Biomedical subjects

D Agrawal

Publications and source records attributed to D Agrawal.

At least 37 records · Page 2Linked to original sources

PubMed1997

An improved flow evaluation scheme in orifices of different aspect ratios.

An accurate and reliable method of regurgitant flow calculation is currently unavailable. The goal of this study was to define a new general method of flow calculation for orifices of different aspect ratios. The success of the method relies on matching the imaged flow field distribution obtained by color flow mapping (CFM) to a three-dimensional (3D) numerical flow field distribution of known geometry. The flow field in three orifices of identical cross-sectional area with aspect ratios of 1 (circular), 2 and 4 (elliptical) was evaluated by: (a) CFM, (b) 3D echocardiographic imaging, and (c) 3D finite element modeling (FEM). The orifice shape and size were accurately estimated by 3D echocardiographic imaging. FEM showed that the normalized centerline velocity profile of the flow field depends on the orifice aspect ratio. CFM provided a good description of the centerline profile for each case. For a given distance from the orifice center, the equivelocity contour surface area increases with increasing aspect ratio. A simple flow calculation scheme was developed to calculate regurgitant flow independent of orifice shape. This improved method showed better results than previous studies and may prove to be advantageous when analyzing in vivo flow fields with complex geometries.

Computer Simulation↗

PubMed1997

The role of p27kip1 in the in vitro differentiation of murine keratinocytes.

We have studied the regulation of cyclins and cyclin-dependent kinase activities during differentiation of primary mouse keratinocytes. Differentiation was induced by placing primary murine keratinocytes into suspension culture, under conditions which prevent cells from attaching to any surface. This treatment induces synthesis of keratin 1, one of the earliest known markers of keratinocyte differentiation, and also results in a profound change in the regulation of G1 and S-phase cyclins and their associated proteins as well as their activities. The placement of cells in suspension culture reduced cyclin A, D1, and E kinase activity within 6 h, accompanied by the cessation of DNA synthesis. K1 mRNA levels were observed to increase after this period, supporting the hypothesis that cell cycle withdrawal precedes the differentiation program. Our data further revealed that the p27kip1 protein level and associated cyclin-dependent kinase inhibitory activity increased when keratinocytes were induced to differentiate. Pretreatment of adherent keratinocytes with p27kip1 antisense oligonucleotides dramatically reduced the accumulation of p27kip1 protein upon subsequent suspension culturing and prevented the onset of differentiation independently of the loss of cyclin-dependent kinase activities. Although antisense oligonucleotide treatment inhibited differentiation, it did not prevent growth arrest. Therefore, the differentiation of primary mouse keratinocytes required a function of Kip other than the inhibition of cyclin-associated activities, and we suggest that this requirement may reflect a novel Rb kinase activity present in Kip immune complexes, which is dependent on the presence of cyclin D3. Thus, the placement of keratinocytes in suspension induces a program that includes loss of cyclin activity, which is linked to terminal growth arrest, and an induction of p27kip1, which is linked to the differentiation program.

Animals↗

PubMed1996

Right ventricular regional wall motion abnormality in congenital heart disease.

Four cases of congenital heart disease with right ventricular overload and echocardiographic evidence of persistent right ventricular regional wall motion abnormalities are presented. Right ventricular infarction could be a possibility. Such regional wall motion abnormalities could add to overall right ventricular dysfunction in these cases. Echocardiography is useful in diagnosis. Right ventricular wall motion abnormalities should be studied in detail in all cases of congenital heart disease with right ventricular overload.

Adult↗

PubMed1996

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells.

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.

3T3 Cells↗

PubMed1995

Influence of hexachlorocyclohexane on phosphoinositides in rat erythrocyte membranes and brain.

Single exposure of rats to hexachlorocyclohexane (100 mg/kg) did not cause any significant change in phosphoinositide levels in rat erythrocyte membrane and cerebrum (fore brain) 2 or 24 h after exposure. However, the phosphoinositide turnover and generation of second messengers from phosphoinositides were increased in the treated erythrocyte membranes as judged from a marked increase in the incorporation of [2-3H]inositol into phosphoinositides 24 h after the treatment. A significant decrease in phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was observed in the erythrocyte membrane and cerebrum of rats repeatedly exposed to the pesticide for 3 or 6 months. This drastic reduction in phosphoinositide levels suggests adverse effects on vital membrane and cell functions modulated by phosphoinositides.

Animals↗

PubMed1995

Regulation of cyclin E and p27kip during mitosis in BALB/c 3T3 cells.

