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Biomedical subjects

R Bhandari

Publications and source records attributed to R Bhandari.

At least 19 recordsLinked to original sources
PubMed2002

Recent advances in tissue engineering: an invited review.

Tissue formation within the body, as part of a development or repair process, is a complex event in which cell populations self-assemble into functional units. There is intense academic, medical, and commercial interest in finding methods of replicating these events outside the body. This interest has accelerated with the demonstration of the engineering of skin and cartilage tissue in the laboratory and there is now worldwide activity in the in vitro regeneration of tissues including nerve, liver, bone, heart valves, blood vessels, bladder, and kidney. Approaches to tissue engineering center on the need to provide signals to cell populations to promote cell proliferation and differentiation. This review considers recent advances in methods of providing these signals to cells using examples of progress in the engineering of complex tissues.

Absorbable Implants↗
PubMed2001

Functional inactivation of the human guanylyl cyclase C receptor: modeling and mutation of the protein kinase-like domain.

Receptor guanylyl cyclases possess an extracellular ligand-binding domain, a single transmembrane region, a region with sequence similar to that of protein kinases, and a C-terminal guanylyl cyclase domain. ATP regulates the activity of guanylyl cyclase C (GC-C), the receptor for the guanylin and stable toxin family of peptides, presumably as a result of binding to the kinase homology domain (KHD). Modeling of the KHD of GC-C indicated that it could adopt a structure similar to that of tyrosine kinases, and sequence comparison with other protein kinases suggested that lysine(516) was positioned in the KHD to interact with ATP. A monoclonal antibody GCC:4D7, raised to the KHD of GC-C, did not recognize ATP-bound GC-C, and its epitope mapped to a region in the KHD of residues 491--568 of GC-C. Mutation of lysine(516) to an alanine in full-length GC-C (GC-C(K516A)) dramatically reduced the ligand-stimulated activity of mutant GC-C, altered the ATP-mediated effects observed with wild-type GC-C, and failed to react with the GCC:4D7 monoclonal antibody. ATP interaction with wild-type GC-C converted a high-molecular weight oligomer of GC-C to a smaller sized oligomer. In contrast, GC-C(K516A) did not exhibit an alteration in its oligomeric status on incubation with ATP. We therefore suggest that the KHD in receptor guanylyl cyclases provides a critical structural link between the extracellular domain and the catalytic domain in regulation of activity in this family of receptors, and the presence of K(516) is critical for the possible proper orientation of ATP in this domain.

Adenosine Triphosphate↗
PubMed2001

Isolation and structure elucidation of vicenistatin M, and importance of the vicenisamine aminosugar for exerting cytotoxicity of vicenistatin.

A new analogue of vicenistatin was isolated from the producing strain Streptomyces sp. HC-34. A characteristic of the elucidated structure involved the existence of a neutral sugar mycarose instead of an aminosugar vicenisamine of vicenistatin. The absolute stereochemistry of the new analogue (named as vicenistatin M) was determined by the synthesis of D-mycarose and of vicenistatin M itself. Biological testing of vicenistatin M suggested the importance of vicenisamine for exerting the cytotoxicity of vicenistatin.

Amino Sugars↗
PubMed2000

Biochemical characterization of the intracellular domain of the human guanylyl cyclase C receptor provides evidence for a catalytically active homotrimer.

Guanylyl cyclase C (GCC) is the receptor for the family of guanylin peptides and bacterial heat-stable enterotoxins (ST). The receptor is composed of an extracellular, ligand-binding domain and an intracellular domain with a region of homology to protein kinases and a guanylyl cyclase catalytic domain. We have expressed the entire intracellular domain of GCC in insect cells and purified the recombinant protein, GCC-IDbac, to study its catalytic activity and regulation. Kinetic properties of the purified protein were similar to that of full-length GCC, and high activity was observed when MnGTP was used as the substrate. Nonionic detergents, which stimulate the guanylyl cyclase activity of membrane-associated GCC, did not appreciably increase the activity of GCC-IDbac, indicating that activation of the receptor by Lubrol involved conformational changes that required the transmembrane and/or the extracellular domain. The guanylyl cyclase activity of GCC-IDbac was inhibited by Zn(2+), at concentrations shown to inhibit adenylyl cyclase, suggesting a structural homology between the two enzymes. Covalent cross-linking of GCC-IDbac indicated that the protein could associate as a dimer, but a large fraction was present as a trimer. Gel filtration analysis also showed that the major fraction of the protein eluted at a molecular size of a trimer, suggesting that the dimer detected by cross-linking represented subtle differences in the juxtaposition of the individual polypeptide chains. We therefore provide evidence that the trimeric state of GCC is catalytically active, and sequences required to generate the trimer are present in the intracellular domain of GCC.

