Biomedical subjects
Publications and source records attributed to R K Bhatnagar.
Schwannomas of the nasal cavity and paranasal sinuses are very rare. We report the case of a 54-year-old woman with a schwannoma arising from the nasal septum. We discuss the clinical presentation, differential diagnosis, imaging characteristics and treatment of this rarely encountered lesion.
Rod lens endoscopes provide residents in otolaryngology a means of more accurately identifying the site of bleeding and, when possible, cauterizing the bleeding vessel. Identification of a posterior bleeding point is often difficult and sometimes impossible. Intranasal manipulation for electrocautery is painful, may require general anesthesia, and is associated with complications. We describe a pilot study designed to evaluate selectively packing the bleeding site with Surgicel (oxidized cellulose) to control the hemorrhage without packing the nasal cavity and to reduce patient morbidity and length of stay in the hospital. We describe the technique and present the results of treating 8 patients admitted with acute posterior epistaxis over a 10-month period in 1995-1996.
Vegetative insecticidal protein (VIP) is a class of insecticidal proteins produced by some strains of Bacillus thuringiensis during the vegetative stage of their growth. Unlike delta-endotoxins which are produced as parasporal inclusion bodies within the cell during sporulation, VIP is secreted into the culture medium. Here we report the relocation of the expression of VIP into the mother cell compartment in a manner similar to well-characterized Cry proteins. Relocation of VIP is directed to mother cell by placing its synthesis under sporulation-dependent promoters, BtI and BtII. The insertion of cry preferred transcription termination sequence at the 3(') region and a STAB-SD sequence at the 5(') region of the gene provided stability to the vip transcript and enhanced its yield. The demonstrated expression of VIP within the cells in the form of inclusion bodies would facilitate development of a suitable formulation for the application of this class of insecticidal proteins in the field.
A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.
A highly efficient somatic embryo production and maturation procedure has been developed to regenerate plantlets from cotton ( Gossypium hirsutum). This procedure involves the acceleration of differentiation through manipulations of nutrient and microenvironment conditions. Embryogenic calli, initiated from hypocotyls or cotyledonary leaf sections on MS medium containing 0.1 mg/l 2,4 dichlorophenoxyacetic acid, 0.5 mg/l kinetin, and 3% maltose produced globular-stage somatic embryos when transferred to hormone-free MS medium supplemented with high concentrations of nitrate. Subculture of globular embryos on hormone-free MS medium led to the development of torpedo- and cotyledonary-stage at a low frequency (two to four per plate) with the majority of embryos lacking further growth or entering into the dedifferentiation stage. Significant improvement in embryogenesis (two- to threefold) was achieved when calli were cultured on 1/5-strength MS medium irrespective of stress treatment. However, the frequency of globular embryos developing into normal plantlets improved considerably (20-24 per plate) when cultured on filter paper placed on MS medium. In this procedure, about 33% of globular embryos not only developed into the cotyledonary stage but rooted simultaneously, eliminating a separate rooting step. More than 70% of cotyledonary embryos developed into normal plantlets when cultured on full- strength MS medium containing 0.05 mg/l gibberellic acid.
The objective was to test the hypothesis that dopamine regulates prolactin (PRL) secretion by determining acute changes in catecholamine concentrations in hypophyseal portal blood of cattle, and their relation to peripheral blood concentration of PRL in hypophyseal stalk-transected (HST) and sham-operated controls (SOC). Holstein heifers (606 +/- 21 kg BW; mean +/- SE) were subjected to neurosurgery for 8 h to collect hypophyseal portal blood with a stainless steel cannula designed with a cuff placed under the pituitary stalk and peripheral blood via a jugular vein catheter. PRL plasma concentration was measured by radioimmunoassay, and dopamine and norepinephrine in portal plasma by radioenzymatic assay. During anesthesia before HST or SOC, PRL plasma concentration ranged from 20-40 ng/ml throughout 255 min. PRL abruptly increased and remained above 90 ng/ml after HST compared with a steady decrease to <20 ng/ml in SOC heifers throughout 440 min. Within 5 min after severing the hypophyseal stalk, dopamine in portal blood (>8 ng/ml) was significantly increased (P < 0.05) compared with peripheral blood (<2 ng/ml). Norepinephrine concentration in portal blood was significantly greater (P < 0.05) than in peripheral blood during the first 60 min. The sustained high PRL level in peripheral plasma after severing the hypophyseal stalk stimulated hypothalamic dopamine secretion from hypophyseal portal vessels during the prolonged period of blood collection. Norepinephrine concentration in these cattle was greater in hypophyseal portal than in peripheral blood, implicating both an important hypothalamic source of the catecholamine as well as an adrenal gland contribution during anesthesia.
A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.
