with increment in the OLR. The average concentrations of total phenols in the affluent were of 48 and 163mg L-1, and the removal efficiencies in the UASB reactors in two stages of 92 and 90%, respectively, and increased to 97% with post-treatment. The average values of the removal efficiencies of total nitrogen and phosphorus were of 57 to 80% and 44 to 60%, respectively, in the UASB reactors in two stages and increased to 91 and 84% with the post-treatment.

Potential of biogas production from swine manure supplemented with glycerine waste

In this study, was studied the biogas generation from swine manure, using residual glycerine supplementation. The biogas production by digestion occurred in the anaerobic batch system under mesophilic conditions (35°C), with a hydraulic retention time of 48 days. The experiment was performed with 48 samples divided into four groups, from these, one was kept as a control (without glycerin) and the other three groups were respectively supplemented with residual glycerine in the percentage of 3%, 6% and 9% of the total volume of the samples. The volume of biogas was controlled by an automated system for reading in laboratory scale and the quality of the biogas (CH4) measured from a specific sensor. The results showed that the residual glycerine has high potential for biogas production, with increases of 124.95%, 156.98% and 197.83% in the groups 3%, 6% and 9%, respectively, relative to the sample control. However, very high organic loads can compromise the process of digestion affecting the quality of the biogas generated in relation to methane.

Cassava starch extraction effluent treatment in a one phase tubular horizontal pilot reactor with support medium

The cassava starch industries generate a large volume of wastewater effluent that, stabilized in ponds, wastes its biogas energy and pollutes the atmosphere. To contribute with the reversion of this reality, this manipueira treatment research was developed in one phase anaerobic horizontal pilot reactor with support medium in bamboo pieces. The reactor was excavated into the ground and sealed with geomembrane in HDPE, having a volume equal to 33.6 m³ and continuous feeding by gravity. The stability indicators were pH, volatile acidity/total alkalinity ratio and biogas production. The statistical analyses were performed by a completely randomized design, with answers submitted to multivariate analysis. The organical loads in COD were 0.556; 0.670; 0.678 and 0.770 g L-1 and in volatile solids (VS) of 0.659; 0.608; 0.570 and 0.761 g L-1 for the hydraulic retention times (HRT) of 13.0; 11.5; 10.0 and 7.0 days, respectively. The reductions in COD were 88; 80; 88 and 67% and for VS of 76; 77; 65 and 61%. The biogas productions relatively to the consumed COD were 0.368; 0.795; 0.891 and 0.907 Lg-1, for the consumed VS of 0.524; 0.930; 1.757 and 0.952 Lg-1 and volumetric of 0.131; 0.330; 0.430 and 0.374 L L-1 d-1. The reactor remained stable and the bamboo pieces, in visual examination at the end of the experiment, showed to be in good physical conditions.

A rapid and sensitive diagnosis of bovine leukaemia virus infection using the nested shuttle polymerase chain reaction

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). In Argentina, where a program to eradicate EBL has been introduced, sensitive and reliable diagnosis has attained high priority. Although the importance of the agar gel immunodiffusion test remains unchanged for routine work, an additional diagnostic technique is necessary to confirm cases of sera with equivocal results or of calves carrying maternal antibodies.Utilizing a nested shuttle polymerase chain reaction, the proviral DNA was detected from cows experimentally infected with as little as 5 ml of whole blood from BLV seropositive cows that were nonetheless normal in haematological terms. It proved to be a very sensitive technique, since it rapidly revealed the presence of the provirus, frequently at 2 weeks postinoculation and using a two-round procedure of nested PCR taking only 3 hours. Additionally, the primers used flanked a portion of the viral genome often employed to differentiate BLV type applying BamHI digestion. It is concluded that this method might offer a highly promising diagnostic tool for BLV infection.

Comparative pathogenicity of bovine herpesvirus 1 (BHV-1) subtypes 1 (BHV-1.1) and 2a (BHV-1.2a)

The study aimed to examine the capacity of two bovine herpesvirus type 1 (BHV-1) isolates of different subtypes (EVI 123/96, BHV-1.1; SV265/98, BHV-1.2a) to induce respiratory disease in calves. These two isolates are representative of the BHV-1 subtypes prevalent in Brazil. Viral subtypes were confirmed by monoclonal antibody analysis and by restriction enzyme digestion of viral genomes. The viruses were inoculated intranasally into seven 3 months old calves (four with BHV-1.1, three with BHV-1.2a). Three other calves of identical age and condition were kept as uninfected controls. In both groups of infected calves, the clinical signs observed were consistent with typical infectious bovine rhinothracheitis (IBR), including pyrexia, apathy, anorexia, nasal and ocular mucopurulent discharges, erosions on the nasal mucosa, conjunctivitis, lachrymation, redness of nasal mucosa, dyspnoea, coughing, tracheal stridor and enlargement of retropharingeal, submandibular and cervical lymphnodes. No significant differences were observed between the clinical scores attributed to both groups. Virus shedding in nasal and ocular secretions were also similar, apart from a significant difference in nasal virus shedding on day 1 to 3 post-inoculation, which was higher for BHV-1.1 than for BHV-1.2a. Following corticosteroid induced reactivation of the latent infection, recrudescence of clinical signs was also observed, with no significant differences on both groups. It was concluded that both subtypes BHV-1.1 and BHV-1.2a were able to induce clinically undistinguishable respiratory disease in calves, either subsequent to a primary infection or following reactivation.

