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S R Barnum

Publications and source records attributed to S R Barnum.

50 records · Page 3Linked to original sources
PubMed1990

Purification and characterization of acyl carrier protein from two cyanobacteria species.

The acyl carrier protein (ACP), an essential protein cofactor for fatty acid synthesis, has been isolated from two cyanobacteria: the filamentous, heterocystous, Anabaena variabilis (ATCC 29211) and the unicellular Synechocystis 6803 (ATCC 27184). Both ACPs have been purified to homogeneity utilizing a three-column procedure. Synechocystis 6803 ACP was purified 1800-fold with 67% yield, while A. variabilis ACP was purified 1040-fold with 50% yield. Yields of 13.0 micrograms ACP/g Synechocystis 6803 and 9.0 micrograms ACP/g A. variabilis were achieved. Amino acid analysis indicated that these ACPs were highly charged acidic proteins similar to other known ACPs. Sequence analysis revealed that both cyanobacterial ACPs were highly conserved with both spinach and Escherichia coli ACP at the phosphopantetheine prosthetic group region. Examining the probability of alpha-helix and beta-turn regions in various ACPs, showed that cyanobacterial ACPs were more closely related to E. coli ACP than spinach ACP I. Immunoblot analysis and a competitive binding assay for ACP illustrated that both ACPs bound poorly to spinach ACP I antibody. SDS/PAGE and native PAGE of Synechocystis 6803 ACP and A. variabilis ACP showed that cyanobacteria ACPs co-migrated with E. coli ACP and had relative molecular masses of 18,100 and 17,900 respectively. Both native and urea gel analysis of acyl-ACP products from fatty acid synthase reactions demonstrated that bacterial ACPs and plant ACP gave essentially the same metabolic products when assayed using either bacterial or plant fatty acid synthase. A. variabilis and Synechocystis 6803 ACP could be acylated using E. coli acyl ACP synthetase.

Acyl Carrier Protein↗
PubMed1990

C4b-binding protein, a regulatory component of the classical pathway of complement, is an acute-phase protein and is elevated in systemic lupus erythematosus.

A radioimmunoassay using monoclonal and polyclonal antihuman C4b-binding protein (C4BP) antibody was developed to quantitate C4BP in serum. Using the assay, the levels of C4BP in healthy individuals, in patients with systemic lupus erythematosus (SLE), and in acute-phase individuals were determined. The levels of C4BP are significantly elevated in individuals with SLE (186%; p = 0.0001) and are even higher in individuals during the acute phase (286%; p = 0.0001). To confirm whether or not individuals were in the acute-phase response, serum C-reactive protein (CRP) levels were assessed. In the acute-phase response, CRP levels were 100-fold elevated over normals, but did not correlate with increases in C4BP (r = -0.031; p = 0.899). In SLE patients, the CRP levels were significantly, but moderately, elevated (5-fold; p = 0.028). The data indicate that C4BP is an acute-phase reactant and is differentially regulated from CRP during the acute-phase response.

Acute-Phase Proteins↗
PubMed1989

Molecular analysis of the murine C4b-binding protein gene. Chromosome assignment and partial gene organization.

Murine C4b-binding protein (C4BP) is a regulatory molecule in the classical pathway of complement. C4BP is composed predominantly of short consensus repeats (SCRs) approximately 60 amino acids in length, which contain a framework of conserved residues. The SCRs are found in many complement molecules and a growing number of noncomplement molecules as well and are a major structural feature of some of these molecules. To characterize the structure of the murine C4BP gene, a cosmid library constructed from Balb/c liver DNA was screened. Several nearly identical, overlapping clones were identified; however, none of the clones, alone or in combination, covered the entire C4BP gene. One clone (D26) was chosen for detailed analysis and found to contain all but the leader region, the first SCR, and the first half of the second SCR. The SCRs three through six were each encoded by single exons. Only the latter half of the second SCR was present on the clone, and it was encoded by a single exon, demonstrating that murine C4BP has a split SCR at the genomic level. Structural mapping of this portion of the gene demonstrates that the region extending from the second half of the second SCR through the nonrepeat and untranslated region spans approximately 12 kb; however, genomic Southern blot analysis suggests that the gene is between 20 and 30 kb in length. Analysis of the 3' genomic sequence demonstrates that this region of the gene has homology with SV-40 late (class II) RNA sequences. These sequences may play a role in 3' cleavage of the precursor RNA prior to polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Complete intron/exon organization of DNA encoding the alpha' chain of human C3.

