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Biomedical subjects

G Dawson

Publications and source records attributed to G Dawson.

At least 91 records · Page 5Linked to original sources
PubMed1996

Triglyceride dependence of factor VII coagulant activity in deep venous thrombosis.

The effect of incubation of plasma with lipoprotein lipase on factor VII coagulant (FVII:C) activity was examined in 40 patients, 22 male and 18 female, aged 28 to 77 years, with history of venographically proven deep venous thrombosis (DVT). While the mean (+/-SEM) FVII:C activity of the 40 patients was 100.9 +/- 4.1%, 19 patients had FVII:C activity less than 100%, 11 had 100 to 120% activity and 10 patients had greater than 120% FVII:C activity. The mean triglyceride level of all the patients was 84.0 +/- 6.5 mg/dl. The FVII:C activity correlated significantly with triglyceride (r = 0.36; n = 40; p = 0.021). There was about 30% average loss of FVII:C activity upon incubation of plasma with lipoprotein lipase. The mean activity loss increased from 23.8% to 31.5% and 42.6% in patients whose FVII:C activity levels were less than 100%, between 100 and 120% and more than 120% respectively, the variation in the means being statistically significant (p < 0.001). While according to current opinion, FVII:C activity represents the total FVII mass (FVII plus FVIIa) and activity state, the present findings demonstrate a lipid dependence of FVII:C activity, and raises the possibility of a therapeutic option of controlling FVII:C by controlling triglyceride levels.

Adult↗
PubMed1995

Glycosphingolipid composition of murine neuroblastoma cells: O-acetylesterase gene downregulates the expression of O-acetylated GD3.

We have studied the glycosphingolipid composition in an F-11 neuroblastoma cell line originated from hybridization of a mouse neuroblastoma cell line (N18TG-2) with rat dorsal root ganglion cells. The total lipid-bound glucose of F-11 cells was estimated to be 0.28 micrograms/mg of protein and the total lipid-bound sialic acid was 0.82 micrograms/mg of protein. The major neutral glycosphingolipids were Gb4 (37% of the total neutral glycosphingolipids), Gb3 (15%), LacCer (21%), and GlcCer (15%). The major gangliosides were found to be GM3 (37% of the total gangliosides), GD3 (27%), O-acetylated GD3 (18%), and GD1a (4%), with trace amounts of GD2. The unusually high concentration of O-acetylated GD3 is consistent with its putative role as a tumor marker. Immunocytochemical localization studies of GD3 and O-acetylated GD3, examined by mouse monoclonal antibodies R24 and D1.1, respectively, revealed that the cell bodies and processes were all positively stained. To elucidate the role of O-acetylated GD3 in tumorigenesis, we transfected F-11 cells with the O-acetylesterase gene from influenza C virus. Compared with the original cell line, the transfected cells showed a dramatic increase in the level of GD3 (150% of that in the control cells) and a significant decrease of the concentration of O-acetylated GD3 (27% of control cells). In addition, the transfected F-11 cells exhibited a morphology different from the parental cells with enlarged cell bodies and elongated neurites. We conclude that alteration of ganglioside composition, particularly the expression of GD3 and O-acetylated GD3, may be associated with the morphological changes observed in this cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1995

Differential solubilization of lipids along with membrane proteins by different classes of detergents.

