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R V Rao

Publications and source records attributed to R V Rao.

At least 19 recordsLinked to original sources
PubMed2006

Coupling endoplasmic reticulum stress to the cell-death program: a novel HSP90-independent role for the small chaperone protein p23.

The endoplasmic reticulum (ER) is the principal organelle for the biosynthesis of proteins, steroids and many lipids, and is highly sensitive to alterations in its environment. Perturbation of Ca(2+) homeostasis, elevated secretory protein synthesis, deprivation of glucose or other sugars, altered glycosylation and/or the accumulation of misfolded proteins may all result in ER stress, and prolonged ER stress triggers cell death. Studies from multiple laboratories have identified the roles of several ER stress-induced cell-death modulators and effectors through the use of biochemical, pharmacological and genetic tools. In the present work, we describe the role of p23, a small chaperone protein, in preventing ER stress-induced cell death. p23 is a highly conserved chaperone protein that modulates HSP90 activity and is also a component of the steroid receptors. p23 is cleaved during ER stress-induced cell death; this cleavage, which occurs close to the carboxy-terminus, requires caspase-3 and/or caspase-7, but not caspase-8. Blockage of the caspase cleavage site of p23 was associated with decreased cell death induced by ER stress. Immunodepletion of p23 or inhibition of p23 expression by siRNA resulted in enhancement of ER stress-induced cell death. While p23 co-immunoprecipitated with the BH3-only protein PUMA (p53-upregulated modulator of apoptosis) in untreated cells, prolonged ER stress disrupted this interaction. The results define a protective role for p23, and provide further support for a model in which ER stress is coupled to the mitochondrial intrinsic apoptotic pathway through the activities of BH3 family proteins.

Animals↗
PubMed2004

Coupling endoplasmic reticulum stress to the cell death program.

The endoplasmic reticulum (ER) regulates protein synthesis, protein folding and trafficking, cellular responses to stress and intracellular calcium (Ca(2+)) levels. Alterations in Ca(2+) homeostasis and accumulation of misfolded proteins in the ER cause ER stress that ultimately leads to apoptosis. Prolonged ER stress is linked to the pathogenesis of several different neurodegenerative disorders. Apoptosis is a form of cell death that involves the concerted action of a number of intracellular signaling pathways including members of the caspase family of cysteine proteases. The two main apoptotic pathways, the death receptor ('extrinsic') and mitochondrial ('intrinsic') pathways, are activated by caspase-8 and -9, respectively, both of which are found in the cytoplasm. Recent studies point to the ER as a third subcellular compartment implicated in apoptotic execution. Here, we review evidence for the contribution of various cellular molecules that contribute to ER stress and subsequent cellular death. It is hoped that dissection of the molecular components and pathways that alter ER structure and function and ultimately promote cellular death will provide a framework for understanding degenerative disorders that feature misfolded proteins.

Apoptosis↗
PubMed2002

A ligand-receptor pair that triggers a non-apoptotic form of programmed cell death.

Several receptors that mediate apoptosis have been identified, such as Fas and tumor necrosis factor receptor I. Studies of the signal transduction pathways utilized by these receptors have played an important role in the understanding of apoptosis. Here we report the first ligand-receptor pair-the neuropeptide substance P and its receptor, neurokinin-1 receptor (NK(1)R)-that mediates an alternative, non-apoptotic form of programmed cell death. This pair is widely distributed in the central and peripheral nervous systems, and has been implicated in pain mediation and depression, among other effects. Here we demonstrate that substance P induces a non-apoptotic form of programmed cell death in hippocampal, striatal, and cortical neurons. This cell death requires gene expression, displays a non-apoptotic morphology, and is independent of caspase activation. The same form of cell death is induced by substance P in NK(1)R-transfected human embryonic kidney cells. These results argue that NK(1)R activates a death pathway different than apoptosis, and provide a signal transduction system by which to study an alternative, non-apoptotic cell death program.

Animals↗
PubMed2001

Double-stranded RNA-dependent protein kinase, PKR, binds preferentially to Huntington's disease (HD) transcripts and is activated in HD tissue.

