Preliminary comparison of three lightcycler pcr assays for the detection of herpes simplex virus in swab specimens

Three Herpes Simplex Virus LightCycler polymerase chain reaction assays were compared for the detection of herpes simplex virus in 48 swab specimens. The assays comprised of one in-house assay and two commercial kits: the Artus HSV LC RealArt PCR kit and the Roche LightCycler HSV 1/2 Detection kit. On the whole, the three assays had comparable sensitivities. However, differentiation of herpes simplex virus types 1 and 2 by melting curve analysis was problematic in all assays. Overall, the results highlight the limitations of typing herpes simplex virus by melting curve analysis.

Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to West Nile virus in multiple avian species

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WW or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.11126 potentially discriminated between WW and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2132 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.676, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.

Epitope-blocking enzyme-linked immunosorbent assays for detection of West Nile virus antibodies in domestic mammals

We evaluated the ability of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) to detect West Nile virus (WNV) antibodies in domestic mammals. Sera were collected from experimentally infected horses, cats, and pigs at regular intervals and screened in ELISAs and plaque reduction neutralization tests. The diagnostic efficacies of these techniques were similar.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1 alpha subunit

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B-1 therapy. T231A has very low activity and a greatly elevated K-m for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.

Biochemical and morpho-anatomical analyses of strawberry vitroplants hyperhydric tissues affected by BA and gelling agents

In vitro propagation has become an effective practice for large-scale production of strawberry plants. The objective of this study was to evaluate the hyperhydricity and the multiplication capacity of two strawberry varieties (Fragaria x ananassa Duch. 'Dover' and 'Burkley') propagated in vitro. Plants maintained in MS medium supplemented with 1.0 mg L-1 BA were individualized and transferred to the same medium solidified with Agar (6.5 g L-1) or Phytagel® (2.5 g L-1) and BA at different concentrations (0; 0.5; 1.0; 2.0 and 3.0 mg L-1). Biochemical and anatomical analyses were carried out, as well as the analysis of the morphological hyperhydricity characteristics. The analysis of data showed: a) the increase in cytokinin concentration increased hyperhydricity frequency in both varieties; b) at concentrations up to 2.0 mg L-1 BA, the replacement of Agar by Phytagel® induced a higher formation of hyperhydric shoots; and c) the addition of BA induced oxidative stress, which is characterized by increased antioxidant activity and lipid peroxidation, as well as alterations at the cellular level, such as malformation of stomata and epidermal cells. In conclusion, the culture medium containing 0.5 mg L-1 BA solidified with Agar provided lower hyperhydricity percentages in association with higher rates of shoot proliferation in strawberry.

Biochemical and structural characterization of β-lactamases tem-180 and tem-201

With the constant development of new antibiotics, selective pressure is a force to reckon when investigating antibiotic resistance. Although advantageous for medical treatments, it leads to increasing resistance. It is essential to use more potent and toxic antibiotics. Enzymes capable of hydrolyzing antibiotics are among the most common ways of resistance and TEM variants have been detected in several resistant isolates. Due to the rapid evolution of these variants, complex phenotypes have emerged and the need to understand their biological activity becomes crucial. To investigate the biochemical properties of TEM-180 and TEM-201 several computational methodologies have been used, allowing the comprehension of their structure and catalytic activity, which translates into their biological phenotype. In this work we intent to characterize the interface between these proteins and the several antibiotics used as ligands. We performed explicit solvent molecular dynamics (MD) simulations of these complexes and studied a variety of structural and energetic features. The interfacial residues show a distinct behavior when in complex with different antibiotics. Nevertheless, it was possible to identify some common Hot Spots among several complexes – Lys73, Tyr105 and Glu166. The structural changes that occur during the Molecular Dynamic (MD) simulation lead to the conclusion that these variants have an inherent capacity of adapting to the various antibiotics. This capability might be the reason why they can hydrolyze antibiotics that have not been described until now to be degraded by TEM variants. The results obtained with computational and experimental methodologies for the complex with Imipenem have shown that in order to this type of enzymes be able to acylate the antibiotics, they need to be capable to protect the ligand from water molecules.

Subacute effects of the thiodicarb pesticide on target organs of male wistar rats: biochemical, histological, and flow cytometry studies

