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Biomedical subjects

A Subramanian

Publications and source records attributed to A Subramanian.

At least 19 recordsLinked to original sources
PubMed2002

Metastasis to and from the central nervous system--the 'relatively protected site'.

The brain has long been recognised as a site with a very low rate of metastases, despite the potential for cancers to be extremely locally aggressive. This feature contrasts with most of the rest of the body, where metastatic spread is much more common. The pathological behaviour of any tumour is governed by both its inherent composition and the composition of the matrix in which it is sited. Much work has been done in recent years to elucidate the factors within the central nervous system (CNS) that give the brain its unique properties. Tumour interactions with the blood-brain barrier, microglia, and various matrix proteins, cytokines, and growth factors have a central role. This review concentrates mainly on the process of tumour spread from the CNS and explores how the brain is a protected site. CNS metastases from extraneural sites are also briefly covered.

Animals↗
PubMed2001

Separation of chromium (III) and chromium (VI) by capillary electrophoresis using 2,6-pyridinedicarboxylic acid as a pre-column complexation agent.

A simple method was developed for the simultaneous determination of Cr(III) and Cr(VI) by capillary zone electrophoresis (CZE), where Cr(III) was chelated with ligands to form anionic complexes. Nitrilotriacetic acid, N-2-hydroxyethylenediaminetriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, and 2,6-pyridinedicarboxylic acid (PDCA) were investigated as Cr(III) complexing ligands. Of all the ligands studied, 2,6-PDCA with Cr(III) gave the largest UV response and high selectivity for Cr(III). In addition, the condition for pre-column derivatization, including pH, concentration ratio [Cr(III)/2,6-PDCA] and the stability of Cr(III) complexes were also examined. The separation of anionic forms of Cr(III) and Cr(VI) was achieved using co-CZE with UV detection at 185 nm. The electrolyte contained 30 mM phosphate, 0.5 mM tetradecyltrimethylammonium bromide, 0.1 mM 2,6-PDCA and 15% (v/v) acetonitrile at pH 6.4. The detection limits were 2 microM for Cr(III) and 3 microM for Cr(VI) and linear plots were obtained in a concentration range of 5-200 microM. The utility of the method was demonstrated for the determination of Cr(III) and Cr(VI) in contaminated soils.

Chromium↗
PubMed2001

p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasia and coronary artery occlusion.

Inhibition of proliferative neointima formed by vascular smooth muscle cells is a potential target in preventing angioplasty-induced restenosis. We have created a potent antiproliferative by fusing the active regions of the p27 and p16 cell cycle inhibitors. Intravascular delivery of a replication-deficient adenoviral vector (AV) encoding this p27-p16 fusion protein, named W9, inhibited balloon injury-induced neointimal hyperplasia in rabbit carotid arteries. In a therapeutically more relevant model, AV-W9 was delivered to balloon-injured porcine coronary arteries in vivo using an infusion catheter. Of the three coronary arteries, two were injured with a 15-mm balloon catheter and either were left untreated or were treated with 10(12) viral particles of either AV-W9 or a control null virus. AV-W9 treatment significantly inhibited neointimal hyperplasia in this porcine arterial balloon injury model compared with untreated or control virus-treated vessels. The average intimal area of the AV-W9-treated group 10 days after balloon injury and treatment was 0.42+/-0.36 mm(2), whereas the AV-null group demonstrated an intimal area of 0.70+/-0.52 mm(2). At day 10 the average intimal thickness of the AV-W9-treated vessels was 9.1 microm (n=5, x 20 magnification) compared with 21.2 microm (n=5, x 20 magnification) in control virus-treated vessels. This trend was also observed at 28 days after balloon injury and gene transfer during which AV-W9-treated vessels demonstrated an average intimal thickness of 4.7 microm (n=8, x 20 magnification) compared with 13.3 microm (n=3, x 20 magnification) in control virus-treated vessels and 7.3 microm (n=5, x 20 magnification) in the sham-treated vessels. The AV-W9 treatment was safe and well tolerated. These data suggest that AV-W9 gene therapy may be useful in preventing angioplasty-induced intimal hyperplasia in the coronary artery.