Fourteen neurological diseases have been associated with the expansion of trinucleotide repeat regions. These diseases have been categorized into those that give rise to the translation of toxic polyglutamine proteins and those that are untranslated. Thus far, compelling evidence has not surfaced for the inclusion of a model in which a common mechanism may participate in the pathobiology of both translated and untranslated trinucleotide diseases. In these studies we show that a double-stranded RNA-binding protein, PKR, which has previously been linked to virally-induced and stress-mediated apoptosis, preferentially binds mutant huntingtin RNA transcripts immobilized on streptavidin columns that have been incubated with human brain extracts. These studies also show, by immunodetection in tissue slices, that PKR is present in its activated form in both human Huntington autopsy material and brain tissue derived from Huntington yeast artificial chromosome transgenic mice. The increased immunolocalization of the activated kinase is more pronounced in areas most affected by the disease and, coupled with the RNA binding results, suggests a role for PKR activation in the disease process.

Animals↗
PubMed2001

Coupling endoplasmic reticulum stress to the cell death program. Mechanism of caspase activation.

The endoplasmic reticulum (ER) is the site of assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. It also regulates cellular responses to stress and intracellular Ca(2+) levels. A variety of toxic insults can result in ER stress that ultimately leads to apoptosis. Apoptosis is initiated by the activation of members of the caspase family and serves as a central mechanism in the cell death process. The present study was carried out to determine the role of caspases in triggering ER stress-induced cell death. Treatment of cells with ER stress inducers such as brefeldin-A or thapsigargin induces the expression of caspase-12 protein and also leads to translocation of cytosolic caspase-7 to the ER surface. Caspase-12, like most other members of the caspase family, requires cleavage of the prodomain to activate its proapoptotic form. Caspase-7 associates with caspase-12 and cleaves the prodomain to generate active caspase-12, resulting in increased cell death. We propose that any cellular insult that causes prolonged ER stress may induce apoptosis through caspase-7-mediated caspase-12 activation. The data underscore the involvement of ER and caspases associated with it in the ER stress-induced apoptotic process.

Animals↗
PubMed2001

Concurrent infection of the central nervous system by Borrelia burgdorferi and Bartonella henselae: evidence for a novel tick-borne disease complex.

OBJECTIVES: To investigate Bartonella henselae as a potential human tick-borne pathogen and to evaluate its role as a coinfecting agent of the central nervous system in the presence of neuroborreliosis. DESIGN: Case report study. SETTING: A primary health care center in Flemington, NJ, and the Department of Research and Development at Medical Diagnostic Laboratories LLC in Mt Laurel, NJ. SUBJECTS: Two male patients (aged 14 and 36 years) and 2 female patients (aged 15 and 30 years, respectively) with a history of tick bites and Lyme disease. MAIN OUTCOME MEASURES: Laboratory and diagnostic findings before and after antimicrobial therapy. RESULTS: Patients residing in a Lyme-endemic area of New Jersey with ongoing symptoms attributed to chronic Lyme disease were evaluated for possible coinfection with Bartonella species. Elevated levels of B henselae-specific antibodies were found in these patients using the immunofluorescent assay. Bartonella henselae-specific DNA was detected in their blood. None of these patients exhibited the clinical characteristics of cat-scratch disease. Findings of cerebrospinal fluid analysis revealed the presence of both B henselae- and Borrelia burgdorferi-specific DNA. Bartonella henselae-specific DNA was also detected in live deer ticks obtained from the households of 2 of these patients. CONCLUSIONS: Our data implicate B henselae as a potential human tick-borne pathogen. Patients with a history of neuroborreliosis who have incomplete resolution of symptoms should be evaluated for B henselae infection.

Adult↗
PubMed2001

Clear cell carcinoma of the larynx--a case report.

Clear cell carcinoma (CCC) of the larynx is extremely rare and to the best of our knowledge no such case has been reported in the paediatric age group. These tumours resemble the clear cell carcinoma of the major and minor salivary glands. We report such a lesion in a 15 years old boy presenting with hoarseness and an exophytic growth in the glottis. A frontolateral conservation laryngectomy with laryngoplasty was done and over 2 years follow-up has shown no evidence of loco-regional recurrence and he enjoys an acceptable voice.