Amino Acid Sequence↗
PubMed2000

Tyrosine phosphorylation of the human guanylyl cyclase C receptor.

Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

Animals↗
PubMed2000

Printing patterns of biospecifically-adsorbed protein.

The advancement of elastomeric patterning techniques in recent years has significantly enhanced our ability to spatially control biomaterial surface chemistry at the micrometre level. The application of this technology to the patterning of biomolecules onto solid surfaces has created many potential applications including the development of advanced biosensors, combinatorial library screening and the formation of tissue engineering templates. In this paper, we describe the direct patterning of protein by microcontact printing. An important consideration for the fabrication of protein micropatterns intended for these applications is the nature of the protein immobilization to a substrate. To date, the patterning of proteins by direct microcontact printing (microCP) has relied on the non-covalent adsorption to a substrate. Ideally, the proteins need to be firmly anchored onto a surface without adversely effecting their activity. Here, the high affinity avidin-biotin receptor-ligand interaction has been exploited to form arrays of avidin molecules onto a polymeric substrate expressing biotin moieties. This has created a generic technique by which any biotinylated species can be subsequently immobilized into defined patterns. Utilizing atomic force microscopy (AFM), the patterned surfaces have been characterized to molecular resolution. The micropatterned sample supported cell adhesion when biotin-(G)11-GRGDS was bound to the avidin bearing arrays.

3T3 Cells↗
PubMed1999

Guanylyl cyclase C receptor: regulation of catalytic activity by ATP.

Guanylyl cyclase C (GCC), a member of the family of membrane bound guanylyl cyclases is the receptor for the heat-stable enterotoxin (ST) peptides and the guanylin family of endogenous peptides. GCC is activated upon ligand binding to increase intracellular cGMP levels, which in turn activates other downstream signalling events in the cell. GCC is also activated in vitro by nonionic detergents. We have used the T84 cell line as a model system to investigate the regulation of GCC activity by ATP. Ligand-stimulated GCC activity is potentiated in the presence of ATP, whereas detergent-stimulated activity is inhibited. The potentiation of GCC activity by ATP is dependent on the presence of Mg2+ ions, and is probably brought about by a direct binding of Mg-ATP to GCC. The protein kinase-like domain of GCC, which has earlier been shown to play a critical role in the regulation of GCC activity, may be a possible site for the binding of Mg-ATP to GCC.

Adenosine Triphosphate↗
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↗
PubMed1995

A pharmacological approach to secretory diarrhea.

Secretory diarrhea is characterized by painless, large volume, watery diarrhea (usually > 1 L), minimal effect on fasting, and a stool osmolality gap of less than 40. There are numerous causes of secretory diarrhea, including secretagogues, laxatives, medications, endogenous detergents, and bacterial enterotoxins. There are no controlled data on the efficacy of any specific therapy. Conservative measures, including fluid and electrolyte replacement, should be provided for all patients, and antidiarrheal therapy is indicated in more severe cases. No specific pharmacological therapy has proved effective in most cases, and antidiarrheal therapy is relatively nonspecific.

Adrenergic alpha-Agonists↗
PubMed1994

Bupivacaine 0.125% improves continuous postoperative epidural fentanyl analgesia after abdominal or thoracic surgery.