The third component of human complement (C3), a central molecule in both the classical and alternative pathways of complement, is comprised of two polypeptides, termed the alpha and beta chains. Activation of C3 cleaves the alpha chain into two fragments, C3a, an inflammatory peptide, and the alpha' chain which remains covalently linked to the beta chain. Proteolytic fragments derived from the alpha' chain during activation and regulation of complement play a significant role in host defense and regulation of the immune response. Two cosmid clones covering the alpha' chain region were used to characterize the structure of this portion of the C3 gene. The alpha' chain is encoded by 24 exons, which range in size from 52 to 213 base pairs (bp) with an average size of 115 bp. The splice donor sequence at the beginning of intron 12 has a rare sequence variant of GC instead of the usual GT sequence. Ten introns have been completely sequenced and were surprisingly short, ranging in size from 85 to 242 bp with an average of 140 bp. Other introns range in size from 250 bp to over 4 kilobases in length. The gene size for this portion of C3 is estimated to be 23-24 kilobases. Comparison of exon structure with protein domains and with peptide mapping studies demonstrates that several binding sites on C3 are encoded by single exons. These data support the hypothesis that individual exons can code for functional protein domains.

Base Sequence↗
PubMed1989

Synthesis of radiolabeled acetyl-coenzyme A from sodium acetate.

The synthesis of high specific radioactivity [14C]-acetyl-Coenzyme A from [14C]sodium acetate, 2,6-dichlorobenzoic acid, 1,1'-carbonyldiimidazole, and CoA is reported. Starting with 1 mumol of [14C]sodium acetate, this method yields pure [14C]acetyl-CoA in yields approaching 40%. Chromatography on a reversed-phase ODS column was used to separate acetyl-CoA from Coenzyme A and side products. The acetylating agent is apparently a reaction intermediate, acetylimidazole.

Acetates↗
PubMed1989

A Cerulenin Insensitive Short Chain 3-Ketoacyl-Acyl Carrier Protein Synthase in Spinacia oleracea Leaves.

A cerulenin insensitive 3-ketoacyl-acyl carrier protein synthase has been assayed in extracts of spinach (Spinacia oleracea) leaf. The enzyme was active in the 40 to 80% ammonium sulfate precipitate of whole leaf homogenates and catalyzed the synthesis of acetoacetyl-acyl carrier protein. This condensation reaction was five-fold faster than acetyl-CoA:acyl carrier protein transacylase, and the initial rates of acyl-acyl carrier protein synthesis were independent of the presence of cerulenin. In the presence of fatty acid synthase cofactors and 100 micromolar cerulenin, the principal fatty acid product of de novo synthesis was butyric and hexanoic acids. Using conformationally sensitive native polyacrylamide gel electrophoresis for separation, malonyl-, acetyl-, butyryl-, hexanoyl, and long chain acyl-acyl carrier proteins could be detected by immunoblotting and autoradiography. In the presence of 100 micromolar cerulenin, the accumulation of butyryl- and hexanoyl-acyl carrier protein was observed, with no detectable long chain acyl-acyl carrier proteins or fatty acids being produced. In the absence of cerulenin, the long chain acyl-acyl carrier proteins also accumulated.

Journal Article↗
PubMed1988

Molecular analysis of human complement component C5: localization of the structural gene to chromosome 9.