We have examined the level of cyclin E, a G1 cyclin, and p27kip, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells. Cell populations stimulated to undergo cell cycle traverse displayed little change in the level of cyclin E, while p27kip was found to be reduced 50-80% in proliferating cells. Analysis of mitotic cells, however, revealed that cyclin E is virtually absent and begins to reaccumulate soon after mitosis is complete, as does p27kip. Immunoprecipitation experiments revealed that p27kip is associated with cyclin E in quiescent but not proliferating cells, indicating that it may function to prevent development of cyclin E activity in the absence of growth factors. Based on our characterization of cyclin E and p27kip in BALB/c 3T3 cells during progression of the cell cycle, we propose a model of growth regulation controlling the G1 phase of growing and quiescent cells involving mitotic degradation of cyclin E and recovery of p27kip inhibitory activity in a late or postmitotic interval.

3T3 Cells↗

PubMed1994

Production and crystallization of virus-like particles assembled in a heterologous protein expression system.

It is of considerable interest to separate the processes of viral infectivity and virion assembly. Until recently this has only been possible with viruses that could be disassembled and reassembled in vitro. Even in these cases it was difficult to establish the authenticity of reassembled capsid protein because of possible irreversible damage that may have occurred to the protein during disassembly. An ideal method for the study of virus assembly is a protein expression system in which conditions are appropriate for spontaneous particle formation from freshly synthesized polypeptides. The baculovirus expression system has proven to be an excellent means to this end. Recently, this approach has been used to study the T = 3 Flock House insect virus and it has been demonstrated that subunits with the wild-type protein sequence, and with site-specific mutations that prevent particle maturation, will assemble and crystallize. This same approach has now been used at Purdue to study the T = 4 Nudaurelia omega capensis insect virus. There is no cell culture system currently available for the study of NomegaV, thus the expression system provides the first opportunity to study assembly under controlled conditions.

Journal Article↗

PubMed1994

Comparative studies of T = 3 and T = 4 icosahedral RNA insect viruses.

Crystallographic and molecular biological studies of T = 3 nodaviruses (180 identical subunits in the particle) and T = 4 tetraviruses (240 identical subunits in the particle) have revealed similarity in both the architecture of the particles and the strategy for maturation. The comparative studies provide a novel opportunity to examine an apparent evolution of particle size, from smaller (T = 3) to larger (T = 4), with both particles based on similar subunits. The BBV and FHV nodavirus structures are refined at 2.8 A and 3 A respectively, while the N omega V structure is at 6 A resolution. Nevertheless, the detailed comparisons of the noda and tetravirus X-ray electron density maps show that the same type of switching in subunit twofold contacts is used in the T = 3 and T = 4 capsids, although differences must exist between quasi and icosahedral threefold contacts in the T = 4 particle that have not yet been detected. The analyses of primary and tertiary structures of noda and tetraviruses show that N omega V subunits undergo a post assembly cleavage like that observed in nodaviruses and that the cleaved 76 C-terminal residues remain associated with the particle.

Crystallography, X-Ray↗

PubMed1994

In vivo phosphorylation of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP): CNP in brain myelin is phosphorylated by forskolin- and phorbol ester-sensitive protein kinases.

2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was phosphorylated in vivo, in brain slices and in a cell free system. Phosphoamino acid analysis of immunoprecipitated CNP labeled in vivo and in brain slices revealed phosphorylation of phosphoserine (94%) and phosphothreonine (5%) residues. Phosphorylation of CNP increased by 3-fold after brain slices were incubated with forskolin. Similarly, incubation of isolated myelin with [gamma-32]ATP with cAMP (5 microM) and cAMP (5 microM)+catalytic unit of cAMP dependent protein kinase dramatically increased CNP2 phosphorylation by 4- and 6-fold, respectively. It is feasible that CNP2 was predominantly phosphorylated on serine and/or threonine residues of the amino terminal peptide of CNP2, and this phosphorylation was catalyzed by protein kinase A. Phosphorylation of CNP1 and CNP2 increased 2-fold by incubating brain slices with phorbol ester. Forskolin and phorbol ester increased the phosphorylation of single, but distinct, CNP peptides. We present the first biochemical evidence that CNP2, on a protein mass basis, is far more heavily phosphorylated than CNP1, suggesting there are more phosphorylation sites on CNP2 than CNP1 and that at least one site is located on the 20-amino acid terminus of CNP2 and that it is likely a PKA site.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1993

Biochemical and structural alterations in rat erythrocytes due to hexachlorocyclohexane exposure.

