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Biomedical subjects

B R Singh

Publications and source records attributed to B R Singh.

At least 55 records · Page 3Linked to original sources
PubMed1998

Coagglutination test: a simple and rapid immunodiagnostic test for parvovirus infection in dogs.

The coagglutination test (COAT) was developed and standardized to detect canine parvovirus (CPV) antigen in faeces of infected dogs. Anti-parvovirus serum was raised in dogs for coating protein-A containing Staphylococcus aureus Cowan I strain. Agglutination of antibody coated bacteria invariably occurred within 2-3 min when mixed with standard CPV antigen or faecal supernatants of dogs having 8 or more haemagglutination (HA) titre of parvovirus antigen. The test had a perfect correlation with HA test and was found to be slightly more sensitive than agar gel precipitation test (AGPT) in detecting CPV antigens. As COAT is easy and needs no specific equipment or much technical know how to perform, it can be used as a field test for rapid clinical diagnosis of parvovirus infection in dogs.

Agglutination Tests↗
PubMed1998

Isolation of synaptotagmin as a receptor for types A and E botulinum neurotoxin and analysis of their comparative binding using a new microtiter plate assay.

Clostridium botulinum neurotoxin acts on nerve endings to block acetylcholine release. Binding of the neurotoxin to a membrane receptor through its heavy chain is the first essential step in its mode of toxin action. Type E botulinum neurotoxin (BoNT/E) or type A botulinum neurotoxin (BoNT/A) receptor was purified from rat brain synaptosomes employing a neurotoxin affinity column chromatography. The protein fraction eluted from the affinity column with 0.5 M NaCl contained a 57 kDa protein as a major eluant. Immunoblotting the eluant with anti-synaptotagmin antibodies revealed that the 57 kDa protein was synaptotagmin I. Rat synaptotagmin I has been suggested as the receptor for BoNT/B (Nishiki et al., J. Biol. Chem. 269, 10498-10503, 1994) in rat brain. In this study, binding of BoNT/A and BoNT/E to synaptotagmin I was studied by a microtiter plate-based method. Binding of synaptotagmin I to BoNT/A coated on the plate was competitively reduced upon preincubation of the proteins with BoNT/E, suggesting a competitive binding of BoNT/A and BoNT/E to the receptor. Taken together, these results suggest that the same receptor protein binds to all three BoNT serotypes tested.

Animals↗
PubMed1998

Hemagglutinin binding mediated protection of botulinum neurotoxin from proteolysis.

Type A Clostridium botulinum, the causative agent of the food poisoning botulism disease, secretes botulinum neurotoxins along with seven neurotoxin associated proteins (NAPs). The function of NAPs has been shown to protect the neurotoxin from acidity, heat, and proteolytic attack in the environmental and gastrointestinal tract during the toxicogenesis of the botulism disease. One of the NAPs, purified from type A botulinum neurotoxin complex, showed hemagglutination activity. A direct interaction has been demonstrated between purified NAP, a 33-kDa hemagglutinin or Hn-33, and the neurotoxin by using Sephadex G-200 column chromatography. Furthermore, Hn-33 has complete resistance against proteolytic attack at pH 2.0 as well as at normal physiological pH. We have investigated digestion of the neurotoxin in the presence and absence of Hn-33. The neurotoxin alone has been found to be more susceptible to the enzymatic digestion than neurotoxin with Hn-33. The presence of Hn-33 changes the proteolytic fragmentation pattern of the neurotoxin. It seems that Hn-33 protects the neurotoxin from proteolysis either by structural modification of the neurotoxin or by blocking the protease accessible sites of the neurotoxin.

Botulinum Toxins, Type A↗
PubMed1997

Characterisation of virulence factors of Serratia strains isolated from foods.

Out of 21 Serratia strains isolated from fresh juice and fish samples, five S. marcescens and two S. rubidaea caused lethality in mice on parenteral inoculation, but none through oral feeding. Three S. marcescens and one S. rubidaea produced heat-labile enterotoxin, detectible with the rabbit ligated ileal loop test, the mouse foot pad test and the vasopermeability factor test. Cell free culture filtrate (CFCF) of two enterotoxigenic S. mearcescens strains induced cytotoxic effect on a monolayer of Vero-cells, but CFCF of other enterotoxigenic strains could only induce cytotonic changes in Vero-cells. All strains possessed fimbriae type 3 while, only pathogenic strains had type 4 fimbriae and a colonization factor. All pathogenic Serratia strains were agglutinated at comparatively lower salt concentrations than non pathogenic strains (< 1.3 M), and had multiple drug resistance. Their public health significance is also discussed.

