A novel cell line (Caco-2) for the study of intestinal lipoprotein synthesis

Lipoprotein synthesis by the colonic adenocarcinoma cell line Caco-2 was investigated to assess the utility of this cell line as a model for the in vitro study of human intestinal lipid metabolism. Electron micrographic analysis of conditioned medium revealed that under basal conditions of culture post-confluent Caco-2 cells synthesize and secrete lipoprotein particles. Lipoproteins of density (d) less than 1.063 g/ml consist of a heterogeneous population of particles (diameter from 10 to 90 nm). This fraction consists of very low density lipoproteins (d less than 1.006 g/ml) and low density lipoproteins (d = 1.019-1.063 g/ml). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled Caco-2 lipoproteins revealed that very low density lipoproteins contain apolipoprotein E (apoE) and C apolipoproteins, while low density lipoproteins contained apoB-100, apoE, apoA-I, and C apolipoproteins. The 1.063-1.21 g/ml density fraction contained two morphological entities, discoidal (diameter 15.6 +/- 3.9 nm) and round high density lipoprotein particles (diameter 10.2 +/- 2.3 nm). The high density lipoproteins contained apoA-I, apoB-100, apoB-48, apoE, and the C apolipoproteins. Using isoelectric focusing polyacrylamide gel electrophoresis newly secreted apoA-I was identified as pro-apoA-I. ApoE and apoC-III released by Caco-2 cells were highly sialylated. mRNA species for apoA-I, apoC-III, and apoE, but not apoA-IV were identified by Northern blot analysis. ApoA-I, apoB, and apoE were visualized in Caco-2 cells by immunolocalization analysis. This intestinal cell line may be useful for in vitro studies of nutritional and hormonal regulation of lipoprotein synthesis.     

Apolipoprotein E isoform phenotyping methodology and population frequency with identification of apoE1 and apoE5 isoforms

Minigel methodology has been utilized for apolipoprotein (apo) E isoform phenotyping and criteria for distinguishing the apoE4/4 phenotype (mean apoE4/apoE3 ratio: 5.81) from the apoE4/3 phenotype (mean ratio: 1.01) and the apoE3/3 phenotype (mean apoE3/apoE2 ratio: 2.67) from the apoE3/2 phenotype (mean ratio: 0.76) based on gel scanning were developed. ApoE allele frequencies in 1209 subjects were: apoE3, 0.786; apoE4, 0.135; apoE2, 0.075; apoE5, 0.002; and apoE1, 0.002. Subjects with the apoE2 allele tended to have higher plasma very low density lipoprotein (VLDL) cholesterol and lower low density lipoprotein (LDL) cholesterol concentrations than subjects with the apoE3 allele, while the converse was true for subjects with the apoE4 allele. Subjects with the rare apoE1 allele had values similar to those with the apoE2 allele, while subjects with the rare apoE5 allele had values similar to those with the apoE4 allele.     

Solid-state NMR studies of regulation of N-(phosphonomethyl)glycine and glycine metabolism in Pseudomonas sp. strain PG2982

Lyophilized samples of Pseudomonas sp. PG2982 grown on 13C- and 15N-labeled glyphosate have been analyzed by single and double cross-polarization 13C NMR. Both the carbon and nitrogen metabolism of glyphosate are significantly influenced by the nitrogen source used for the growth of the organism. When ammonium sulfate is the source of nitrogen, the glycyl moiety of glyphosate is utilized intact for the biosynthesis of purines and proteins. But when the organism is grown on glycine as the source of nitrogen, the carbons and nitrogen of glyphosate are scrambled, consistent with incorporation into serine and pyruvate, and hence participation in general metabolism. When both ammonium and glycine are present in the growth medium, regulation of the metabolic fluxes along each of the two major pathways appears to be determined by the intracellular glycine concentration.     

