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Biomedical subjects

N Ramakrishna

Publications and source records attributed to N Ramakrishna.

At least 19 recordsLinked to original sources
PubMed2002

Plasticizer effect and comparative evaluation of cellulose acetate and ethylcellulose-HPMC combination coatings as semipermeable membranes for oral osmotic pumps of naproxen sodium.

The objective of this study was to compare the performance of cellulose acetate (CA) and ethylcellulose (EC)-HPMC combination coatings as semipermeable membranes (SPMs) for osmotic pump tablets (OPTs) of naproxen sodium (NPS) so as to deliver a constant, predetermined amount of drug in solution form over a fixed span of time, independent of external environmental conditions. Osmotic pump tablets were designed with different coating variables and optimized in terms of nature of plasticizer, membrane thickness, and orifice diameter. The effect of insertion of an inner microporous film around the NPS core to minimize deformation of the SPM due to peristaltic movement of the gut was also studied. Osmotic pump tablets composed of membranes with water-soluble plasticizer, propyleneglycol (PG), released drug mainly through diffusion, whereas those designed with CA and EC-HPMC (4:1) coats containing water-insoluble plasticizer, castor oil, released their contents by perfect zero-order kinetics over a prolonged period of time, though the average release rate that could be achieved with the EC-HPMC (4:1) membrane was only about half the rate achieved with the CA membrane for the same membrane thickness. Release rates for both the membranes decreased with increasing membrane thickness and were found to be independent of orifice diameter, agitation intensity, and pH of the dissolution medium.

Administration, Oral↗
PubMed2001

Design and evaluation of osmotic pump tablets of naproxen sodium.

Osmotic pump tablets (OPTs) deliver a constant, predetermined amount of drug in solution form over a fixed span of time, independent of external environmental conditions. OPTs of naxopren sodium (NPS) were made and evaluated with the principal aim of limiting the drug's frequently experienced gastrointestinal side effects, by preventing the undissolved drug from coming in to contact with the gastric mucosa. OPTs with and without the osmotic driving agent, sodium chloride (SCL), with different membrane thicknesses, and with and without an orifice were designed. In vitro release profiles of the OPTs, when compared to a conventional marketed tablet (Xenobid 275), were highly controlled and exhibited an almost perfect zero-order release pattern. The magnitudes of lag times and average release rates were found to depend on the amount of SCL present in the formulation which decreased the drug's solubility within the system. The release rates were found to be independent of stirring rate and decreased with increasing membrane thickness. Release profiles of the OPTs with and without an orifice were found to be comparable.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

Apolipoprotein E alters metabolism of AbetaPP in cells engaged in beta-amyloidosis.

Canine smooth muscle cells (SMCs), cultured from amyloid-affected brain blood vessels accumulate Alzheimer amyloid-beta peptide (Abeta) intracellularly, either spontaneously or after treatment with apolipoprotein E (apoE). ApoE is codeposited with Abeta, which suggests that apoE participates in Abeta accumulation. We tested the hypothesis that apoE-induced accumulation of Abeta in SMCs is caused by an increased production of amyloid-beta precursor protein (AbetaPP) and/or its altered metabolism. We found that 24 hours of treatment with apoE3 or apoE4 induced intracellular accumulation of Abeta-immunoreactive deposits in SMCs but did not influence AbetaPP production and processing. The treatment with apoE3 or E4 for 3 days resulted in the following: increased Abeta-accumulation; reduced levels of secreted Abeta; increased production and cellular retention of mature AbetaPP770; and reduced culture growth, cell proliferation, and viability. ApoE4, but not apoE3, increased cellular levels of mRNA AbetaPP 770 (the main form produced in SMCs) about ninefold. ApoE3 stimulated production and cellular retention of endogenous apoE. We hypothesize that Abeta accumulation is triggered by apoE, which may bind and immobilize soluble Abeta produced in SMCs. The newly formed Abeta deposits may further accelerate Abeta accumulation by altering metabolism of AbetaPP.

Amyloid beta-Protein Precursor↗
PubMed1997

Reduction of histone cytotoxicity by the Alzheimer beta-amyloid peptide precursor.

