PCR Detection of a Newly Emerged Pandemic Vibrio parahaemolyticus O3:K6 Pathogen in Pure Cultures and Seeded Waters from the Gulf of Mexico

This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 10² ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10³ cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.     

Real-time PCR detection of Vibrio vulnificus in oysters: comparison of oligonucleotide primers and probes targeting vvhA

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C(T)) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10(3) V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 x 10(3) CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.     

Chemopreventive effect of orange oil on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine in rats: an ultrastructural study

Orange peel oil is used extensively as an approved flavour enhancer in fruit drinks, carbonated beverages and as a scenting agent in soaps and cosmetics. Limonene, which is a monocyclic monoterpene is present in orange peel oil from 90 to 95% (w/w). Monoterpenes have been shown to be very effective chemopreventive agents against several rodent tumors and are currently in clinical trials. However, not much information is available regarding the ultrastructural changes associated with the chemopreventive effects of the monoterpenes. The effect of orange oil on the suppression of preneoplastic hepatic lesions during N-nitrosodiethylamine (DEN) induced hepatocarcinogenesis was studied electron microscopically. Rats were administered 200 ppm DEN through drinking water for a period of 1 month. After an interval of 2 weeks, the animals were administered orange oil by gavage for a period of 5 1/2 months. The chemopreventive effect of orange oil was monitored on the basis of liver weight profile, histological pattern by light microscopy and ultrastructural alterations by electronmicroscopy. Orange oil administration following DEN treatment showed decreased liver weights, increased intercellular gap junctional complexes, cell density and polarity when compared with only the DEN treated rats. In the present study chemopreventive effect of orange oil on DEN-induced hepatic preneoplasia in rats which is associated with the restoration of the normal phenotype and upregulation of junctional complexes has been demonstrated.     

Production of Polyclonal Antisera to Choline Acetyltransferase Using a Fusion Protein Produced by a cDNA Clone Victor

A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a lambda gtll lysate of Escherichia coli. The cDNA insert, which contained a 728-amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti-ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75-kilodalton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75- and 67-kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75- and 67-kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37 degrees C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple Laminin Binding Proteins in Human Fetal Heart

The possibility of occurrence of laminin binding proteins in cardiac tissue under different stages of growth was examined by affinity chromatography of the soluble fraction of human fetal myocardial plasma membrane over Ln-Sepharose. A 67 kDa protein was isolated by elution with glycine/HCl buffer containing 1 M NaCl and visualized as a coomassie stainable band on SDS gel electrophoresis under reducing conditions. Dot blot assays of the radioiodinated protein revealed the binding of 67 kDa protein with high affinity to laminin in a cation independent manner. This protein appears to be present in relatively higher amounts in tissues from early stage fetus. The occurrence of cation dependent laminin binding proteins was also examined by affinity chromatography. Electrophoresis of the EDTA eluate under reducing conditions followed by silver staining showed two prominent bands with average molecular size 130 and 174 kDa which under non-reducing conditions appeared as two bands with average molecular weight of 115 and 135 kDa. Using radioiodinated protein in dot blot assays, its binding to Ln was found to be maximum in the presence of Mn⁺⁺ ions. Immunoblotting using anti-β₁ integrin antibodies showed that 115 kDa protein is a β₁ integrin suggesting the possibility of this protein belonging to the integrin group of receptors. The occurrence of multiple laminin binding proteins and the relative abundance of one of these proteins viz. the 67 kDa protein during early stages than in late stage tussue suggest a possible role for these proteins in cellular interactions with laminin during myocardial tissue development.     

Detection of pandemic Vibrio parahaemolyticus O3:K6 serovar in Gulf of Mexico water and shellfish using real-time PCR with Taqman fluorescent probes

We describe a real-time multiplexed PCR method using Taqman probes for the detection of total and pandemic Vibrio parahaemolyticus O3:K6 serovar in oysters and Gulf of Mexico water (gulf water). The specificity of these primers and probes was tested for amplification of a 450 bp thermolabile hemolysin (tlh) and a 369 bp ORF8 amplicon representing all V. parahaemolyticus and post-1996 clinical isolates of pandemic serovar O3:K6, respectively. The sensitivity of detection was 10 pg purified DNA or 10(3) CFU in 1 mL pure culture. Enrichment of this pathogen in oyster tissue homogenate or gulf water for 5 or 8 h resulted in the detection of an initial inoculum of 1 CFU in 1 mL or 1 g of samples. Application of the Taqman PCR assay on natural oysters exhibited a positive detection of V. parahaemolyticus, ranging from 16% to 100% of the samples collected primarily during the summer months. None of the samples exhibited a positive detection of O3:K6 serovar. Rapid and sensitive detection of this pathogen will help shellfish industry and Interstate Shellfish Sanitation Conference (ISSC) undertake appropriate measures to monitor this pathogen in oysters and oyster-growing waters, thereby preventing disease outbreaks and consequently protecting consumer health.     