The dnaK operon in gram-positive bacteria includes grpE, dnaJ and, in some members, hrcA as well. Both DnaK and DnaJ have been utilized for constructing phylogenetic relationships among various organisms. Multiple copies exist for dnaK and dnaJ genes in some bacterial genera, as opposed to a single gene copy for grpE and for hrcA, according to the currently available data. Here, we present a partial protein-based phylogenetic tree for gram-positive bacteria, derived by using the amino acid sequence identity of GrpE; the results are compared with the phylogenetic trees generated from 5S rRNA, 16S rRNA, dnaK and dnaJ sequences. Our results indicate three main groupings: two are within low-G+C DNA gram-positive bacteria comprising Bacillus species and Staphylococcus aureus on the one hand and Streptococcus species/Lactococcus lactis/Enterococcus faecalis/Lactobacillus sakei on the other hand; the Mycobacterium species and Streptomyces coelicolor, belonging to the high-G+C DNA gram-positive bacteria, form the third cluster. This hierarchical arrangement is in close agreement with that obtained with 16S rRNA and DnaK sequences but not DnaJ-based phylogeny.
The dnaK gene region of Bacillus sphaericus was cloned as a 3.8 kb HindIII fragment and an overlapping 1.7 kb EcoRI fragment by using an internal B. sphaericus specific dnaK gene probe generated by polymerase chain reaction (PCR). Complete DNA sequencing of the two fragments revealed three complete open reading frames (ORFs). These ORFs exhibited a high degree of identity to the grpE dnaK, and dnaJ heat shock genes from other gram-positive bacteria. The order of the genes was found to be grpE-dnaK-dnaJ. Additionally, the 5'-end and 3'-end contained amino acid sequences that were homologous to the C-terminal sequence of the hrcA gene and the N-terminal sequence of ORF35 (yqeT), respectively, from Bacillus subtilis. The entire hrcA gene from B. sphaericus was then isolated by high-fidelity PCR and completely sequenced. A transcription stop site is located between the dnaK and dnaJ genes but not after the dnaJ gene. Consistent with this observation, the dnaJ gene is immediately followed by an ORF that shows a high degree of identity to ORF35 from B. subtilis, Staphylococcus aureus, and Clostridium acetobutylicum. The presence of ORF35 is not indicated in other genera representing the gram-positive bacteria. The amino acid sequence of ORF35 exhibited nearly 30% identity with the methyltransferase for large subunit ribosomal protein L11 from gram-negative Proteobacteria and the related protein from cyanobacteria, other gram-negative bacteria, and Archaea, suggesting the presence of the gene for this protein in the common ancestor of Bacteria and Archaea. The absence of the ORF35 gene in Mycobacterium tuberculosis and other gram-positive bacteria indicates that the loss of this gene must have occurred in an ancestor of other gram-positive bacteria following their divergence from the ancestor of Bacillus/Clostridium/staphylococcus lineage.
The dnaK operon from Bacillus subtilis and other Gram-positive bacteria with low G + C DNA content contains additional heat-shock genes, including hrcA. The hrcA gene encodes a transcription factor that negatively regulates heat-shock genes and is uniformly present in all Gram-positive bacteria studied to date. An hrcA homologue is also present in Synechocystis species, Leptospira interrogans, Chlamydia trachomatis, Caulobacter crescentus and Methanococcus jannaschii, organisms that diverged early on from the common ancestor of all Gram-positive bacteria and Proteobacteria, according to 16S rRNA phylogeny. A partial, protein-based phylogenetic tree, derived using amino acid sequence homology of hrcA proteins from Gram-positive bacteria, is presented here, and the results are compared with the phylogenetic trees generated from 16S rRNA, dnaK and dnaJ sequences. The location of the hrcA gene and the genome organization of the dnaK operon support the division of all Gram-positive bacteria into three major groups: one group contains high-G + C Gram-positive bacteria, and two others contain low-G + C Gram-positive bacteria. Among the Gram-positive bacteria with low G + C DNA content, the results indicate that there is a close phylogenetic relationship between Bacillus species and Clostridium species on the one hand and between Lactococcus lactis and Streptococcus mutans on the other. Streptomyces and Mycobacterium species also exhibited a close relationship. A hierarchical arrangement of Gram-positive bacteria based on HrcA sequences is proposed as an additional refinement of the phylogenetic relationships within this important bacterial group.
The binary mosquitocidal genes of 51-kDa and 42-kDa proteins isolated from Bacillus sphaericus 1593 have been expressed at moderate levels in Escherichia coli employing the pQE expression system. The expressed proteins are readily visible in Coomassie blue-stained protein gels. The recombinant E. coli cells expressing toxic proteins were toxic towards Culex larvae. During the assembly of crystals in B. sphaericus, the 42-kDa toxin is first cleaved at the N-terminal end by a specific B. sphaericus protease. To express the toxins in E. coli the B. sphaericus specific protease-recognition site was deleted at the N-terminal end of the 42-kDa toxin, thereby mimicking the structure of the toxin as present in the crystal. This modification resulted in a twofold increase in the toxicity of the E. coli cells expressing the modified 42-kDa toxin as a constituent of the binary toxin. Our results demonstrate the utility of this modification for heterologous expression of the binary toxin genes from B. sphaericus.
