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Biomedical subjects

S Pandian

Publications and source records attributed to S Pandian.

At least 19 recordsLinked to original sources
PubMed2002

A rapid and improved method for the detection of Vibrio parahaemolyticus and Vibrio vulnificus strains grown on hydrophobic grid membrane filters.

DNA probe-based detection methods were developed and characterized as an alternative to time-consuming and less specific conventional protocols. Digoxigenin-labeled probes were prepared by polymerase chain reaction amplification of the targeted sequences in the specific amplicons generated from genomic DNA. Specific probes with high yields were generated for the detection of the tlh gene of Vibrio parahaemolyticus and the cth gene of V. vulnificus. Colony (Southern) hybridization analyses were carried out using hydrophobic grid membrane filters (HGMFs) to allow biotype-specific differentiation of the two species. Eight strains of V. vulnificus and five strains of V. parahaemolyticus, including one standard (ATCC) strain of each biotype, were examined. Colony lysis, hybridization, and nonradioactive detection parameters were optimized for identification of the target biotypes arranged on the same HGMF and also on a conventional nylon membrane, thereby confirming the specificity of the probes and the comparative usefulness of the HGMFs. The experimental procedure presented here can be completed in 1 day. The protocol was designed specifically to identify the target Vibrio spp. and could potentially be used for the enumeration and differentiation of V. parahaemolyticus and V. vulnificus in foods.

DNA Probes↗
PubMed1996

Phaeohyphomycosis caused by Exophiala dermatitidis following intra-articular steroid injection.

A patient with long-standing rheumatoid arthritis presented with a painful pigmented chronic nodule on the dorsum of the right hand, at the site of intra-articular steroid injections undertaken 5 years previously. Histology showed pigmented fungal elements consistent with phaeohyphomycosis. Cultures yielded black yeast-like colonies, identified as Exophiala dermatitidis and sensitive to itraconazole and amphotericin. A 1-month course of itraconazole resulted in marked clinical improvement but surgical excision and skin grafting were required for complete resolution. Phaeohyphomycosis has been related to inoculation injury but association with intra-articular steroid injection appears hitherto to be unreported.

Dermatomycoses↗
PubMed1995

Influence of Ammonium Salts and Cane Molasses on Growth of Alcaligenes eutrophus and Production of Polyhydroxybutyrate.

The production of polyhydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 was studied in a synthetic medium with 3% glucose at pH 7.0 supplemented with several ammonium substrates and cane molasses. Growth was measured by dry cell weight, and the PHB content was measured by gas chromatography. The effects of ammonium sources such as sulfate, nitrate, phosphate, and chloride salts and those of different ammonium sulfate concentrations were evaluated. The best growth and PHB production were obtained with ammonium sulfate; however, NH(inf4)(sup+) concentrations between 0.5 and 1.5 g/liter showed no significant difference. Ammonium sulfate was therefore used as the sole source of NH(inf4)(sup+) for experiments with cane molasses as the growth activator. Optimal growth and PHB production were obtained with 0.3% molasses. However, the yields of biomass (39 to 48%) and PHB (17 to 26%) varied significantly among the different ammonium substrates and cane molasses concentrations.

Journal Article↗
PubMed1994

Evolution of a Lytic Bacteriophage via DNA Acquisition from the Lactococcus lactis Chromosome.

We discovered a phage-host interaction in which the lytic phage ul36, in response to pressure exerted by an abortive phage resistance mechanism, acquired a large DNA fragment from the chromosome of Lactococcus lactis NCK203 to form a new phage, ul37. Phage ul37 was characterized at morphological, phenotypic, and genotypic levels and was found to be a member of the P335 species. Although it exhibits a high level of DNA homology with ul36, phage ul37 is resistant to the abortive mechanism and has a longer tail, a different base plate, and apparently a different origin of replication. The chromosomal DNA implicated in the formation of new phage ul37 was disrupted by site-specific integration in NCK203. This strategy prevented the appearance of ul37 during subsequent infections with ul36.

Journal Article↗
PubMed1993

Restriction/Modification systems and restriction endonucleases are more effective on lactococcal bacteriophages that have emerged recently in the dairy industry.

Recently, eight lytic small isometric-headed bacteriophages were isolated from cheese-manufacturing plants throughout North America. The eight phages were different, but all propagated on one strain, Lactococcus lactis NCK203. On the basis of DNA homology, they were classified in the P335 species. Digestion of their genomes in vitro with restriction enzymes resulted in an unusually high number of type II endonuclease sites compared with the more common lytic phages of the 936 (small isometric-headed) and c2 (prolate-headed) species. In vivo, the P335 phages were more sensitive to four distinct lactococcal restriction and modification (R/M) systems than phages belonging to the 936 and c2 species. A significant correlation was found between the number of restriction sites for endonucleases (purified from other bacterial genera) and the relative susceptibility of phages to lactococcal R/M systems. Comparisons among these three phage species indicate that the P335 species may have emerged most recently in the dairy industry.

