Biomedical subjects

N Arora

Publications and source records attributed to N Arora.

At least 55 records · Page 3Linked to original sources

PubMed1996

Evidence for the shikimate-3-phosphate interacting site in the N-terminal domain of 5-enolpyruvyl shikimate-3-phosphate synthase of Bacillus subtilis.

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.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1996

Argyrophilic nucleolar organizer regions in soft tissue tumors.

Argyrophilic nucleolar organizer region (AgNOR) staining was employed on 51 apparently normal representative soft tissues, 53 benign soft tissues tumors and 52 malignant soft tissue tumors with an aim to study the sensitivity and specificity of method in differentiating between the benign and malignant soft tissue tumors. The mean AgNORs count in apparently normal fibrous tissue was 1.02, whereas it was 0.94 in adipose tissue, 1.14 in smooth muscle tissue, 1.115 in skeletal muscle tissue, 1.025 in blood vessels endothelial lining cells and 1.04 in nerve tissue. The mean AgNOR count was found to be higher in benign soft tissue tumors as compared to respective apparently normal soft tissue and was found to be statistically significant. The mean AgNOR count in soft tissue sarcomas was found to be higher as compared to both apparently normal soft tissue and benign soft tissue tumors. An increase AgNOR score in both benign and malignant soft tissue tumors as compared to apparently normal soft tissue indicates high proliferative activity. The neurofibrosarcoma showed low AgNOR count as compared to other soft tissues sarcomas. The fibrohistiocytic sarcoma, leiomyosarcoma and angiosarcoma showed a mean AgNOR score of 4 or more than four. The mean AgNOR score was found to increase with high grade of the tumor. The AgNOR staining is simple and useful method in estimating tumor cell proliferation thereby differentiating normal soft tissue from non-neoplastic proliferative growth, benign and malignant soft tissue tumors. It may help in differentiating fibromatosis from fibrosarcoma, dermatofibrosarcoma protuberans of low grade malignancy from high grade malignant fibrous histiocytoma and benign hemangiopericytoma from malignant hemangiopericytoma.

Adult↗

PubMed1995

Point mutation of a conserved arginine (104) to lysine introduces hypersensitivity to inhibition by glyphosate in the 5-enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis.

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.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1995

5-Enolpyruvylshikimate-3-phosphate synthase of Bacillus subtilis is an allosteric enzyme. Analysis of Arg24-->Asp, Pro105-->Ser and His385-->Lys mutations suggests a hidden phosphoenolpyruvate-binding site.

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.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1995

Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases.

Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC50] = 12 ng/ml) > or = RAAR (EC50 = 18 ng/ml) > FTKR (EC50 = 24 ng/ml) > STRR (EC50 = 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC50 = 2.9 ng/ml) and PA (EC50 = 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA.

ADP Ribose Transferases↗

PubMed1994

The chymotrypsin-sensitive site, FFD315, in anthrax toxin protective antigen is required for translocation of lethal factor.

The protective antigen (PA) component of anthrax toxin contains two sites that are uniquely sensitive to proteolytic cleavage. Cleavage at the sequence RKKR167 by the cellular protease furin is absolutely required for toxicity, whereas cleavage by chymotrypsin or thermolysin at the sequence FFD315 inactivates the protein, apparently by blocking the ability of PA to translocate the catalytic moieties of the toxins, lethal factor (LF) and edema factor (EF), to the cytosol of eukaryotic cells. To specify the role of the chymotrypsin-sensitive site of PA in the translocation of LF, we altered residues 313-315. None of the mutations in this region interfered with the ability of PA to bind to its cellular receptor, be cleaved by cell surface furin, and bind LF. Substitution of Ala for Asp315 or for both Phe313 and Phe314 reduced the ability of PA to intoxicate cells in the presence of LF by 3- and 7-fold, respectively. Substitution of Phe313 by Cys greatly reduced the rate of LF translocation and delayed toxicity. The rate at which the Cys-substituted PA killed cells was increased significantly by blocking the sulfhydryl group with iodoacetamide, suggesting that this added Cys interacts with cellular proteins and slows translocation of LF. Deletion of the 2 Phe rendered PA completely non-toxic. This deleted PA protein lacked the ability shown by native PA to form oligomers on cells and in solution and to induce release of 86Rb from Chinese hamster ovary cells. These results suggest that the chymotrypsin-sensitive site in PA is required for membrane channel formation and translocation of LF into the cytosol. PA double mutants were constructed that cannot be cleaved at either the furin or chymotrypsin sites. These PA proteins were more stable in Bacillus anthracis culture supernatants and may therefore be useful as a replacement for PA in anthrax vaccines.

Amino Acid Sequence↗

PubMed1994

Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells.

