Caracterização fenotípica e genotípica de cepas de Salmonella spp. e subtipagem molecular de plasmídeos carreando genes de beta-lactamases de espectro estendido e AmpC / Phenotypic and genotypic characterization of strains of Salmonella spp. and molecular subtyping of plasmids carrying extended-spectrum beta-lactamase and AmpC genes

Salmonella spp. são reconhecidas como um dos mais importantes patógenos que ocasionam doença entérica, sendo a terceira causa de morte entre as doenças transmitidas por alimentos (DTAs). A maior preocupação atual deve-se ao aumento da resistência aos beta-lactâmicos, e a produção de beta-lactamases mediadas por plasmídeos dentro do gênero. O objetivo deste trabalho foi caracterizar fenotipicamente e genotipicamente 44 isolados de Salmonella spp. produtores de beta lactamases além de, realizar a subtipagem dos plasmídeos de resistência aos beta lactâmicos de cepas de origem humana e não humana. Foi realizado o teste de sensibilidade aos antimicrobianos e a determinação da Concentração Inibitória Mínima (CIM). A PCR foi utilizada para a identificação dos genes blaESBL e blaAmpC, em seguida, confirmados por sequenciamento de Sanger. Para a subtipagem plasmídial dos isolados foram utilizadas as técnicas de PBRT e pMLST. Os resultados do teste de sensibilidade aos antimicrobianos mostrou que 97,7% dos isolados foram resistentes à cefotaxima, 86,3% resistentes à sulfonamida e tetraciclina, 81,9% resistentes ao ácido nalidíxico, 68,1% resistentes a ceftriaxona e 56,8% a estreptomicina. De acordo com a PCR, 24 isolados (54,5%) foram positivos para ESBLs (CTX-M), e 20 isolados (45,5%) positivos para beta-lactamase do tipo AmpC, CMY-2. A variante CTX-M-2 foi encontrada em 27,2% dos isolados, seguido da variante CTX-M-8 (22,7%). As variantes CTX-M-15 e CTX-M-65 foram detectadas em um isolado respectivamente. De acordo com a tipagem plasmidial foram encontrados os grupos de incompatibilidade IncI1/ST12 e IncA/C/ST2 em cepas positivas para blaCMY-2. Para as variantes de CTX-M foram detectados os grupos de incompatibilidade IncHI2 em cepas produtoras de CTX-M-2, IncI1, IncF e IncL/M identificados em cepas CTX-M-8, e dois isolados não tipáveis nas variantes CTX-M-15 e CTX-M-65. A análise do perfil genético dos isolados por PFGE sugerem a circulação de cepas geneticamente relacionadas entre fontes humanas e não humanas no sorotipo Muenchenn, e um perfil de similaridade de 82,3% no sorotipo Heidelberg, em cepas provenientes de fontes de origem e anos de isolamento diferentes. Os resultados encontrados neste estudo revelam o cenário atual da resistência aos beta-lactâmicos e reforçam a necessidade de intensificar o monitoramento de isolados portadores de plasmídeos epidêmicos carreadores de beta-lactamases (ESBL e AmpC) na clínica, ambiente e na cadeia produtiva. (AU)

Salmonella spp. are recognized as one of the most important pathogens that cause enteric disease, being the third leading cause of death among foodborne diseases. The greatest current concern should be the increase in resistance to beta-lactams and the production of plasmidmediated beta-lactamases within the genus. The objective of this work was to characterize phenotypically and genotypically 44 isolates of Salmonella spp. beta-lactamase producers, in addition to subtyping plasmids resistant to beta-lactams from human and non-human strains. The antimicrobial sensitivity test was performed and the Minimum Inhibitory Concentration (MIC) was determined. A PCR was used to identify the blaESBL and blaAmpC genes, followed by Sanger sequencing. For plasmid subtyping were used PBRT and pMLST techniques. The results of the antimicrobial sensitivity test showed that 97.7% of the strains were resistant to cefotaxime, 86.3% resistant to sulfonamide and tetracycline, 81.9% resistant to nalidic acid, 68.1% resistant to ceftriaxone and 56.8% streptomycin. According to the PCR, 24 (54.5%) were positive for ESBLs (CTX-M) and 20 (45.5%) positive for AmpC-type beta-lactamase, CMY-2. A CTX-M-2 variant was found in 27.2% of isolates followed by the CTX-M-8 variant (22.7%). The variants, CTX-M-15 and CTX-M-65 were detected in one isolate, respectively. According to plasmid typing, the incompatibility groups IncI1/ST12 and IncA/C/ST2 were found in positive strains for blaCMY-2. For the CTX-M variants, the IncHI2 incompatibility groups were detected in the CTX-M-2, IncI1, IncF and IncL/M were found in the CTX-M-8 strains, and CTX-M-15 and CTX-M-65 variants were not typeable. The analysis of the genetic profile by PFGE suggests the circulation of strains related between human and non-human sources in the Muenchen serotype, and a similarity index of 82.3% in the Heidelberg serotype, in strains from different sources and years. The results found in this study reveal the current scenario of resistance to betalactams and reinforce the need to intensify the monitoring of epidemic plasmids carrying beta-lactamases (ESBL and AmpC) in the clinic, environment and productive chain. (AU)