Adenoviridae↗
PubMed2001

Initial sequencing and analysis of the human genome.

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

Animals↗
PubMed2001

Inverted meso-aryl porphyrins with heteroatoms; characterization of thia, selena, and oxa N-confused porphyrins.

Synthesis and characterization of inverted porphyrins containing S, Se, and O are reported. A simple 3 + 1 MacDonald-type condensation using modified tripyrrane containing the N-confused ring and diols afforded various N-confused porphyrins 6a-f in 19-30% yield. The single-crystal X-ray structure of 6b shows a ruffled conformation with tilt angles of 21.11 degrees and 31.23 degrees for the N-confused ring and the adjacent pyrrole ring III, respectively, revealing its severe nonplanarity. Significant changes in C alpha-C beta, C beta-C beta, and C alpha-X bond lengths are observed in 6b relative to free thiophene and pyrrole, suggesting the altered delocalization pathway in the modified N-confused porphyrins. The two molecules in the unit cell show a cyclophane-type noncovalent dimer with a face to face orientation of two N-confused pyrrole rings as a result of the presence of weak N-H...N and C-H...N intermolecular hydrogen bonds involving pyrrole-NH, the N atom of the N-confused ring, and the C atom of the pyrrole ring. A detailed 1H and 13C NMR study by 1D and 2D methods allowed assignments of all the peaks in the free base and protonated forms. NMR studies reveal the presence of three different tautomeric forms in solution for 6c in CDCl3 at low temperature. UV-visible studies reveal absorption band shifts upon heteroatom substitution, and the magnitudes of these shifts are dependent on the nature of the heteroatom. In all cases both monoprotonated and diprotonated species have been identified, and on addition of acid, the first proton goes to the outer N2 atom of the N-confused ring.

Journal Article↗
PubMed2001

Accumulation of organochlorine pesticides and polychlorinated biphenyls in sediments, aquatic organisms, birds, bird eggs and bat collected from south India.

Concentrations of polychlorinated biphenyls (PCBs), DDT and its metabolites (DDTs), HCH isomers (HCHs), chlordane compounds (CHLs) and hexachlorobenzene (HCB) were determined in sediment, soil, whole body homogenates of resident and migratory birds and their prey items (including fish, green mussel, snail, earthworm, crabs, prawn, lizard and frogs), bird eggs and bats collected from southern India during 1995 and 1998. Accumulation pattern of organochlorines (OCs) in biota was, in general, in the order, HCHs > DDTs > PCBs > CHLs = HCB. Magnitude of OC concentrations increased in the order of sediments < green mussel < earthworm < from < lizard < fish < bird egg < bats < birds tissues. Biomagnification features of OCs were examined in resident and migrant birds to evaluate the exposure levels of these chemicals in wintering grounds of migrant birds. Accumulation of DDTs in migratory birds during wintering in India may be of concern due to the great biomagnification potential of DDTs. Eggs of some resident species contained noticeable concentrations of OCs. Concentrations of OCs in three species of bats analyzed in this study were lower than that found in passerine birds. In addition to OCs, butyltin compounds were also detected at low concentrations in bats.

Animals↗
PubMed2000

Use of spray-dried zirconia microspheres in the separation of immunoglobulins from cell culture supernatant.

A method suitable for the isolation of monoclonal antibodies (MAbs) on novel zirconia microspheres (20-30 microm) is described. Zirconia microspheres were generated by spray drying colloidal zirconia. Spray-dried zirconia microspheres were further classified and characterized by X-ray diffraction, BET porosimetry and scanning electron microscopy. Spray-dried zirconia microspheres were modified with ethylenediamine-N,N'-tetra(methylenephosphonic) acid (EDTPA) to create a cation-exchange chromatographic support. The chromatographic behavior of a semi-preparative column packed with EDTPA-modified zirconia microspheres was evaluated and implications for scale-up are provided. EDTPA-modified zirconia microspheres were further used to purify MAbs from cell culture supernatant. Analysis by enzyme linked immunosorbent assay and gel electrophoresis demonstrate that MAbs can be recovered from a cell culture supernatant at high yield (92-98%) and high purity (>95%) in a single chromatographic step.