Membrane proteins are typically extracted by detergent concentrations of 0.5-2.0%, using detergent/protein ratios of 1:1 to 3:1. We have compared the ability of 14 different detergents from seven different structural and ionic classes, at a concentration of 2.0% and a detergent/protein ratio of 2:1, to extract an integral membrane protein (the serotonin 5-HT1A receptor) in active form and have observed profound differences in both lipids and proteins. All extracts were freed from detergents and dialyzed to form vesicles containing 95-100% of the extracted lipids, prior to [3H]8-hydroxy-2-(N,N-di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding. The most efficient detergents in extracting active 5-HT1A receptor protein were the zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 3-[(cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), followed by the neutral n-dodecyl-beta-D-maltoside. Zwitterionic detergents also produced the highest solubilized lipid/protein ratio (3.0 and 2.5, respectively) and in general the relative amounts of extracted lipids and proteins followed inverse profiles. Thus, hydrophobic detergents such as Tritons (with critical micelle concentrations similar to CHAPS) and Thesit (structurally similar to Lubrol) extracted the most protein, but relatively little lipid (ratios of less than 0.2) and very little active 5-HT receptor. Dramatic differences were also observed in the ratios of individual lipids extracted by the same concentrations of different detergents and resolved by high-performance thin-layer chromatography. For example, galactosylceramide (GalCer) content ranged from 2.7% (CHAPSO) to 13.4% (sodium cholate) of the total lipid extract and cholesterol ranged from 0% (digitonin) to 17.9% (Triton X-100). The detergent-extractability profile for phosphatidylethanolamine (PE) (range 15-40% of total lipid) paralleled that of phosphatidylinositol (PI) (range 4-10%), but was inverse to that for GalCer and cholesterol. Detergent-extractability profiles for phosphatidylcholine (PC) and phosphatidylserine (PS) also followed inverse profiles, with zwitterionic detergents giving high PS/PC and high PE/PC ratios (approximately 2:1), whereas the Tritons and digitonin gave ratios of 1:2. We believe that differential solubilization of lipids, as well as proteins, by detergents is important for the biological activity of the extracted proteins, and lipid extractability should be taken into account when purifying membrane proteins.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1995

Phorbol esters and PKC signaling regulate proliferation, vimentin cytoskeleton assembly and glutamine synthetase activity of chick embryo cerebrum astrocytes in culture.

We have recently shown that expression of specific protein kinase C (PKC) isoforms correlates with cell fate in neural chicken embryo cells. Therefore we investigated the effects of PKC activation by phorbol esters on acquisition of the astrocytic phenotype, using cultured embryonic cortical astrocytes, derived from 15-day-old chick embryos (E15CH), as a model. Short term treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA), which activates PKC-alpha/beta in E15CH, caused association of PKC with the cytoskeleton. In vitro kinase assays of cytoskeleton-associated PKC demonstrated phosphorylation of many cytoskeletal proteins. Phosphorylation was blocked by protein kinase inhibitors (H8), and enhanced by phosphatase inhibitors (calyculin A). Among these PKC substrates, a most prominent 60-kDa protein was identified as vimentin. Assembly of vimentin into the cytoskeleton depends on cell type and state of differentiation. To establish that TPA (PKC) regulates assembly of vimentin into the cytoskeleton of astrocytes, we used pulse-chase (20/5 min) labeling with [35S]methionine, and immunoprecipitations with an anti-vimentin mAb from extractable and cytoskeletal fractions. These studies revealed that 20 min treatment with TPA leads to a 3-fold increase in the rate of newly synthesized full-length vimentin assembly (posttranslational assembly). Furthermore, TPA increased cotranslational assembly of vimentin. The protein kinase A activator forskolin, did not have such effects on vimentin assembly. Long-term TPA treatment, which correlates with a prolonged phospholipase D (PLD) activation, was mitogenic and caused dramatic changes in the morphology of astrocytes. In addition these fibrous, polarized astrocytes had decreased activity of the astrocyte specific enzyme, glutamine synthetase, but had increased abundance of vimentin protein. These studies provide biochemical evidence on acquisition of a different astrocytic phenotype after activation of the PKC/PLD pathway, in the chick embryo. Therefore PKC and PLD activation is pivotal for the acquisition and maintenance of phenotypes in chick embryonic astrocytes.

Animals↗
PubMed1995

Differential regulation of phospholipases C and D by phorbol esters and the physiological activators carbachol and glutamate in astrocytes from chicken embryo cerebrum and cerebellum.

