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E J Neufeld

Publications and source records attributed to E J Neufeld.

At least 55 records · Page 3Linked to original sources
PubMed1995

P-selectin expression in myocardium of children undergoing cardiopulmonary bypass.

Cardiopulmonary bypass is a planned support technique that results in a period of myocardial ischemia and reperfusion. In addition, it is associated with an inflammatory response likely involving endothelial cell activation. In previous studies, we showed that E-selectin and intercellular adhesion molecule-1 (ICAM-1) messenger ribonucleic acid (mRNA) are increased in human myocardium after cardiopulmonary bypass. We have now examined the expression of P-selectin mRNA by ribonuclease protection in paired atrial biopsy specimens from 12 patients before and after cardiopulmonary bypass. By means of immunocytochemistry, we have also examined the endothelial cell surface expression of P-selectin protein, as well as that of E-selectin and ICAM-1 in three additional patients. Patient ages ranged from 1 day to 8.5 years (median 12 months), and cardiopulmonary bypass times ranged from 46 to 196 minutes (median 144 minutes). By ribonuclease protection, there was marked variability in the expression of P-selectin in biopsy specimens before bypass. However, when compared with prebypass levels, P-selectin mRNA decreased modestly in 10 of 12 patients after bypass (median decrease 1.5-fold, p = 0.016). As seen with immunocytochemistry, P-selectin protein was distributed diffusely through the vascular bed on large vessels and small vessels before bypass but was virtually absent on capillaries in specimens taken after bypass. E-selectin, which was absent in prebypass biopsy specimens, was induced in one of the three specimens after bypass, but no change in ICAM-1 protein expression above baseline was noted. We also find that cultured human endothelial cells treated with tumor necrosis factor-alpha in doses which induce ICAM-1 mRNA simultaneously decrease their expression of P-selectin mRNA as compared with untreated cells. These observations suggest that endothelial P-selectin is transcriptionally downregulated after cardiopulmonary bypass at times when E-selectin and ICAM-1 are induced. Furthermore, we find that E-selectin and ICAM-1 are expressed at times and at sites where P-selectin is absent. Although it is possible that P-selectin may have been induced and lost at early times before reperfusion, these data suggest that endothelial P-selectin plays a limited role in the inflammatory response that ensues after cardiopulmonary bypass.

Adolescent↗
PubMed1995

Severe haemophilia A in a female resulting from two de novo factor VIII mutations.

A 2-year-old girl is described with severe haemophilia A (factor VIII: C < 0.01 units/ml). Both of her parents were phenotypically normal. Cytogenetic analysis on the proband demonstrated an interstitial X chromosome deletion encompassing Xq26-q28. Molecular studies with several polymorphic markers close to and within the factor VIII gene showed that the proband had inherited only the paternal factor VIII gene, indicating that the X chromosome deletion had occurred de novo in the maternal germ line. Further study of the factor VIII gene inherited by the proband from her father showed the presence of a de novo gene inversion mutation (a type 1, distal pattern inversion). Neither parent showed any evidence of the factor VIII inversion in their somatic DNA. The severe haemophilia A documented in this girl is therefore the result of two de novo mutations affecting the factor VIII gene, a maternally derived X chromosome deletion and a paternal factor VIII inversion mutation.

Blotting, Southern↗
PubMed1994

Sequence-specific DNA binding of individual cut repeats of the human CCAAT displacement/cut homeodomain protein.

CCAAT displacement protein (CDP), a nuclear protein of 180-190 kDa, contains a triplicated motif, the cut domain, similar (80-90% conserved) to three repeats of 60-65 amino acids first identified in Drosophila cut, a homeo-domain protein involved in cell-fate decisions in development. Cut repeats bind DNA and exhibit subtle differences in target-site recognition. DNA sequences specifically bound by cut repeats were isolated by PCR-mediated DNA target-site selection. Sequences selected for cut repeat 2 and 3 (CR2 and CR3) binding are A+T-rich and favor an ATA motif with similar, but not identical, flanking base preferences. CR2 and CR3 discriminate among similar target sequences. CR1, which is more divergent from CR2 and CR3, displays the most restricted pattern of DNA sequence recognition. Methylation interference analysis demonstrates different protein-DNA contacts for CR1 and CR3 binding to a target sequence. Thus, CDP/cut is a complex protein whose DNA-binding properties reflect the combinatorial interaction of four domains (three cut repeats and one homeodomain) with target DNA sequences.

