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Biomedical subjects

A Nandi

Publications and source records attributed to A Nandi.

At least 19 recordsLinked to original sources
PubMed2004

Molecular remission of CML after autotransplantation followed by adoptive transfer of costimulated autologous T cells.

Four patients with chronic myelogenous leukemia (CML) that was refractory to interferon alpha (two patients) or imatinib mesylate (two patients), and who lacked donors for allogeneic stem cell transplantation, received autotransplants followed by infusions of ex vivo costimulated autologous T cells. At day +30 (about 14 days after T-cell infusion), the mean CD4+ cell count was 481 cells/microl (range 270-834) and the mean CD8+ count was 516 cells/microl (range 173-1261). One patient had a relative lymphocytosis at 3.5 months after T-cell infusion, with CD4 and CD8 levels of 750 and 1985 cells/microl, respectively. All the four patients had complete cytogenetic remissions early after transplantation, three of whom also became PCR negative for the bcr/abl fusion mRNA. One patient, who had experienced progressive CML while on interferon alpha therapy, became PCR- post transplant, and remained in a molecular CR at 3.0 years of follow-up. All the four patients survived at 6, 9, 40, and 44 months post transplant; the patient who remained PCR+ had a cytogenetic and hematologic relapse of CML, but entered a molecular remission on imatinib. Autotransplantation followed by costimulated autologous T cells is feasible for patients with chronic phase CML, who lack allogeneic donors and can be associated with molecular remissions.

Adoptive Transfer↗
PubMed2001

A recessive mutation in the Arabidopsis SSI2 gene confers SA- and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens.

The Arabidopsis thaliana NPR1 gene is required for salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance. However, loss-of-function mutations in NPR1 do not confer complete loss of PR gene expression or disease resistance. Thus these responses also can be activated via an NPR1-independent pathway that currently remain to be elucidated. The ssi2-1 mutant, identified in a genetic screen for suppressors of npr1-5, affects signaling through the NPR1-independent defense pathway(s). In comparison with the wild-type (SSI2 NPR1) plants and the npr1-5 mutant (SSI2 npr1-5), the ssi2-1 npr1-5 double mutant and the ssi2-1 NPR1 single mutant constitutively express PR genes [PR-1, BGL2 (PR-2) and PR-5]; accumulate elevated levels of SA; spontaneously develop lesions; and possess enhanced resistance to a virulent strain of Peronospora parasitica. The ssi2-1 mutation also confers enhanced resistance to Pseudomonas syringae pv. tomato (Pst); however, this is accomplished primarily via an NPR1-dependent pathway. Analysis of ssi2-1 NPR1 nahG and ssi2-1 npr1-5 nahG plants revealed that elevated SA levels were not essential for the ssi2-1-conferred phenotypes. However, expression of the nahG transgene did reduce the intensity of some ssi2-1-conferred phenotypes, including PR-1 expression, and disease resistance. Based on these results, SSI2 or an SSI2-generated signal appears to modulate signaling of an SA-dependent, NPR1-independent defense pathway, or an SA- and NPR1-independent defense pathway.

Arabidopsis↗
PubMed2000

Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44.

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-alpha stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation.

Animals↗
PubMed2000

Expression of GC-C, a receptor-guanylate cyclase, and its endogenous ligands uroguanylin and guanylin along the rostrocaudal axis of the intestine.

Members of the receptor-guanylate cyclase (rGC) family possess an intracellular catalytic domain that is regulated by an extracellular receptor domain. GC-C, an intestinally expressed rGC, was initially cloned by homology as an orphan receptor. The search for its ligands has yielded three candidates: STa (a bacterial toxin that causes traveler's diarrhea) and the endogenous peptides uroguanylin and guanylin. Here, by performing Northern and Western blots, and by measuring [125I]STa binding and STa-dependent elevation of cGMP levels, we investigate whether the distribution of GC-C matches that of its endogenous ligands in the rat intestine. We establish that 1) uroguanylin is essentially restricted to small bowel; 2) guanylin is very low in proximal small bowel, increasing to prominent levels in distal small bowel and throughout colon; 3) GC-C messenger RNA and STa-binding sites are uniformly expressed throughout the intestine; and 4) GC-C-mediated cGMP synthesis peaks at the proximal and distal extremes of the intestine (duodenum and colon), but is nearly absent in the middle (ileum). These observations suggest that GC-C's activity may be posttranslationally regulated, demonstrate that the distribution of GC-C is appropriate to mediate the actions of both uroguanylin and guanylin, and help to refine current hypotheses about the physiological role(s) of these peptides.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Thermodynamic analyses reveal role of water release in epitope recognition by a monoclonal antibody against the human guanylyl cyclase C receptor.