Petroleum contamination and bioaugmentation in bacterial rhizosphere communities from Avicennia schaueriana

ABSTRACT Anthropogenic activity, such as accidental oil spills, are typical sources of urban mangrove pollution that may affect mangrove bacterial communities as well as their mobile genetic elements. To evaluate remediation strategies, we followed over the time the effects of a petroleum hydrocarbon degrading consortium inoculated on mangrove tree Avicennia schaueriana against artificial petroleum contamination in a phytoremediation greenhouse experiment. Interestingly, despite plant protection due to the inoculation, denaturing gradient gel electrophoresis of the bacterial 16S rRNA gene fragments amplified from the total community DNA indicated that the different treatments did not significantly affect the bacterial community composition. However, while the bacterial community was rather stable, pronounced shifts were observed in the abundance of bacteria carrying plasmids. A PCR-Southern blot hybridization analysis indicated an increase in the abundance of IncP-9 catabolic plasmids. Denaturing gradient gel electrophoresis of naphthalene dioxygenase (ndo) genes amplified from cDNA (RNA) indicated the dominance of a specific ndo gene in the inoculated petroleum amendment treatment. The petroleum hydrocarbon degrading consortium characterization indicated the prevalence of bacteria assigned to Pseudomonas spp., Comamonas spp. and Ochrobactrum spp. IncP-9 plasmids were detected for the first time in Comamonas sp. and Ochrobactrum spp., which is a novelty of this study.

pMEX01, a 70kb Plasmid Isolated From Escherichia Coli That Confers Resistance to Multiple ß-Lactam Antibiotics

Abstract Multidrug-resistant microorganisms are of great concern to public health. Genetic mobile elements, such as plasmids, are among the most relevant mechanisms by which bacteria achieve this resistance. We obtained an Escherichia coli strain CM6, isolated from cattle presenting severe diarrheic symptoms in the State of Querétaro, Mexico. It was found to contain a 70 kb plasmid (pMEX01) with a high similarity to the pHK01-like plasmids that were previously identified and described in Hong Kong. Analysis of the pMEX01 sequence revealed the presence of a blaCTX-M-14 gene, which is responsible for conferring resistance to multiple β-lactam antibiotics. Several genes putatively involved in the conjugative transfer were also identified on the plasmid. The strain CM6 is of high epidemiological concern because it not only displays resistance to multiple β-lactam antibiotics but also to other kinds of antibiotics.

Survey on pathogenic Escherichia coli and Salmonella spp. in captive cockatiels (Nymphicus hollandicus)

Abstract We surveyed healthy captive cockatiels (Nymphicus hollandicus) for Escherichia coli and Salmonella spp. Cloacal swabs were collected from 94 cockatiels kept in commercial breeders, private residencies and pet shops in the cities of São Paulo/SP and Niterói/RJ (Brazil). Three strains of E. coli from each individual were tested for the presence of ExPEC-, APEC- and DEC-related genes. We evaluated the blaTEM, blaSHV, blaOXA, blaCMY, blaCTX-M, tetA, tetB, aadA, aphA, strAB, sul1, sul2, sul3, qnrA, qnrD, qnrB, qnrS, oqxAB, aac (6)′-Ib-cr, qepA resistance genes and markers for plasmid incompatibility groups. Salmonella spp. was not detected. E. coli was isolated in 10% of the animals (9/94). Four APEC genes (ironN, ompT, iss and hlyF) were detected in two strains (2/27-7%), and iss (1/27-4%) in one isolate. The highest resistance rates were observed with amoxicillin (22/27-82%), ampicillin (21/27-79%), streptomycin (18/27-67%), tetracycline (11/27-41%). Multiresistance was verified in 59% (16/27) of the isolates. We detected strAB, bla TEM, tetA, tetB, aadA, aphaA, sul1, sul2, sul3 resistance genes and plasmid Inc groups in 20 (74%) of the strains. E. coli isolated from these cockatiels are of epidemiological importance, since these pets could transmit pathogenic and multiresistant microorganisms to humans and other animals.