Solid-state NMR studies of Klebsiella pneumoniae grown under nitrogen-fixing conditions

The carbon and nitrogen metabolism of Klebsiella pneumoniae M5a1 has been characterized using 13C and 15N labeling with detection by cross-polarization magic-angle spinning solid-state NMR. Cells grown on ammonium typically require some 20 h to derepress fully for nitrogenase when transferred to medium devoid of any source of fixed nitrogen. We have established that during this period some cellular proteins are catabolized with the liberated nitrogen being used for the synthesis of purines needed for formation of ribosomal RNA. The 20-h derepression period can be shortened to 6 h by the introduction of fixed nitrogen in certain specific forms. Serine is the most successful agent we have examined for shortening the derepression period and glycine among the least successful. We attribute this difference to the advantage of serine over glycine in providing both specific and nonspecific carbon and nitrogen sources for complete purine synthesis. These determinations were made by tracing the metabolism of 13C- and 15N-labeled chemical bonds from the 2 amino acids during derepression.     

Amino acid sequence of a novel calmodulin from Paramecium tetraurelia that contains dimethyllysine in the first domain

A class of Paramecium behavioral mutants called pantophobiacs have a deficiency in calcium-dependent potassium efflux, and this deficiency can be corrected by the microinjection of wild-type Paramecium calmodulin (Hinrichsen, R. D., Burgess-Cassler, A., Soltvelt, B. C., Hennessey, T., and Kung, C. (1986) Science 232, 503-506). As a starting point in investigations of which features allow wild-type Paramecium calmodulin to fully restore this behavior while other calmodulins are inactive or poorly effective, we elucidated the amino acid sequence of the wild-type calmodulin. We utilized an approach that combined Edman chemistry with mass spectrometry. This approach resulted in the identification of a new post-translational modification in calmodulin: N epsilon,N epsilon-dimethyllysine at residue 13. This particular modification has not been described for calmodulins studied previously. The only other first-domain modification that has been described for any calmodulin is acetylation of the amino terminus (Watterson, D. M., Sharief, F., and Vanaman, T. C. (1980) J. Biol. Chem. 255, 962-975). These results along with analyses of pantophobiac calmodulin and calmodulin binding proteins will provide insight into calmodulin's role in a well-defined behavioral mutant.     

Modulation of a respiratory motor program by peptide-secreting neurons in Aplysia

Respiratory pumping of the gill and siphon of Aplysia californica is a fixed-action pattern coordinated by a defined set of interneurons and motor neurons. In semi-intact preparations of the gill and siphon innervated by the abdominal ganglion, respiratory pumping is facilitated for a prolonged period following activation of the peptidergic bag cell neurons. The induced changes in contractile behavior of the gill and siphon correlate with cell-specific actions of the bag cells on motor neurons regulating these organs. Our results suggest that peptidergic neurons can alter the expression of a fixed pattern of behavior by modulating the excitability of motor neurons controlling the behavior.     

Continuous monitoring of plant water potential

Plant water potential was monitored continuously with a Wescor HR-33T dewpoint hygrometer in conjunction with a L51 chamber. This commercial instrument was modified by replacing the AC-DC mains power converter with one stabilized by zener diode controlled transistors. The thermocouple sensor and electrical lead needed to be thermally insulated to prevent spurious signals. For rapid response and faithful tracking a low resistance for water vapor movement between leaf and sensor had to be provided. This could be effected by removing the epidermis either by peeling or abrasion with fine carborundum cloth. A variety of rapid plant water potential responses to external stimuli could be followed in a range of crop plants (sunflower (Helianthus annuus L., var. Hysun 30); safflower (Carthamus tinctorious L., var. Gila); soybean (Glycine max L., var. Clark); wheat (Triticum aestivum L., var. Egret). These included light dark changes, leaf excision, applied pressure to or anaerobiosis of the root system. Water uptake by the plant (safflower, soybean) mirrored that for water potential changes including times when plant water status (soybean) was undergoing cyclical changes.     

Transplantation of bone marrow with constitutional chromosomal anomalies. Cytogenetic studies and clinical implications

We report on two cases of transplantation of bone marrow with constitutional chromosomal anomalies. A female patient with acute myelocytic leukemia (FAB, M 3) in first complete remission received a bone marrow graft from her sister with the karyotype 47 XXX (triple-X-syndrome). A male patient with Ph-positive CML and a constitutional Robertsonian t(14; 15) received HLA and MLC loci compatible bone marrow from his sister who was also a carrier of the Robertsonian t(14; 15). Our findings indicate that transplantation of marrow from donors with balanced chromosomal translocation is possible, although no conclusion can be made regarding long term results as both recipients died early from infectious complications.     