Animals↗
PubMed1996

Immunochemical characterization of type A botulinum neurotoxin in its purified and complexed forms.

Immunochemical reactivities of type A botulinum neurotoxin to polyclonal antibodies raised against the neurotoxin complex toxoid have been investigated using enzyme-linked immunosorbent assay (ELISA) and with a fiberoptic immunosensor. The complex contains a group of complexing proteins in addition to the neurotoxin itself. Neurotoxin specific immunoglobulin G (IgG), purified from the IgG raised against the complex using an affinity column, showed a two-fold increase in reactivity with purified neurotoxin compared to the neurotoxin in the complex. Antibodies against the whole complex reacted approximately five times better with the complex than with the neurotoxin, suggesting that the detection of the neurotoxin complex may be more sensitive. Considering the fact that the amount of the complexing proteins and neurotoxin appears to be in a 4:1 ratio, a five-fold higher reactivity could suggest a 25-fold higher detectability of the neurotoxin in the complex. ELISA binding curves of complexing proteins and purified neurotoxin with antibodies raised against the whole complex indicated the complexing proteins to have significantly higher antigenicity. Furthermore, IgG fraction with or without the neurotoxin specific antibodies reacted almost equally to the neurotoxin complex, again suggesting higher immunogenicity of the complexing proteins. Increased binding of the complexing proteins versus the purified neurotoxin to antibodies against the complex and thus immunogenicity was also observed in the binding curves generated using a fiberoptic immunosensor.

Animals↗
PubMed1996

Gene probe-based detection of type E botulinum neurotoxin binding protein using polymerase chain reaction.

Using primers based on the nucleotide sequence of a neurotoxin binding protein from Type E Clostridium botulinum cultures, an amplified DNA product was obtained through polymerase chain reaction. The 400 base pair amplified DNA fragment was detectable with as low as 0.1 pg template DNA from Type E C. botulinum, and its fidelity was confirmed by Southern blotting using a DNA probe designed to detect the expected amplified DNA fragment. On the other hand, no DNA amplification was observed with as high as 10 ng template DNA from related Types A and B C. botulinum or from C. tetani, indicating the specificity of the probe.

Botulinum Toxins↗
PubMed1996

Toxigenicity of Edwardsiella tarda isolates of fish and pig origin in experimental models.

Indian isolates of E. tarda from fish (7) and pigs (2) were examined for their enterotoxigenicity/cytotoxigenicity in rabbit ligated ileal loop (RLIL), suckling mouse, rabbit skin and Vero cell monolayers. Cell free culture filtrates (CFCF) of isolates from fish and pigs, induced blood tinged fluid accumulation in RLIL, increased vasopermeability in rabbit skin, and caused cytopathic effect in Vero cells but could not induce fluid accumulation in suckling mouse. CFCFs lost their activity on heating at 63 degrees C for 30 min or 72 degrees C for 15 sec, and also at pH < or = 4.5 or > or = 8.5. The toxic factor was released extracellularly and was nondializable.

Animals↗
PubMed1995

Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified from Clostridium botulinum.

Type E botulinum neurotoxin is produced by Clostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adverse pH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% alpha-helix, 50% beta-sheets, 28% random coils, and 3% beta-turns. This compared to 22% alpha-helix, 44% beta-sheets, 34% random coils, and no beta-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25% alpha-helix, 45% beta-sheets, 27% random coils, and 3% beta-turns, suggesting a significant alteration at least in the alpha-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at low pH as indicated by an initial binding rate of 8.4 min-1 at pH 5.7 compared to 4.0 min-1 at pH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.

Animals↗
PubMed1995

Botulinum versus tetanus neurotoxins: why is botulinum neurotoxin but not tetanus neurotoxin a food poison?