Animals↗
PubMed2000

Structural analysis of alpha-enolase. Mapping the functional domains involved in down-regulation of the c-myc protooncogene.

Myc-binding protein-1 (MBP-1) is a 37-kDa protein with sequence homology to the 3' portion of the alpha-enolase gene. alpha-Enolase is a 48-kDa protein, which plays a critical role in the glycolytic pathway. MBP-1 binds to the c-myc P2 promoter and down-regulates c-myc expression. We have investigated the role of alpha-enolase in regulation of the c-myc protooncogene. RNase protection assay shows that alpha-enolase is transcribed into a single RNA species in HeLa cells. A start codon, 400 base pairs downstream of the alpha-enolase ATG, corresponds to the MBP-1 ATG, suggesting that MBP-1 is an alternative translation initiation product of the alpha-enolase RNA. Domain mapping was performed using constructs containing truncations of the alpha-enolase gene. In vitro binding to the c-myc gene was abolished after deletion of the N-terminal portion of alpha-enolase. In order to determine the relationship between DNA binding activity and transcription inhibition, we performed co-transfection assays in HeLa cells. These studies confirmed that an N-terminal deletion of alpha-enolase is unable to down-regulate c-myc promoter activity. Our data suggest that alpha-enolase plays an important role in regulation of c-myc promoter activity in the form of an alternative translation product MBP-1, which is distinct from its role as a glycolytic enzyme.

Biomarkers, Tumor↗
PubMed2000

Isomer-specific accumulation and toxic assessment of polychlorinated biphenyls, including coplanar congeners, in cetaceans from the North Pacific and Asian coastal waters.

To elucidate the global distribution and toxicological impacts of polychlorinated biphenyls (PCBs) on cetaceans, the present study determined the concentrations of individual PCB congeners, including toxic non-ortho (IUPAC Nos. 77, 126, 169) and mono-ortho (IUPAC Nos. 105, 118, 156) coplanar congeners, in the blubber of 10 species of adult male odontocetes collected from several locations in the North Pacific Ocean and along coastal waters of Japan, Hong Kong, the Philippines, and India during 1985-1997. Total PCB concentrations in cetaceans from temperate and cold waters were higher than those in cetaceans from tropical regions. Residue levels were found to be the highest in Fraser's dolphins collected off Kii Peninsula, Japan, and hump-backed dolphins from Hong Kong, reflecting serious marine pollution by PCBs in industrialized Asian countries. Penta- and hexa-chlorobiphenyls were the predominant PCB congeners, accounting for about 70% of the total PCBs. 2,3,7,8-Tetrachlorodibenzo-p-dioxin equivalents (TEQs) of non- and mono-ortho coplanar PCBs in the blubber of cetaceans ranged from 36 (in spinner dolphins from the Philippines) to 510 pg/g wet weight (in hump-backed dolphins from Hong Kong). Toxic evaluation of coplanar PCBs using the TEQ concept indicates an greater impact on cetaceans from mid-latitudes. Toxicity contribution of mono-ortho congener IUPAC 118 was prominent in species from high latitude oceans, such as the Bering Sea and the North Pacific, whereas non-ortho congener IUPAC 126 accounted for the highest contribution in cetaceans from lower latitude regions, such as the Philippines and India. The estimated TEQ concentrations in the blubber of some cetacean species, such as northern right whale dolphin and Pacific white-sided dolphin from the northern North Pacific, Dall's porpoise from the Japan Sea, striped dolphin off Sanriku and Fraser's dolphin off Kii Peninsula, Japan, hump-backed dolphin and finless porpoise from Hong Kong, exceeded the levels associated with immunosuppression in harbour seals.

Animals↗
PubMed2000

Particle image velocimetry investigation of intravalvular flow fields of a bileaflet mechanical heart valve in a pulsatile flow.