In a search for Alzheimer beta-amyloid peptide precursor ligands, Potempska et al. (Arch. Biochem. Biophys. (1993) 304, 448) found that histones bind with high affinity and specificity to the secreted precursor. Because exogenous histones can be cytotoxic, we compared the effects of histones on the viability of cells which produce little beta-amyloid peptide precursor (U-937) to those on cells that produce twenty times as much precursor (COS-7). Addition of purified histones caused necrosis of U-937 cells (histone H4, LD50 = 1.5 microM). Extracellular A beta precursor in the submicromolar range prevented histone-induced U-937 cell necrosis. Cell-surface precursor also reduced histone toxicity: COS-7 cells were less sensitive to the toxic effects of histone H4 (LD50 = 5.4 microM). COS-7 cells in which the expression of an APP mRNA-directed ribozyme reduced the synthesis of the protein by up to 80% were more sensitive to histone H4 (LD50 = 3.2 microM) than cells that expressed the vector alone. Histone H4 binds to cell-associated A beta precursor. Cells expressing the A beta precursor-directed ribozyme bound less 125I-labeled histone H4 than those expressing the vector alone. In the limited extracellular space of tissues in vivo, both secreted and cell-surface A beta precursor protein may play significant roles in trapping chromatin or histones and removing them from the extracellular milieu.

Amyloid beta-Protein Precursor↗
PubMed1997

Upregulation of actin gene expression in cells expressing exogenous beta-amyloid precursor protein.

We investigated the effects of beta-amyloid peptide precursor (APP) overexpression upon the levels of other mRNAs. Using quantitative slot-blot hybridization and immunoblot analysis we observed that enhanced levels of APP elevated the levels of beta-actin and beta-actin mRNA. Our results also suggest that the cytoplasmic domain of APP is crucial for the elevation in beta-actin gene expression.

Actins↗
PubMed1997

Effects of cadmium, copper, and zinc and beta APP processing and turnover in COS-7 and PC12 cells. Relationship to Alzheimer disease pathology.

The effects of cadmium, copper, and zinc on beta APP metabolism were investigated in COS-7 and PC12 cells. Cadmium chloride (CdCl2) increased beta APP steady-state protein levels and decreased beta APP posttranslational processing. These changes were not accompanied by alterations in beta APP mRNA levels or splicing. In addition, cytosolic alpha-actin and G3PDH levels were not affected. Further, neither zinc (ZnCl2) nor copper (CuSO4) altered beta APP levels or affected its normal processing. Pulse-chase studies revealed that the rate of beta APP maturation decreased twofold in the presence of 25 microM CdCl2 compared to untreated controls. beta APP secretion from the cell also dramatically slowed. These two factors result in the accumulation of partially processed beta APP inside cells. The presence of CdCl2 also decreased the amount of an 8-kDa beta APP C-terminal fragment, indicating that the cellular compartment in which beta APP accumulates is not accessible to alpha-secretase. Studies using Brefeldin A suggest that this compartment may be the cis or medial Golgi. However, A beta production was proportionately increased. These data show that CdCl2 can modulate the beta APP cleavage to favor A beta. Finally, beta APP mis- metabolism was shown to be unrelated to the hsp70 induction elicited by CdCl2; both heat shock and CuSO4 induced hsp70 but had no effect on steady-state levels of beta APP, although heat shock did slow beta APP maturation. These data indicate that hsp70 alone cannot chaperone beta APP through an alternate processing pathway leading to A beta production.

Amyloid Precursor Protein Secretases↗
PubMed1996

Suppression of Alzheimer amyloid precursor protein (APP) expression by exogenous APP mRNA.

To establish a cell line expressing enhanced levels of beta-amyloid precursor protein (beta-APP), we constructed plasmid DNAs expressing beta-APP-751 mRNA and transfected them into COS-1 cells. Using a modified version of the reverse transcriptase polymerase chain reaction which is RNA sensitive to study the beta-APP iso-RNAs, we have made the unexpected observation that enhanced expression of beta-APP-751 mRNA resulted in a significant reduction of beta-APP-770 and -695 mRNA levels. Suppression of beta-APP-770 and -695 was also observed in cells expressing truncated and chimeric beta-App-751 mRNAs. Similar observations were made in P19 cells expressing a chimeric beta-APP-751 mRNA where endogenous beta-APP-751 mRNA levels also were decreased. Also, suppression of beta-APP-770 and -751 mRNAs was observed in human kidney cells expressing exogenous beta-APP-695 mRNA.