Base Sequence↗
PubMed1994

Induction of intercellular adhesion molecule-1 and E-selectin mRNA in heart and skeletal muscle of pediatric patients undergoing cardiopulmonary bypass.

Leukocyte adhesion to vascular endothelium is an early step in inflammatory damage to tissues. To investigate the expression of endothelial adhesion molecules in the inflammatory response associated with cardiopulmonary bypass, we measured messenger ribonucleic acid (mRNA) encoding the adhesion molecules E-selectin and intercellular adhesion molecule-1 in intraoperative samples of cardiac tissue and skeletal muscle from infants undergoing cardiopulmonary bypass. Atrial tissue samples were obtained before and after bypass from 11 children and paired samples of rectus abdominis muscle from 15. mRNA was analyzed by ribonuclease protection with the use of nonmuscle actin as an internal control. Atrial E-selectin mRNA levels increased from before to after bypass (median increase 3.5-fold, p = 0.0002) in each of nine patients tested, and atrial intercellular adhesion molecule-1 mRNA increased in seven of nine patients (median, 2.1-fold, p = 0.025). In skeletal muscle, E-selectin mRNA increased in 11 of 12 patients (median 4.3-fold, p = 0.0018), and intercellular adhesion molecule-1 mRNA levels increased in 13 of 13 patients (median 3.2-fold, p = 0.013). E-selectin and intercellular adhesion molecule-1 induction in skeletal muscle occurred with or without circulatory arrest. We conclude that adhesion molecule mRNA induction occurs in cardiac and noncardiac tissue during cardiopulmonary bypass in man.

Cardiopulmonary Bypass↗
PubMed1993

Effects of nutritional counseling on lipoprotein levels in a pediatric lipid clinic.

OBJECTIVE: To determine the impact of nutritional counseling on lipoprotein profiles in dyslipoproteinemic children. DESIGN: Retrospective case review. SETTING: An academic hospital-based pediatric lipid clinic in Boston, Mass. PARTICIPANTS: One hundred four newly referred children with primary dyslipoproteinemia. INTERVENTIONS: Nutritional recommendations were adapted from the National Cholesterol Education Program's step 2 diet. Three-day diet records were used to assess baseline and follow-up diets. RESULTS: Two thirds of the children continued to have excellent diets or improved their diets after counseling, but low-density lipoprotein cholesterol (LDL-c) values decreased by 15% or more in only 19% of children. The observed change in LDL-c was not significantly associated with nutritional counseling. However, a strong correlation was evident between dietary interventions and concentration of high-density lipoprotein cholesterol (HDL-c) values in serum. Marked fat restriction lowered HDL-c levels, while liberalization of use of fat, with emphasis on monounsaturates, in a subset of children following an excessively fat-restricted diet on presentation, appeared to improve HDL-c levels. CONCLUSIONS: After nutritional counseling, LDL-c levels decreased by 15% or more in only 19% of dyslipoproteinemic children referred for treatment. There were no clear predictors of LDL-c responsiveness, but changes in dietary fat intake appeared to significantly influence HDL-c levels.

Child↗
PubMed1993

Inherited dyslipidemias in childhood.

This review touches on three areas of recent research on genetic lipid disorders. The first is the advent of a trial of gene therapy for homozygous familial hypercholesterolemia. Affected individuals are at risk for myocardial infarction by adolescence. The experimental therapeutic protocol is aimed at restoring function of the defective low density lipoprotein (LDL) receptor gene in patients by introducing complementary DNA encoding normal LDL receptor into a patient's hepatocytes ex vivo. The second area reviewed concerns an important but lesser known relative of LDL, lipoprotein (a). Concentrations of lipoprotein (a) vary a thousandfold among humans. High levels pose a strong, independent risk for early heart disease, but lipoprotein (a) determinations are not yet routine. Finally, an update is presented on the controversy over cholesterol screening and treatment in childhood.