The light chain of tetanus toxin is a zinc endoprotease that inhibits neurotransmitter release by selective proteolysis of the synaptic vesicle-associated protein synaptobrevin/vesicle-associated membrane protein. Cellubrevin is a homologue of synaptobrevin that is found in most cell types and is also a substrate for tetanus toxin. The lack of receptors for tetanus toxin on most cell types has made studies of tetanus toxin action in non-neuronal cells difficult. To characterize tetanus toxin effects in non-neuronal cells, a fusion protein consisting of the 254 amino-terminal amino acids of lethal factor (LF) of anthrax toxin and tetanus toxin light chain (LC) was prepared. This protein (LF-LC) inhibited evoked glycine release from primary spinal cord neurons at concentrations between 1.0 and 100 ng/ml. LF-LC was cytotoxic to RAW 264.7, ANA-1 cells (mouse macrophage cell lines), and Chinese hamster ovary cells in a dose-dependent manner. These effects required the presence of protective antigen, the receptor binding component of anthrax toxin. In contrast, LF-LC was not cytotoxic to RBL-2H3, Vero, or mouse hybridoma cell lines. Mutagenesis of conserved amino acids (His237 and Glu234) in the zinc-binding motif of LC resulted in fusion proteins having no biological activity. LF-LC did not inhibit regulated secretion of serotonin in RBL-2H3 cells or constitutive secretion in any non-neuronal cell lines as measured in several different assays. We suggest that the cytotoxic effects of LF-LC result from inhibition of a specific intracellular membrane fusion event mediated by cellubrevin.

Animals↗

PubMed1994

Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity.

Comparison of the anthrax toxin lethal factor (LF) amino acid sequence with sequences in the Swiss protein database revealed short regions of similarity with the consensus zinc-binding site, HEXXH, that is characteristic of metalloproteases. Several protease inhibitors, including bestatin and captopril, prevented intoxication of macrophages by lethal toxin. LF was fully inactivated by site-directed mutagenesis that substituted Ala for either of the residues (H-686 and H-690) implicated in zinc binding. Similarly, LF was inactivated by substitution of Cys for E-687, which is thought to be an essential part of the catalytic site. In contrast, replacement of E-720 and E-721 with Ala had no effect on LF activity. LF bound 65Zn both in solution and on protein blots. The 65Zn binding was reduced for several of the LF mutants. These data suggest that anthrax toxin LF is a zinc metallopeptidase, the catalytic function of which is responsible for the lethal activity observed in cultured cells and in animals.

Amino Acid Sequence↗

PubMed1994

Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells.

To investigate the ability of anthrax toxin lethal factor (LF) to translocate foreign proteins into the cytosol of eukaryotic cells and to characterize the structural requirements of this process, fusion proteins containing a portion of LF and the catalytic domains of either diphtheria toxin or Shiga toxin were constructed. Previous work showed that residues 1 to 254 of anthrax toxin lethal factor (LF1-254) are sufficient for binding to the protective antigen component of the toxin and that portions of Pseudomonas exotoxin A fused to LF1-254 are efficiently translocated to the cytosol of eukaryotic cells (N. Arora and S. H. Leppla, J. Biol. Chem. 268:3334-3341, 1993). In this study, it was found that fusion proteins containing the ADP-ribosylation domain of diphtheria toxin fused at either the amino end or the carboxyl end of LF1-254 are highly toxic to Chinese hamster ovary (CHO) cells, indicating that translocation does not strictly require that the amino terminus of LF be free. A fusion protein containing the ribosome-inactivating A1 subunit of Shiga toxin fused to the carboxyl terminus of LF1-254 was also highly toxic for CHO cells. All fusion proteins were toxic only when administered with the anthrax toxin protective antigen component. The data show that the combination of protective antigen and LF fusion proteins can efficiently import polypeptides from diverse bacterial sources to the cytosol of eukaryotic cells and that LF fusion proteins may have the passenger polypeptides fused at either the amino terminus or the carboxyl terminus of LF1-254. These LF fusion proteins could potentially be used as components of a therapeutic agent when the destruction of certain types of cells is desired (e.g., in treating cancer).

Amino Acid Sequence↗

PubMed1993

Residues 1-254 of anthrax toxin lethal factor are sufficient to cause cellular uptake of fused polypeptides.