Amyloid beta-Protein Precursor↗
PubMed1994

Induction of kidney epithelial morphogenesis by cells expressing Wnt-1.

During kidney development, unknown signals derived from the ureteric bud induce metanephric mesenchymal cells to differentiate into nephron epithelia. In addition to the ureteric bud, a number of other tissues can act as heterologous inducers of this process in vitro, including embryonic spinal cord. In this report we demonstrate that Wnt-1, a gene that encodes a secreted glycoprotein expressed in embryonic spinal cord, is capable of conferring nephron-inducing activity to fibroblast cell lines. When cocultured with cells expressing exogenous Wnt-1, metanephric mesenchyme differentiated into glomerular and renal tubular epithelia. No such effect was observed using control cells. These data imply that the ability of embryonic spinal cord to act as an inducer of nephrogenesis may result from its production of Wnt-1 protein and suggest that a member of the Wnt gene family may be a mediator of renal epithelial morphogenesis in vivo.

Animals↗
PubMed1994

The expression and processing of human beta-amyloid peptide precursors in Saccharomyces cerevisiae: evidence for a novel endopeptidase in the yeast secretory system.

In mammalian cells, the transmembrane beta-amyloid peptide precursor (beta-APP) undergoes a complex series of alternative proteolytic processing steps that result in the secretion of varying proportions of its extra-cellular domain (protease nexin II) and beta-amyloid peptide. The protein is also reinternalized and degraded in the endosomal-lysosomal system. The relative efficiencies of these competing processes determine the yield of beta-amyloid peptide. Several proteases have been implicated in this complex processing pathway, although none has been identified to date. The yeast secretory system contains proteases homologous to mammalian pro-hormone convertases and is susceptible to genetic manipulation. We therefore investigated the expression and processing of the beta-amyloid peptide precursors (beta-APP-695 and beta-APP-751) in Saccharomyces cerevisiae transformed with human beta-APP cDNA's. beta-APP (695 or 751) cDNA either with its authentic signal sequence or the yeast-derived prepro-alpha-factor leader, was inserted into a glucose-regulated expression vector and transfected into a protease-deficient yeast strain. In all instances, expression of beta-APP was about 1% of total protein. Protease protection studies indicated that either the natural human signal sequence or the alpha-factor leader sequence targetted beta-APP to the endoplasmic reticulum and inserted it with the amino-terminal domain in the lumen. All of the beta-APP fused to the alpha-factor leader proceeded to the trans-Golgi, where Kex2 endopeptidase removed the leader and released the normal amino-terminus of beta-APP. About one-half of the beta-APP was also cleaved at the "alpha-secretase" site in the middle of the beta-peptide sequence, 12 residues before the membrane-spanning sequence. A fraction of the alpha-secretase-cleaved beta-APP appeared in the culture medium; however, most of it associated with the exterior of the cells. The carboxyl-terminal fragments formed by cleavage at the alpha-secretase site accumulated in the membranes. Other proteolytic processes generated membrane-associated carboxyl-terminal fragments that also resembled those found in mammalian cells. These results indicate that the secretory system of S. cerevisiae possesses proteases with specificities similar to the mammalian enzymes that process beta-APP.

Amino Acid Sequence↗
PubMed1993

Interaction between the beta-amyloid peptide precursor and histones.

The function of the beta-amyloid peptide precursors (beta-APPs) remains undefined. In a search for ligands of beta-APP we found that the most prominent beta-APP-binding proteins were histones. To measure the specificity of binding the secreted or extracellular form of beta-APP(751), beta-APP-S, was purified from recombinant baculovirus-infected Sf-9 cells and was labeled with iodine-125. The labeled beta-APP-S bound to each of the histones, which had previously been adsorbed to nitrocellulose membranes. Differences in their apparent affinities were small. beta-APP-S did not bind to histones incorporated into chromatin. beta-APP-S bound relatively weakly to fibronectin, laminin, collagen type I, or collagen type IV. It did not bind to other basic proteins tested, myelin basic protein, or cytochrome c. The beta-APP-S-histone complex adhered to most filters and gel media, which precluded a direct determination of its stability. The apparent dissociation constant of beta-APP-S bound to histone-4-Sepharose was about 30 nM. Although beta-APP normally does not contact histones, it may bind those that are released from damaged cells. A function of beta-APP may be to bind and recycle substances such as histones, proteases, and matrix proteins from the extracellular medium.