Thiodicarb, a carbamate pesticide widely used on crops, may pose several environmental and health concerns. This study aimed to explore its toxicological profile on male rats using hematological, biochemical, histopathological, and flow cytometry markers. Exposed animals were dosed daily at 10, 20, or 40 mg/kg/body weight (group A, B, and C, respectively) during 30 d. No significant changes were observed in hematological parameters among all groups. After 10 d, a decrease of total cholesterol levels was noted in rats exposed to 40 mg/kg. Aspartate aminotransferase (AST) activity increased (group A at 20 d; groups A and B at 30 d) and alkaline phosphatase (ALP) (group B at 30 d) activity significantly reduced. At 30 d a decrease of some of the other evaluated parameters was observed with total cholesterol and urea levels in group A as well as total protein and creatinine levels in groups A and B. Histological results demonstrated multi-organ dose-related damage in thiodicarb-exposed animals, evidenced as hemorrhagic and diffuse vacuolation in hepatic tissue; renal histology showed disorganized glomeruli and tubular cell degeneration; spleen was ruptured with white pulp and clusters of iron deposits within red pulp; significant cellular loss was noted at the cortex of thymus; and degenerative changes were observed within testis. The histopathologic alterations were most prominent in the high-dose group. Concerning flow cytometry studies, an increase of lymphocyte number, especially T lymphocytes, was seen in blood samples from animals exposed to the highest dose. Taken together, these results indicate marked systemic organ toxicity in rats after subacute exposure to thiodicarb.

Biotoxicity assays of quantum dots in in vitro cultures of Medicago sativa and Medicago truncatula

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Biotecnologia

Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brasilian sera

Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA), as well as of four alternative assays, namely indirect immunofluorescence (IIF), passive hemagglutination (PHA), dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA) to 84.2% (PHA) and the specificities ranged from 99.3% (IIF) to 80.2% (PHA). The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered.

Characterization of the Toxicological Effects of Aminocarb on Rats: Hematological, Biochemical, and Histological Analyses

Aminocarb is a widely applied carbamate insecticide with action of controlling pests such as Lepidoptera and Coleoptera. In this study, subchronic effects on Wistar rats were investigated using hematological, biochemical, and histological techniques. Rats were exposed orally at sublethal levels of 10, 20, or 40 mg/kg body weight (groups A, B, and C, respectively) for 14 d. Hematological results revealed no statistical differences after 1 d of exposure but significant reduction in white blood cells detected after 7 d of exposure in group C, as well as, in all treated groups after 14 d of exposure. Biochemical data showed a decrease of acetylcholinesterase activity in all groups after 1 d of exposure with a return to normal after 7 and 14 d. Significant increase in alkaline phosphatase activity of rats exposed to aminocarb was noted after 7 d of treatment. The levels of triglycerides were also significantly decreased. The present investigation also showed a significant increase in content of serum urea and creatinine in animals from group A (14 d), and from groups B and C (7 and 14 d). Histological results demonstrated hemorrhagic focus on hepatic and renal parenchyma in all exposed groups. Taken together, the attained results were dose dependent and indicated adverse effects of aminocarb on hepatic and renal functions, as well as on immune responsiveness at sublethal tested doses.

Biochemical analysis of the methylic antigen of Paracoccidioides brasiliensis

Yeast forms of five strains of Paracoccidioides brasiliensis (SN, 2, 18, 192 and JT- 1) were cultured in a synthetic medium for obtaining methylic antigens. These antigens were lyophilized and studied for each strain, to determine their partial biochemical composition, through measurements of total lipid, protein and carbohydrate contents. Lipids of methylic antigens were purified and analysed for sterols, phospholipids, glycolipids, li-poproteins, and partial characterization of sterols. Significant differences were found among antigenic preparations derived from distinct P. brasiliensis strains, in relation to the quantitative determinations. On the other hand, sterol analysis revealed the presence of ergosterol, lanosterol and squalene in all samples. The diversity verified in the biochemical characteristics of antigens derived from different P. brasiliensis strains, confirm the need of using a pool of fungal samples in order to produce antigen preparations for serological procedures without hampering their sensitivity.

Trypanosoma cruzi strains: behavior after passage into authoctonous or foreign species of triatomine (biological and biochemical patterns)

The behavior of T. cruzi strains from S. Felipe - BA (19 SF, 21 SF and 22 SF) classified as Type II Zymodeme 2, was investigated after passage through the authoctonous (P. megistus) and foreign vectors (T. infestans and R. prolixus). For each strain Swiss mice were infected: I - with blood forms (control); II - with metacyclic forms (MF) from P. megistus; III - with MF from T. infestans; IV - with MF from R. prolixus. Inocula: MF from the three species of triatomine, 60 to 120 days after feeding in infected mice, adjusted to 10 4. Biological behavior in mice (parasitemia, morphology, mortality, virulence and pathogenicity) after passage through triatomine was compared with data from the same strain in control mice. Isoenzymic electrophoresis (ASAT, ALAT, PGM, GPI) were also performed after culture into Warren medium. The three strains maintained the isoenzyme profiles (zymodeme 2), in the control groups and after passages through different species of triatomine. Biological characterization disclosed Type II strains patterns for all groups. An increased virulence was observed with the 22 SF strain isolated from P. megistus and T. infestans and higher levels of parasitemia and predominance of slender forms in mice inoculated with the 19 SF and 21 SF from these same species. Results indicate that the passage through the two species T. infestans and P. megistus had a positive influence on the virulence of the regional strains of S. Felipe, regardless of being autocthonous (P. megistus) or foreign to the area (T. infestans).