Botulinum and tetanus neurotoxins, produced by Clostridium botulinum and Clostridium tetani, respectively, are the most poisonous poisons known to mankind. Although botulinum and tetanus neurotoxins share several characteristics, such as similar mol. wts, similar macrostructure, virtually identical mode of action, and a strong amino acid sequence homology, the two neurotoxins differ in one very significant way; only botulinum neurotoxin is a food poison. Factors responsible for the food poisoning potential of botulinum neurotoxins seem to be a group of complexing proteins that are also produced by C. botulinum, and are known to associate with the neurotoxin. Translation products of nucleotide sequences upstream to the neurotoxin genes of serotypes A, B, C, D, E and F botulinum neurotoxin reveal the location of genes for one of the complexing proteins that could be transcribed as polycistronic mRNA to include neurotoxin sequences. No such protein seems to be present in C. tetani, suggesting that the lack of complexing proteins might be responsible for tetanus not being a food poison.

Amino Acid Sequence↗
PubMed1995

Survivability of Salmonella paratyphi B var Java on experimentally infected cockroaches.

Cockroaches are inhabitant of sewers and frequent visitors of kitchen and stores in the night to feed on left over. It is liable to disseminate a number of pathogens by contaminating kitchen surface, feeding vessels and food items left open. Salmonella paratyphi B var Java a common pathogen of man and animal was used in the study to evaluate its survival and excretion in cockroaches. The host when fed on semisolid feed containing 1 x 10(7) CFU of S. paratyphi B var Java g-1, it was found that the pathogen was eliminated earlier from the live than euthanized cockroaches.

Animals↗
PubMed1994

Quaternary structure of botulinum and tetanus neurotoxins as probed by chemical cross-linking and native gel electrophoresis.

Botulinum and tetanus neurotoxins are water-soluble proteins (mol. wt 150,000) produced by Clostridium botulinum and Clostridium tetani, respectively. It is believed that these neurotoxins, once internalized via receptor-mediated endocytosis, form membrane channels in order to traverse the endosomal membrane and enter the cytoplasm of the nerve terminal. Investigation of the associative properties between neurotoxin molecules could yield an understanding of this channel formation. That is, an association between neurotoxin monomers could result in an oligomeric form of the neurotoxin necessary for assembly of a channel through the hydrophobic interior of the endosomal membrane, thereby allowing passage of the neurotoxin or its active fragment through the resulting pore. Based on the native gel electrophoresis and chemical cross-linking experiments, tetanus neurotoxin exists as a dimer and a trimer, type A botulinum neurotoxin exists as a dimer, trimer, and a larger species, type E botulinum neurotoxin exists as a monomer and dimer, and type B botulinum neurotoxin appears to exist as a dimer in aqueous solution. The results imply that quaternary structures of these neurotoxins may play an important role in their mode of action during neuronal poisoning.

Amino Acids↗
PubMed1992

Detection of Clostridium botulinum toxin A using a fiber optic-based biosensor.

A rapid, sensitive, analytical method for the detection of Clostridium botulinum toxin has been developed. The fiber optic-based biosensor utilizes the evanescent wave of a tapered optical fiber for signal discrimination. A 50 mW argon-ion laser, which generates laser light at 514 nm, is used in conjunction with an optical fiber probe that is tapered at the distal end. Antibodies specific for C. botulinum are covalently attached to the surface of the tapered fiber. The principle of the system is a sandwich immunoassay using rhodamine-labeled polyclonal anti-toxin A immunoglobin G (IgG) antibodies for generation of the specific fluorescent signal. Various anti-toxin antibodies were immobilized to the fibers. Affinity-purified polyclonal horse anti-toxin A antibodies performed better than the IgG fraction from the same horse serum or than the monoclonal anti-toxin A antibody BA11-3. Botulinum toxin could be detected within a minute, at concentrations as low as 5 ng/ml. The reaction was highly specific and no response was observed against tetanus toxin.

Animals↗
PubMed1992

Preliminary examinations on the enterotoxigenicity of isolates of Klebsiella pneumoniae from seafoods.