Complete genome sequence of a phthalic acid esters degrading Mycobacterium sp. YC-RL4

ABSTRACT Mycobacterium sp. YC-RL4 is capable of utilizing a broad range of phthalic acid esters (PAEs) as sole source of carbon and energy for growth. The preliminary studies demonstrated its high degrading efficiency and good performance during the bioprocess with environmental samples. Here, we present the complete genome of Mycobacterium sp. YC-RL4, which consists of one circular chromosome (5,801,417 bp) and one plasmid (252,568 bp). The genomic analysis and gene annotation were performed and many potential genes responsible for the biodegradation of PAEs were identified from the genome. These results may advance the investigation of bioremediation of PAEs-contaminated environments by strain YC-RL4.

Consumption and expenditure on food prepared away from home among Mexican adults in 2006 / Consumo y gasto en alimentos preparados fuera de casa en adultos mexicanos en 2006

Objective. To describe food expenditure and consumption of foods prepared away from home among Mexican adults. Materials and methods. Data were from 45 241 adult participants in the National Health and Nutrition Survey 2006, a nationally-representative, cross-sectional survey of Mexican households. Descriptive statistics and multivariable linear and logistic regression were used to assess the relationship between location of residence, educational attainment, socioeconomic status and the following: 1) expenditure on all food and at restaurants, and 2) frequency of consumption of comida corrida or restaurant food and street food. Results. Food expenditure and consumption of food prepared away from home were positively associated with socioeconomic status, educational attainment, and urban vs. rural residence (p<0.001 for all relationships in bivariate analyses). Conclusions. Consumption of food prepared outside home may be an important part of the diet among urban Mexican adults and those with high socioeconomic status and educational attainment.

Objetivo. Describir los gastos en alimentos y el consumo de alimentos preparados fuera de casa en población mexicana. Material y métodos. Los datos fueron de 45 241 adultos mexicanos en la Encuesta Nacional de Salud y Nutrición de 2006, representativa al nivel nacional. Se utilizaron estadísticas descriptivas y regresión linear y logística para estimar la relación entre el lugar de residencia, el nivel educativo y el nivel socioeconómico, con el gasto en todos los alimentos y en restaurantes, y con la frecuencia de consumo de comida corrida, en restaurantes y de la calle. Resultados. El gasto en alimentos y el consumo de alimentos preparados se asociaron positivamente con el nivel socioeconómico, el nivel educativo y la residencia rural (p<0,001 para todas las relaciones). Conclusiones. El consumo de alimentos preparados puede ser una parte importante de la dieta de los adultos urbanos y de aquéllos con altos niveles socioeconómicos y educativos.

Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure

The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.

Identification of ACT-1 Plasmid-Mediated AmpC beta-Lactamase Producing Citrobacter freundii from a Chinese Patient

No abstract available.

DNA degradation by aqueous extract of Aloe vera in the presence of copper ions.

The plant Aloe vera has long been used in medicine, as dietary supplements and for cosmetic purposes. Aloe vera extracts are a rich source of polyphenols, such as aloin and aloe emodin and have shown a wide range of pharmacological properties, including anti-inflammatory and anti-cancer properties. The bioactive component aloe emodin has been reported to induce apoptosis in various cancer cell lines. Many of the biological activities of Aloe vera have been attributed to its antioxidant properties. However, most plant-derived polyphenols that are also present in Aloe vera may exhibit pro-oxidant properties either alone or in the presence of transition metals, such as copper. Previous reports from this laboratory have implicated the pro-oxidant action as one of the mechanisms for their anti-cancer properties. In the present paper, we show that aqueous extract of Aloe vera is also able to cause DNA degradation in the presence of copper ions. Further, the extract is also able to reduce Cu(II) to Cu(I) and generate reactive oxygen species, such as superoxide anion and hydroxyl radicals in a dose-dependent manner, which correlates with ability of the extract to cause DNA breakage. Thus, the study shows that in addition to antioxidant activity, Aloe vera extract also possess pro-oxidant properties, leading to oxidative DNA breakage.

DNA damage by ribose: Inhibition at high ribose concentrations.

