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Biomedical subjects

R Bhasin

Publications and source records attributed to R Bhasin.

17 recordsLinked to original sources
PubMed1998

Brain mast cells lack the c-kit receptor: immunocytochemical evidence.

Mast cells are reported to differ from other cells of the hematopoietic lineage in that as mature cells, they retain the c-kit receptor, and are thus capable of responding to the stem cell factor (SCF) ligand. SCF is important for development and survival of mast cells. In this study, c-kit expression was examined immunocytochemically in the brains of mice, rats and doves. The results indicate that brain mast cells lack the c-kit receptor; those of the leptomeninges and other connective tissues are a mixed population of c-kit positive and negative cells. The mechanisms whereby brain mast cells might survive in the absence of SCF-c-kit signaling are discussed.

Animals↗
PubMed1994

Ubiquitin-mediated degradative pathway degrades the extracellular but not the intracellular form of amyloid beta-protein precursor.

The presence of ubiquitin and ubiquitin conjugates has been detected in patients affected by neurodegenerative diseases such as Alzheimer's disease. We investigated the role of ubiquitin in the degradation of amyloid beta-protein precursor (APP) and its participation in the process of amyloid beta-protein formation. APP was tested as a substrate for ubiquitin-mediated degradation, using both the extracellular and the intracellular forms of APP770, APP751 and APP695. The intracellular APP forms did not show appreciable ubiquitin-mediated degradation. In contrast, the three extracellular forms of APP were degraded in vitro by this proteolytic pathway, with similar degradation rates. Our results suggest a potential regulatory role for the ubiquitin-dependent degradation mechanism in the in vivo APP metabolic pathway.

Alzheimer Disease↗
PubMed1994

Transthyretin sequesters amyloid beta protein and prevents amyloid formation.

The cardinal pathological features of Alzheimer disease are depositions of aggregated amyloid beta protein (A beta) in the brain and cerebrovasculature. However, the A beta is found in a soluble form in cerebrospinal fluid in healthy individuals and patients with Alzheimer disease. We postulate that sequestration of A beta precludes amyloid formation. Failure to sequester A beta in Alzheimer disease may result in amyloidosis. When we added A beta to cerebrospinal fluid of patients and controls it was rapidly sequestered into stable complexes with transthyretin. Complexes with apolipoprotein E, which has been shown to bind A beta in vitro, were not observed in cerebrospinal fluid. Additional in vitro studies showed that both purified transthyretin and apolipoprotein E prevent amyloid formation.

Amino Acid Sequence↗
PubMed1993

Sequestration of amyloid beta-peptide.

Amyloid beta-protein, or beta/A4, is a 4-kilodalton peptide that forms poorly soluble extracellular depositions of amyloid in brains and leptomeninges of patients with Alzheimer's disease (AD), Down's syndrome (DS), and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). beta/A4 peptide is a derivative of a large transmembrane glycoprotein (APP) and is found in the extracellular space, i.e., in the cerebrospinal fluid and serum of individuals with and without AD and in the conditioned media of many different cells grown in culture. The mechanism by which normally produced amyloid beta peptide forms extracellular aggregates in patients is unknown. One possible explanation is a failure of a mechanism for removal of the beta/A4 peptide that prevents this highly aggregating peptide from forming extracellular amyloid depositions.

Alzheimer Disease↗
PubMed1993

Proteolytic processing of human amyloid beta protein precursor in insect cells. Major carboxyl-terminal fragment is identical to its human counterpart.

