Biomedical subjects

D Agrawal

Publications and source records attributed to D Agrawal.

At least 55 records · Page 3Linked to original sources

PubMed1990

Effect of methyl isocyanate (MIC) on rat erythrocytes.

Changes in surface morphology of rat erythrocytes were studied by scanning electron microscopy after a single and repeated exposure of methyl isocyanate under static conditions. Marked morphological changes (echinocytes and spherocytes) were observed, followed by an increase in osmotic fragility of the erythrocyte membrane. The activity of the membrane bound enzyme adenosine triphosphatase (Na+, K+, Mg++) was inhibited.

Adenosine Triphosphatases↗

PubMed1989

Effect of cycloheximide on palmitylation of PO protein of the peripheral nervous system myelin.

Incubation of rat sciatic nerve slices with Krebs-Ringer bicarbonate buffer containing [3H]palmitic acid resulted in the acylation of the PO glycoprotein and a 24 kDa protein of the peripheral nerve myelin. Radioactivity was removed from PO after treating PO with hydroxylamine (83%) and methanolic KOH (97%). These results provided evidence that the radioactivity incorporated into PO was not due to the metabolic conversion of [3H]palmitic acid into amino acids or sugars. PO was more heavily labelled in the homogenate than in the myelin membrane in 8-day-old rat nerve between 5 min and 2 h of incubation. These results suggested that PO may be primarily acylated in the cell body. Incubation of purified myelin with [1-14C]palmitoyl-CoA resulted in the non-enzymic acylation of PO. This provided evidence of the absence of fatty acyltransferase from the purified peripheral nerve myelin. Glycosylation of PO has been shown to occur in the Golgi complex, and monensin inhibited glycosylation of PO in the homogenate and myelin by 53 and 61% respectively. These results suggest that the processing of PO in the Golgi complex and the assembly of PO into myelin is impaired by monensin. However, fatty acylation of PO was unimpaired by monensin, suggesting that the addition of fatty acids may not occur in the Golgi complex. There was a progressive decrease in the acylation of PO between 5 min (28%) and 2 h (61%) in the presence of cycloheximide, as the pool of previously synthesized PO was gradually depleted. These results also provide evidence that palmitylation of PO is not coupled to protein synthesis, and acylation of this protein probably occurs in the early subcompartment of the Golgi complex, which appears to be insensitive to monensin.

Acylation↗

PubMed1989

Tumor promoters accentuate phosphorylation of PO: evidence for the presence of protein kinase C in purified PNS myelin.

The effects of carbon tetrachloride, methylene chloride and chloroform on phosphorylation of PO was examined. The results of the dose response curve revealed that carbon tetrachloride (0.67%), methylene chloride (2%) and chloroform (1%) induced phosphorylation of PO by approximately 4, 6, and 12-fold, respectively. PO was found to be phosphorylated on the serine residue, and the phosphorylation of the serine residue was markedly increased when PO was phosphorylated in the presence of these compounds. Since tumor promoters, carbon tetrachloride and chloroform, have been shown to activate protein kinase C in platelets it is postulated that the increased phosphorylation of PO may result from the activation of myelin associated protein kinase C. The presence of phospholipid sensitive Ca2+-dependent protein kinase (protein kinase C) in in purified nerve myelin was demonstrated by increased phosphorylation of PO in the presence of Ca2+ and phosphatidylserine.

Animals↗

PubMed1987

Cell-free acylation of rat brain myelin proteolipid protein and DM-20.

Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective labelling of 'PLP' [proteolipid protein; Folch & Lees (1951) J. Biol. Chem. 191, 807-817] and 'DM-20' [Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J. Neurochem. 19, 2083-2089] which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography. These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin. Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively. Incubation of myelin with [3H]palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively. The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids. The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of [3H]palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with [3H]palmitic acid and [14C]palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin. We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin. Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.

Acylation↗

PubMed1987

In vitro acylation of myelin PLP and DM-20 in the quaking mouse brain.