Primary astrocytic cultures derived from day-15 chick embryo (E15) cerebral hemispheres (CH) or cerebellum (CB) express a calcium/phospholipid-dependent isoform as the major protein kinase C (PKC-alpha/beta). PKC was activated (translocation of activity from cytosol to membrane) following stimulation with carbachol, so we tested for activation of phospholipase C (PLC) as the source of diacylglycerol released from polyphosphoinositide (PIP2) hydrolysis. Carbachol activated PLC (inositol phosphate release) 4-fold in a time- and dose-dependent manner in cortical (CH) astrocytes, but there was no activation of PLC in astrocytes from cerebellum (CB). Pirenzepine, but not gallamine, attenuated both carbachol-induced PKC translocation and PIP2 hydrolysis in E15CH astrocytes, arguing for contribution of M1 subtype. The phorbol ester TPA completely inhibited PIP2 hydrolysis, both basal and carbachol-stimulated, and elicited a stronger, but shorter (10 min) activation of PKC than that observed with carbachol. We investigated phospholipase D (PLD) activation as an alternate source of diacylglycerol in astrocytes, since the ratio of PLC to PKC activation by carbachol was lower in astrocytes than observed in neurons. We observed a dramatic (10-fold) time- and dose-dependent activation of PLD by TPA in CH and a 3-fold increase in CB. The duration of TPA-dependent PLD activation correlated well with increased cell proliferation and changes in astrocytic phenotype markers. Carbachol-stimulated PLD activation was observed in CH but not in CB astrocytes, being mostly dependent on the M3 receptor subtype in the former. In contrast, glutamate elicited a greater PLD activation in CB astrocytes, than in CH astrocytes. TPA activation of PLD was totally blocked by staurosporine (PKC inhibitor) and genistein (a tyrosine kinase inhibitor) in cerebellar (CB) astrocytes; however, total inhibition of TPA-dependent PLD activation was only achieved in cortical (CH) astrocytes after addition of EGTA. Thapsigargin activated PLD in both populations, further emphasizing the PLD activation dependency on [Ca2+]i. Taken together with our previous observations that TPA induces proliferation, cytoskeleton changes, and decreases of glutamine synthetase activity, these data suggest that phospholipase D is a differential but important participant in the regulation of the signalling of mitosis and differentiation in astrocytes during their development.

Animals↗
PubMed1995

Hyperammonemia increases serotonin 1A receptor expression in both rat hippocampus and a transfected hippocampal cell line, HN2-5.

Hyperammonemia is an important cause of cerebral dysfunction in liver failure. We used two well-established models to induce hyperammonemia in rats, injection of urease and injection of methionine sulfoximine (MSO). Urease gave a 10-fold increase in blood ammonia while MSO, a glutamine synthetase inhibitor, gave a 4-fold increase in blood ammonia with no increase in brain glutamine levels. We observed a 2-fold increase in 5-HT1A receptor (5-HT1A-R) expression ([3H] 8-OH-DPAT binding) in hippocampus, and little change elsewhere, including thalamus in both models, thus eliminating a role for increased glutamine in the receptor induction. In contrast, a 4 to 8-fold increase in 5-HT1A-R mRNA was observed both in hippocampus and thalamus, suggesting some post-transcriptional regulation. In the absence of glutamine, ammonium acetate treatment of a hippocampal cell line which had been engineered to stably express the 5-HT1A-R (HN2-5) gave a 1.5-fold increase in [3H] 8-OH-DPAT binding and a 4-fold increase in the mRNA levels for the 5-HT1A-R. We conclude that the cell line HN2-5 is a good model for studying some of the biochemical sequelae of hyperammonemia and that changes in brain function are not only at the metabolic level, as thought earlier, but can also occur at the transcriptional level.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1995

Resistance to anti-IgM-induced apoptosis in a WEHI-231 subline is due to insufficient production of ceramide.

We describe the properties of a physiological cell death (PCD)-resistant subline of WEHI-231 generated from the PCD-susceptible WEHI-231.7 JM cell line maintained in our laboratory. The PCD-resistant WEHI-231.7 JMRE subline was uniquely resistant to anti-immunoglobulin (Ig)M-induced PCD but not to irradiation and etoposide. In these sublines, we compared the expression of genes implicated in regulating PCD. Northern analysis of c-myc, c-fos, egr-1, Fas, p53 and retinoblastoma revealed similar basal levels of expression in all sublines tested and comparable responses to anti-IgM treatment. Similarly, the expression of bcl-2, bcl-x, bax and IL-1 beta converting enzyme did not correlate with susceptibility to anti-IgM-induced PCD. Next, we systematically studied signal transduction events including: tyrosine phosphorylation, Ca++ flux, and ceramide production in the Jm and JMRE sublines. The tyrosine phosphorylation patterns and the Ca++ influx generated following sIgM engagement were very similar in the JM and JMRE sublines. In contrast, the generation of ceramide differed in the PCD-resistant and PCD-susceptible sublines. Ceramide is produced following cross-linking sIgM on WEHI-231.7 JM cells and causes PCD. Ceramide levels in anti-IgM-treated WEHI-231.7 JMRE cells are low and appear to be insufficient to induce PCD.