The changes in biochemical constituents of rat erythrocyte membranes were observed after a single ip exposure to 300 mg commercial HCH/kg body weight (one-third of the LD50). The phospholipid:cholesterol ratio was altered, and there were changes in the activities of the membrane-bound enzymes ATPase and acetylcholinesterase. The phospholipid content was increased while cholesterol and ATPase and acetylcholinesterase activities were significantly decreased. The erythrocytes also showed morphological changes (cell deformity and echinocyte formation).

Animals↗

PubMed1993

Bilateral anterior tibial compartment syndrome in association with hypothyroidism.

Local vascular and traumatic involvement are well-recognized causes of anterior tibial compartment syndrome (ATCS) terminating as myoneural ischaemia. However, in a large number of patients the cause of ATCS remains unidentified. We document the occurrence of bilateral ATCS in a patient of hypothyroidism, to our knowledge a previously unrecorded association.

Adult↗

PubMed1991

Proteolipid protein and DM-20 are synthesized by Schwann cells, present in myelin membrane, but they are not fatty acylated.

Proteolipid protein (PLP) and DM-20 were intensely labeled after immunoprecipitation of total cellular proteins and myelin proteins labeled with [35S]methionine in nerve slices. These results provided evidence that PLP and DM-20 are incorporated into the myelin membrane following their synthesis in Schwann cells. In contrast, PLP and DM-20 were not fatty acylated after incubation of the nerve slices with [3H]palmitic acid, however, P0 glycoprotein and 24 kDa protein were heavily fatty acylated. The lack of fatty acylation of PLP and DM-20 in the peripheral nervous system suggests that fatty acyltransferase responsible for their acylation is absent or non-functional in the peripheral nervous system.

Acylation↗

PubMed1991

Oxidative damage and changes in the glutathione redox system in erythrocytes from rats treated with hexachlorocyclohexane.

The concentration of reduced glutathione in the erythrocytes of rats was significantly decreased 24-72 hr after the rats were treated with 300 mg commercial hexachlorocyclohexane/kg body weight (one-third of the LD50), given ip. The activities of glutathione reductase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase were also significantly decreased 24 hr after treatment but there was no change in glutathione peroxidase activity. The results suggest that hexachlorocyclohexane produces significant changes in the glutathione redox system of rat erythrocytes leading to oxidative membrane damage.

Animals↗

PubMed1991

Influence of mancozeb on mitogenically responsive lipids in rat cerebrum and liver.

Mancozeb, a commonly used fungicide, has been shown to induce tumours in mouse skin and maneb, unit constituent of mancozeb, is reported to induce tumours in rats. The mechanism by which mancozeb induced tumorigenicity is not known. Since the levels of inositol phospholipids and phosphatidic acid have roles in the regulation of cell proliferation, the effects of mancozeb on the levels of these lipids were studied in rats. Daily oral administration of commercial grade mancozeb at a concentration of 50 mg/kg body wt for 30 days (5 days a week) caused no significant change in the levels of inositol phospholipids and phosphatidic acid (PA) in both cerebrum and liver, while at high concentration (250 mg/kg body wt) under the same treatment schedule mancozeb increased the levels of these lipids. In cerebrum, the levels of phosphatidylinositol (PI) and PA were increased by 36 and 43% respectively without affecting the levels of polyphosphoinositides, whereas in liver the levels of not only PI (50%) and PA (49%) but also those of polyphosphoinositides were increased. These results show that mancozeb influences the levels of PA and inositol phospholipids, involved in phospholipase C-pathway of signalling.

Animals↗

PubMed1990

2',3'cyclic nucleotide-3'-phosphodiesterase in peripheral nerve myelin is phosphorylated by a phorbol ester-sensitive protein kinase.

2',3' cyclic nucleotide-3'-phosphodiesterase (CNP) is phosphorylated in the peripheral nervous system after immunoprecipitation of myelin proteins radiolabeled in vivo, in nerve slices and in a cell-free system. Only radiolabeled phosphoserine was detected after partial acid hydrolysis of immunoprecipitated CNP. Two major phosphopeptides were resolved by two dimensional electrophoresis-chromatography after digestion with trypsin of CNP phosphorylated in the nerve slices. Phosphorylation of CNP was not stimulated a) by forskolin in the nerve slices and b) after incubation of purified nerve myelin with cAMP. However, CNP phosphorylation was increased after incubation of PNS myelin with catalytic unit of protein kinase A. Phosphorylation of the central nervous system myelin CNP was dramatically stimulated by cAMP. These results suggest that PKA may be absent from peripheral nerve myelin or CNP may not be accessible to this enzyme in the PNS. Incubation of nerve slices with phorbol 12 myristate-13-acetate caused a marked increase in the phosphorylation of CNP. These results provide strong evidence that CNP is phosphorylated in the PNS and its phosphorylation in vivo is in all probability regulated by protein kinase C.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗

PubMed1990

2',3'-cyclic nucleotide-3'-phosphodiesterase in the central nervous system is fatty-acylated by thioester linkage.

