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G C Chen

Publications and source records attributed to G C Chen.

At least 55 records · Page 3Linked to original sources

Adsorption of proteins onto glass surfaces and its effect on the intensity of circular dichroism spectra.

Errors in analyzing CD spectra of proteins arising from adsorption loss onto glass surfaces were examined for six proteins: apolipoproteins A-I and E, fibronectin, bovine serum albumin, insulin, and glucagon. Among these, the glycoproteins, apolipoprotein E and fibronectin, adsorbed most onto glass surfaces. Their CD intensities decreased by about 50% when proteins were diluted serially from 1 to 0.01 mg/ml in regular glass-ware and CD was measured in uncoated cells. The other proteins, except glucagon, also showed a certain degree of adsorption. Thus, adsorption loss of proteins onto glass surfaces is common and may lead to serious errors in experimental results. Adsorption can be minimized by using plastic containers and pipet tips, coating the cell with silicone, and wetting the cell before adding the protein solution.

Adsorption↗

[Continuous production of anti-hepatitis B surface antigen monoclonal antibody in hollow fiber perfusion bioreactor].

Hybridoma cell line H440Ac was cultivated continuously in hollow fiber perfusion bioreactor for fifty days to produce anti-hepatitis B surface antigen (anti-HBsAg) monoclonal antibody. A total of 90 mg anti-HBsAg antibody was produced in the hollow fiber bioreactor with 1000 cm2 membrane surface area. The collected production medium has an average antibody concentration of 0.45 mg/ml with a high concentration of 2.3 mg/ml. This compares with 0.016 and 1.1 mg/ml antibody concentration as collected from spinner flask and mouse ascites. The purity of the produced antibody is 0.085, 0.0028 and 0.026 mg antibody per mg of total harvested protein in the culture media produced from the hollow fiber bioreactor, spinner flasks and mouse ascites, respectively.

Animals↗

Structural comparison of human apolipoproteins B-48 and B-100.

In this study we have investigated the structural relationship between human apolipoproteins B-48 and B-100 by comparing protein structure and by comparing nucleotide sequence from intestinal and hepatic cDNA clones. Sequences from intestinal and hepatic cDNA were identical over the entire distance analyzed (7194 bases), which is more than required to code for B-48. The amino-terminal amino acid sequences from intact B-48 and B-100 proteins were also identical over the entire distance analyzed (16 residues). Additional protein homology was evaluated by the combined techniques of peptide mapping and immunoblotting. Purified B-48 and B-100 were each digested with three different endoproteases, and the resulting peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide bands were then detected by silver stain and by Western blotting with antisera against specific regions of B-48 and B-100. The resulting patterns suggest that B-48 is extensively homologous with the amino-terminal portion of B-100. We have identified only four peptides from B-48 (at least one in each digest) that are absent from the parallel digests of B-100. These peptides appear to arise from the ultimate carboxyl terminus of B-48 and appear to be totally homologous with a region located near the center of B-100. Our observations suggest that mature, circulating B-48 is homologous over its entire length (estimated to be between 2130 and 2144 amino acid residues) with the amino-terminal portion of B-100 and contains no sequence from the carboxyl end of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Analysis of cDNA clones encoding the entire B-26 region of human apolipoprotein B.

We report the characterization of intestine and liver cDNA clones for human apolipoprotein B (apoB) that map to the 5' end of the mRNA. The protein sequence encoded by the 5011 nucleotides derived from sequence analysis of these clones includes 1643 amino acid residues of the mature protein of Mr 184,000. The amino acid sequence at the amino terminus of B-74 peptide was determined and mapped to residue 1298. The size (Mr 145,700) and amino acid composition of the B-26 region encoded by these clones (including amino acid residues 1-1297) closely match the values obtained from the B-26 peptide. The amino acid sequence of peptide B-100 at the junction of peptides B-26 and B-74 (Phe-Lys decreases- Ser) shows structural homology to the site on human kininogen (Phe-Arg decreases- Ser) that is cleaved by the protease plasma kallikrein. The encoded protein contains five potential N-glycosylation sites and several regions in which the hydroxyamino acids, serine and threonine, are present in high abundance. The protein sequence presented in this report represents approximately 30% of the total B-100 protein and will aid in the characterization of additional cDNA clones.