The predominant component of amyloid plaques of Alzheimer's disease is the amyloid beta protein (A beta), a 39-42-amino-acid peptide derived by proteolysis of a family of precursors known as amyloid precursor proteins (APP). In mammalian brain and in cultured mammalian cells, the release of APP amino-terminal fragments into the extracellular medium occurs by a proteolytic cleavage within the A beta domain, thereby precluding amyloidogenesis. Infection of Sf9 insect cells with baculovirus vectors containing APP cDNAs results in high levels of APP expression. The concomitant release of amino-terminal fragments of APP and the production of carboxyl-terminal, cell-associated cleavage products are observed. Here we demonstrate by direct protein microsequencing that the proteolytic processing of APP in the Sf9 cells generates a prominent carboxyl-terminal species that is identical to that produced in human cells, suggesting that the major pathway for proteolytic processing of APP is conserved among metazoans.

Amino Acid Sequence↗
PubMed1993

Avid binding of beta A amyloid peptide to its own precursor.

The senile plaque and congophilic angiopathy in brains of patients with Alzheimer's disease contain abnormal extracellular depositions. These depositions have very complex molecular structure, some elements of which have been identified. The most abundant protein in these structures is beta A amyloid peptide, which is produced by proteolytic processing of a larger protein, the amyloid precursor protein. It is not known how plaques and angiopathy are formed. Binding of proteins with the beta A peptide may be critical in this process. Using synthetic beta A peptides we show that the amyloid precursor protein found in cerebrospinal fluid binds beta A peptide with high avidity. Analysis of recombinant amyloid precursor proteins containing deletions of various domains demonstrate that the beta A peptide binds to the amino terminus of its own precursor. A model of plaque assembly is presented.

Amino Acid Sequence↗
PubMed1992

Alzheimer beta/A4-amyloid precursor protein: evidence for putative amyloidogenic fragment.

Recombinant baculovirus was used to overexpress human Alzheimer beta/A4-amyloid precursor protein (APP) in Spodoptera frugiperda (Sf9) cells. Lysates of these cells were then analyzed for the presence of carboxyl-terminal fragments of APP by an immunoblotting assay using either an antibody against the APP cytoplasmic domain (rabbit anti-human 695APP645-694) or an antibody against the amino terminus of beta/A4-amyloid (rabbit anti-human 695APP586-606). Anti-human 695APP645-694 identified APP holoprotein, a 25-kDa species, and a prominent group of carboxyl-terminal fragments of 17, 16, and 14 kDa, whereas anti-human 695APP586-606 identified APP holoprotein and a single prominent low-molecular-mass protein species comigrating with the 17-kDa carboxyl-terminal fragment identified by anti-human 695APP645-694. No immunoreactive species was detected at these molecular mass positions when either antibody was used for analysis of lysates of either uninfected Sf9 cells or Sf9 cells infected with wild-type Autographa californica baculovirus. For each antibody, specific immunoreactivity was abolished by preabsorption with the corresponding peptide immunogen. The incorporation of a beta/A4-amyloid amino-terminal epitope into a 17-kDa fragment of APP suggests that, in the baculoviral overexpression system, the electrophoretic microheterogeneity of APP carboxyl-terminal fragments is due, at least in part, to alternative proteolysis of APP. If such carboxyl-terminal fragments of APP containing an intact beta/A4-amyloid domain are produced in human brain, then they may represent intermediates in the conversion of APP to deposited beta/A4-amyloid.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗
PubMed1991

The nucleotide sequence of the Escherichia coli crp divergent RNA and an overlapping ORF.

The nucleotide sequence specifying the crp divergent RNA of Escherichia coli was determined. An open reading frame (ORF) is located at +135 to +536 relative to the initiation site of the divergent RNA. Potential factor independent transcription terminators were found at +257 to +294 and +544 to +576. These putative termination sites may account for the two RNAs of approximately 300 and 550 nucleotides previously identified as originating from the crp divergent promoter.

Amino Acid Sequence↗
PubMed1991

The predominant form of the amyloid beta-protein precursor in human brain is protease nexin 2.