Journal Article↗
PubMed1993

Differentiation of Two Abortive Mechanisms by Using Monoclonal Antibodies Directed toward Lactococcal Bacteriophage Capsid Proteins.

Monoclonal antibodies were used to monitor the accumulation of the major capsid protein of the lactococcal small isometric bacteriophage u136 (P335 species) over the course of a one-step growth curve. A sandwich enzyme-linked immunosorbent assay was then used to distinguish two abortive phage resistance mechanisms, Hsp and Prf. Capsid protein production of u136 was almost totally inhibited by the Hsp-induced abortive mechanism, supporting previous data that this mechanism blocks phage DNA replication. Prf-induced abortive infection only partially (50%) inhibited capsid protein production, suggesting that this mechanism targets some other point, perhaps within transcription or translation processes. The results confirmed that Hsp and Prf act at different targets in the phage lytic cycle. Use of monoclonal antibodies also demonstrated that production of the major capsid protein is a nonlimiting step in the lytic cycle of lactococcal phage u136.

Journal Article↗
PubMed1993

Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk.

The only major structural protein (35 kDa) of the lactococcal small isometric-headed bacteriophage ul36, a member of the P335 species, was isolated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and beta-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 10 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages.

Journal Article↗
PubMed1993

A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate.

Base Sequence↗
PubMed1993

Production and characterization of anti-DNA-RNA monoclonal antibodies and their application in Listeria detection.

Murine monoclonal antibodies (MAbs) specific for DNA-RNA hybrids were successfully produced with two different heteropolymers as antigens, cDNA-mRNA and phi X174 DNA-RNA heteroduplexes. The former was simpler to prepare. Both had shown similar immunogenicities. Two different immunoglobulin M MAbs were isolated. The 20D3 MAb, generated with the phi X174 DNA-RNA hybrid, showed association constants of 1.05 x 10(12), 2.12 x 10(10), and 1.68 x 10(7) for the antigens phi X174 DNA-RNA, cDNA-mRNA, and poly(rA)-poly(dT), respectively. The 6B5 MAb, obtained with the cDNA-mRNA hybrid, showed association constants of 1.59 x 10(5), 5 x 10(12), and 7.1 x 10(8) for the above-described antigens, respectively. With the 20D3 MAb, an immunoassay was developed for the detection of Listeria DNA-RNA hybrids. In brief, a biotinylated rRNA gene probe specific for the genus Listeria was hybridized with rRNA in the solution phase. The hybrids thus formed were then captured in microtiter plate wells precoated with the purified 20D3 MAb, and the probe-target hybrids were detected with a streptavidin-alkaline phosphatase conjugate. This assay was shown to be specific for the genus Listeria and highly sensitive, allowing the detection of as little as 2.5 pg of target rRNA.

Animals↗
PubMed1993

Biological inactivation of adhering Listeria monocytogenes by listeriaphages and a quaternary ammonium compound.

The use of listeriaphages as a means of disinfecting contaminated stainless-steel and polypropylene surfaces was investigated. Surfaces artificially contaminated with L. monocytogenes 10401 and 8427 were sanitized with suspensions of listeriaphages (H387, H387-A, and 2671), all belonging to the Siphoviridae family. Phage suspensions at concentrations of up to 3.5 x 10(8) PFU/ml were at least as efficient as a 20 ppm solution of a quaternary ammonium compound (QUATAL) in reducing L. monocytogenes populations. A synergistic activity was observed when two or more phages were used in combination and when phages were suspended in QUATAL. The biological activity of the three phages was not affected by QUATAL concentrations of 50 ppm and a contact time of 4 h.

Bacterial Adhesion↗
PubMed1991

Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides.

Leuconostoc mesenteroides UL5 was found to produce a bacteriocin, referred as mesenterocin 5, active against Listeria monocytogenes strains but with no effect on several useful lactic acid bacteria. The antimicrobial substance is a protein, since its activity was completely destroyed following protease (pronase) treatment. However, it was relatively heat stable (100 degrees C for 30 min) and partially denaturated by chloroform. The inhibitory effect of the bacteriocin on sensitive bacterial strains was determined by a critical-dilution micromethod. Mutants of L. mesenteroides UL5 which had lost the capacity to produce the bacteriocin were obtained. The mutant strain was stable and phenotypically identical to parental cells and remained resistant to the bacteriocin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect bacteriocin activity corresponding to an apparent molecular mass of about 4.5 kDa.

Bacteriocins↗
PubMed1991

A rapid and efficient method of lysis of Listeria and other gram-positive bacteria using mutanolysin.

A rapid and simple procedure is described for cell lysis for preparation of nucleic acids and intact ribosomal RNA from Gram-positive bacteria. Commercial mutanolysin (purified from Streptomyces globisporus) was used for inducing lysis. Listeria, Lactobacillus and Lactococcus strains were very sensitive to mutanolysin when compared to lysozyme. Susceptibility to mutanolysin was improved by a preliminary treatment with acetone, and sodium dodecyl sulfate reduced the efficiency of lysis when used together with mutanolysin. The procedure was also effective for recovering plasmids from these bacteria.