Nucleobases and DNA react non-enzymatically with sugars, and generate DNA-advanced glycation end products (DNA-AGEs). Incubation of plasmid pBr322 with ribose for 3-7 days caused the transformation of the supercoiled plasmid to the open circular and linear forms. Removal of sugar after an initial 24 h incubation resulted in marked enhancement in the transformation rate. Enhancement in transformation was also observed when bovine serum albumin (BSA) exposed to ribose for short durations was incubated with plasmid in absence of the sugar. The effect on DNA was attenuated when excess ribose remained in the incubation mixture of ribose and protein. The data suggested that an increase in ribose concentration in the vicinity of DNA could be damaging and the damage exacerbated, if sugar levels fell subsequently. Incubation of herring sperm DNA with ribose resulted in a concentration and time-dependent increase of N2-carboxyethyl-2’-deoxyguanosine (CEdGA,B). The concentration of CEdGA,B, however, did not increase further when ribose was removed from the reaction mixture.

Effect of p27Kip1 inhibition on proliferation of bovine corneal endothelial cells by RNA interference / 华中科技大学学报（医学）（英德文版）

Three plasmids (pGenesil-P1, pGenesil-P2, pGenesil-P3) with different p27Kip1-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endothelial cells (bCEC) were investigated. Plasmid expressing irrelevant shRNA with a random combination was used as negative control (pGenesil-HK). The recombination of four plamids was confirmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kip1 mRNA and p27Kip1 protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group (non-transfected group). pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The proliferation rates of the pGenesil-P3 group, the pGenesil-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was detected by flow cytometry (FCM). Compared with the control groups, the proliferation rate of the pGenesil-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kip1 could down-regulate the expression of p27Kip1 effectively and increase the proliferation of bCEC. RNA interference (RNAi) may be an effective means to promote the proliferation of CEC.

Comparison of anticoagulant effects on vein grafts between human TFPI gene transfection and aspirin oral administration / 华中科技大学学报（医学）（英德文版）

To develop a more efficient antithrombotic way after coronary artery bypass grafting (CABG), the anticoagulant effects were compared of human tissue factor pathway inhibitor (TFPI) gene transfection and aspirin oral administration (traditional method) on vein grafts. An eukaryotic expression plasmid pCMV-(Kozak) TFPI was prepared. Animal model of carotid artery bypass grafting was constructed. In operation, endothelial cells of vein grafts in TFPI group and empty plasmid control group were transfected with pCMV-(Kozak) TFPI and empty plasmid pCMV respectively, while no transfection was conducted in aspirin control group. After operation, aspirin (2 mg.kg(-1).(-1)) was administered (i.g.) in aspirin control group. Three days later, grafts (n=10) were harvested for RT-PCR, Western blotting and immunohistochemical analyses of exogenous gene expression and for pathological, scanning electron microscopic observation of thrombus. Thirty days later, the patency rates of remnant grafts (n=10) were recorded by vessel Doppler ultrasonography. Human TFPI gene products were detected in gene transferred vein grafts. Three days later, thrombi were found in 7 animals of aspirin control group and in 8 animals of empty plasmid control group, but in only 1 of TFPI group (P<0.01). Thirty days later, 5 grafts were occluded in empty plasmid control group, but none of grafts was occluded in the other groups (P<0.05). The endothelial surfaces of grafts in both of the control groups were covered with aggregated erythrocytes and platelets, and it were not seen in TFPI group. It was suggested that the anticoagulant effects on vein grafts of human TFPI gene transfection are better than those of aspirin.

Characterization of carotenoid hydroxylase gene promoter in Haematococcus pluvialis.

Astaxanthin, a high-value ketocarotenoid is mainly used in fish aquaculture. It also has potential in human health due to its higher antioxidant capacity than beta-carotene and vitamin E. The unicellular green alga Haematococcus pluvialis is known to accumulate astaxanthin in response to environmental stresses, such as high light intensity and salt stress. Carotenoid hydroxylase plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, we report the characterization of a promoter-like region (-378 to -22 bp) of carotenoid hydroxylase gene by cloning, sequence analysis and functional verification of its 919 bp 5'-flanking region in H. pluvialis. The 5'-flanking region was characterized using micro-particle bombardment method and transient expression of LacZ reporter gene. Results of sequence analysis showed that the 5'-flanking region might have putative cis-acting elements, such as ABA (abscisic acid)-responsive element (ABRE), C-repeat/dehydration responsive element (C-repeat/DRE), ethylene-responsive element (ERE), heat-shock element (HSE), wound-responsive element (WUN-motif), gibberellin-responsive element (P-box), MYB-binding site (MBS) etc., except for typical TATA and CCAAT boxes. Results of 5' deletions construct and beta-galactosidase assays revealed that a highest promoter-like region might exist from -378 to -22 bp and some negative regulatory elements might lie in the region from -919 to -378 bp. Results of site-directed mutagenesis of a putative C-repeat/DRE and an ABRE-like motif in the promoter-like region (-378 to -22 bp) indicated that the putative C-repeat/DRE and ABRE-like motif might be important for expression of carotenoid hydroxylase gene.