The thermodynamics of a monoclonal antibody (mAb)-peptide interaction have been characterized by isothermal titration microcalorimetry. GCC:B10 mAb, generated against human guanylyl cyclase C, a membrane-associated receptor and a potential marker for metastatic colon cancer, recognizes the cognate peptide epitope HIPPENIFPLE and its two contiguous mimotopes, HIPPEN and ENIFPLE, specifically and reversibly. The exothermic binding reactions between 6.4 and 42 degrees C are driven by dominant favorable enthalpic contributions between 20 and 42 degrees C, with a large negative heat capacity (DeltaC(p)) of -421 +/- 27 cal mol(-1) K(-1). The unfavorable negative value of entropy (DeltaS(b)(0)) at 25 degrees C, an unusual feature among protein-protein interactions, becomes a positive one below an inversion temperature of 20.5 degrees C. Enthalpy-entropy compensation due to solvent reorganization accounts for an essentially unchanged free energy of interaction (DeltaDeltaG(b)(0) congruent with 0). The role of water molecules in the recognition process was tested by coupling an osmotic stress technique with isothermal titration microcalorimetry. The results provide direct and compelling evidence that GCC:B10 mAb recognizes the peptides HIPPENIFPLE, HIPPEN, and ENIFPLE differentially, with a concomitant release of variable and nonadditive numbers of water molecules (15, 7, and 3, respectively) from the vicinity of the binding site.

Antibodies, Monoclonal↗
PubMed1999

Interleukin 15 induces endothelial hyaluronan expression in vitro and promotes activated T cell extravasation through a CD44-dependent pathway in vivo.

T cell recruitment to extralymphoid tissues is fundamental to the initiation and perpetuation of the inflammatory state during immune and autoimmune responses. Interleukin (IL)-15 is a proinflammatory cytokine whose described functions largely overlap with those of IL-2. The latter is attributable in large part to its binding of the heterotrimeric receptor that contains the beta and gamma chains of the IL-2R in combination with an unique IL-15R alpha chain. However, unlike IL-2, IL-15 and its receptor have a wide tissue and cell type distribution, including endothelial cells. Here, we examine the effect of IL-15 on hyaluronan expression by endothelial cells, and investigate its role in vivo in promoting the extravasation of antigen-activated T cells through a CD44-dependent pathway. The expression of hyaluronan on primary endothelial cells and microvascular endothelial cell lines is induced by IL-15, whereas IL-2 has no such activity. Moreover, intraperitoneal administration of IL-15 or TNF-alpha in the absence of other exogenous proinflammatory stimuli allows the extravasation of superantigen-stimulated T cells into this site in vivo in a CD44-dependent manner. T cell recruitment induced by IL-15 requires expression of an intact IL-2R beta chain, indicating that IL-15 operates in this context through the traditional IL-15R. The results suggest that IL-15 can regulate endothelial cell function and thereby enables a CD44-initiated adhesion pathway that facilitates entry of activated T lymphocytes into inflammatory sites. They further demonstrate a novel role for IL-15 (distinct from any of IL-2) in regulating microvascular endothelial cell adhesive function help to understand the role of IL-15R expression on endothelium, and further support a central position for this cytokine in orchestrating multiple sequential aspects of T cell effector function and therefore chronic inflammatory processes.

Animals↗
PubMed1998

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor.

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.

Amino Acid Sequence↗
PubMed1998

Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.

Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

Adenosine Triphosphate↗
PubMed1997

ATPase gene transfer and mutational analysis of the cation translocation mechanism.