Amino Acid Sequence↗

Isolation of a cDNA clone encoding the amino-terminal region of human apolipoprotein B.

A partial cDNA clone for the B-26 region of apolipoprotein B was isolated from an adult human liver DNA library by screening with an oligonucleotide probe derived from amino-terminal protein sequence obtained from purified B-26 peptide. Antisera against a synthetic 17-residue peptide whose amino acid sequence was encoded by the clone cross-reacts with apolipoproteins B-26, B-100, and B-48, but not with B-74. The nucleotide sequence immediately upstream from the amino terminus of B-26 codes for an apparent signal sequence, implying that the B-26 moiety is in an amino-terminal locus in the B-100 protein. That this sequence represents a 5' end region is further supported by primer extension analysis using a fragment of the cDNA clone and by S1 nuclease protection experiments using the corresponding region in a genomic clone.

Amino Acid Sequence↗

Thermoluminescence of 7LiF in therapeutic high linear energy transfer (LET) charged-particle beams.

The thermoluminescence of the 200 and 260 degrees C peaks of 7LiF has been measured and compared with off-axis dose and depth-dose distributions for three therapeutic high-LET beams: negative pions, helium ions and neon ions. The methods of analysis consisted of both a single-peak analysis and the dual-peak analysis methods of Hogstrom and Irifune and Hoffmann et al. The results indicate that 7LiF, analysed by the dual-peak analysis methods, is potentially useful for extracting total dose, high-LET dose, and beam quality of helium-ion and negative-pion beams. For the higher-LET neon beam, differences in sensitivity between the 200 and 260 degrees C peaks of 7LiF were found to be independent of variations in LET within the beam; consequently, only the single-peak analysis method is applicable in which case the dosemeter at best can only estimate total dose.

Fluorides↗

Restriction endonuclease activities in the legionellae.

Fifteen legionellae isolates were studied for the presence of restriction endonucleases. At least three different specificities were found in nine of these isolates. One particular activity was present in two groups of isolates isolated from widely separated geographic areas.

Bacteriophage lambda↗

Circular dichroic spectra of apolipoprotein E in model complexes and cholesterol-rich lipoproteins: lipid contribution.

Lipid-free apolipoprotein E (apo E) and canine apo E HDLc, a cholesterol-rich lipoprotein containing apo E as the only apolipoprotein, show very different circular dichroism (CD) spectra. To determine the cause of the spectral difference, we estimated the CD contribution of phospholipid, cholesterol, and cholesteryl ester in liposomes and microemulsions. We prepared microemulsions, containing nearly equal amounts of egg phosphatidylcholine (PC) and cholesteryl oleate (mean diameter 320 A), by an injection technique. Both microemulsions and cholesterol-containing liposomes exhibit intense negative CD bands in the far-ultraviolet region. Lipids contribute about 20% of the spectral difference between apo E and apo E HDLc at 222 nm, and about 60% of the spectral difference at 208 nm. The remainder of the spectral difference is attributable to lipid-protein interaction corresponding to a 15-30% increase in helicity of apo E. CD analysis indicates that the helical content of apo E in apo E HDLc resembles that in the ternary complex apo E-PC-cholesterol (or apo E-PC-cholesteryl ester) more than that in the binary complex apo E-PC, suggesting that cholesterol affects the conformation of apo E. Our data indicate that in going from a lipid-free state to a lipid environment, apo E undergoes a random to helix transition, assuming the maximal helicity predicted from its primary structure.