The addition of 0.125% and 0.25% bupivacaine to continuous postoperative epidural infusions of fentanyl, in a 10 micrograms.ml-1 concentration, were studied in 39 patients following abdominal or thoracic surgery in prospective, random, double-blind fashion. Patients received an initial bolus of 0.1 ml.kg-1 of the the study solution and an infusion of 6 ml.hr-1 which was titrated to maintain analgesia (VAS < 40). Assessments of pain (VAS), pulmonary function (pH, PaCO2), and bowel function (time to flatus or po fluids) were made until the second post-operative morning. There was a difference among the three groups in analgesia (means VAS scores) over time (P < 0.01), with the fentanyl-alone group producing less analgesia than the 0.125% bupivacaine group (P < 0.01). There was no difference in the average infusion rates, postoperative pulmonary function, or bowel function. The incidence of side effects including somnolence, nausea and vomiting, and pruritus was also similar. Fewer patients in the 0.125% bupivacaine group than in the 0.25% group developed a transient sensory loss to pinprick and ice (3 vs 10, P < 0.001). Four patients in both bupivacaine groups had leg weakness, those in the 0.125% were all a Bromage 1 score, while in the 0.25% group one had a Bromage 1, one a Bromage 2, and two Bromage 3 scores. The addition of 0.125% bupivacaine improves the analgesia of epidural infusions of fentanyl (10 microgms.ml(-1)) when used following abdominal or thoracic surgery and results in minimal sensorimotor disturbance.

Abdomen↗
PubMed1994

Fatal air embolism during radical retropubic prostatectomy.

Air embolism during urological surgery only rarely has been reported. We report a case of fatal air embolism occurring during radical retropubic prostatectomy. This entity is discussed in an attempt to raise the level of awareness of this rare but potentially lethal complication, especially in view of the ever increasing popularity of radical retropubic prostatectomy as the treatment for organ confined carcinoma of the prostate.

Aged↗
PubMed1993

Alveolar type II cell-fibroblast interactions, synthesis and secretion of surfactant and type I collagen.

During alveolar development and alveolar repair close contacts are established between fibroblasts and lung epithelial cells through gaps in the basement membrane. Using co-culture systems we have investigated whether these close contacts influence synthesis and secretion of the principal surfactant apoprotein (SP-A) by cultured rat lung alveolar type II cells and the synthesis and secretion of type I collagen by fibroblasts. The alveolar type II cells remained cuboidal and grew in colonies on fibroblast feeder layers and on Matrigel-coated cell culture inserts but were progressively more flattened on fixed fibroblast monolayers and plastic. Alveolar type II cells cultured on plastic released almost all their SP-A into the medium by 4 days. Alveolar type II cells cultured on viable fibroblasts or Matrigel-coated inserts above fibroblasts accumulated SP-A in the medium at a constant rate for the first 4 days, and probably recycle SP-A by endocytosis. The amount of mRNA for SP-A was very low after 4 days of culture of alveolar type II cells on plastic, Matrigel-coated inserts or fixed fibroblast monolayers: relatively, the amount of mRNA for SP-A was increased 4-fold after culture of alveolar type II cells on viable fibroblasts. Co-culture of alveolar type II cells with confluent human dermal fibroblasts stimulated by 2- to 3-fold the secretion of collagen type I into the culture medium, even after the fibroblasts' growth had been arrested with mitomycin C. Collagen secretion, by fibroblasts, also was stimulated 2-fold by conditioned medium from alveolar type II cells cultured on Matrigel. The amount of mRNA for type I collagen increased only modestly when fibroblasts were cultured in this conditioned medium. This stimulation of type I collagen secretion diminished as the conditioned medium was diluted out, but at high dilutions further stimulation occurred, indicating that a factor that inhibited collagen secretion also was being diluted out. The conditioned medium contained low levels of IGF-1 and the stimulation of type I collagen secretion was abolished when the conditioned medium was pre-incubated with antibodies to insulin-like growth factor 1 (IGF-1). There are important reciprocal interactions between alveolar type II cells and fibroblasts in co-culture. Direct contacts between alveolar type II cells and fibroblasts appear to have a trophic effect on cultured alveolar type II cells, increasing the levels of mRNA for SP-A. Rat lung alveolar type II cells appear to release a factor (possibly IGF-1) that stimulates type I collagen secretion by fibroblasts.

Animals↗