Conversion of apolipoprotein-specific high-density lipoprotein populations during incubation of human plasma

Incubation studies were performed on plasma obtained from subjects selected for relatively low levels of high-density lipoprotein cholesterol (HDL-C) (no greater than 30 mg/dl) and particle size distributions enriched in the HDL3 subclass. Incubation (12 h, 37 degrees C) of plasma in the presence or absence of lecithin: cholesterol acyltransferase activity produces marked alteration in size profiles of both major apolipoprotein-specific HDL3 populations (HDL3(AI w AII), HDL3 species containing both apolipoprotein A-I and apolipoprotein A-II, and HDL3(AI w/o AII), HDL3 species containing apolipoprotein A-I) as isolated by immunoaffinity chromatography. In the presence or absence of lecithin: cholesterol acyltransferase activity, plasma incubation results in a shift of HDL3(AI w AII) species (initial mean sizes of major components, approx. 8.8 and 8.0 nm) predominantly to larger particles (mean size, 9.8 nm). A less prominent shift to smaller particles (mean size, 7.8 nm) accompanies the conversion to larger particles only when the enzyme is active. Combined shifts to larger (mean size, 9.8 nm) and smaller (mean size, 7.4 nm) particles are observed for HDL3(AI w/o AII) particles (mean size, 8.3 nm) also only in the presence of enzyme activity. However, in the absence of enzyme activity, HDL3(AI w/o AII) species, unlike the HDL3(AI w AII) species, are converted to smaller (mean size 7.4 nm) rather than to larger particles. Like native HDL2b(AI w/o AII) particles, the larger HDL3(AI w/o AII) conversion products exhibit a protein moiety with molecular weight equivalent to four apolipoprotein A-I molecules per particle; small HDL3(AI w/o AII) products are comprised predominantly of particles with two apolipoprotein A-I per particle. Incubation-induced conversion of HDL3 particles in the presence of lecithin: cholesterol acyltransferase activity is associated with increased binding of both apolipoprotein-specific HDL populations to low-density lipoproteins (LDL). The present studies indicate that, in the absence of lecithin: cholesterol acyltransferase activity, the two HDL3 populations follow different conversion pathways, possibly due to apolipoprotein-specific activities of lipid transfer protein or conversion protein in plasma. Our studies also suggest that lecithin: cholesterol acyltransferase activity may play a role in the origins of large HDL2b(AI w/o AII) species in human plasma by participating in the conversion of HDL3(AI w/o AII) particles, initially with three apolipoprotein A-I, to larger particles with four apolipoprotein A-I per particle.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Factors affecting adhesion of Fibrobacter succinogenes subsp. succinogenes S85 and adherence-defective mutants to cellulose.

Fibrobacter succinogenes subsp. succinogenes S85, formerly Bacteroides succinogenes, adheres to crystalline cellulose present in the culture medium. When the cells are suspended in buffer, adhesion is enhanced by increasing the ionic strength. Heat, glutaraldehyde, trypsin, and pronase treatments markedly reduce the extent of adhesion. Treatment with dextrinase, modification of amino and carboxyl groups with Formalin or other chemical agents, and inclusion of either albumin (1%) or Tween 80 (0.5%) do not decrease the degree of adhesion. Adherence-defective mutants isolated by their inability to bind to cellulose exhibited different growth characteristics. Class 1 mutants grew on glucose, cellobiose, amorphous cellulose, and crystalline cellulose. Class 3 mutants grew on glucose and cellobiose but not on amorphous or crystalline cellulose. No substantial changes were detected in the endoglucanase, cellobiosidase, and cellobiase activities of the wild type and the mutants. These data suggest that adhesion to crystalline cellulose is specific and that it involves surface proteins.     

Effects of wearing two different types of clothing on body temperatures during and after exercise

The experiment was conducted to investigate the human thermoregulatory responses during rest, exercise and recovery atT _a 20°C and 60% R.H. under the conditions of wearing two different types of clothing. Six healthy men wore two types of clothing: one covering the whole body area except the head (Type A, weight 1656 g), and the other covering only the trunk, upper arms and thighs (Type B, weight 996 g). The level of rectal temperature was kept significantly higher in Type B than in Type A during rest and recovery. The increased and decreased rates of rectal temperature during exercise and recovery were significantly greater in Type A than in Type B, respectively. These findings are discussed from the viewpoint of the differences of skin temperatures of the extremities between Type A and Type B.     