Animals↗

Brewers' yeast pyruvate decarboxylase produces acetoin from acetaldehyde: a novel tool to study the mechanism of steps subsequent to carbon dioxide loss.

A gas-liquid chromatographic technique was developed for the determination of both acetaldehyde and the 3-4% acetoin side product that results from the brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) catalyzed reaction of pyruvic acid. Employing this method enabled the demonstration of the catalysis of acetaldehyde condensation to acetoin by the enzyme. It was found that the acetoin produced enzymatically from pyruvic acid or from acetaldehyde was optically active, thus providing stereochemical information about the reaction. Deuterium kinetic isotope effects (employing CH3CHO and CH3CDO) were determined on the steady-state kinetic parameters to be 4.5 (Vmax) and 3.2 (Vmax/Kappm), respectively. This enabled, for the first time, the estimation of relative kinetic barriers for steps past decarboxylation. It could be concluded that (a) C-H bond scission was part of rate limitation in the enzyme-catalyzed condensation of acetaldehyde to acetoin and that (b) among the steps leading to the release of acetaldehyde, protonation of the key enamine intermediate was part of rate limitation. This latter finding is also directly applicable to the mechanism of pyruvate decarboxylation.

Acetaldehyde↗

Thermal behavior of cores of human serum triglyceride-rich lipoproteins: a study of induced circular dichroism of beta-carotene.

Induced circular dichroism (CD) of beta-carotene has been used to study the physical state in the cores of three classes of triglyceride-rich lipoproteins from human serum: intermediate-density lipoproteins (IDL) (1.006 less than d less than 1.019 g/mL) and subfractions of the d less than 1.006 g/mL lipoproteins of beta and pre-beta electrophoretic mobility. Effects on the physical state in the cores attributable to the ratio of triglycerides to cholesteryl esters and particle diameters were assessed by comparing the temperature-dependent CD spectra of beta-carotene with those of low-density lipoproteins (LDL). Lipoproteins were prepared from serum by sequential ultracentrifugation after the donors were given supplemental dietary beta-carotene (60 mg/day) for 2 weeks. The beta- and pre-beta-migrating d less than 1.006 g/mL lipoproteins were separated by starch block electrophoresis and were then individually separated into subfractions by agarose gel filtration chromatography. Between 7 and 30 degrees C, four subfractions of the beta-migrating d less than 1.006 g/mL lipoproteins and IDL exhibited reversible, temperature-dependent induced CD of beta-carotene, with contours similar to those of LDL but with smaller magnitudes and much broader transitions of the CD bands than those of LDL. In contrast, subfractions of the pre-beta-migrating d less than 1.006 g/mL lipoproteins showed no detectable induced CD of beta-carotene. These results show that the cores of triglyceride-rich lipoproteins can exist in some ordered state between 7 and 30 degrees C if they have a relatively low ratio of triglycerides to cholesteryl esters (mass ratio less than 1.6) and relatively small particle diameter (less than 60 nm).

Carotenoids↗

Plasmid transfer into members of the family Legionellaceae.

The plasmids RP4 and pPH1JI::Mu::Tn5 were transferred with moderate frequency from Escherichia coli to Legionella pneumophila (strain Bloomington 2) and Fluoribacter (Legionella) bozemanae (strain WIGA). The frequency of transfer to Tatlockia (Legionella) micdadei (strain Tatlock) was much lower. In these transconjugant strains the plasmids were maintained following at least 10 nonselective passages. However, the introduced plasmids could not be transferred from these strains to several other strains of Legionella or Tatlockia with a detectable frequency, yet they could be transferred to a restriction-modification deficient (rm-) E. coli strain with moderate frequency. This suggests not only that the expression of transfer genes may be repressed but that the legionellae are poorer recipients than are the rm- E. coli.

Bacteria↗

Secondary structure and thermal behavior of trypsin-treated low-density lipoproteins from human serum, studied by circular dichroism.