Apoptosis↗
PubMed1995

Subgroups of autistic children based on social behavior display distinct patterns of brain activity.

Two questions were addressed in the present study: (1) Do autistic and normally developing children exhibit regionally specific differences in electroencephalographic (EEG) activity? (2) Do subgroups of autistic children classified according to Wing and Gould's (1979) system which emphasizes degree of social impairment exhibit distinct patterns of EEG activity? Twenty-eight children with autism (5 to 18 years of age) and two groups of normally developing children (one matched on chronological age and the other on receptive language level) participated. EEG was recorded from left and right frontal, temporal, and parietal regions during an alert baseline condition. Compared to normally developing children, autistic children exhibited reduced EEG power in the frontal and temporal regions, but not in the parietal region. Differences were more prominent in the left than the right hemisphere. Furthermore, subgroups of autistic children based on Wing and Gould's system displayed distinct patterns of brain activity. Compared to autistic children classified as "active-but-odd," "passive" autistic children displayed reduced alpha EEG power in the frontal region.

Adolescent↗
PubMed1995

Hypoxia alters iron homeostasis and induces ferritin synthesis in oligodendrocytes.

Both iron and the major iron-binding protein ferritin are enriched in oligodendrocytes compared with astrocytes and neurons, but their functional role remains to be determined. Progressive hypoxia dramatically induces the synthesis of ferritin in both neonatal rat oligodendrocytes and a human oligodendroglioma cell line. We now report that the release of iron from either transferrin or ferritin-bound iron, after a decrease in intracellular pH, also leads to the induction of ferritin synthesis. The hypoxic induction of ferritin synthesis can be blocked either with iron chelators (deferoxamine or phenanthroline) or by preventing intracellular acidification (which is required for the release of transferrin-bound iron) with weak base treatment (ammonium chloride and amantadine). Two sources of exogenous iron (hemin and ferric ammonium citrate) were able to stimulate ferritin synthesis in both oligodendrocytes and HOG in the absence of hypoxia. This was not additive to the hypoxic stimulation, suggesting a common mechanism. We also show that ferritin induction may require intracellular free radical formation because hypoxia-mediated ferritin synthesis can be further enhanced by cotreatment with hydrogen peroxide. This in turn was blocked by the addition of exogenous catalase to the culture medium. Our data suggest that disruption of intracellular free iron homeostasis is an early event in hypoxic oligodendrocytes and that ferritin may serve as an iron sequestrator and antioxidant to protect cells from subsequent iron-catalyzed lipid peroxidation injury.

Acids↗
PubMed1994

Ceramide mediates the apoptotic response of WEHI 231 cells to anti-immunoglobulin, corticosteroids and irradiation.

We demonstrate for the first time how immature B cells kill themselves. Ceramide is identified as the mediator of apoptosis in the murine B lymphoma line WEHI 231 commonly used as a model to study clonal deletion in B lymphocytes. We show that exogenous ceramide induces apoptosis in WEHI 231 cells. To maintain self tolerance, immature lymphocytes readily undergo apoptotic death in response to the cross-linking of their antigen-specific receptors. We demonstrate that endogenously produced ceramide accumulates in WEHI 231 cells exposed to anti-IgM, an antigen surrogate before the onset of apoptosis. We also show that two other inducers of apoptosis, irradiation and dexamethasone, cause intracellular accumulation of ceramide.

Animals↗
PubMed1994

Preferential beta-hexosaminidase (Hex) A (alpha beta) formation in the absence of beta-Hex B (beta beta) due to heterozygous point mutations present in beta-Hex beta-chain alleles of a motor neuron disease patient.