The amyloid beta protein and the amyloid beta-protein precursor (APP) are major constituents of senile plaques and cerebrovascular deposits in patients with Alzheimer disease and Down syndrome. Most human tissues contain mRNA that encodes forms of APP that contain the Kunitz protease inhibitor (KPI+) domain. A major 120-kDa protein corresponding to this KPI+ mRNA is also found in these tissues. This protein is identical to the protease inhibitor protease nexin 2. Brain contains an additional mRNA species that encodes a form of APP that lacks the KPI domain (KPI-). This latter mRNA has been suggested to encode a 105-kDa KPI- form of APP protein also found in brain. Using protease inhibitory functional assays, we show that both the 105-kDa and 120-kDa APP proteins in normal and Alzheimer disease brain contain the KPI domain. Moreover, KPI domain-specific precipitation assays reveal that KPI- forms of APP protein represent less than 14% of total brain APP. Lastly, an enriched fraction from total brain homogenate contains proteolytic activity that can process the purified 120-kDa KPI+ form of APP into a 105-kDa form, resulting in a high-molecular-mass doublet identical to that seen in brain. These findings indicate that although KPI- APP mRNA is abundant in brain, little corresponding protein is present. Thus, KPI+ APP protein (equivalent to protease nexin 2) is the predominant form of APP in human brain.

Alzheimer Disease↗
PubMed1991

Expression of active secreted forms of human amyloid beta-protein precursor by recombinant baculovirus-infected insect cells.

Three alternatively spliced forms of the amyloid precursor protein (APP), APP-695, APP-751, and APP-770, were expressed in the baculovirus expression vector system. The recombinant proteins were secreted into the culture medium by infected insect cells, and APP molecules were detected in insect cells and medium 2 days after infection with the recombinant APP-baculoviruses. A partial sequence of the NH2 terminus of the secreted protein revealed identity with the native secreted protein and showed that the signal peptide was recognized and properly cleaved in insect cells. Purified secreted recombinant APP-751 comigrated with protease nexin 2 purified from platelets and fibroblasts. A 15-kDa COOH-terminal fragment of APP was also detected in cells infected with recombinant baculoviruses, suggesting that recombinant APP proteins were cleaved at the COOH-terminal end like native APP protein. Recombinant APP-751 and APP-770 formed complexes with epidermal growth factor-binding protein, whereas APP-695 did not. In addition, recombinant APP-751 and APP-770 inhibited trypsin and chymotrypsin activity, whereas APP-695 did not. Growth of a human fibroblast cell line, A-1, that required APP for complete growth, was restored upon addition of secreted recombinant APP-695 or APP-751. Thus, the appropriately sized, secreted recombinant APP proteins produced in this expression system are biologically active.

Amino Acid Sequence↗
PubMed1990

Human immunodeficiency virus infection in the blood donors of Delhi, India.

In the present study, blood from paid and unpaid donors was screened for the presence of HIV infection using a competitive ELISA test. None of the 8,000 unpaid donors but 4 (0.23%) of the 1,700 paid donors showed evidence of HIV infection. All of these four ELISA-positive samples were confirmed by the Western blot test. The present study indicates that HIV infection has entered the group of paid donors in the Delhi metropolitan area. Careful donor screening, discouraging use of paid donors, and stringent screening of donated units of blood has become mandatory in India to prevent HIV transmission through blood and blood products.

Adult↗
PubMed1988

Evidence in vivo for autogenous control of the cyclic AMP receptor protein gene (crp) in Escherichia coli by divergent RNA.

Control of crp expression in vivo was studied by using the cloned crp gene. The synthesis of the product of this gene, cyclic AMP (cAMP) receptor protein (CRP), was strongly reduced by exogenous cAMP. This regulation was completely abolished by the inactivation of a divergent promoter located within the crp promoter region. These data are consistent with our in vitro studies (Okamoto and Freundlich, Proc.Natl. Acad. Sci. USA 83:5000-5004, 1986), which showed that crp autoregulation is due to the inhibition of crp transcription by divergent (antisense) RNA produced by cAMP-CRP activation of the divergent promoter.

Cloning, Molecular↗