The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector

The yellow fever (YF) virus is the prototype flavivirus. The use of molecular techniques has unraveled the basic mechanisms of viral genome structure and expression. Recent trends in flavivirus research include the use of infectious clone technology with which it is possible to recover virus from cloned cDNA. Using this technique, mutations can be introduced at any point of the viral genome and their resulting effect on virus phenotype can be assessed. This approach has opened new possibilities to study several biological viral features with special emphasis on the issue of virulence/attenuation of the YF virus. The feasibility of using YF virus 17D vaccine strain, for which infectious cDNA is available, as a vector for the expression of heterologous antigens is reviewed

Genetic engineering of baker's and wine yeasts using formaldehyde hyperresistance-mediating plasmids

Yeast multi-copy vectors carrying the formaldehyde-resistance marker gene SFA have proved to be a valuable tool for research on industrially used strains of Saccharomyces cerevisiae. The genetics of these strains is often poorly understood, and for various reasons it is not possible to simply subject these strains to protocols of genetic engineering that have been established for laboratory strains of S. cerevisiae. We tested our vectors and protocols using 10 randomly picked baker's and wine yeasts all of which could be transformed by a simple protocol with vectors conferring hyperresistance to formaldehyde. The application of formaldehyde as a selecting agent also offers the advantage of its biodegradation to CO2 during fermentation, i.e., the selecting agent will be consumed and therefore its removal during down-stream processing is not necessary. Thus, this vector provides an expression system which is simple to apply and inexpensive to use

Molecular epidemiology of type 1 and 2 dengue viruses in Brazil from 1988 to 2001

Dengue is a mosquito-borne viral infection that in recent decades has become a major international public health concern. Epidemic dengue fever reemerged in Brazil in 1981. Since 1990 more than one dengue virus serotype has been circulating in this tropical country and increasing rates of dengue hemorrhagic fever and dengue shock syndrome have been detected every year. Some evidence supports the association between the introduction of a new serotype and/or genotype in a region and the appearance of dengue hemorrhagic fever. In order to study the evolutionary relationships and possible detection of the introduction of new dengue virus genotypes in Brazil in the last years, we analyzed partial nucleotide sequences of 52 Brazilian samples of both dengue type 1 and dengue type 2 isolated from 1988 to 2001 from highly endemic regions. A 240-nucleotide-long sequence from the envelope/nonstructural protein 1 gene junction was used for phylogenetic analysis. After comparing the nucleotide sequences originally obtained in this study to those previously studied by others, and analyzing the phylogenetic trees, we conclude that, after the initial introduction of the currently circulating dengue-1 and dengue-2 genotypes in Brazil, there has been no evidence of introduction of new genotypes since 1988. The increasing number of dengue hemorrhagic fever cases seen in Brazil in the last years is probably associated with secondary infections or with the introduction of new serotypes but not with the introduction of new genotypes.

Encapsulated specialty oils commercialized in São Paulo state, Brazil: evaluation of identity (fatty acid profile) and compliance of fatty acids and Vitamin E contents with nutrition labeling

Encapsulated specialty oils commercialized in São Paulo state, Brazil, were evaluated for their identity (fatty acids profile) and compliance with nutrition labeling (fatty acids and Vitamin E (alpha tocopherol) contents). Twenty one samples [flaxseed oil (6), evening primrose (5), safflower (8), borage (1), and black currant (1)] purchased from local markets or collected by the health surveillance agency were analyzed. The fatty acids and vitamin E contents were analyzed by gas chromatography with flame ionization detector and liquid chromatography with UV detector, respectively. Nine samples were adulterated (5 samples of safflower oil, 3 of flaxseed oil, and one of evening primrose). Among them, 3 flaxseed and 2 safflower oil samples were probably adulterated by the addition of soybean oil. Conjugated linoleic acid (CLA) was found in two safflower oils samples although the sale of oils with conjugated linoleic acid (CLA) is not permitted by the National Health Surveillance Agency in Brazil (ANVISA). Only two samples presented all values in compliance with nutrition labeling (one safflower oil sample and one borage oil sample). The results show that a continuous monitoring of encapsulated specialty oils commercialized in Brazil is necessary including a greater number of samples and sanitary surveillance.