BACKGROUND AND AIM OF THE STUDY: Our previous studies of bileaflet mechanical heart valves (MHV) explanted from sheep revealed patterns of localized platelet aggregation on valve surfaces, which may have clinical relevance. Since flow phenomena may promote localized platelet aggregation, an evaluation of flow within a valve lumen was conducted. METHODS: Phase-locked particle image velocimetry (PIV) measurements were obtained within the lumen of a 'mitral' model bileaflet MHV with transparent acrylic leaflets and housing, in a pulsatile flow loop. Instantaneous, two-dimensional flow maps of a central plane, perpendicular to the flow and leaflet pivot axes, were obtained at discrete times during the simulated cardiac cycle. Flow conditions were cardiac output, 3.5 l/min; rate, 72 beats/min; and systolic duration, 300 ms, using blood analog fluid refractive index-matched to acrylic. Leaflet closing velocities and angles were found using double-exposure imagery, and maximum leaflet closing velocity was extrapolated from regression analysis. RESULTS: During full opening, flow within the three lumenal orifices formed a three-peak axial velocity profile. Vorticity was concentrated in shear layers adjacent to downstream leaflet surfaces and in downstream wakes. Forward flow peak velocity was 90 cm/s, with a steep velocity gradient in the central orifice. During closing, the central-gap regurgitant flow formed a jet (peak velocity, 144 cm/s). High vorticity occurred near leaflet leading and trailing edges. During full closure, first a transient (<3 ms) 'stopping vortex' developed near the leaflet trailing edge, followed by a wall jet which formed at the leaflet-housing junction. Maximum leaflet closing velocity was 1.4 m/s. CONCLUSION: Localized jets, steep velocity gradients, high vorticity and vortex recirculation have been observed in vitro near model MHV surfaces. In vivo, each of these flow phenomena, when occurring near valve surfaces, may promote localized platelet aggregation. For the acrylic leaflets, maximum velocity was comparable with results reported for pyrolytic carbon leaflets. PIV of fully transparent models is a promising method for evaluating lumenal flows.

Cardiac Output↗
PubMed1999

A red-dot-blot protein assay technique in the low nanogram range.

A simple, sensitive, rapid (3 min), and highly reproducible solid-phase assay for the detection of proteins in the low nanogram range (4 ng) is described. The assay is based on differential Ponceau S staining of the protein spots on nitrocellulose and quantification of the protein-dye complexes on lubricated membranes using a densitometer. The dye solution used for protein staining contained 0.1% Ponceau S in 15% phosphoric acid and 10% ethanol. Proteins were directly spotted onto pre-Ponceau S-stained nitrocellulose membranes, cross-linked with glutaraldehyde, rinsed in NaOH, restained with Ponceau S, and finished by rinsing in acid water at pH 3. Dry membranes were lubricated with mineral oil to achieve brightness of the colored spots before scanning with a densitometer at 560 nm. The assay shows tolerance to extreme acidic and basic buffer conditions and no significant protein-to-protein variations were observed. The effects of detergent contaminants and various other reagents such as polyethylene glycol, mercaptoethanol, and urea were also tested in the assay. The nonionic detergent, digitonin, and the anionic detergent, sodium dodecyl sulfate, up to 1%, and Triton X-100 up to 0.25% do not interfere with the assay. Efficacy of the assay was tested for five different proteins and the sensitivity was compared with the most widely used method of Bradford's.

Azo Compounds↗
PubMed1999

Purification of monoclonal antibodies from cell culture supernatants using a modified zirconia based cation-exchange support.

A method suitable for the isolation of monoclonal antibodies (Mabs) is described. The protocol utilizes a zirconia based column modified with ethylenediamine-N,N'-tetra(methylenephosphonic) acid to create a novel cation-exchange chromatographic support. Initial experiments using a linear salt gradient demonstrate the ability of this support to efficiently separate Mab from transferrin and bovine serum albumin in a model matrix. Results of the purification of Mab from an actual cell culture supernatant over a range in protein concentrations are also shown. Analyses by enzyme-linked immunosorbent assay and gel electrophoresis demonstrate that Mabs can be recovered from a cell culture supernatant at high yield (92-98%) and high purity (> 95%) in a single chromatographic step.