Low-density lipoproteins (LDL) were prepared from the serum of normolipidemic men on normal diets with or without supplemental beta-carotene. LDL were subjected to limited hydrolysis (5 h at 37 degrees C) with trypsin (enzyme:protein, 1:40 w/w), and their digested products separated by gel filtration. The trypsin-treated LDL contained about 80% of the original protein and essentially all of the original lipids of native LDL. The circular dichroic spectrum of trypsin-treated LDL below 240 nm resembled that of native LDL, except that the magnitudes of the ellipticity were smaller, corresponding to 25 and 33% helical content, respectively. The lower content of helix in trypsin-treated LDL suggests that certain helical regions in apolipoprotein B are sensitive to tryptic attack; however, a major portion of the helical structure of the apolipoprotein is resistant. The thermal stability of helix in trypsin-treated LDL resembled that of native LDL, suggesting that removal of the trypsin-accessible regions of the apolipoprotein has little or no effect on the forces stabilizing the remaining helices. Data on the induced circular dichroism of beta-carotene, an intrinsic probe of the neutral lipid core, showed a reduced transition temperature for cholesteryl esters after trypsin treatment. This finding suggests that the trypsin-accessible regions of apolipoprotein B may influence the fluidity of the core.

Adult↗

Improved colorimetric determination of cell wall chitin in wood decay fungi.

The Svennerholm modification of the Elson-Morgan method for glucosamine analysis was evaluated for its applicability to the rapid determination of chitin in wood decay fungi. The evaluation included extent of chromogen interference, sensitivity, color stability, and hydrolysis conditions for maximum release of glucosamine from fungal cell walls. With our further modification, the Svennerholm method was shown to be suitable for rapid quantitative determination of fungal chitin without chromatographic separation of hydrolysate chromogens.

Journal Article↗

Estradiol receptors of two distinct populations of Leydig cells.

UNLABELLED: The optimal concentrations of molybdate (Mo) to prevent thermal inactivation of estrogen receptor (ER) were found to be 5-20 mM. Using metrizamide density centrifugation, interstitial cells of adult Sprague-Dawley rat testes (2-3 months of age) can be separated into 5 distinct bands. Band 2 (B2) and 3 (B3) cells represented functionally different Leydig cells. For measurement of cytosolic ER, purified Leydig cells (B2 and B3 cells) were homogenized in 10 mM Tris-EDTA buffer with 5 mM of Mo (pH 7.4) and centrifuged at 105,000xg for 60 min. Cytosols were used immediately for ER determination. ER of B2 cells was 24.0 +/- 3.1 fmol/mg protein (mean +/- SE, n = 7), which was significantly higher than that of B3 cells, 18.6 +/- 3.2 fmol/mg protein (n = 7), p less than 0.005. The association constants of these two populations of Leydig cells for E2 receptor were comparable, 5.1 X 10(10)M-1 for B2 cells and 4.2 x 10(10)M-1 for B3 cells. In response to hCG 100 IU s.c., ER of B2 cells was reduced to 2.0 +/- 0.38 fmol/mg protein (n = 5), which was 8.4% of the control B2 cells. ER of B3 cells was almost completely depleted by the same dose of hCG, to 0.70 +/- 0.38 fmol/mg protein, 3.7% of the control B3 cells. IN CONCLUSION: B2 cells contained higher amounts of ER under basal conditions than B3 cells, and hCG-induced desensitization caused depletion of ER of both B2 and B3 cells.

Animals↗

Characterization and quantitation of apolipoproteins A-I and E of normal and cholesterol-fed guinea pigs.