Genes involved in the metabolism of fatty acids and risk for Crohn's disease in children: a candidate gene study

Contexte - La prévalence de la maladie de Crohn (MC), une maladie inflammatoire chronique du tube digestif, chez les enfants canadiens se situe parmi les plus élevées au monde. Les interactions entre les réponses immunes innées et acquises aux microbes de l'hôte pourraient être à la base de la transition de l’inflammation physiologique à une inflammation pathologique. Le leucotriène B4 (LTB4) est un modulateur clé de l'inflammation et a été associé à la MC. Nous avons postulé que les principaux gènes impliqués dans la voie métabolique du LTB4 pourrait conférer une susceptibilité accrue à l'apparition précoce de la MC. Dans cette étude, nous avons exploré les associations potentielles entre les variantes de l'ADN des gènes ALOX5 et CYP4F2 et la survenue précoce de la MC. Nous avons également examiné si les gènes sélectionnés montraient des effets parent-d'origine, influençaient les phénotypes cliniques de la MC et s'il existait des interactions gène-gène qui modifieraient la susceptibilité à développer la MC chez l’enfant. Méthodes – Dans le cadre d’une étude de cas-parents et de cas-témoins, des cas confirmés, leurs parents et des contrôles ont été recrutés à partir de trois cliniques de gastro-entérologie à travers le Canada. Les associations entre les polymorphismes de remplacement d'un nucléotide simple (SNP) dans les gènes CYP4F2 et ALOX5 ont été examinées. Les associations allélique et génotypiques ont été examinées à partir d’une analyse du génotype conditionnel à la parenté (CPG) pour le résultats cas-parents et à l’aide de table de contingence et de régression logistique pour les données de cas-contrôles. Les interactions gène-gène ont été explorées à l'aide de méthodes de réduction multi-factorielles de dimensionnalité (MDR). Résultats – L’étude de cas-parents a été menée sur 160 trios. L’analyse CPG pour 14 tag-SNP (10 dans la CYP4F2 et 4 dans le gène ALOX5) a révélé la présence d’associations alléliques ou génotypique significatives entre 3 tag-SNP dans le gène CYP4F2 (rs1272, p = 0,04, rs3093158, p = 0.00003, et rs3093145, p = 0,02). Aucune association avec les SNPs de ALOX5 n’a pu être démontrée. L’analyse de l’haplotype de CYP4F2 a montré d'importantes associations avec la MC (test omnibus p = 0,035). Deux haplotypes (GAGTTCGTAA, p = 0,05; GGCCTCGTCG, p = 0,001) montraient des signes d'association avec la MC. Aucun effet parent-d'origine n’a été observé. Les tentatives de réplication pour trois SNPs du gene CYP4F2 dans l'étude cas-témoins comportant 225 cas de MC et 330 contrôles suggèrent l’association dans un de ceux-ci (rs3093158, valeur non-corrigée de p du test unilatéral = 0,03 ; valeur corrigée de p = 0.09). La combinaison des ces deux études a révélé des interactions significatives entre les gènes CYP4F2, ALOX et NOD2. Nous n’avons pu mettre en évidence aucune interaction gène-sexe, de même qu’aucun gène associé aux phénotypes cliniques de la MC n’a pu être identifié. Conclusions - Notre étude suggère que la CYP4F2, un membre clé de la voie métabolique LTB4 est un gène candidat potentiel pour MC. Nous avons également pu mettre en évidence que les interactions entre les gènes de l'immunité adaptative (CYP4F2 et ALOX5) et les gènes de l'immunité innée (NOD2) modifient les risques de MC chez les enfants. D'autres études sur des cohortes plus importantes sont nécessaires pour confirmer ces conclusions.

Studies on Methylene Blue Sensitized Poly(Vinyl Alcohol): Effect of Molecular Weight of the Polymer and Crosslinking Agents on its Holographic Properties

Dept.of Polymer Science and Rubber Technology,Cochin University of Science and Technology

Biochemical and molecular studies of cadmium resistance and metal biosorption in bacterial species isolated from cochin environment

Cochin, commercial capital of Kerala, located on the west-coast of South India has a large number of chemical and sea food industries. Earlier studies in the past indicated that these industries contribute to heavy metal pollution, particularly mercury, copper, and cadmium, in Cochin backwater. Hence, in the present study, it was desired to isolate cadmium resistant bacteria from effluent discharged by chemical industry with a view to develop an ideal bioremediation process for safe discharge of industrial effluent in to the nearby aquatic environment. Effluent from three industries, located in the industrial belt of Cochin, were collected from the discharge point and cadmium resistant bacteria were screened using standard microbiological techniques

Molecular structures of benzoic acid and 2-hydroxybenzoic acid, obtained by gas-phase electron diffraction and theoretical calculations