Both proteolipid proteins (PLP) and DM-20 were found to be present by the immunoblot technique in myelin isolated from quaking mouse brain; however, the relative concentration of these proteins in myelin from quaking brain was substantially reduced when compared to the control. Brain slices from littermate control and quaking mice were incubated with [3H]palmitic acid to determine the incorporation of fatty acid into myelin proteolipid proteins. Fluorography of gels containing myelin proteins from control and quaking mice brain revealed that both PLP and DM-20 were acylated. The incorporation of [3H]palmitic acid into quaking myelin PLP and DM-20 was reduced by 75% and 20% respectively of those in control brain. The significance of differential acylation of quaking myelin PLP and DM-20 is discussed with respect to availability of non-acylated pools of proteolipid proteins and the activities of acylating enzymes.

Acylation↗

PubMed1987

Lindane-induced changes in glucose and glutathione levels in cats.

Cats were given lindane (1 and 2 mg/kg i.v.) and the levels of glucose in blood and glutathione in blood and liver were measured at 30 min, 1, 2, 4 and 8 h. The results showed a sharp decrease in blood glutathione at an early interval with subsequent recovery, whereas there was no change in liver up to 2 h followed by a significant decrease at later intervals with no sign of recovery. The pesticide also showed a hypoglycemic effect at all intervals.

Animals↗

PubMed1986

Immunochemical evidence of phosphorylation of a new 23K basic protein in rat brain myelin.

Myelin from developing rat brain (8-44 day-old rat) was incubated in vitro with [gamma-32P]ATP to determine how many basic proteins were phosphorylated. Myelin proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets. The nitrocellulose sheets were stained with antisera to human basic protein by the immunoblot technique. Five basic proteins with molecular weights of 23K, 21.5K, 18.5K, 17K, and 14K were distinctly immunostained. These basic proteins were found to be phosphorylated when the same nitrocellulose sheets were exposed to x-ray film. The in vitro phosphorylation of 23K and 21.5K basic proteins appear to decrease with maturation of the brain. The result of this study suggests that intense phosphorylation of various forms of basic proteins, in particular 23K and 21.5K basic proteins, during the initial stages of myelin formation, may play a pivotal role in the compaction of myelin membrane.

Adenosine Triphosphate↗

PubMed1986

Expression of myelin proteolipid protein and basic protein in normal and dysmyelinating mutant mice.

Expression of myelin proteins was studied in the brains of 21-day-old normal mice and three dysmyelinating mutants-jimpy, quaking, and shiverer. Total brain polyribosomes and poly(A)+ mRNA were translated in two cell-free systems and the levels of synthesis of the myelin basic proteins (MBPs) and proteolipid protein (PLP) were determined. Synthesis of the MBPs in quaking homozygotes was at or above normal levels but PLP synthesis was significantly reduced to approximately 15% of control values, indicating independent effects on the expression of these proteins in this mutant. Immunoblot analysis of 21-day-old quaking brain homogenates showed a reduction in the steady-state levels of MBPs and PLP, suggesting a failure of newly synthesized MBPs to be incorporated into a stable membrane structure such as myelin. In the shiverer mutant very little synthesis of MBPs was observed, whereas greater synthesis of PLP occurred (approximately 50% of control). Almost no MBP, and low levels of PLP, were detected in the immunoblots, suggesting the possibility of a partial failure of PLP to be assembled into myelin in shiverer. In the jimpy mutant, low levels of MBP synthesis were observed in vitro (approximately 26% of controls) and very little synthesis of PLP was evident. The immunoblots of 21-day jimpy brain homogenates revealed no appreciable steady-state levels of PLP or MBP, again indicating that most newly synthesized MBPs were not incorporated into a stable membrane structure in this mutant. In sum, the data show that in the three cases examined, the mutation appears to affect the expression of the MBPs and PLP independently. Furthermore, regardless of their absolute levels of synthesis these proteins may or may not be assembled into myelin.

Animals↗

PubMed1985

Electron microscopic immunocytochemical localization of myelin proteolipid protein and myelin basic protein to oligodendrocytes in rat brain during myelination.

Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti-large basic protein serum was shown by the immunoblot technique to cross-react with all five forms of basic protein present in the myelin of 8-day-old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.