Adenocarcinoma, Clear Cell↗
PubMed2000

Impaired resensitization and recycling of the cholecystokinin receptor by co-expression of its second intracellular loop.

Intermolecular interaction represents an important theme in regulation of intracellular trafficking of organelles that can be interrupted by competitive overexpression of a relevant molecular domain. We attempted to identify the functional importance of intracellular domains of the cholecystokinin (CCK) receptor by their over-expression in receptor-bearing Chinese hamster ovary (CHO-CCKR) cell lines. Although clathrin-dependent endocytosis and recycling of this receptor are well-established (J Cell Biol 128:1029-1042, 1995), any influence of distinct receptor domains is not understood. In this work, constructs representing each of the intracellular domains of the CCK receptor were coexpressed with wild-type receptor, and stable clonal cell lines were selected. Each was characterized for ligand binding and agonist-stimulated biological activity (inositol 1,4,5-trisphosphate generation), desensitization, resensitization, receptor internalization, and recycling. Each cell line expressed normal CCK radioligand binding, signaling, internalization, and desensitization. Three independent cell lines that coexpressed the 25-residue second intracellular loop domain exhibited deficient resensitization. In morphological assessment of receptor trafficking, this construct was also shown to interfere with receptor recycling to the plasma membrane. As a control, recycling of an unrelated G protein-coupled receptor was demonstrated to occur normally in this cell line. These observations suggest that rather than representing passive cargo within an endosome, a receptor can influence its own trafficking within the cell.

Amino Acid Sequence↗
PubMed2000

CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking.

Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.

Animals↗
PubMed2000

Primary choriocarcinoma of stomach.

A 45 year old patient wad admitted with pain abdomen and a palpable mass in the epigastrium of 3 months duration. Endoscopy revealed growth in the stomach and biopsy showed poorly differentiated Carcinoma. Distal radical subtotal gastrectomy was done. Histopathology revealed choriacarcinoma with Syncytiotrophoblastic and cytotrophoblastic and foci of adenocarcinoma. Postoperatively urine and serum had very high levels of beta-human chorionic gonogotrophins (B-HCG). Immunochemistry showed positivity for B-HCG. Clinically and on scan both the testis were normal. Because of its rarity, we are presenting this case with brief review of literature.

Adult↗
PubMed1999

Connective tissue stromal changes in tumours and tumour-like lesions of the breast.

Between June 1994 and December 1995, one hundred and fifteen tumours and tumour-like lesions of the breast were studied in the Department of Pathology, Kasturba Medical College, Mangalore. Neoplasms constituted 80.9% and tumour like lesions accounted for 19.13%. Among the tumours, 51.6% were benign and 48.4% were malignant. Tumour-like lesions included the entire spectrum of fibrocystic disease. Tumours were common between 2nd and 8th decades while tumour-like lesions were uncommon above 6th decade. Whereas benign tumours exhibited ground substance, collagen and reticulin fibres in varying amounts, tumour-like lesions and carcinoma showed more of collagen fibres and less of ground substance. Elastosis and lymphoplasmacytic infiltrate were prominent features in carcinomas rather than in benign lesions.

Adult↗
PubMed1998

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells.

Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840-G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 muM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 muM) and cholecystokinin-(26-33)-peptide amide (CCK8, 0.1 muM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK8-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-alpha and, to a lesser extent, PKC-epsilon. In addition, TPA-induced inhibition of the increase in cytosolic free Ca2+ concentration ([Ca2+]i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-alpha. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca2+]i. Taken together, the present findings are in agreement with the idea that PKC-alpha-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca2+]i oscillations and eventually to inhibition of high-affinity Ca2+ signalling through the native CCK receptor in pancreatic acinar cells.

Animals↗
PubMed1998

Identification of a domain in the carboxy terminus of CCK receptor that affects its intracellular trafficking.