One hundred and eighty-five seafood samples, consisting of 96 freshwater fish, 37 marine fish, 13 freshwater prawn, 13 marine prawn and 26 molluscs were screened for presence of Klebsiella. Out of these, 12 isolates of Klebsiella were identified, Four K. pneumoniae var. ozaenae were isolated from marine fish samples and eight K. pneumoniae var. pneumoniae, six from freshwater fish and two from freshwater prawns. All 12 isolates were tested for enterotoxigenicity by the vasopermeability factor test in rabbits, the mouse foot pad test, the latex agglutination test and the coagglutination test. One isolate of K. pneumoniae var pneumoniae, isolated from fresh water prawn was found enterotoxigenic.

Animals↗
PubMed1991

Comparative molecular topography of botulinum neurotoxins from Clostridium butyricum and Clostridium botulinum type E.

Production of botulinum-like neurotoxin by a non-Clostridium botulinum organism has profound implications in the epidemiology of the disease botulism. Molecular topography of the approximately 150 kDa neurotoxic protein produced by Clostridium butyricum (strain 5839) and its activation kinetics were examined and compared with a serologically related botulinum neurotoxin produced by C. botulinum type E to further characterize the butyricum neurotoxin. Botulinum neurotoxin was fully activated within 30 min of incubation with trypsin, whereas butyricum neurotoxin achieved maximum activation within 5 min of incubation. Molecular topography of the two neurotoxins was analyzed in terms of secondary structures and the surface accessibilities of the polypeptide domains containing aromatic amino acids. The secondary structure parameters of the butyricum neurotoxin (alpha-helix 22%, beta-sheet 41% and random coil 37%), as estimated from the far ultraviolet circular dichroic spectra, appeared similar to that of botulinum neurotoxin. (Singh, B.R. and DasGupta, B.R., (1989) Mol. Cell. Biochem. 86, 87). Second derivative ultraviolet spectral analysis revealed 37 and 41 Tyr residues exposed on the surface of butyricum and botulinum neurotoxins, respectively, suggesting a differential surface accessibility of polypeptide segments containing Tyr residues. Fluorescent Trp residues in both the botulinum type E and butyricum neurotoxins were in a relatively hydrophobic environment as indicated by the blue-shifted emission maxima (334 nm). About half of the fluorescent Trp residues of both proteins were accessible to acrylamide, a neutral fluorescence quencher, and appeared to be in a similar molecular environment. The ionic surface probe, I-, quenched the Trp fluorescence of botulinum significantly, but not that of butyricum neurotoxin. Thus, a considerable number of fluorescent Trp residues were apparently located on the surface of the botulinum, but not on that of the butyricum neurotoxin. Botulinum and butyricum neurotoxins, indistinguishable by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, migrated differently in the absence of sodium dodecyl sulfate suggesting difference(s) in their surface charge distribution. These results provide the first report of the secondary and tertiary structure parameters of the neurotoxin produced by a non-botulinum species and comparison of the molecular topography of the neurotoxin with the antigenically related botulinum neurotoxin type E.

Animals↗
PubMed1991

Botulinum neurotoxin type A: structure and interaction with the micellar concentration of SDS determined by FT-IR spectroscopy.

Secondary structures of botulinum neurotoxin type A have been determined using Fourier transform infrared spectroscopy in the amide I and amide III frequency regions. Using Fourier self-deconvolution, second derivatization, and curve-fit analysis, the amide I frequency contour was resolved into Gaussian bands at 1678, 1654, 1644, and 1634 cm-1. In the amide III frequency region, several small bands were resolved between 1320 and 1225 cm-1. Assignments of the bands in both amide I and amide III frequency regions to various types of secondary structures and the estimation of spectral band strengths by integrating areas under each band suggested that the neurotoxin contains 29% alpha-helix, 45-49% beta-sheets and 22-26% random coils. These values agreed very well with those determined earlier from CD spectra. The neurotoxin was treated with a micellar concentration of sodium dodecyl sulfate to simulate interaction between the protein and the amphipathic molecules. Sodium dodecyl sulfate micelles induced significant alterations both in the spectral band positions, and their strengths suggest refolding of the neurotoxin polypeptides. However, these changes were not entirely reversible, which could implicate the role of the altered structures in the function of the neurotoxin.

Botulinum Toxins↗