Animals↗

PubMed1985

Myelin basic protein and P2 protein are not immunohistochemical markers for Schwann cell neoplasms. A comparative study using antisera to S-100, P2, and myelin basic proteins.

Immunohistochemical localization of tissue specific or cell-specific antigenic markers in neoplastic cells has become an increasingly important tool in the pathologic diagnosis of tumors. The myelin-specific proteins of peripheral nervous system myelin, because they are normally synthesized in Schwann cells, are potentially useful markers for neoplasms arising from peripheral nerves. The authors carried out immunohistochemical studies on 18 cases of Schwann cell neoplasms, including schwannomas, neurofibromas, and granular cell tumors, to determine whether two myelin-specific proteins, myelin basic protein and P2 protein, were present in neoplastic Schwann cells. None of these tumors showed immunostaining for either myelin basic protein or P2 protein in neoplastic cells. In contrast, S-100 protein, which is a well established marker for normal and neoplastic Schwann cells, was localized by immunohistochemistry to neoplastic cells in all 18 neoplasms. Therefore, although myelin basic protein and P2 protein are known to be Schwann-cell-specific proteins, they do not appear to be expressed commonly in neoplastic Schwann cells.

Histocytochemistry↗

PubMed1984

Identification of antibodies in anti-CNS and anti-PNS myelin sera by immunoblot, characterization by immunohistochemistry, and their effect in tissue culture.

Immunoblot analysis of antiserum to rat central nervous system (CNS) myelin revealed antibodies to myelin basic protein (MBP), proteolipid protein (PLP), and numerous high molecular weight proteins. In addition, anti-CNS myelin serum exclusively immunostained 4 basic proteins of rat peripheral nervous system (PNS) myelin. Similarly, anti-PNS myelin sera immunostained many high molecular weight proteins in both CNS and PNS myelin in addition to P0 and 4 basic proteins. Purified MBP and PLP were immunostained by anti-CNS myelin sera and MBP and P0 by anti-PNS myelin sera, indicating that antigenic sites are preserved during protein purification. Immunohistochemical localization with antisera was confined to the myelin sheath except that antisera to CNS myelin also stained oligodendrocytes during the active period of myelination. While anti-CNS myelin sera specifically demyelinated centrally myelinated fibers in culture, none of the anti-PNS myelin sera used here demyelinated organotypic spinal cord-dorsal root ganglion cultures.

Animals↗

PubMed1984

Immunoblot identification of 13.5 kilodalton myelin basic protein in goldfish brain myelin.

Myelin isolated from goldfish brain shows a multilamellar structure with a major dense line and two intraperiod lines. Sodium dodecyl sulfate gel electrophoresis revealed that the protein profile of goldfish brain myelin is distinctly different from that of rat brain myelin. No protein migrating to the position of proteolipid protein or DM-20 was seen in goldfish myelin. Goldfish acclimated to 5 degrees, 15 degrees, and 30 degrees C showed no qualitative differences in myelin proteins. The 13.5 kD protein in goldfish brain myelin and brain homogenate was intensely immunostained with the antiserum to human basic protein by the immunoblot technique. In contrast, none of the proteins of goldfish myelin were immunostained with antiproteolipid protein serum; however, both proteolipid protein and DM-20 of rat brain myelin were immunostained. The significance of the synthesis of myelin proteins by astrocytes in the goldfish brain is discussed.

Adaptation, Physiological↗

PubMed1983

In vivo incorporation of [3H]palmitic acid into PO protein, the major intrinsic protein of rat sciatic nerve myelin.