A human C5 clone (pC5HG2) was isolated from a cDNA library constructed from Hep G2 mRNA. The DNA sequence showed that the pC5HG2 insert was comprised of 3309 base pairs of pro-C5 coding sequence and 404 base pairs of 3'-untranslated sequence. The derived amino acid sequence contained the entire coding sequence of the C5 alpha-chain, the beta-alpha-chain junction region, and 100 amino acids (approximately 50%) of the beta-chain. Protein sequences of four C5 tryptic peptides were aligned exactly to this sequence and demonstrated that C5 synthesized and secreted by Hep G2 cells is probably identical with plasma-derived C5. Coding sequence alignment of the human C5 sequences with those of murine C5 indicated that 80% of the nucleotides and 79% of the amino acids were placed identically in the two species. Amino acid sequence alignment of the homologous family members C3, C4, and alpha 2-macroglobulin with that of C5 demonstrated 27%, 25%, and 19% identity, respectively. As was found in murine C5, the corresponding thiol ester region of human C5 contained several conserved amino acids, but the critical cysteine and glutamine residues which give rise to the intramolecular thiol ester bond in C3, C4, and alpha 2-macroglobulin were absent in C5, having been replaced by serine and alanine, respectively. With the use of a panel of hamster-human somatic cell hybrids, the C5 gene was mapped to human chromosome 9. In situ chromosomal hybridization studies employing metaphase cells further localized the gene to bands 9q32-34, with the largest cluster of grains at 9q34.1.

Amino Acid Sequence↗
PubMed1985

Renal filtration and catabolism of complement protein D.

Complement protein D, a serine protease participating in the formation of the C3 convertase of the alternative complement pathway, has the lowest molecular weight (23,750) and serum concentration of all complement proteins. In normal serum, D is the rate-limiting protease of the alternative pathway of complement activation. We report that the serum concentrations of D in 20 patients with chronic renal failure (mean +/- S.D., 0.42 +/- 0.28 mg per deciliter) and in 16 patients on long-term dialysis (1.53 +/- 0.39 mg per deciliter) were significantly higher (P less than 0.001) than in 22 healthy adults (0.18 +/- 0.04 mg per deciliter). In chronic renal failure the serum concentration of D correlated with that of creatinine (r = 0.75, P less than 0.001). The serum concentrations of D found in patients with renal failure reached and in some cases exceeded those at which the protease is no longer rate-limiting. Thus, enhanced activity of the alternative pathway of complement should be expected in patients with advanced renal failure. Urinary D was undetectable (less than 0.2 micrograms per deciliter) in 17 normal adults and either undetectable or below the concentration expected from the degree of proteinuria in 10 patients with nephrotic syndrome. However, in a patient with Fanconi's syndrome the urinary concentration of D (1.3 mg per deciliter) was an order of magnitude higher than the serum concentration, representing 0.5 per cent of the total protein. The urinary D in this patient had normal hemolytic activity, antigenicity, and size. These results indicate that D is filtered through the glomerular membrane and is probably catabolized in the proximal renal tubules.

Adult↗
PubMed1985

Biosynthesis of complement protein D by HepG2 cells: a comparison of D produced by HepG2 cells, U937 cells and blood monocytes.

The biosynthesis of complement protein D of the alternative pathway by HepG2 cells, a human hepatocyte cell line, was studied and compared to the biosynthesis of D by U937 cells and blood monocytes. Increasing amounts of antigenic D were detected in HepG2 cell culture supernatants by radioimmunoassay. The kinetics of D synthesis and secretion by HepG2 cells was followed in a pulse-chase study using [35S]cysteine. As analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography, only a single D band was seen intra- and extracellularly and both forms had the same apparent molecular weight as D synthesized by U937 cells or purified from serum. Treatment of HepG2 and U937 cells with canavanine, an arginine amino acid analog, to inhibit intracellular processing resulted in slight depression of the apparent molecular weight of D synthesized by these cells. D synthesized by blood monocytes had an apparent molecular weight similar to that synthesized by HepG2 and U937 cells, suggesting that these cell lines do not synthesize and process D differently than normal monocytes. The data demonstrate that the hepatocyte is a site of D synthesis and suggest that D is not synthesized as a precursor molecule.