Colocalization of Interferon Regulatory Factor 7 (IRF7) with Latent Membrane Protein 1 (LMP1) of Epstein-Barr Virus

Interferon regulatory factor 7 (IRF7) is one of the transcriptional factors for the activation of type I Interferon (IFN) genes. It is known that IRF7 and the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) are highly expressed in EBV type III latency cells, and LMP1 induces mRNA expression of IRF7. In this study, the expression pattern of endogenous IRF7 was observed in several B cell lines with or without EBV infection by immunofluorescence staining. IRF7 was localized in the cytoplasm of EBV-negative B cells and EBV type I latency B cell lines. However, IRF7 was located both in the cytoplasm and nucleus of EBV type III latency cell lines. In the Jijoye cell (type III latency cell), IRF7 was colocalized with LMP1 in the cytoplasm in a capping configuration, and their interaction was confirmed by co-immunoprecipitation of LMP1 and IRF7. This colocalization was confirmed by co-transfection of IRF7 and LMP1 plasmids in EBV-negative B cells. These results suggest that the IRF7 and LMP1 interact with each other, and this may relate to the mechanism whereby LMP1 exerts functional effects in B-lymphocytes.

Cloning and sequencing of 28 kDa outer membrane protein gene of Brucella melitensis Rev. 1.

Brucella melitensis is an organism of paramount zoonotic importance. The 28 kDa outer membrane protein (OMP) is one of the immunodominant antigens of B. melitensis. The gene encoding 28 kDa OMP (omp28) has been amplified from B. melitensis Rev. 1 strain. A PCR product of 753 bp, encoding complete omp28 gene of B. melitensis, was obtained. The gene was further cloned and sequenced. The nucleotide sequence of B. melitensis Rev. 1 strain showed substitution of 2 nucleotides from that of 16M strain.

Infectivity analysis of two variable DNA B components of Mungbean yellow mosaic virus-Vigna in Vigna mungo and Vigna radiata.

Mungbean yellow mosaic virus-Vigna (MYMV-Vig), a Begomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85.6%) for KA27 DNA B. The Rep-binding domain had three complete 11-nt (5'-TGTATCGGTGT-3') iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first iteron in KA27 DNA B (5'-ATCGGTGT-3') had a 3-nt deletion. KA27 DNA B, which exhibited 93.9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first iteron besides having an 18-nt insertion between the third iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94.1%) and BC1 (97.6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMVVig is an important determinant of host-range between V. mungo and V. radiata.

Antibacterial and antidiarrhoeal effects of alkaloids of Holarrhena antidysenterica WALL.

The alkaloids from the ethanolic extract of H. antidysenterica seeds were evaluated for their antibacterial activity against clinical isolates of enteropathogenic Escherichia coli (EPEC) in vitro, and their antidiarrhoeal activity on castor oil-induced diarrhoea in rats, in vivo. The plasmid DNA, whole cell lysate and outer membrane protein profile of a clinical isolate of EPEC was determined in presence of alkaloids of H. antidysenterica. The disc diffusion and agar well diffusion methods were used to evaluate the antibacterial efficacy. The alkaloids showed strong antibacterial activity against EPEC strains. In castor oil-induced diarrhoea, alkaloids reduced the diarrhoea with decrease in the number of wet faeces in pretreated rats at a dose of 200-800 mg/kg. The loss of plasmid DNA and suppression of high molecular weight proteins were observed on alkaloids treatment. Taking into account the multiple antibiotic resistance of EPEC, the results suggest usefulness of alkaloids of H. antidysenterica seeds as antibacterial and antidiarrhoeal agents.

Genetics of metal resistance in acidophilic prokaryotes of acidic mine environments.