The peptide segment interposed between cation binding and phosphorylation domains retains a high degree of homology in all cation transport ATPases. Mutational analysis and chimeric replacements of Ca2+ ATPase components with corresponding Na+,K(+)-ATPase components indicate that this segment is utilized by various cation ATPases as a common structural device for a long-range functional linkage of enzyme phosphorylation and cation transport. Vectorial displacement of bound cation is rendered possible by a transmembrane channel formed by four clustered helices (M4, M5, M6, and M8). Originating from the four helices, the oxygen functions of Glu309, Glu771, Thr799, Asp800, and Glu908 form a duplex Ca2+ binding site in the middle of the channel, while Lys297 seals the luminal end of the channel with its positively charged side chain. The perturbation triggered by enzyme phosphorylation is apparently transmitted through the linkage segment to produce rotational displacement of the M4 helix with minimal change of secondary structure. The cation binding site is thereby disrupted and the Lys297 side chain removed, permitting Ca2+ to dissociate in exchange for H+ and to flow through the luminal end of the channel.

Amino Acid Sequence↗
PubMed1997

Epitope conservation and immunohistochemical localization of the guanylin/stable toxin peptide receptor, guanylyl cyclase C.

The heat-stable enterotoxins (ST) are a family of cysteine-rich low-molecular weight peptides produced by pathogenic bacteria, and are one of the major causes of watery diarrhea all over the world. These toxins mediate their action by binding to an intestinal cell surface receptor that is a membrane-associated guanylyl cyclase (GCC). This receptor also serves as the receptor for the recently characterised endogenous ligand, guanylin. We have expressed various domains of the receptor in Escherichia coli and used purified proteins for the generation of both polyclonal and monoclonal antibodies. While polyclonal antibodies were able to partially inhibit ST binding to the native receptor present in the T84 human colonic cell line, GCC:B10 monoclonal antibody did not interfere with ligand binding. Western blot analysis, using membranes prepared from human colonic T84 cells, detected two bands of size 160 and 140 kDa, representing alternately glycosylated forms of the receptor. Using the recombinant proteins, we could map the epitope of GCC:B10 monoclonal antibody to the intracellular domain of the receptor. We used the antibody to localize the receptor throughout the rat intestine, and in the porcine and bonnet monkey colon. We could detect receptor expression in the villus and the crypts of the duodenum, jejunum, ileum, and caecum, and in the crypts of the colon. Receptor expression was observed in cells that had earlier been shown to express cGMP-dependent kinase, but not the cystic fibrosis transmembrane regulator, a known downstream target of cGMP/G-kinase, which suggests that GCC/ cGMP could regulate additional cellular signal transduction machinery.

Amino Acid Sequence↗
PubMed1997

Evolutionary significance of vitamin C biosynthesis in terrestrial vertebrates.

Evolution of vertebrates from aquatic medium to the terrestrial atmosphere containing high concentration of environmental oxygen was accompanied by tissue-specific expression of the gene for L-gulonolactone oxidase (LGO). LGO is the terminal enzyme in the pathway of biosynthesis of ascorbic acid in animals. In this paper we present data to indicate that emergence of LGO is apparently to provide the terrestrial vertebrates with adequate amount of ascorbic acid and thereby protect their tissues against oxygen toxicity. Superoxide dismutase (SOD) was not induced in the early tetrapods. However, SOD activity has increased in the mammals which is accompanied by a decrease in the LGO activity. In fact, there has been an inverse relationship between LGO and SOD in the progress of evolution. SOD activity is markedly high in the guinea pig, flying mammal, monkey and man, the species those lack LGO. The inverse relationship between LGO and SOD is also observed in rats during postnatal development, that is when the new born rats are exposed to high concentration of atmospheric oxygen. Recent results from our laboratory indicate that ascorbic acid is specifically needed for protection of microsomal membranes against cytochrome P450-mediated lipid peroxidation and protein oxidation, where SOD is ineffective. Data presented in this paper also indicate an apparent tissue-specific correlation among LGO activity, P450 level and O2.- production during phylogenetic evolution.

Animals↗
PubMed1996

Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies.

The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions. Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor. Purified antibodies recognized a single protein of Mr 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.

Antibodies↗
PubMed1996

Mesenchymal cell activation is the rate-limiting step of granulation tissue induction.