Canavanine↗
PubMed1985

In vitro biosynthesis of complement protein D by U937 cells.

Preliminary studies demonstrating the secretion of antigenic D by blood monocytes/macrophages led us to study the biosynthesis of D by U937 cells, a human monocyte cell line. The kinetics of secretion of D into cell culture supernatants were followed by a solid-phase radioimmunoassay and by hemolytic assay. Daily synthesis of antigenic D was nearly linear (mean +/- 1 SD = 5.3 +/- 2.2 ng D/10(6) cells) over a 6-day period. The D produced after day 2 was hemolytically active, with a specific hemolytic activity greater than (although in the same range as) D in normal serum. Cycloheximide (10(-7) M) inhibited D synthesis, which returned to the levels found in untreated cells after removal of the inhibitor. Supernatants and lysates of cells grown in the presence of [35S]methionine were incubated with rabbit anti-D serum or FD10-1, a monoclonal anti-D antibody, bound to protein A-agarose. Autoradiograms of SDS-PAGE analysis of the precipitates demonstrated a main band of an approximate m.w. of 24,000, co-migrating with purified 125I-D. Identity of this band with D was established by blocking with excess purified D. Pulse-chase studies with the use of [35S]cysteine demonstrated a single D band both intra and extracellularly. Both forms of D had the same apparent m.w. which was approximately 3000 heavier than control 125I-D. These data demonstrate that U937 cells synthesize functionally active D, which appears to be structurally and antigenically similar to D in serum.

Amino Acids↗
PubMed1984

Quantitation of complement factor D in human serum by a solid-phase radioimmunoassay.

A sensitive solid-phase radioimmunoassay is described which quantitates human D to 1-2 ng/ml. The assay was used to measure the concentration of D in normal and acute-phase sera and sera from individuals with systemic lupus erythematosus. All 3 groups of sera had comparable levels of D with mean values of 1.8, 2.3 and 2.5 micrograms/ml, respectively. Also tested were sera decomplemented in vitro by activators of the classical and alternative pathways. The results indicated that D is not depleted by alternative or classical pathway activation. However, heat inactivation (56 degrees C, 30 min) of serum resulted in almost complete loss of antigenic D.

C-Reactive Protein↗
PubMed1983

Effect of lectins and sugars on primary sperm attachment in the horseshoe crab, Limulus polyphemus L.

The apical regions of motile Limulus spermatozoa readily adhere to the outer layer of the egg envelope. Shortly after this adherence or primary attachment, the sperm acrosome reaction occurs, resulting in a stronger adhesion (secondary attachment). A sperm attachment assay that quantified the number of spermatozoa attaching to egg sections was utilized to identify components involved in primary attachment. The number of spermatozoa attached was examined after treatment of either egg sections or spermatozoa with various compounds. Egg sections treated with asparagus pea lectin (250 micrograms/ml) bound significantly fewer spermatozoa as compared to those exposed to wheat germ agglutinin, concanavalin A, and garden pea lectin. Furthermore, sperm attachment was also greatly reduced when egg sections were first incubated with the glycosidase, alpha-L-fucosidase (less than or equal to 5% of controls). Treatment of spermatozoa with alpha-L-fucose, fucoidan, or p-aminophenyl fucoside also reduced sperm attachment when compared to Millipore-filtered artificial seawater controls. Egg sections were treated with fluorescein-conjugated lectins to confirm that the lectins actually bound to portions of the egg envelope and that various sugars are present in the egg envelope. Evidence suggests that the methylpentose, alpha-L-fucose, plays an important role in primary sperm attachment in Limulus.

Animals↗