We have characterized and quantified the two major plasma apoproteins of high density lipoproteins (HDL), apolipoproteins A-I (apoA-I) and E (apoE), of guinea pigs fed standard chow (normal) or chow supplemented with 1% cholesterol (cholesterol-fed). ApoA-I isolated from plasma HDL of the normal guinea pig exists in six polymorphic forms (pI 5.75-5.40). A similar isoform pattern of this apoprotein was present in nascent HDL isolated from perfused livers of normal and cholesterol-fed animals. This apoprotein contains cysteine and isoleucine and is slightly different in overall amino acid composition from apoA-I of human and rat, but activates lecithin:cholesterol acyltransferase from human plasma with an activation curve almost identical to that obtained with human apoA-I. ApoE present in nascent VLDL and HDL from perfused liver of normal animals contains three isoforms (pI 5.42-5.34). Following cholesterol feeding, the numbers of apoE isoforms from perfused livers were increased from three to five or more by shifting the major component (pI 5.42) to more acidic isoforms (pI 5.28-5.17). This shifting was mostly reversible when apoE was treated with neuraminidase, suggesting that cholesterol feeding leads to a modification of apoE by increasing its content of sialic acid. Similar changes of apoE isoforms were also observed in plasma lipoproteins as early as 10 days after cholesterol feeding. The amino acid compositions of four apoE isoform fractions isolated from plasma HDL of cholesterol-fed guinea pigs were similar to that of parent apoE. The plasma concentrations of apoA-I and apoE, measured by electroimmunoassay, were 6.2 +/- 2.0 and 2.2 +/- 0.5 mg/dl, respectively, in guinea pigs fed standard chow. In animals that had been fed 1% cholesterol, plasma levels of apoA-I slightly increased in 1 week and showed a twofold increase in 8-10 weeks. Plasma levels of apoE, on the other hand, sharply increased by 10-fold in 1 week and up to 22-fold in 8-10 weeks on the cholesterol diet.-Guo, L. S. S., R. L. Hamilton, J. P. Kane, C. J. Fielding, and G. C. Chen. Characterization and quantitation of apolipoproteins A-I and E of normal and cholesterol-fed guinea pigs.

Amino Acids↗

The aging Leydig cell: 2. Two distinct populations of Leydig cells and the possible site of defective steroidogenesis.

Using metrizamide gradient centrifugation two populations of Leydig cells were found in both 60-90 day-old and 24 month-old rats. Cells from both Band 2 (B2) and Band 3 (B3) responded to LH stimulation with increased cyclic AMP formation; however, only B3 cells produced significant amounts of testosterone. Cells from both B2 and B3 of the old rats synthesized less cyclic AMP and testosterone than cells from their younger counterparts. In response to LH stimulation, 0.01 - 1.0 mIU/ml, no appreciable difference of cyclic AMP formation could be detected between young and old Leydig cells. Maximal testosterone production occurred when 1 mIU/ml LH was used. Only when LH concentration was increased to 10 and 100 mIU/ml, did young Leydig cells produce significantly more cyclic AMP than old Leydig cells. After addition of 5X10(-7)M of pregnenolone or progesterone to the incubation medium, both young and old Leydig cells produced comparable amounts of testosterone. These results demonstrate no impairment of old rat Leydig cells to synthesize testosterone from pregnenolone and progesterone.

Aging↗

The aging Leydig cell: VII. Cytoplasmic estradiol receptors.

Plasma estradiol and cytosolic estradiol receptor levels of testes were determined in a group of young (2-3 months) and old (24 months) Sprague-Dawley rats. Estradiol binding sites for the young rats averaged 5.6 +/- 0.3 fmol/mg protein (x +/- SE, n=12), which was comparable to that of the old rats, 5.7 +/- 0.3 fmol/mg protein (n=12). Using Scatchard analyses, the association constants at equilibrium of estradiol receptor binding of the old and young rats were the same, 6.1 x 10 10 M-1. Plasma estradiol levels were also similar in both groups-19.6 +/- 2.8 pg/ml (n=14) for the young and 19.2 +/- 2.6 pg/ml (n=10) for the old rats. Our results suggest that impaired testosterone biosynthesis in old rats was not due to elevated plasma estradiol levels or to differences in testicular estradiol receptor content.

Aging↗