The display of proteins or peptides on the surface of filamentous phages or phagemids has been shown to be a very powerful technology for the rescue of specific binders from large combinatorial libraries, as well as to select derivatives of known proteins with altered binding properties. The Bacillus thuringiensis (Bt) crystal proteins are a large family of insecticidal toxins which bind to receptors found on the brush border of larval midgut cells, different crystal toxins having different larval specificities. Here we describe the display of different CryIA(a) toxin regions on the surface of phagemids using the display vector pHEN1, the purpose being the identification of toxin sequences suitable for mutagenesis and selection using phage display. We show that CryIA(a) domain II, in which the receptor binding activity is located, is efficiently displayed as well as being secreted as soluble protein into the periplasm of bacterial cell. This forms the basis of a simple means for the modification of toxin specificity and the selection of toxin proteins with novel or expanded host ranges.
The role of basic amino acid residues that are highly conserved in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPs) in the binding of the substrate, shikimate-3-phosphate, has been assessed. Lys 19 and Arg 24 in the Bacillus subtilis EPSPs were substituted by glutamic acid and aspartic acid residues respectively by site-directed mutagenesis. Native and the mutant proteins were expressed using a two-vector system and the expressed proteins were purified to near homogeniety. The replacement of either Lys 19 or Arg 24 with a negatively charged residue nearly completely abolished the enzyme activity. The kinetic characterization of the purified wild type and the mutant proteins revealed that the substitution of positively charged residues in the N-terminal domain (K19 and R24) results in reduced affinity for shikimate-3-phosphate (S3P). The results suggest the involvement of these residues in the binding of S3P during enzyme catalysis.
Alport's syndrome (hereditary nephritis with deafness), is an uncommon disease and is seen very infrequently in India. We report a fatal case in a young girl with characteristic ultrastructural changes in the kidney thus emphasising the exception noted in the observation that females have a better prognosis compared to males.
The role of a conserved arginine (R104) in the putative phosphoenol pyruvate binding region of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis has been investigated. Employing site directed mutagenesis arginine was substituted by lysine or glutamine. Native and mutant proteins were expressed and purified to near homogeneity. Estimation of Michaelis and inhibitor constants of the native and mutant proteins exhibited altered substrate-inhibitor binding mode and constants. Mutation R104K hypersensitized the enzyme reaction to inhibition by glyphosate. The role of R104 in discriminating between glyphosate and phosphoenol pyruvate is discussed.
Explore the source record for details and available documents.
5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis has been cloned, expressed and purified to near homogeneity. Clustal alignment of the amino acid sequences from different bacteria revealed several conserved residues located in the N-terminal, middle and C-terminal domains. The role of conserved Arg24, Pro105, and His385 residues has been examined by site-directed mutagenesis. Steady-state kinetic analysis of the native synthase exhibited allosteric behaviour, a feature thought to be unique amongst bacterial and plant 5-enolpyruvylshikimate-3-phosphate synthase enzymes investigated so far. Both substrates, phosphoenolpyruvate (P-pyruvate) and shikimate 3-phosphate have multiple interaction sites. There are two sites for P-pyruvate binding, catalytic and non-catalytic. Glyphosate (N-phosphonomethyl glycine) competes for binding at the catalytic site and does not interact at the secondary site. Glyphosate in the absence of ammonium ions increases cooperativity of P-pyruvate binding and favors dimerization of the enzyme through an interaction between P-pyruvate-binding sites. The ammonium-ion-activated 5-enolpyruvylshikimate-3-phosphate synthase displays no cooperativity with respect to P-pyruvate. Absence of ammonium ions decreases affinity for substrates and introduces cooperativity. Cooperativity was also introduced in the enzyme by point mutations, Arg24-->Asp and His385-->Lys. The latter mutant of the native enzyme exists as a dimer and aggregates to a tetrameric form in the presence of glyphosate. The occurrence of multimeric forms of the synthase has been demonstrated by staining for the enzyme activity on the native gel and by resolving purified enzyme preparations on a sucrose density gradient. A model describing the alteration in the aggregation status of the enzyme by the inhibitor, activator and the substrates has been proposed.
An efficient protocol, termed background-minimized cassette mutagenesis (BMCM) by PCR, has been developed for multiple mutagenesis of DNA. This method uses suitable extension primers for incorporating various mutation(s) and is not limited by either the nature of the mutation or the size and spatial location of mutational loci. Minimization of the wild type background clone and mutant selection at very high frequency were easily achieved through a two-step process. First, a deletion of a unique restriction site within the cassette was introduced through additional silent mutation(s). Then, the recombinant clones were digested with the corresponding enzyme followed by transformation when selective linearization of wild-type clone led to its near total removal leaving the mutant clones as the only practicable transformants. Because it is generally possible to design several such cassette-specific unique background minimization markers for any gene, for multiple mutagenesis involving distally located portions of the gene the present protocol is superior to other currently available methods. The efficiency of BMCM-PCR has been demonstrated here by using the multisubstrate enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs) of Bacillus subtilis as a model system. Three different sets of cassettes of varying sizes were generated to encompass the three putative active/binding regions in the beginning, middle, and the end of the gene encoding EPSPs. Very high efficiency of mutation incorporation and selection were obtained in all cases. Furthermore, by taking advantage of the unique cassette-specific background elimination markers, it was possible to generate a nested set of double and/or triple mutants. The mutant enzymes were overexpressed in Escherichia coli and purified to near homogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)