The carboxy-terminal region of many guanine nucleotide-binding protein (G protein)-coupled receptors contains important regulatory sequences such as an NP(x)2-3Y motif, a site of fatty acid acylation, and serine- and threonine-rich domains. The type A CCK receptor contains all of these, yet their significance has not been examined. We have, therefore, constructed a series of receptor site mutants and truncations that interfere with each of these motifs and expressed each in Chinese hamster ovary cells where they were studied for radioligand binding, cell signaling, receptor internalization, and intracellular trafficking. Each construct was synthesized and transported appropriately to the cell surface, where CCK bound with high affinity, elicited an inositol 1,4, 5-trisphosphate response, and resulted in internalization and normal trafficking. Thus modification or elimination of each of these established sequence motifs had no substantial effect on any of these parameters of receptor and cellular function. However, an additional construct that truncated the carboxy terminus, eliminating an additional 15-amino-acid segment devoid of any currently recognized sequence motifs, resulted in a marked change in receptor trafficking, with all other parameters of receptor function normal. This mutant receptor construct was delayed at the stage of early endosomes, delaying its progress to the lysosome-enriched perinuclear compartment from the rapid time course followed by wild-type receptor and all of the other constructs. It is proposed that this region of the CCK receptor tail contains a new motif important for intracellular receptor trafficking.

Amino Acid Sequence↗
PubMed1998

Serum butyrylcholinesterase of non-human primate shows amine sensitive aryl acyl amidase and metallopeptidase activities and characteristics similar to those of the human serum enzyme.

Butyrylcholinesterase (BChE) was purified from monkey serum and the catalytic activities were examined. The enzyme has a molecular mass of approximately equal to 74 kDa as seen by SDS-gel electrophoresis. Monkey serum BChE also exhibits an amine sensitive aryl acylamidase (AAA) and a metallocarboxypeptidase activity. The tyramine activation of the aryl acylamidase activity and the metal chelator inhibition of the peptidase activity were characteristics similar to those of the human enzyme. Studies on 65Zn2+ binding and zinc chelate Sepharose chromatography showed that monkey serum BChE and human serum BChE have similar characteristics. Limited alpha chymotrypsin digestion of monkey serum BChE followed by Sephadex gel chromatography cleaved the enzyme into a 36 kDa fragment exhibiting peptidase activity. However the 20 kDa fragment corresponding to cholinesterase and aryl acylamidase activity was not detectable possibly due to the unstable nature of the fragment. Immunological studies showed that a polyclonal antibody against human serum BChE cross reacted with monkey serum BChE. The identical nature of the catalytic activities of human serum BChE and monkey serum BChE supports the postulate that all three catalytic activities co-exist in the same enzyme. This is the first time that purification and characterisation of the monkey serum BChE which has the highest sequence identity and immunological identity with that of human serum BChE, is being reported.

Amidohydrolases↗
PubMed1997

Quantification of trace elements in normal human brain by inductively coupled plasma atomic emission spectrometry.

Eight normal human brain autopsy samples were analyzed for Na, K, P, Ca, Mg, Si, Cr, Cu, Ni, Zn, Fe, Al, Cd, Pb and As in 12 regions of brain (frontal cerebrum, temporal cerebrum, parietal cerebrum, somatosensory cortex, occipital cerebrum, cerebellum, mid-brain, pons, hypothalamus, thalamus, hippocampus and medulla oblongata) using inductively coupled plasma atomic emission spectrometry (ICPAES). The distribution of these 15 elements varied significantly from region to region of the brain. Potassium was most abundant in nearly all regions of the brain, followed by sodium and phosphorus (mg/g). The concentration of Al was found to be comparatively high and varied in different areas of the brain (58-196 microg/g). Moderate levels of Pb, Cd and As were observed in different regions. Ratios of Al to Fe were found to be high in temporal cerebrum (8.07) and hippocampus (9.05) and these two regions are significantly involved in Alzheimer's disease. The concentration of Na in mole percentage showed an inverse correlation with that of K, Ca, Mg, Fe and Cr. Direct correlation was observed in the concentration of all analyzed elements, which indicated for the first time the direct dependency of concentration of trace elements in one brain region to other regions. The mole ratios between different elements in different brain regions and total amounts of the elements in an average weight of 1.4 kg human brain were also computed. The present study provides new and in-depth data which may be used as base line data for normal human brains.