Acidophilic bacteria inhabiting acidic mine regions cause natural leaching of sulphidic ores. They are now exploited in industrial operations for leaching of metals and beneficiation of low-grade and recalcitrant ores. Recent trends emphasize application of thermoacidophiles and genetic engineering of ore-leaching bacteria for greater success in this area. This requires an in-depth understanding on the molecular genetics of these bacteria and construction of cloning vectors for them. Metal resistance is considered as the most suitable phenotypic trait for cloning vectors of bio-mining chemolithoautotrophic (viz. Acidithiobacillus ferrooxidans) and heterotrophic (Acidiphilium and Acidocella species) bacteria of mine environments. These bacteria take part in ore-leaching either directly or indirectly, exhibit low to high level of resistance/tolerance to various metals under different conditions. Majority of these bacteria contain one or more plasmids--the genetic elements that usually carry metal resistant genes. But none of the At. ferrooxidans plasmids has been definitely proved to harbour metal-resistant genes which have mostly been found in the chromosome of this bacterium. Plasmids of acidophilic heterotrophs of the genera Acidiphilium and Acidocella, on the other hand, carry metal resistant genes. While genes bestowing arsenic resistance in Acidiphilium multivorum are similar to those analyzed from other sources, the metal (Cd and Zn)-resistance conferring cloned plasmid DNA fragments from Acidiphilium symbioticum KM2 and Acidocella GS19h strains were found to have no sequence similarity with the reported Cd- and Zn-resistant genes. Such observations indicate some novel aspects of metal resistance in acidophilic bacteria.

Effects of HMGB-1 Overexpression on Cell-Cycle Progression in MCF-7 Cells

High mobility group-1 (HMGB-1) enhances the DNA interactions and possesses a transcriptional activation potential for several families of sequence-specific transcriptional activators. In order to examine the effect of HMGB-1 on the cell cycle progression in MCF-7 cells, the HMGB-1 expression vector was transfected into synchronized MCF-7 cells, and the effect of HMGB-1 overexpression on the cell cycle was examined. The HMGB-1 protein level in the transfected cells increased 4.87-fold compared to the non-transfected cells. There were few changes in the cell cycle phase distribution after HMGB-1 overexpression in the MCF-7 cells. Following the estrogen treatment, the cell cycle progressed in both the HMGB-1 overexpressed MCF-7 and the mock-treated cells. However, a larger proportion of HMGB-1 overexpressing MCF-7 cells progressed to the either S or G2 phase than the mock-treated cells. The mRNA levels of the cell cycle regulators changed after being treated with estrogen in both the HMGB-1 overexpressing MCF-7 and the mock-treated cells, but the changes in the expression level of the cell cycle regulator genes were more prominent in the HMGB-1 overexpressing MCF-7 cells than in the mock-treated cells. In conclusion, HMGB-1 overexpression itself does not alter the MCF-7 cell cycle progression, but the addition of estrogen to the HMGB-1 overexpressing MCF-7 cells appears to accelerate the cell cycle progression.

Expression & immunogenicity of malaria merozoite peptides displayed on the small coat protein of chimaeric cowpea mosaic virus.

BACKGROUND & OBJECTIVES: Foreign peptide sequences can be inserted into the betaB-betaC loop of the cowpea mosaic virus (CPMV) small coat protein (SCP) to yield functional chimaeric viruses. Immunisation with chimaeric CPMV elicits immune responses that protect against human immunodeficiency and mink enteritis viruses. The present study was undertaken to investigate the expression of a B cell epitope from the merozoite surface antigen-1 of the malaria parasite Plasmodium falciparum (PfMSP1) in CPMV for an epitope based vaccine. METHODS: DNA encoding a 19 aa sequence (VTHESYQEL VKKLEALEDA, termed P109), the N-terminus of the mature PfMSP1, was cloned into SCP gene yielding a chimaeric virus CPMV-P109. CPMV-P109 was propagated in cowpea plants. The immunogenicity of purified recombinant virus in rabbits was investigated. RESULTS: CPMV-P109 developed a systemically spreading infection in cowpea, with normal viral morphology. The P109 epitope was detected on CPMV-P109 by ELISA with an antiserum produced against homopolymeric P109. Immunisation of rabbits with CPMV-P109 yielded antibodies that, although were predominantly directed against virus-specific epitopes, also recognized the P109 peptide on the recombinant virus and free P109 peptide. These antibodies however, did not react with the native antigen on merozoite by immunofluorescence. INTERPRETATION & CONCLUSION: The results indicate that selecting immunodominant peptide epitopes and presenting them in a near native conformation are important for generating biologically relevant antibodies in the CPMV expression system. Further, the findings draw attention to the importance of measuring immune responses to the viral vector antigens, a preponderance of which can result in undesirable effects such as autoimmunity and hypersensitivity in immunized hosts.