The effect of timing temporary cements to treat induced pulp necrosis in the teeth of dogs

During endodontic therapy (pulpectomy, root canal debridement and root canal filling) microbiological management is a major concern. Bacteria present in dentine tubules, apical foramina and apical delta are causally related to failure of the procedure. Studies have shown that during single session endodontic treatment bacteria remain within dental structures. The aim of the present study was to evaluate endodontic treatment performed as two sessions, using temporary endodontic dressing materials for different periods in four groups of experimental dogs. A total of 80 roots of second and third upper premolar teeth and second, third and fourth lower premolar teeth were divided into four groups. The pulp chamber was opened with burrs and the pulp exposed for 60 days to induce pulpal inflammation and necrosis. Groups II, III and IV were treated with calcium hydroxide plus camphorated paramono-chlorophenol (PMCC) for 7, 15 and 30 days, respectively. In all groups, the root canals were filled with zinc oxide-eugenol and gutta-percha cones. Clinical and radiographical measurements were performed every 2 weeks. After 60 days a small block section containing the teeth, surrounding periapical tissues and the periodontium was removed for histological and microbiological study. Histological analysis revealed intense inflammatory response in all groups. Microbiological analysis showed microbial reduction inversely proportional to the period of time that the intracanal temporary medicament was left in place.

Diagnose of the ingestion of Asclepias mellodora St. Hil. by sheep through microhistological analysis of their digestive contents

Asclepias mellodora St. Hil. is a native acute toxic species frequent in the grasslands of the Buenos Aires province, Argentina, whose toxicity had not been assessed until now. This study evaluates the minimal lethal dose of this species for sheep, and the possibility of microscopically recognizing its fragments in gastrointestinal contents as a complementary diagnostic tool in necropsies. Three Frisona sheep (average LW=55±4.5 kg) were dosed via an esophageal tube with each one of the following doses of asclepias: 8.0, 5.0, 2.0 and 0.8 g DM.kg LW-1. Sheep poisoned with the three higher doses died between 10 and 85 h after intoxication, but those receiving the lower dose did not. During necropsies we: 1) determined the dry weight of the contents of rumen+reticulum, omasum+abomasum, and large intestine, 2) estimated the percentages of asclepias fragments by microanalysis correcting for digestion effects on fragment recognition, and 3) calculated the total mass of asclepias in the digestive tract of each animal. For the three higher doses, the mass of asclepias identified in the total ingesta was 12.3±3.4% of the amount supplied, possibly because of the strong diarrhea its ingestion produced. The percentages of asclepias in rumen+reticulum did not differ from the average quantified for the entire tract. The results of this study indicate that the minimal lethal doses of asclepias for sheep is between 2.0 and 0.8g DM·kg LW-1, and that the microhistological analysis of the rumen+reticulum, the easiest region to sample, can be used to confirm the ingestion of this toxic species, although the estimated percentage will be not a good estimator of the ingested percentage.

Vitamin D receptor alleles and bone mineral density in a normal premenopausal Brazilian female population

Studies on the association between vitamin D receptor (VDR) polymorphism and bone mineral density (BMD) in different populations have produced conflicting results probably due to ethnic differences in the populations studied. The Brazilian population is characterized by a very broad genetic background and a high degree of miscegenation. Of an initial group of 164, we studied 127 women from the city of São Paulo, aged 20 to 47 years (median, 31 years), with normal menses, a normal diet and no history of diseases or use of any medication that could alter BMD. VDR genotype was assessed by PCR amplification followed by BsmI digestion of DNA isolated from peripheral leukocytes. BMD was measured using dual energy X-ray absorptiometry (Lunar DPX) at the lumbar site (L2-L4) and femoral neck. Most of the women (77.6%) were considered to be of predominantly European ancestry (20.6% of them reported also native American ancestry), 12.8% were of African-Brazilian ancestry and 9.6% of Asian ancestry, 41.0% (52) were classified as bb, 48.8% (62) as Bb and 10.2% (13) as BB. The BB, Bb and bb groups did not differ in age, height, weight, body mass index or age at menarche. Lumbar spine BMD was significantly higher in the bb group (1.22 ± 0.16 g/cm²) than in the BB group (1.08 ± 0.14; P<0.05), and the Bb group presented an intermediate value (1.17 ± 0.15). Femoral neck BMD was higher in the bb group (0.99 ± 0.11 g/cm²) compared to Bb (0.93 ± 0.12) and BB (0.90 ± 0.09) (P<0.05). These data indicate that there is a significant correlation between the VDR BsmI genotype and BMD in healthy Brazilian premenopausal females.

Cloning, expression and purification of recombinant streptokinase: partial characterization of the protein expressed in Escherichia coli

We cloned the streptokinase (STK) gene of Streptococcus equisimilis in an expression vector of Escherichia coli to overexpress the profibrinolytic protein under the control of a tac promoter. Almost all the recombinant STK was exported to the periplasmic space and recovered after gentle lysozyme digestion of induced cells. The periplasmic fraction was chromatographed on DEAE Sepharose followed by chromatography on phenyl-agarose. Active proteins eluted between 4.5 and 0% ammonium sulfate, when a linear gradient was applied. Three major STK derivatives of 47.5 kDa, 45 kDa and 32 kDa were detected by Western blot analysis with a polyclonal antibody. The 32-kDa protein formed a complex with human plasminogen but did not exhibit Glu-plasminogen activator activity, as revealed by a zymographic assay, whereas the 45-kDa protein showed a Km = 0.70 µM and kcat = 0.82 s-1, when assayed with a chromogen-coupled substrate. These results suggest that these proteins are putative fragments of STK, possibly derived from partial degradation during the export pathway or the purification steps. The 47.5-kDa band corresponded to the native STK, as revealed by peptide sequencing