Acetone↗
PubMed1985

Effects of trichothecenes (T-2 toxin) on protein synthesis in vitro by brain polysomes and messenger RNA.

The effects of T-2 toxin on protein synthesis were tested in two reticulocyte lysate in vitro systems pretreated with micrococcal nuclease. One of the test systems contained purified globin mRNA and was initiation dependent. The other contained rat brain polysomes and incorporated amino acids by an elongation dependent process. T-2 toxin inhibited the translation of globin mRNA at all concentrations tested, from 10(-8) M to 10(-4) M. Rat brain polysomes were much less sensitive to T-2 toxin than globin mRNA. While high concentrations of the toxin (10(-4) M) led to partial inhibition of protein synthesis by polysomes, low concentrations (10(-8) M and 10(-6) M) stimulated protein synthesis. Comparison of the above results with those obtained by other workers suggest that the T-2 toxin may inhibit not only the initiation step of translation, but also elongation and termination, depending upon the concentration of the toxin and the nature of the translation system. A similar mechanism may operate for all the trichothecene toxins that exert their effect through binding to ribosomal peptidyl transferase.

Animals↗
PubMed1983

Purification of phosphofructokinase from rabbit skeletal muscle based on its ability to aggregate in absence of its effectors.

Rabbit muscle phosphofructokinase was purified to homogeneity based on its property to form large aggregates with time at high concentration of its protein in absence of its effectors. The method involves no heat step or treatment with organic solvent or any ion-exchange columns. The enzyme thus prepared, however, exhibits the same kinetic properties as the enzyme purified by more drastic methods.

Adenosine Triphosphate↗
PubMed1982

Perturbed angular correlation studies of 111In-labelled platelets.

Indium-111, as the 8-hydroxyquinoline and acetylacetone complexes, has been used to label cellular blood components. Because of the gamma-ray cascade emitted following the decay of indium-111, perturbed angular correlation studies can be used to investigate the environment of the indium nucleus. Perturbed angular correlation studies have been carried out on the indium at various steps in the procedure used to label platelets with indium using both 8-hydroxyquinoline and acetylacetone as the ligand. Similar values of the integral correlation coefficient were obtained in both cases, suggesting identical labeling mechanisms. Following lysis of the platelets the values of (G22(infinity)) changed, suggesting that the intracellular complex is relatively weak.

Blood Platelets↗
PubMed1981

An alternative pathway for the biosynthesis of isoprenoid compounds in bacteria.

The pattern of incorporation of radioactivity from [1-14C]acetate and [2-14C]acetate into the polyprenyl side-chain of ubiquinones in bacteria (Azotobacter vinelandii, Pseudomonas sesami, Escherichia coli and Rhodopseudomonas capsulata) was studied. For this purpose, a new degradation method involving a modified Barbier-Wieland reaction of laevulinic acid was developed, and used along with the iodoform reaction. Both C-1 and C-2 of acetate were incorporated exclusively into C-2 of laevulinic acid suggesting that the well-known pathway through acetoacetyl-CoA ('acetoacetate pathway') was not operative in these bacteria. An alternative pathway ('acetolactate pathway'), starting with pyruvate and acetaldehyde as the distal precursors, and utilizing the reactions of leucine and valine metabolism, was postulated. It was also postulated that C-1 of acetate is incorporated not directly, but after oxidation to CO2. The pattern of incorporation of radioactivity from [U-14C]valine, [U-14C]alanine and NaH14CO3 into the side-chain of ubiquinone of R. capsulata was in agreement with the operation of the 'acetolactate pathway'.

Acetates↗
PubMedDate not supplied

Anti-DNA.RNA antibodies: an efficient tool for non-isotopic detection of Listeria species through a liquid-phase hybridization assay.

This study was undertaken to evaluate the potential of a new approach using anti-DNA.RNA monoclonal antibodies to detect Listeria in both pure culture and inoculated meat and meat products. A sensitive liquid-phase assay was first developed, based on the formation in solution of a hybrid between a 784-bp DNA probe, specific for the genus Listeria, and target rRNA. Monoclonal antibody and antisera raised against hybrid nucleic acids were then used in various immunoenzymatic assays to detect specific hybrids formed in solution. System 2, using a double sandwich enzyme-linked immunosorbent assay, and system 1, using a biotinylated probe, proved to be very effective. The method using biotin-streptavidin complex, however, resulted in a higher background signal. System 2 described here, using unlabeled probe, was more effective. This strategy allowed the detection of as little as 2.5 pg target RNA from pure culture and 500 cells from inoculated meat homogenate, even in the presence of other contaminating bacteria. The assay was more sensitive and could be completed within 3 h, as opposed to several days when conventional culture methods were used.

Antibodies, Monoclonal↗