During wound repair a 3-day lag occurs between injury and granulation tissue development. When full-thickness, 8-mm-round, excisional wounds were made in the paravertebral skin of outbred Yorkshire pigs and harvested at various times, no granulation tissue was observed before day 4. Day 4 wounds were 3% filled with granulation tissue, day 5 wounds 48% filled, and day 7 wounds 88% filled. The prerequisites for granulation tissue induction are not known but hypothetically include fibrin matrix maturation or cell activation. To examine whether matrix maturation was necessary, wounds were allowed to heal for 5 or 7 days and then aggressively curetted, resulting in the formation of fresh fibrin clots in the newly formed wound spaces. In contrast to original wounds, no lag phase was observed; wounds curetted on day 5 were 23% filled with granulation tissue 1 day later and 99% filled 3 days later, whereas wounds curetted on day 7 were 47% filled 1 day later and completely filled within 2 days. Thus, granulation tissue formation resumed promptly and independently of fibrin clot matrix maturation. This observation suggested that mesenchymal cell activation might be the rate-limiting step in granulation tissue formation. To address this hypothesis more directly, cultured porcine or human fibroblasts, grown to 80% confluence in Dulbecco's minimal essential medium plus 10% fetal calf serum, were added to new wounds. These wounds were sealed with a freshly made exogenous fibrin clot. In some wounds, platelet releasate was added to the fibrin clot. Granulation tissue did not form in day 3 wounds, which had received either fibrin alone, fibrin and platelet releasate, or fibrin and fibroblasts. In contrast, granulation tissue was observed in wounds receiving fibrin, human fibroblasts, and platelet releasate. By day 4, wounds receiving cultured human fibroblasts, fibrin, and platelet releasate were 14% filled with granulation tissue compared with less than 4% granulation tissue in control wounds. Thus, fibroblast activation is a limiting step of granulation tissue formation, and continued cell stimulation is required for accelerated development.

Animals↗
PubMed1994

Ascorbate protects guinea pig tissues against lipid peroxidation.

In recent years we and others have shown that ascorbic acid (AH2) is a potential scavenger of superoxide (O2.-) and peroxyl (LOO.) radicals, the species involved in lipid peroxidation (LPO) in animal tissues. In this paper we have demonstrated that AH2 protects guinea pig tissues from LPO both in vivo and in vitro. The extent of LPO has been determined by estimating malonaldehyde using the thiobarbituric acid test and HPLC and also by measuring the accumulation of fluorescent pigment and occurrence of protein changes in the microsomal membranes. In AH2-deficiency, LPO occurs progressively in guinea pig tissues, despite the presence of adequate levels of antioxidants like alpha-tocopherol, GSH, protein thiols, and scavenging enzymes, namely, superoxide dismutase, catalase, and glutathione peroxidase. In a model in vitro system, microsomal LPO initiated by O2.- is completely prevented by AH2 but not by alpha-tocopherol, GSH, uric acid, and catalase. AH2 is also the most effective antioxidant in preventing microsomal LPO mediated by tert-butylhydroperoxide or the chain propagating species LOO., generated from 2,2'-azobis (2-amidinopropane) hydrochloride. The results obtained with guinea pigs may be applicable to humans, because humans are also dependent on dietary AH2. Our data suggest that an adequate vitamin C nutrition may prevent common cellular degenerative diseases associated with LPO.

Adrenal Glands↗
PubMed1992

Protective role of ascorbic acid against lipid peroxidation and myocardial injury.

Ascorbic acid (AH2) is a potential scavenger of superoxide radical and singlet oxygen. In the guinea pig, marginal AH2 deficiency results in intracellular oxidative damage in the cardiac tissue as evidenced by lipid peroxidation, formation of fluorescent pigment and loss of structural integrity of the microsomal membranes. The oxidative damage does not occur due to lack of enzymatic scavengers of reactive oxygen species such as superoxide dismutase, catalase and glutathione peroxidase. Also, glutathione transferase activity is not decreased in AH2 deficiency. Lipid peroxidation, fluorescent pigment formation and protein modification disappear after AH2 therapy. These results, if extra-polated to human beings, would indicate that chronic subclinical AH2 deficiency may result in progressive oxidative damage which in the long run may lead to permanent degenerative diseases in the heart.

Animals↗