Separation of rat sciatic nerve myelin proteins by sodium dodecyl sulfate-slab gel electrophoresis 18 h after injection of [3H]palmitic acid into the nerve demonstrated acylation of the PO protein. When sciatic nerve myelin labeled with [3H]palmitic acid was extracted with acidified chloroform/methanol or chloroform/methanol, the radioactivity associated with PO was retained. These results provided evidence that the radioactivity derived from [3H]palmitic acid in PO protein was not due to labeled phospholipid bound to PO protein by strong physical interaction. Furthermore, the radioactivity associated with purified preparations of PO remained after dialysis and re-electrophoresis, providing additional evidence that [3H]palmitic acid was firmly bound to PO. Treatment of myelin proteins with hydroxylamine at pH 6.6 released most of the radioactivity, indicating that [3H]palmitic acid was covalently bound by ester linkage to PO. Cleavage of purified acylated PO with methanolic NaOH released 85% of the protein-bound radioactivity. Gas-liquid chromatography of the fatty acids released from PO showed labeling of methyl esters of palmitate, stearate, and oleate (56, 16, and 5%, respectively). In addition, a small unlabeled peak with retention time identical with methyl linoleate was also observed. Intraneural injection of a mixture of [3H]palmitic acid and [14C]fucose or [3H]palmitic acid and [35S]sulfate in vivo followed by disc gel electrophoresis and determination of incorporated radioactivity clearly showed that PO was glycosylated, sulfated, and acylated. The potential significance of fatty acids linked to PO is discussed.

Acylation↗

PubMed1982

In vitro acylation of rat brain myelin proteolipid protein.

The incorporation of fatty acid into myelin proteolipid protein (PLP) was studied by incubating brain slices from 17-day-old rats with [9,10-3H]palmitic acid. Myelin proteins were analyzed by gel electrophoresis. As in vivo (Agrawal, H. C., Randle, C. L., and Agrawal, D. (1982) J. Biol. Chem. 257, 4588-4592) most of the radioactivity co-migrated with PLP, with radioactivity also appearing in the regions of the DM-20 and Wolfgram proteins. Treatment of myelin proteins or purified PLP with 1 M hydroxylamine released most of the radioactivity, indicating that [3H]palmitic acid was covalently bound by ester linkage to the proteins. Cleavage of PLP with methanolic NaOH and examination of the released fatty acids by thin layer chromatography showed radioactivity associated with the methyl esters of palmitate, stearate, and oleate (76, 23, and 1%, respectively). A linear increase in radioactivity from [3H]palmitic acid was observed in PLP in both myelin and myelin-like fractions, while appearance of [14C]glycine-labeled PLP showed a lag of 45-60 minutes. These results indicate that lipid is added to PLP in the myelin and myelin-like membranes, or immediately prior to the appearance of PLP in these fractions. For both precursors, the specific activity of PLP was greater in the myelin-like fraction than in myelin, indicating that the myelin-like fraction may contain a precursor pool of PLP for myelin.

Acylation↗

PubMed1982

In vivo acylation of rat brain myelin proteolipid protein.

Examination of brain myelin proteins by sodium dodecyl sulfate-gel electrophoresis followed by fluorography clearly showed that both proteolipid protein (PLP) and DM-20 were acylated 24 h after the intracerebral injection of 30-day-old rats with [3H]palmitic acid. The radioactivity associated with PLP remained after purification, re-electrophoresis, and fluorography. Most of the radioactivity associated with PLP was removed when the gels were treated with hydroxylamine and then fluorographed, indicating that fatty acids were bound to PLP by ester linkage. Cleavage of purified PLP with methanolic sodium hydroxide readily released almost all protein-bound radioactivity. Thin layer chromatography of this material on both silver nitrate and reverse-phase plates provided evidence that most of the radioactivity co-migrated with methyl palmitate (77%) and methyl stearate (19%); however, some radioactivity was associated with methyl oleate (4%). Gas-liquid chromatography of the fatty acids associated with PLP distinctly revealed the presence of methyl palmitate and a detectable peak of methyl stearate.

Acylation↗

PubMed1982

Phosphorylation in vivo of four basic proteins of rat brain myelin.