Effect of Moringa oleifera leaves on hematological profile of fluorosis affected rats

Fluorosis is a metabolic disease that is endemic in nearly 25 countries with India being one of the most affected. It primarily affects the bone and the teeth. Moringa oleifera (MO) leaves are known to reduce the effect of fluorosis on various tissues. Therefore, it is of interest to document the effect of Moringa oleifera leaves on the hematological profile of fluorosis affected rats. Twenty four Sprague Dawley rats were housed two per cage in a room with 12 hours light and 12 hours dark cycle. The rats were allowed to adjust to the laboratory environment for about one to two weeks before the beginning of the study. This study reveals that MO leaves is effective in reducing the plasma fluoride content. It also helps in improving the Hb % and RBC count in fluorosis affected rats. Data shows that Moringa olifera leaves powder is effective in reducing the plasma fluoride content. It also helps in improving the Hemoglobin percentage & Red Blood Cell count in fluorosis affected rats.     

Does proliferation capacity of lymphocytes depend on human blood types?

In vitro human lymphocyte culture methodology is well established yet certain confounding factors such as age, medical history as well as individual’s blood type may potentially modulate in vitro proliferation response. These factors have to be carefully evaluated to release reliable test report in routine cytogenetic evaluation for various genetic conditions, radiation biodosimetry, etc. With this objective, the current study was focused on analyzing the proliferation response of lymphocytes drawn from 90 individuals (21-29 years) with different blood types. The proliferation response was assessed in the cultured lymphocytes by cell cycle, mitotic index (MI), and nuclear division index (NDI) after stimulation with phytohaemagglutinin (PHA). To investigate the toxic effect on proliferation, MI was calculated in representative samples of each blood type were X-irradiated. The results showed that there was no significant difference among the cell cycle phases of lymphocytes in different blood types (P > 0.05). Similarly, both MI and NDI of lymphocytes derived from different blood types also did not show significant difference ( P > 0.05). The extensive interindividual variation within and among the blood types is likely responsible for the lack of significant difference in lymphocyte proliferation. Although spontaneous proliferation efficiency of lymphocytes of different blood types after PHA stimulation was grossly similar, the MI observed after radiation exposure showed a significant difference ( P < 0.05) indicating a differential proliferation response among the blood types. Our results suggest that the blood types did not have any impact on PHA-induced proliferation; however, a specific differential lymphocyte proliferation observed after radiation exposure needs to be considered.     

Antiperoxidative and Antiinflammatory Effect of <Emphasis Type="Italic">Sida Cordifolia</Emphasis> Linn. on Quinolinic Acid Induced Neurotoxicity

Sida cordifolia is a plant belonging to the Malvaceae family used in many ayurvedic preparations. This study aimed at assessing the effects of ethanolic extract of Sida cordifolia root on quinolinic acid (QUIN) induced neurotoxicity and to compare its effect with the standard drug deprenyl in rat brain. Rats were divided into six groups: (1) control group (2) QUIN (55 μg/100 g bwt/day) (3) 50% ethanolic plant extract treated group (50 mg/100 g bwt/day) (4) Deprenyl (100 μg/100 g bwt/day) (5) QUIN (55 μg/100 g bwt/day) + 50% ethanolic plant extract treated group (50 mg/100 g bwt/day) (6) QUIN (55 μg/100 g bwt/day) + Deprenyl (100 μg/100 g bwt/day). At the end of the experimental period a status of lipid peroxidation products, protein peroxidation product, activities of the scavenging enzymes and the activities of the inflammatory markers were analyzed. Results revealed that the lipid peroxidation products decreased and the activities of the scavenging enzymes increased significantly in the brain of the plant extract treated group, deprenyl treated group and also in the coadminstered groups. The activities of markers of inflammatory responses such as cyclooxygenase and lipoxygenase were found to be significantly increased in the QUIN treated rats and this was decreased upon the administration of plant extract and deprenyl. In short, the study revealed that 50% ethanolic extract of Sida cordifolia has got potent antioxidant and antiinflammatory activity and the activity is comparable with the standard drug deprenyl.