Adolescent↗
PubMed1993

Niacin treatment of hypercholesterolemia in children.

OBJECTIVE: To determine the efficacy and adverse effects of niacin treatment of hypercholesterolemia in children. DESIGN: Retrospective review. SETTING: Two university hospital referral clinics. PATIENTS: All children who received single-drug niacin treatment for severe hypercholesterolemia between 1980 and 1991. RESULTS: Twenty-one children, aged 4 to 14 years, were treated with niacin, 500 to 2250 mg daily. Pretreatment total serum cholesterol value (mean +/- SD) was 7.84 +/- 1.14 mmol/L (303 +/- 44 mg/dL), and low-density lipoprotein cholesterol value was 6.28 +/- 1.16 mmol/L (243 +/- 45 mg/dL). Niacin treatment in daily doses > 1000 mg reduced total cholesterol by 23% and low-density lipoprotein cholesterol by 30% (P < .001) but had no effect on high-density lipoprotein cholesterol and triglycerides. As in adults, reversible adverse effects were common, occurring in 16 (76%) of the 21 children. Six children (29%) had reversible dose-related elevations of serum aminotransferase levels. Niacin therapy was discontinued in 8 children (38%) because of flushing, abdominal pain, vomiting, headache, or elevated serum aminotransferase levels. CONCLUSIONS: This study suggests that although niacin treatment in children is efficacious, adverse effects are common. Until further study demonstrates long-term safety, niacin treatment should be reserved for the closely-supervised treatment of severe hypercholesterolemia by a lipid specialist.

Adolescent↗
PubMed1992

Sequence-specific binding of HMG-I(Y) to the proximal promoter of the gp91-phox gene.

Screening of a cDNA expression library with a CCAAT-box element derived from the myelomonocyte-specific gp91-phox promoter resulted in the isolation of three independent HMG-I(Y) cDNA clones. Filter binding competition studies reveal that HMG-Y binds to this promoter element in a sequence-specific manner and exhibits a gradient of binding affinities for various A/T-rich sequences. Two adjacent A/T-rich regions within the gp91-phox promoter CCAAT-box element are required for maximal binding. In addition, competition experiments demonstrate that the binding affinity of HMG-Y is influenced by sequences that flank A/T-rich core binding sites.

Base Sequence↗
PubMed1992

Human CCAAT displacement protein is homologous to the Drosophila homeoprotein, cut.

Human CCAAT displacement protein (CDP), a putative repressor of developmentally regulated gene expression, was purified from HeLa cells by DNA binding-site affinity chromatography. cDNA encoding CDP was obtained by immunoscreening a lambda gt11 library with antibody raised against purified protein. The deduced primary amino acid sequence of CDP reveals remarkable homology to Drosophila cut with respect to the presence of a unique homeodomain and "cut repeats". As cut participates in determination of cell fate in several tissues in Drosophila, the similarity predicts a broad role for CDP in mammalian development.

Amino Acid Sequence↗
PubMed1985

Phosphoinositide interconversion in thrombin-stimulated human platelets.