When rat brain myelin was examined by sodium dodecyl sulphate/polyacrylamideslab-gel electrophoresis followed by fluorography of the stained gel, it was found that a host of proteins of rat brain myelin were labelled 2, 4 and 24h after the intracerebral injection of H(3) (32)PO(4). Among those labelled were proteins migrating to the positions of myelin-associated glycoprotein, Wolfgram proteins, proteolipid protein, DM-20 and basic proteins. The four basic proteins with mol.wts. 21000, 18000 (large basic protein), 17000 and 14000 (small basic protein) were shown to be phosphorylated after electrophoresis in both acid-urea- and sodium dodecyl sulphate-containing gel systems followed by fluorography. The four basic proteins imparted bluish-green colour, after staining with Amido Black, which is characteristic of myelin basic proteins. The four basic proteins were purified to homogeneity. Fluorography of the purified basic proteins after re-electrophoresis revealed the presence of phosphorylated high-molecular-weight ;polymers' associated with each basic protein. The amino acid compositions of the phosphorylated large basic protein and small basic proteins are compatible with the amino acid sequences. Proteins with mol.wts. 21000 and 17000 gave the expected amino acid composition of myelin basic proteins. Radiolabelled phosphoserine and phosphothreonine were identified after partial acid hydrolysis of the four purified basic proteins. The [(32)P]phosphate-protein bond in the basic protein was stable at an acidic pH but was readily hydrolysed at alkaline pH, as would be expected of phosphoester bonds involving both serine and threonine residues. Double-immunodiffusion analysis demonstrated that the four phosphorylated proteins showed complete homology when diffused against antiserum to a mixture of small and large basic proteins. Since the four basic proteins of rat brain myelin were phosphorylated both in vivo and in vitro it is postulated that the same protein kinase is responsible for their phosphorylation in both conditions.

Amino Acids↗

PubMed1982

Immunoblot identification of phosphorylated basic proteins of rat and rabbit CNS and PNS myelin: evidence for four phosphorylated basic proteins and P2 in rat PNS myelin.

The immunoblot technique permits the visualization of proteins following their separation on acrylamide gels, transfer to cellulose nitrate sheets and subsequent staining with antiserum. We have utilized this technique to demonstrate the presence of four basic proteins in rat PNS myelin with molecular weights of 21K, 18K, 17K, and 14K. Similarly, we demonstrated the presence of two basic proteins in rabbit PNS myelin (molecular weights of 21K and 18K). Exposure of the immunostained cellulose nitrate strips to X-ray film revealed the phosphorylation of four and two basic proteins in rat and rabbit PNS myelin, respectively. These basic proteins were present in the CNS myelin of the two species and were also phosphorylated. Because of the sensitivity of the immunoblot technique, it was also possible for us to visualize the P2 protein in both rat and rabbit PNS myelin.

Animals↗

PubMed1982

Development and maturation of central nervous system myelin: comparison of immunohistochemical localization of proteolipid protein and basic protein in myelin and oligodendrocytes.

The immunohistochemical localization of two myelin specific proteins-basic protein (BP) and proteolipid protein (PLP)-was compared during the process of myelination. Although both proteins were present in oligodendrocytes, (i) neither protein was observed in oligodendrocytes not already closely associated with nerve fibers exhibiting a fluorescent coating; (ii) in any discrete anatomical area oligodendrocytes were positive for BP before PLP was visible; and (iii) as myelination progressed, immunoreactivity for BP in oligodendrocytes appeared to decrease and simultaneously PLP immunofluorescence became visible in this cell type. During the period of active myelination, fibers exhibited a distinct varicose appearance. As myelination progressed, the myelin sheath increased in thickness and these varicosities became less prominent, eventually completely disappearing. Therefore, the nature and the appearance of varicosities can be used as an index of the relative stage of maturation of myelin in an individual fiber. In general, PLP appeared in fibers at a later stage of maturation than did BP based on the above criteria. However, in a relatively small number of fine fibers PLP was observed at a very early stage. In fully mature myelin, very large fibers were frequently more intensely fluorescent for BP than PLP, whereas fine myelinated fibers were more intensely stained for PLP. These observations are consistent with the following interpretations. (i) Substantial differentiation of oligodendrocytes occurs prior to appearance of either of these proteins by immunofluorescence. (ii) BP is added to the myelin sheath prior to PLP and there appears to be a shift in priority of synthesis from BP to PLP in individual oligodendrocytes during the process of myelination. (iii) Very small fibers often contain low concentrations of BP relative to PLP, and conversely, very large fibers may contain a high concentration of BP relative to PLP. Thus, the relative concentration of these proteins in myelin appears not to be constant but may vary as a function of the size of the myelinated fiber.

Aging↗