2',3'-Cyclic nucleotide-3'-phosphodiesterase (CNP1 and CNP2 with Mr of 46,000 and 48,000, respectively) is the major enzyme of central nervous system myelin. It is associated with oligodendroglial plasma membrane and uncompacted myelin (myelin-like fraction), which are in contact with glial cytoplasm. Proteins of the myelin-like fraction were labeled with [3H]palmitic acid in brain slices from 17-day-old rats and immunoprecipitated with anti-CNP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material revealed intense acylation of CNP1 and CNP2, and radioactivity was released by hydroxylamine. Palmitic acid was covalently bound to CNP because radioactivity was not removed by extraction of immunoprecipitated CNP with organic solvent or by boiling in sodium dodecyl sulfate and dithiothreitol. However, treatment of immunoprecipitated CNP with (a) hydroxylamine-released palmitohydroxamate and palmitic acid, (b) sodium borohydride-released hexadecanol, and (c) methanolic-KOH-released methyl palmitate. Synthesis, acylation, or transport of CNP was not affected by monensin or colchicine. However, acylation of CNP was inhibited 24-32% by cycloheximide. These results provide conclusive evidence that CNP1 and CNP2 are fatty acid acylated with palmitate through a thioester linkage and is posttranslationally modified sometime after synthesis.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1990

The myelin-associated glycoprotein is phosphorylated in the peripheral nervous system.

Phosphorylation of the myelin-associated glycoprotein (MAG) in the peripheral nervous system is demonstrated by immunoprecipitation from myelin proteins radiolabeled in vivo, in nerve slices and in a cell-free system. Phosphoamino acid analysis of immunoprecipitated MAG revealed the presence of radioactivity in phosphoserine, but not in phosphothreonine or phosphotyrosine. Only the shorter isoform of MAG (S-MAG) was detected by immunostaining of nitrocellulose sheets with anti-MAG anti-serum after enzymatic deglycosylation of immunoprecipitated MAG labeled in nerve slices. Autoradiography of the same Western blots revealed that most of the radioactive phosphate was in S-MAG, demonstrating that the polypeptide backbone of S-MAG is phosphorylated in the PNS.

Animals↗

PubMed1990

Cleavage of the P0 glycoprotein of the rat peripheral nerve myelin: tentative identification of cleavage site and evidence for the precursor-product relationship.

The incubation of sciatic nerve slices in Krebs Ringer bicarbonate (KRB) buffer (pH 7.4) at 37 degrees C, or the incubation of freshly isolated myelin in ammonium bicarbonate buffer (pH 8), resulted in the generation of a 24 kDa protein with a concomitant decrease of P0 protein. The conversion of P0 into 24 kDa protein was blocked by heating isolated myelin at 100 degrees C for 5 min suggesting that the reaction is enzyme mediated. Inclusion of the protease inhibitors and chelating agent to isolated myelin did not prevent the formation of 24 kDa protein. Similarly, addition of CaCl2 to isolated myelin did not accentuate the formation of 24 kDa protein suggesting that the conversion of P0 into 24 kDa protein may not be due to Ca2+ activated protease. It is postulated that the formation of 24 kDa protein may be due to neutral protease and/or metalloproteinase associated with the PNS myelin. 24 kDa protein was purified and characterized. The N-terminal sequence of 1-17 amino acid residues of 24 kDa protein was identical to P0. 24 kDa protein was immunostained and immunoprecipitated with anti-P0 antiserum indicating the immunological similarities between P0 and 24 kDa protein. Labeling of 24 kDa protein with [35S]methionine provided evidence that P0 may be in all probability cleaved between Met-168 and Met-193. Further studies were carried out to demonstrate that 24 kDa protein was phosphorylated, glycosylated and acylated like P0. Phosphorylation of 24 kDa protein in the nerve slices was increased five-fold by phorbol esters and phosphoserine was the only phosphoamino acid identified after partial acid hydrolysis of 24 kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