The structures of benzoic acid (C6H5COOH) and 2-hydroxybenzoic acid (C6H4OHCOOH) have been determined in the gas phase by electron diffraction using results from quantum chemical calculations to inform restraints used on the structural parameters. Theoretical methods (HF and MP2/6-311+G(d, p)) predict two conformers for benzoic acid, one which is 25.0 kJ mol(-1) (MP2) lower in energy than the other. In the low-energy form, the carboxyl group is coplanar with the phenyl ring and the O-H group eclipses the C=O bond. Theoretical calculations (HF and MP2/6-311+ G(d, p)) carried out for 2-hydroxybenzoic acid gave evidence for seven stable conformers but one low-energy form (11.7 kJ mol-1 lower in energy (MP2)) which again has the carboxyl group coplanar with the phenyl ring, the O-H of the carboxyl group eclipsing the C=O bond and the C=O of the carboxyl group oriented toward the O-H group of the phenyl ring. The effects of internal hydrogen bonding in 2-hydroxybenzoic acid can be clearly observed by comparison of pertinent structural parameters between the two compounds. These differences for 2-hydroxybenzoic acid include a shorter exocyclic C-C bond, a lengthening of the ring C-C bond between the substituents, and a shortening of the carboxylic single C-O bond.

Effect of dietary fish oil on biohydrogenation of fatty acids and milk fatty acid content in cows

Mechanisms underlying milk fat conjugated linoleic acid (CLA) responses to supplements of fish oil were investigated using five lactating cows each fitted with a rumen cannula in a simple experiment consisting of two consecutive 14-day experimental periods. During the first period cows were offered 18 kg dry matter (DM) per day of a basal (B) diet formulated from grass silage and a cereal based-concentrate (0.6 : 0.4; forage : concentrate ratio, on a DM basis) followed by the same diet supplemented with 250 g fish oil per day (FO) in the second period. The flow of non-esterified fatty acids leaving the rumen was measured using the omasal sampling technique in combination with a triple indigestible marker method based on Li-Co-EDTA, Yb-acetate and Cr-mordanted straw. Fish oil decreased DM intake and milk yield, but had no effect on milk constituent content. Milk fat trans-11C(18:1), total trans-C-18:1, cis-9 trans-11 CLA, total CLA, C-18 :2 (n- 6) and total C-18:2 content were increased in response to fish oil from 1.80, 4.51, 0.39, 0. 56, 0.90 and 1.41 to 9.39, 14.39, 1.66, 1.85, 1.25 and 4.00 g/100 g total fatty acids, respectively. Increases in the cis-9, trans-11 isomer accounted for proportionately 0.89 of the CLA response to fish oil. Furthermore, fish oil decreased the flow of C-18:0 (283 and 47 g/day for B and FO, respectively) and increased that of trans-C-18:1 fatty acids entering the omasal canal (38 and 182 g/day). Omasal flows of trans-C-18:1 acids with double bonds in positions from delta-4 to -15 inclusive were enhanced, but the effects were isomer dependent and primarily associated with an increase in trans-11C(18:1) leaving the rumen (17.1 and 121.1 g/day for B and FO, respectively). Fish oil had no effect on total (4.36 and 3.50 g/day) or cis-9, trans-11 CLA (2.86 and 2.08 g/day) entering the omasal canal. Flows of cis-9, trans-11 CLA were lower than the secretion of this isomer in milk. Comparison with the transfer of the trans-9, trans-11 isomer synthesized in the rumen suggested that proportionately 0.66 and 0.97 of cis-9, trans-11 CLA was derived from endogenous conversion of trans-11 C-18:1 in the mammary gland for B and FO, respectively. It is concluded that fish oil enhances milk fat cis-9, trans-11 CLA content in response to increased supply of trans-11 C-18:1 that arises from an inhibition of trans C-18:1 reduction in the rumen.

Sterically hindered metal alkenyls. Part 1. Synthesis, reactions, and crystal and molecular structures of some palladium and platinum σ-alkenyls

The compounds trans-[PtBr{C(C10H15)CH2}(PEt3)2](1)(C10H15= adamant-1-yl), trans-[MBr{C(C10H7)CMe2}(PEt3)2][M = Pd (2) or Pt (3); C10H7= naphth-1-yl], and trans-[MBr{C(Ph)CMe2}(PEt3)2][M = Pd (4) or Pt (5)] have been prepared from Grignard [for (2) and (3)] or lithium reagents [for (1), (4), and (5)] and appropriate dichlorobis(phosphine)metal derivatives. Full single-crystal X-ray data are reported for (1) and (3), and reveal unusually long Pt–C(sp2) bonds. Insertion reactions into these M–C bonds occur with MeNC [for (1), (3), and (5)], and with CO [for (1) and (3)]; the latter, the first reported insertion into a Pt–C(sp2) bond, occurs under mild conditions as expected for the abnormally long M–C bonds.

Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations

Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 angstrom resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis, for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting beta-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.

Molecular characterization of Mycobacterium massiliense and Mycobacterium bolletii in isolates collected from outbreaks of infections after laparoscopic surgeries and cosmetic procedures

An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belem, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and. rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak all of which were originally attributed to a single strain of M. abscessus.