Stimulation of platelets and other secretory cells by agonists results in the degradation of phosphoinositides by phospholipase C. Kinetic studies suggest that hydrolysis of phosphatidylinositol 4,5-diphosphate (PI-4,5-P2) is an initial event in this process. Platelets contain much larger amounts of phosphatidylinositol (PI) than PI-4,5-P2, and approximately 50% of total phosphoinositides are degraded upon stimulation. We have investigated whether degradation of PI occurs by direct phospholipase C hydrolysis or by phosphorylation to PI-4,5-P2 followed by phospholipase C action on the latter compound. When platelets are incubated for 3 min with 32Pi prior to stimulation, the phosphoinositides are labeled to different specific activities. Under these nonequilibrium conditions, the time course of change in specific activity reflects turnover. The rise in specific activity of phosphatidylinositol 4-phosphate (PI-4-P) is similar in stimulated and unstimulated cells, indicating that there is little increase in the conversion of PI to PI-4-P during thrombin stimulation. In addition, the specific activity of the 4-phosphate in PI-4-P during thrombin stimulation is less than both the 5-phosphate of PI-4,5-P2 and the phosphate group of phosphatidic acid, indicating that the 4-phosphate moiety is not labeled to equilibrium with ATP. This finding is inconsistent with a rapid flux of PI via PI-4-P to PI-4,5-P2 during thrombin stimulation, in which case the 4-phosphate would be at maximum specific activity. We, therefore, conclude that the bulk of PI breakdown that occurs in thrombin-stimulated platelets occurs via direct phospholipase C hydrolysis of PI.

Blood Platelets↗
PubMed1985

Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography.

We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDM1C1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDM1C1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 min in HSDM1C1 cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM1C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM1C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM1C1 cells release labeled arachidonate for eicosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of eicosanoid precursors.

Animals↗
PubMed1985

Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography.

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.

Animals↗
PubMed1984

A mutant HSDM1C1 fibrosarcoma line selected for defective eicosanoid precursor uptake lacks arachidonate-specific acyl-CoA synthetase.

Mutagenesis followed by suicide with highly radioactive tritiated arachidonic acid has been used to select for mouse fibrosarcoma (HSDM1C1) cells defective in eicosanoid precursor uptake. Survivors of the selection were screened by replica plating and autoradiographic assay of [3H]arachidonate esterification; a mutant cell line, EPU-1, was established. EPU-1 cells contain one-third as much arachidonate as normal HSDM1C1 cells. The mutant lacks arachidonate-specific acyl-CoA synthetase, which accounts for decreased arachidonate uptake. EPU-1 exhibits enhanced turnover of arachidonoyl- but not linoleoyl-phosphatidylcholine. Bradykinin-induced arachidonate release and prostaglandin E2 synthesis are decreased in EPU-1. Thus, arachidonoyl-CoA synthetase is required for arachidonate homeostasis in HSDM1C1 cells.

Animals↗
PubMed1984

Fatty acid structural requirements for activity of arachidonoyl-CoA synthetase.

We have examined the fatty acid substrate specificity of arachidonoyl-CoA synthetase from human platelet membranes. A variety of positional isomers and chain-length analogs of arachidonic acid [20:4(5, 8, 11, 14)] were synthesized, and assayed for their ability to inhibit arachidonoyl-CoA formation or to serve as substrates for the synthetase. The chain-length specificity of the synthetase for delta 8,11,14 trienoic fatty acids was C19 greater than C18 = C20 much greater than C21 greater C22. Inhibition activity by positional isomers of arachidonate was 20:4(5, 8, 11, 14) approximately equal to 20:4(6, 9, 12, 15) = 20:4(7, 10, 13, 16) much greater than 20:4(4, 7, 10, 13), however, Vmax for arachidonate was greater than that for 20:4(6, 9, 12, 15). The enzyme apparently "counts" double bonds from the carboxyl terminus. As counted from the methyl terminus we found that several n-6,-9,-12 fatty acids were ineffective as inhibitors [18:3(6, 9, 12); 19:4)4, 7, 10, 13); 21:3(9, 12, 15)], whereas all methylene-interrupted tri- and tetraenoic fatty acids which contained delta 8 and delta 11 double bonds were potent inhibitors. The delta 11 double bond was best associated with optimal inhibition: 20:3(5, 11, 14) had a lower Ki than 20:3(5, 8, 14). 13-Methyl-20:3(8, 11, 14) did not inhibit the enzyme. Partially purified enzyme from calf brain, depleted of nonspecific long-chain acyl-CoA synthetase, exhibited the same fatty acid specificity as crude platelet enzyme.

Animals↗