Deficiency of the lysosomal enzyme beta-hexosaminidase B (beta-Hex B) (a homodimer, beta beta), caused by a defect in the HEX B gene encoding the beta-chain, is usually accompanied by an absence of beta-Hex A (a heterodimer, alpha beta), thereby causing Sandhoff disease. However, we have earlier demonstrated the presence of partial beta-Hex A (30-50% of normal) even in the absence of beta-Hex B in an adult with motor neuron disease. The patient is a compound heterozygote with normal beta-chain message and one HEX B point mutation originating from each asymptomatic parent. Since the non-expression of beta-Hex B was post-transcriptional, we transfected COS-7 cells to understand the effect of each mutation on beta-Hex B activity. Transfection of the A1367-->C mutant (maternal allele) construct produced no overexpressed beta-Hex B, indicating that the encoded Tyr456-->Ser beta-chain was non-functional. Chou-Fasman analysis predicted that the Tyr456-->Ser mutation would cause a dramatic change in beta-chain folding (which often inhibits formation of functional dimers). This explains the complete lack of beta-Hex B in the transfectants and a partial deficiency of beta-Hex A and B (50% of normal) in the patient's mother. Since immunoprecipitated beta-Hex A (alpha beta) protein from patient fibroblasts showed the presence of mature beta-chains even though there was no beta-Hex B (beta beta) protein, the mutant beta-chain inherited from the father (who has normal beta-Hex A and B) must undergo preferential association with the normal alpha-chains in the patient, thus producing only beta-Hex A. Transient expression of the A619-->G mutant (paternal allele) construct produced beta-Hex B activity comparable to the wild type (approximately 10-20-fold over mock-transfected) whereas stable expression produced normal message but no beta-Hex B activity (wild type beta-Hex B expression: only 2-fold over mock-transfected). The lack of increased beta-Hex B after stable expression of the Ile207-->Val beta-chains at a lower copy number indicates the absence of self-association at low concentrations of Ile207-->Val beta-chain. In the patient who also has a non-functional Tyr456-->Ser allele, the effective concentration of beta-chains is reduced to 50% of normal and the remaining Ile207-->Val beta-chains fail to self-associate but can still dimerize with the abundant normal alpha-chains thus producing partial beta-Hex A and no beta-Hex B.

Adult↗
PubMed1994

Early recognition of children with autism: a study of first birthday home videotapes.

Coded home videotapes of 11 autistic and 11 normally developing children's first year birthday parties for social, affective, joint attention, and communicative behaviors and for specific autistic symptoms. Autistic children displayed significantly fewer social and joint attention behaviors and significantly more autistic symptoms. In combination, four behaviors correctly classified 10 of 11 autistic children and 10 of 11 normal children. These behaviors consisted of pointing, showing objects, looking at others, and orienting to name.

Anniversaries and Special Events↗
PubMed1994

Hypoxia specifically and reversibly induces the synthesis of ferritin in oligodendrocytes and human oligodendrogliomas.

Neonatal (3 day old) rat oligodendrocytes grown in monolayer culture and exposed to increasingly hypoxic culture conditions showed increased Tran35S-label incorporation into a 22-kDa protein. Reoxygenation of cultures reversed the synthesis of the protein. Amino acid sequencing of a peptide derived from the purified protein revealed a 13 amino acid sequence with complete identity to a human heavy chain subunit of ferritin. This was confirmed by two-dimensional gel electrophoresis, immunoprecipitation, and western blot analysis with antiferritin antibody. In addition, hypoxia was able to induce the synthesis of ferritin in a cell line derived from human oligodendroglioma cells but not in astrocytes or neurons. Actinomycin D (1-15 micrograms/ml) treatment did not block the hypoxic induction of ferritin synthesis, whereas cycloheximide (1 microM) gave complete inhibition. Northern blot analysis showed that ferritin mRNA levels remained unchanged in both control and hypoxic oligodendrocytes and human oligodendroglioma cells, suggesting that the synthesis of ferritin was translationally rather than transcriptionally regulated by hypoxia. In neither oligodendrocytes nor the oligodendroglioma was there any cross-reaction with an antibody to alpha B-crystallin, the 22-kDa protein induced in astrocytes by various types of stress, further suggesting the specificity of hypoxic induction of ferritin in oligodendrocytes.

Amino Acid Sequence↗