Anthrax lethal toxin is a complex of protective antigen (PA, 735 amino acids) and lethal factor (LF, 776 amino acids) that lyses certain eukaryotic cells. LF interacts with PA to gain access to the cytosol to assert its toxicity. The internalization of LF requires that PA bind to a specific membrane receptor and be cleaved by a cell-surface protease (probably furin), so as to expose a site on PA to which LF binds with high affinity. To localize LF functional domains, amino, carboxyl, and internal deletions of LF were made. Toxicity was eliminated by deletion of 40 and 47 residues from the amino and carboxyl termini, respectively. Similarly, deleting the first of the four imperfect repeats of 19 amino acids located at residues 308-383 made LF non-toxic, showing that this region is also essential for activity. To identify the minimum region of LF which is required for binding to PA, varying amino-terminal portions of LF were fused to the ADP-ribosylation domain of Pseudomonas exotoxin A. Fusion proteins containing residues 1-254 of LF were toxic when administered with PA, while those having only residues 1-198 of LF were inactive, showing that the PA-binding domain of LF lies within residues 1-254.

ADP Ribose Transferases↗

PubMed1993

Saliva-binding protein (SsaB) from Streptococcus sanguis 12 is a lipoprotein.

Two lipoprotein consensus sequences (Leu-X-X-Cys) are found in the presumptive signal peptide region (positions 12 to 15 and 17 to 20) of saliva-binding protein (SsaB) from Streptococcus sanguis 12. Three analogs of SsaB containing Cys-->Gly mutations were constructed by site-directed mutagenesis of pSA2, the recombinant plasmid expressing SsaB. [3H]palmitate was incorporated into SsaB only when the native Cys-20 residue was present. These data show that SsaB is a lipoprotein and that Cys-20 is the critical site for acylation.

Adhesins, Bacterial↗

PubMed1993

Immortalization of mouse bone marrow-derived mast cells with Ad12-SV40 virus.

Mast cells arise in cultures of murine bone marrow in medium supplemented with interleukin-3 (IL-3). In the present study, we report the development of long-term mast cell lines from murine bone-marrow-derived cultured mast cells (BMCMC) following inoculation with adenovirus 12-simian virus 40 (Ad12-SV40) hybrid virus. One culture of Ad12-SV40 immortalized BMCMC (designated as MCP-5) was selected for further analysis. These transformed cells appear similar in morphology and histochemistry to the primary BMCMC from which they are derived and did not shed infectious virus into the culture supernatants. In addition, these cells synthesize predominantly chondroitin sulfate proteoglycans and contain histamine which is released following a physiologic stimulus. Limiting-dilution single-cell cloning produced five independent mast cell lines (MCP-5.1 to MCP-5.5). Southern blot analysis of genomic DNA isolated from these single-cell clones demonstrates different patterns of viral integration in all the five clones. All clones retain responsiveness to an exogenous source of IL-3 for growth and proliferation. Each single-cell clone also demonstrates a unique pattern of cytokine gene expression in response to calcium ionophore A23187 and phorbol-12-myristate-13-acetate. This suggests that within a culture of BMCMC there are differences in cytokine gene expression that vary from one cell to another. The availability of immortalized mast cell lines derived from murine bone marrow which retain their growth factor responsiveness and the ability to respond to degranulating stimuli should facilitate future studies of mast cell biology.

Adenoviruses, Human↗

PubMed1993

Serum inorganic phosphate levels in experimental intestinal ischaemia.

Serial serum inorganic phosphate estimates were done following production of experimental intestinal ischaemia in dogs by occlusion of superior mesenteric artery (10 dogs) or vein (10 dogs) and compared to phosphate values in control dogs (5 for each experimental group). Elevated phosphate values were seen within one hour after ischaemia. Elevation was more marked following arterial occlusion, becoming statistically significant within 2 hours. It is suggested that serum inorganic phosphate elevation can be used as important laboratory test in early diagnosis of acute intestinal ischaemia.

Animals↗

PubMed1992

Fusions of anthrax toxin lethal factor to the ADP-ribosylation domain of Pseudomonas exotoxin A are potent cytotoxins which are translocated to the cytosol of mammalian cells.

The lethal factor (LF) and edema factor (EF) components of anthrax toxin are toxic to animal cells only if internalized by interaction with the protective antigen (PA) component. PA binds to a cell surface receptor and is proteolytically cleaved to expose a binding site for LF and EF. To study how LF and EF are internalized and trafficked within cells, LF was fused to the translocation and ADP-ribosylation domains (domains II and III, respectively) of Pseudomonas exotoxin A. LF fusion proteins containing Pseudomonas exotoxin A domains II and III were less toxic than those containing only domain III. Fusion proteins with a functional endoplasmic reticulum retention sequence, REDLK, at the carboxyl terminus of domain III were less toxic than those with a nonfunctional sequence, LDER. The most potent fusion protein, FP33, had an EC50 = 2 pM on Chinese hamster ovary cells, exceeding that of native Pseudomonas exotoxin A (EC50 = 420 pM). Toxicity of all the fusion proteins required the presence of PA and was blocked by monensin. These data suggest that LF and LF fusion proteins are efficiently translocated from acidified endosomes directly to the cytosol without trafficking through other organelles, as is required for Pseudomonas exotoxin A. This system provides a potential vehicle for importing diverse proteins into the cytosol of mammalian cells.