Amyloid beta-Protein Precursor↗
PubMed1991

Expression of human Alzheimer amyloid precursor protein in insect cells.

The amyloid beta-peptide is a major constituent of amyloid deposited in the brains of patients with Alzheimer's disease and is derived from a larger precursor protein/s (APP-695, 751, 770). A human cDNA encoding full-length APP-751 was inserted into the genome of Autographa californica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of APP-751. Analysis of infected cell proteins indicate that APP-751 is localized in the membrane fraction; however, a significant amount of the protein was cleaved and released into the medium. The NH2-terminal sequence of recombinant APP-751 from the membrane fraction was identical to that of mammalian APP. Immunoblot analysis suggests that the secreted form results from cleavage within the beta-peptide.

Alzheimer Disease↗
PubMed1991

Immunolocalization of Alzheimer beta-amyloid peptide precursor to cellular membranes in baculovirus expression system.

One characteristic of Alzheimer's disease (A beta disease) is the accumulation of amyloid deposits within the extracellular space of the brain and meninges. A 40 amino acid peptide called beta-peptide or A4 protein is the subunit of the amyloid fibrils found in these deposits. The sequence of beta-peptide is contained within those of a family of larger proteins called the Alzheimer beta-amyloid peptide precursor (APP). These APPs contain, in addition to a signal sequence, a hydrophobic sequence that is believed to span cell membranes. Although biochemical studies indicate that some APPs have properties of integral membrane proteins, morphological confirmation of this has not been reported. We recently described an expression system in which human APP751 cDNA was placed under the transcriptional regulation of the polyhedrin gene promoter in the baculovirus Autographica californica infecting a Spodoptera frugiperda cell line (Ramakrishna et al., Biochem Biophys Res Commun 174:983-989, 1991). As part of a larger biochemical and molecular biological study of APP, we have carried out an immunocytochemical study using antibodies directed against several epitopes within APP to reveal, at both the light and the electron microscopic levels, the cellular localization of APP in the baculovirus expression system. These studies demonstrate that APP751 is abundantly synthesized and inserted into certain of the membrane compartments of the cell. As early as 24 hr postinfection, APP751 is found associated with all membrane compartments excepting mitochondrial membranes. The patterns of immunolabeling are consistent with our biochemical findings that the protein is processed in these cells so as to release the extracellular domain and to retain a transmembrane and intracellular segment. These data provide the first morphological demonstration of the membrane location of APP751, its posttranslational processing to a secreted fragment, and its exclusion from the mitochondrial membranes. This system is especially valuable for identifying conditions under which antibodies raised against APP or appropriate synthetic peptides will react with native APP.

Amyloid beta-Protein Precursor↗
PubMed1991

Distribution and activity of alternatively spliced Alzheimer amyloid peptide precursor and scrapie PrP mRNAs on rat brain polysomes.

Mammalian brains contain low levels of the Alzheimer amyloid precursor variants (AAPPs) and the normal form of the scrapie agent protease-resistant protein (PrPc); however, their mRNAs are readily detectable. To understand these discrepancies we have investigated some aspects of the translational regulation of these mRNAs. An accurate blot-hybridization procedure was developed to measure absolute amounts of mRNA. Rat brain contains the following mRNA levels (ng/g tissue) AAPP(695), 170; AAPP(751/770), 63; PrPc, 144; actin, 615; glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 359; ferritin, 148. The method was also used to determine the distribution of mRNAs between translationally active polysomes and translationally inactive ribonucleoprotein protein particles (mRNPs). More than 90% of G3PDH and actin mRNAs were associated with polysomal RNA; whereas, ferritin light chain mRNA was predominantly (90%) in mRNP RNA. The degree of cross-contamination of mRNPs with polysomes was less than 10%. Probes specific for the scrapie PrP protein and the AAPP(695) splice junction revealed that 70% of these mRNAs were associated with polysomes. One-half of AAPP(751/770) mRNAs (which comprise 20-30% of all AAPP mRNA in brain) were found in polysomes. We conclude therefore that both scrapie and AAPP mRNAs are subject to translational regulation in rat brain. Evidence from in vitro translational experiments confirm the message distribution determined by blot hybridization and corroborate the hypothesis that AAPP is subject to partial post-transcriptional regulation. Nevertheless, the low tissue levels of AAPP and PrPc must result primarily from their relatively rapid turnover.