Erythroid differentiation and the Na+,K+-pump in ouabain-sensitive and ouabain-resistant Friend erythroleukemia cell lines

The selection and biochemical characterization of ouabain-resistant erythroleukemia cell lines are described. Treatment of ouabain-resistant Friend erythroleukemia cell (FLC) lines with 1 mM ouabain demonstrated a reduced ouabain-sensitive 86Rb+-uptake after Na+-preloading in comparison with ouabain-sensitive cells. The ouabain- and diuretic (piretanide)-insensitive component of the 86Rb+-uptake (residual influx) was significantly enhanced in the ouabain-resistant FLC clones. Measurements of the Na+,K+-ATPase activity (E.C. 3.6.1.3) in plasma membrane preparations of the ouabain-resistant FLC clone B6/2 indicated that a ouabain-resistant Na+,K+-ATPase activity of about 20% of the total enzyme activity existed in the presence of 1 mM ouabain. Further experiments showed that the Na+,K+-ion-gradient in ouabain-resistant B6/2 cells was unaffected by ouabain exposure whereas the gradient collapsed in wild type 12 N cells. Another property of the ouabain-resistant cell lines was a decrease of the 86Rb+-uptake due to the Na+,K+, 2Cl(-)-cotransport system measured as piretanide-sensitive 86Rb+-uptake. The data on ion transport mechanisms in QuaR and QuaS FLC are discussed with respect to mutagen-induced and spontaneous cellular ouabain resistance. In addition, the role of altered ion transport mechanisms is considered for induced erythroid differentiation.     

Postprandial plasma lipoprotein changes in human subjects of different ages

Plasma lipoprotein changes were monitored for 12 hr after a fat-rich meal (1 g of fat/kg body weight) in 22 subjects (9 males, 13 females, 22-79 yr old). Plasma triglyceride, measured hourly, peaked once in some subjects, but twice or three times in others. The magnitude of postprandial triglyceridemia varied considerably between subjects (range: 650-4082 mg.hr/dl). Males tended to have greater postprandial triglyceridemia than females, and elderly subjects had significantly (P less than 0.05) greater postprandial triglyceridemia than younger subjects. Total plasma cholesterol, measured every three hr, increased significantly (6.0 +/- 2.1%) in 7 subjects, decreased significantly (7.1 +/- 1.2%) in 10 subjects, and remained unchanged in the remainder. Single spin ultracentrifugation and dextran sulfate precipitation procedures were used to quantitate triglyceride and cholesterol in triglyceride-rich lipoproteins (TRL, d less than 1.006 g/ml), low density lipoproteins (LDL), and high density lipoproteins (HDL). Plasma TRL and HDL triglyceride increased after the fat meal, while LDL triglyceride decreased at 3 hr but increased at 9 and 12 hr. TRL cholesterol increased postprandially, while LDL and HDL cholesterol decreased. Phospholipid (PL), free (FC) and esterified (EC) cholesterol measurements were carried out on the plasma and lipoprotein fractions of 8 subjects. Plasma PL increased significantly at 3, 6, and 9 hr after the fat-rich meal, due to increases in TRL and HDL PL. TRL CE increased postprandially, but a greater decrease in LDL and HDL CE caused plasma CE to be decreased. Plasma FC increased, predominantly due to an increase in TRL FC. Plasma concentrations of apolipoprotein A-I and apolipoprotein B both decreased after the fat-rich meal. The magnitude of postprandial triglyceridemia was inversely correlated with HDL cholesterol levels (r = -0.502, P less than 0.05) and positively correlated with age (r = -0.449, P less than 0.05), fasting levels of plasma triglyceride (r = 0.636, P less than 0.01), plasma apoB (r = 0.510, P less than 0.05), TRL triglyceride (r = 0.564, P less than 0.01), TRL cholesterol (r = 0.480, P less than 0.05) and LDL triglyceride (r = 0.566, P less than 0.01). Change in postprandial cholesterolemia was inversely correlated with fasting levels of HDL cholesterol (r = -0.451, P less than 0.05) and plasma apoA-I (r = -0.436, P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)