ADP Ribose Transferases↗

PubMed1992

Passive cutaneous anaphylaxis in mouse skin is associated with local accumulation of interleukin-6 mRNA and immunoreactive interleukin-6 protein.

We used a BALB/c model of passive cutaneous anaphylaxis (PCA), an IgE-mediated, mast cell-dependent reaction, to demonstrate the early production of the proinflammatory cytokine interleukin-6 (IL-6) mRNA and protein product. Northern blot analysis detects IL-6 mRNA 1, and 2 hours after antigen challenge (dinitrophenyl30-40 human serum albumin [DNP30-40-HSA]) and in situ hybridization reveals that it is primarily cells with round-to-oval nuclei within the dermis (1 to 3 per high-power field) expressing IL-6 mRNA. Immunohistochemistry revealed perinuclear and cytoplasmic staining for immunoreactive IL-6 in mononuclear dermal cells and also cells within the basal keratinocyte layer. Injection of recombinant murine IL-6 (rmIL-6) either systemically or locally during antidinitrophenyl IgE skin sensitization resulted in increased vasopermeability at the PCA site after DNP30-40-HSA. However, this increased permeability was not associated with a change in the character of the cellular infiltrate at the PCA site 8 hours later. Although the specific role of IL-6 in the generation of the allergic response remains unknown, its detection during PCA unequivocally demonstrates that IL-6 be considered one of the mediators identified in inflammation that follows allergic reactions.

Animals↗

PubMed1992

Efficacy of liposome entrapped allergen in down regulation of IgE response in mice.

The effect of liposome entrapped allergen on primary, secondary and ongoing IgE response was studied in mice. It was observed that mice primed with liposome entrapped allergen maintained significantly lower levels of serum specific IgE as compared to that of controls primed with allergen. Although alum adsorbed allergen could induce IgE synthesis in mice primed with liposome entrapped allergen the increase in serum specific IgE levels was lower than the animals primed and challenged with alum adsorbed allergen. On the contrary when BALB/c mice were primed with alum adsorbed allergen to induce IgE synthesis subsequent challenge by liposome entrapped allergen could down regulate the serum specific IgE response. Serum specific IgG response did not show any significant difference between the two groups. There was an increase in T suppressor cell subpopulation as measured by immunofluorescence technique in mice injected liposome entrapped allergen as compared to that in controls. The results indicate that liposome entrapped allergens may prove to be safe and effective in immunotherapy by reducing the allergenic response of the allergen at the same time increasing the antigenicity.

Allergens↗

PubMed1992

Performance characteristics of contemporary hemodialysis and venovenous hemofiltration in acute renal failure.

UNLABELLED: Modality choice in the treatment of acute renal failure (ARF) should be based on the match between individual patient needs and the characteristics of available therapies. Considerations include access, risk of bleeding, hemodynamic instability, and ability to remove excess volume. We prospectively studied 547 consecutive treatments for ARF in 110 patients to determine the performance features of modalities based on single-vessel venous access: hemodialysis (HD) and venovenous hemofiltration (VVH). All treatments were performed in an 18-month period at a single center. Patients' ages ranged from 16 to 84 years; 26 were trauma cases; 69 patients expired during their hospitalization. Mean number of treatments per patient was 4.97. Mean treatment duration was 197 min. Heparin was used unless active bleeding was present or felt to be a high risk; mean dose required was 2628 units. Pressor therapy was in progress prior to initiation of 260 (48%) of treatments. RESULTS: pretreatment and posttreatment mean systolic and diastolic BP were unchanged for the group. Forty-three (7.9%) treatments were terminated prematurely; of these only 27 (4.9%) were due to hypotension. No patient developed clinically apparent bleeding during any treatment. Contemporary equipment and techniques allow for provision of high-quality intermittent therapy for ARF, with excellent hemodynamic stability. Shorter, single-vein access treatments are advantageous for severely ill or injured patients who often undergo invasive monitoring and multiple studies or procedures.

Acute Kidney Injury↗

PubMed1992

Marked improvement in parameters of renal osteodystrophy with the use of intraperitoneal calcitriol.

Parenteral administration of Calcitriol is felt to be superior to oral Calcitriol in the treatment of renal osteodystrophy in chronic renal failure patients. We analyzed the results of serum calcium, phosphorus, alkaline phosphatase, and N-terminal parathormone level which are the clinical parameters of renal osteodystrophy in twenty-three chronic peritoneal dialysis patients who received varying dosages of intraperitoneal Calcitriol. The results which were analyzed at the end of one to twenty-three months revealed significant increase in serum calcium and a significant decrease in the values of alkaline phosphatase and N-terminal parathormone level.

Alkaline Phosphatase↗