Brain Chemistry↗
PubMed1997

Cholecystokinin-induced desensitization, receptor phosphorylation, and internalization in the CHP212 neuroblastoma cell line.

Agonist stimulation of cells often results in desensitization of the response, to protect the cell from overstimulation. We have previously shown that the type A cholecystokinin (CCK) receptor on the pancreatic acinar cell and on the model CHO-CCKR cell line undergoes desensitization in response to CCK, with receptor phosphorylation and internalization playing key roles. Although these mechanisms contribute in a cell-specific manner, no analogous information exists for the CCK receptor expressed on neuronal cells, where in vivo data demonstrate a particularly sensitive response to CCK. The present study was designed to explore CCK receptor desensitization in the CHP212 neuroblastoma cell line, focusing on inositol 1,4,5-trisphosphate (IP3) responses to CCK and on recognized molecular and cellular mechanisms of desensitization. CCK promptly stimulated IP3 responses in these cells, with hormonal responsiveness rapidly and completely desensitized. Both receptor phosphorylation and internalization were observed to occur, with the former occurring most rapidly and likely being responsible for the earliest desensitization observed. Although the time course of receptor phosphorylation and dephosphorylation, and the groups of kinases involved in the neuroblastoma cell line, were most similar to those in the pancreatic cell, the movement of the agonist-bound receptor in these cells was quite different from that in the pancreatic cell and most similar to that in the CHO-CCKR cell line. This hybrid response supports the cell-specific nature of CCK receptor regulation and provides an important system to explore the molecular determinants of these processes.

Dose-Response Relationship, Drug↗
PubMed1997

Roles of cholecystokinin receptor phosphorylation in agonist-stimulated desensitization of pancreatic acinar cells and receptor-bearing Chinese hamster ovary cholecystokinin receptor cells.

Receptor phosphorylation has been implicated in desensitization responses to some agonist ligands, in which receptors may become uncoupled from G proteins and move into cellular compartments inaccessible to hydrophilic ligands. Understanding of the linkage between these processes, however, has come largely from recombinant receptor-bearing cell systems with consensus sites of kinase action mutagenized. We recently established methodology permitting direct assessment of sites of phosphorylation of the cholecystokinin receptor (CCKR) in its native milieu in the pancreatic acinar cell and in a Chinese hamster ovary (CHO)-CCKR cell line (1, 2). Although CCK binding leads to phosphorylation of serine residues within the third intracellular loop of the receptor in both cell types, there are clear differences in the time course of phosphorylation, in the balance of action of kinases and a receptor phosphatase, and in a few of the distinct sites phosphorylated. In this work, we have directly assessed the inositol 1,4,5-triphosphate responses to CCK and desensitization of these responses in both cells. CHO cell lines expressing receptor mutants with protein kinase C consensus sites modified were also studied. CCK-stimulated inositol 1,4,5-triphosphate responses in both cells expressing wild-type receptors were rapidly and completely desensitized, associated with the onset of receptor phosphorylation. However, despite maintenance of the phosphorylated state of the receptor in the CHO-CCKR cell and its dephosphorylation returning the receptor to its basal state in the acinar cell, desensitization continued to be present in both. Mutagenesis of Ser260 and Ser264 to alanines individually reduced receptor phosphorylation by approximately 50%, whereas the dual mutant completely eliminated agonist-stimulated phosphorylation. Because other sites of phosphorylation were still intact in this construct, this raises the possibility of hierarchical phosphorylation with these two sites key in making other sites accessible to kinases. Constructs modifying Ser264 delayed the onset of desensitization, whereas all constructs proceeded to achieve complete desensitization by 10 min. Receptor internalization occurred independent of its phosphorylation state in the CHO cell lines, explaining the desensitization observed. In the acinar cell in which the receptor remains on the cell surface after agonist occupation, we postulate that receptor insulation achieves similar uncoupling from G protein association as is achieved by receptor phosphorylation early after agonist occupation.

Animals↗