Adsorption↗
PubMed1999

Transgenic pigs as bioreactors: a comparison of gamma-carboxylation of glutamic acid in recombinant human protein C and factor IX by the mammary gland.

The mammary gland of transgenic livestock can be used as a bioreactor for producing complex therapeutic proteins. However, the capacity for making a given post-translational modification upon any given polypeptide is uncertain. For example, the efficiency of gamma-carboxylation of glutamic acid in the amino terminal regions of recombinant human protein C (rhPC) and recombinant human Factor IX (rhFIX) is different at similar expression levels. At an expression level of about 200 microg/ml in the milk of transgenic pigs, rhFIX is highly gamma-carboxylated as indicated by pro-coagulant activity and amino acid sequencing. However, only about 20-35% of rhPC has a native, gamma-carboxyglutamic acid-dependent conformation and anti-coagulant activity. Thus, this work provides an example of apparent differences in substrate specificity between two homologous proteins to the endogenous carboxylase of porcine mammary epithelium which leads to varying degrees of post-translational modification.

Amino Acid Sequence↗
PubMed1999

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.

Animals↗
PubMed1999

Regulation of human CD4(+) alphabeta T-cell-receptor-positive (TCR(+)) and gammadelta TCR(+) T-cell responses to Mycobacterium tuberculosis by interleukin-10 and transforming growth factor beta.

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor beta [TGF-beta]) cytokines. IL-10 and TGF-beta are produced by M. tuberculosis-infected macrophages. The effect of IL-10 and TGF-beta on M. tuberculosis-reactive human CD4(+) and gammadelta T cells, the two major human T-cell subsets activated by M. tuberculosis, was investigated. Both IL-10 and TGF-beta inhibited proliferation and gamma interferon production by CD4(+) and gammadelta T cells. IL-10 was a more potent inhibitor than TGF-beta for both T-cell subsets. Combinations of IL-10 and TGF-beta did not result in additive or synergistic inhibition. IL-10 inhibited gammadelta and CD4(+) T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4(+) T cells and, to a lesser extent, for gammadelta T cells. TGF-beta inhibited both CD4(+) and gammadelta T cells directly and had little effect on APC function for gammadelta and CD4(+) T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis-infected monocytes to a greater extent than TGF-beta. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-beta both inhibited CD4(+) and gammadelta T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis.

Antigens, CD↗
PubMed1998

STAT 5 and NF-Y are involved in expression and growth hormone-mediated sexually dimorphic regulation of cytochrome P450 3A10/lithocholic acid 6beta-hydroxylase.

The level of expression of a number of sexually differentiated liver proteins is primarily determined by plasma growth hormone (GH). Adult males have a pulsatile profile of GH release, while females have a relatively steady-state pattern of GH release. An important subset of these sexually differentiated hepatic proteins is certain cytochrome P450s (P450s). CYP3A10/6beta-hydroxylase is a male-specific P450 that catalyzes 6beta-hydroxylation of lithocholic acid, and the pattern of GH secretion is directly responsible for male-specific expression of this gene. The DNA element involved in GH-mediated regulation of CYP3A10/6beta-hydroxylase promoter activity binds a member of the STAT (signal transducers and activators of transcription) family of proteins. In this study we functionally demonstrate that two members of the STAT family, STAT 5a and STAT 5b, mediate GH-dependent regulation of CYP3A10/6beta-hydroxylase promoter activity. Furthermore, a neighboring DNA element binds NF-Y, a transcription factor involved in maintaining high levels of transcription of many genes and known to functionally interact with other factors. In the CYP3A10/6beta-hydroxylase gene, NF-Y also modulates binding of STAT 5, thereby modulating GH-mediated activation of its transcription.

Animals↗
PubMed1997

Dot-blot analysis of the degree of covalent modification of proteins and antibodies at amino groups.

The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethyl-maleic anhydride prior to processing. Immobilon AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected epsilon-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzene sulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 microliter) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.

Amines↗