Iron kinetics effects of 88 millirads. Partial-versus-total body X-irradiation

Rats were irradiated with one tibia shielded (95% marrow exposure), total body exposed (TBI, 100%), and only one tibia exposed (5%), or they were sham irradiated (SI, 0%). Plasma Fe-59 clearance time (T1/2) and Fe-59 content ratio in the right and left tibia (RT/LT) were assayed to determine the erythroid activity of the overall marrow of the animals and the relative marrow activity in the exposed and shielded tibias, respectively. When a major fraction of the overall marrow was shielded or irradiated, the overall erythroid activity levels were identical to those of the SI and TBI animals, respectively. Interestingly, enhanced normoblastosis was observed in the marrow of the exposed tibia of individual animals exhibiting normal erythroid activity in 95% of the marrow. Conversely, localized marrow with normal erythroid activity was found in a shielded tibia of individual rats, demonstrating an enhanced erythroid activity in a major fraction of the total body. It was concluded that 88 mrad can alter marrow functions in a small isolated skeletal region as effectively as in the whole body, and tandem assays of the Fe-59 T1/2 and Fe-59 RT/LT can facilitate ultra-low-dose X-ray studies involved with partial body exposures.     

Strain-dependent expression of VH gene families

The tremendous diversity of the antibody specificity repertoire stems from the ability of each developing B cell to select one out of many possible variable, diversity, and joining gene segments by specific rearrangement of the DNA. The mechanism by which V region gene segments is selected is not known. Moreover, evidence for both random and nonrandom expression of VH genes in mature B cells has been presented previously. In this report, the technique of in situ hybridization is used to accurately measure at the single cell level VH gene family expression in LPS-induced cells from several strains. In this way, at least one-third of the B cells are stimulated and a large sampling of activated splenocytes from each strain analyzed. The use of in situ hybridization eliminates any potential biases resulting from transformation protocols. In addition, because all populations of cells are analyzed by both in situ hybridization and immunocytochemical staining with anti-IgM, the proportion of cells detected by in situ hybridization could be compared with the proportion of B cells, blasts, and plasma cells in the population. It was concluded from these comparisons that the cells being detected by in situ hybridization under the conditions described are plasmablasts and plasma cells. Therefore, an accurate measure of the functional and expressed VH gene repertoire could be made. The results clearly demonstrate strain-dependent variation in VH gene family expression, particularly VH 7183 and VH J558 with up to three-fold differences observed. Thus, either there is considerable strain variation in the number of functional VH gene family segments or the expression of VH genes is not entirely random.     

用巨细胞病毒抗原致敏的冻干血球作间接血凝试验

本文报告用巨细胞病毒(CMV)抗原致敏的冻干绵羊红细胞(简称冻干血球)做间接血凝试验。用5％正常兔血清和10％蔗糖磷酸盐缓冲液作保护剂,将巨细胞病毒抗原致敏的绵羊红细胞进行真空冷冻干燥,制成了冻干血球。经反复检测发现,用含百万分之一Tween20、2％正常兔血清的磷酸缓冲液稀释冻干血球,可消除冻干过程中产生的非特异性凝集反应。所建立的方法重复性较好,特异性与ELISA相同,敏感性比CF高。     

The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor.     

Changes in pulmonary Cu−Zn contents in superior mesenteric artery occlusion shock of rabbit

Contents of Cu and Zn of into-pulmonary blood (IPB), out-pulmonary blood (OPB), Lung tissue, and supernatant and macrophages of Lung Lavage were determined in superior mesenteric artery occlusion (SMAO) shock of rabbits. Zn of pulmonary tissue was 11.42 +/- 0.60 and 14.52 +/- 1.78 (micrograms/g wet wt) in SMAO shock and control groups, respectively. Content of Zn was found to be lower, Cu was not changed, and Cu/Zn ratio increased in lung tissue in SMAO shock. Contents of Cu and Zn in other samples were not changed. The results suggest that lower Zn in lung tissue related to acute lung injury.