Amyloid beta-Peptides↗
PubMed1991

Effect of surface sterilization, fumigation and gamma irradiation on the microflora and germination of barley seeds.

Sodium hypochlorite (NaOCl) and mercuric chloride (HgCl2) surface sterilization, methyl bromide and propylene oxide fumigation and gamma irradiation treatments were compared for their effectiveness in killing microorganisms on or within barley seeds. Surface sterilization with 12.5, 25 or 50% (v/v) NaOCl for 5, 15 or 30 min, decreased Fusarium spp., Epicoccum purpurascens, and Bacillus spp. but did not kill Alternaria alternata. However, surface sterilization with 0.1 or 0.2% (w/v) HgCl2 for 3 min significantly decreased A. alternata, Fusarium spp. and E. purpurascens but Bacillus spp. were only killed by 0.3% (w/v) HgCl2 used for 10 min, which also decreased seed germination. Aspergillus flavus inoculated onto barley seeds as spores, was completely killed by surface sterilization with NaOCl but not with HgCl2, while Fusarium culmorum was killed by both NaOCl and HgCl2 treatments. Fumigation with methyl bromide yielding a concentration-time product of 3000 mg h l-1 or with propylene oxide giving a concentration-time product of 2400 mg h l-1 eliminated all filamentous fungi but Bacillus spp. and yeasts survived, and both treatments adversely affected seed germination. Gamma irradiation at a dose of 4 kGy eliminated most Alternaria, Fusarium and Epicoccum spp. but a dose of 12 kGy was required to kill Bacillus spp., yeasts and Aureobasidium pullulans. Germination was improved slightly up to a dose of 8 kGy but gradually decreased with increase in dosage to 15 kGy of gamma irradiation.

Alternaria↗
PubMed1991

Immunoassay of ochratoxin and other mycotoxins from a single extract of cereal grains utilizing monoclonal antibodies.

Immunoassays provide rapid, specific, sensitive and inexpensive methods for analysing mycotoxins but have generally been tested individually. Mycotoxigenic fungi rarely occur in pure culture in nature, and different mycotoxins may occur together. It would therefore be advantageous if several immunoassays for different mycotoxins could utilize a single extract. Monoclonal antibodies specific for ochratoxin A, aflatoxin B1 and T-2 toxin, raised at the University of Strathclyde, allow detection limits of 1 ng/ml ochratoxin A, 0.1 ng/ml aflatoxin B1 and 10 ng/ml T-2 toxin, when used in competitive enzyme-linked immunosorbent assays. These antibodies have been used to assay ochratoxin A. aflatoxin B1 and T-2 toxin in a single acetonitrile: 0.5% KCl:6% H2SO4 (89:10:1) extract of cereal grain. Extracts were either diluted 1:10 for direct assay or subjected, before assay, to a simple liquid-liquid clean-up procedure, which removed interfering substances and resulted in a 5:1 concentration. Recoveries from barley to which mycotoxins had been added averaged 95.8% for ochratoxin A, 93.8% for aflatoxin B1 and 80.6% for T-2 toxin, and the detection limits were 5 ng/g for ochratoxin A, 4 ng/g for aflatoxin B1 and 50 ng/g for T-2 toxin. Mean coefficients of variation within and between assays and between subsamples were less than 12% for ochratoxin A and aflatoxin B1 but up to 17% for T-2 toxin in assays of barley inoculated with toxigenic fungi.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetonitriles↗
PubMed1988

Fine mapping of an Alzheimer disease-associated gene encoding beta-amyloid protein.

We have sublocalized an Alzheimer Disease-associated gene, which encodes for cerebrovascular beta-amyloid protein, to the region from the centromere through the proximal half of band 21q21 using both somatic cell and in situ mapping techniques. In addition we found repeatedly significant but weaker hybridization of the beta-amyloid protein probe to the short arm of chromosome 20. 794 cells were analyzed from whole blood, lymphoblastoid and skin cultures. The latter two types of cultures had parts of the 21st chromosome translocated to other chromosomes facilitating sublocalization.

Alzheimer Disease↗