Conversion of human plasma high density lipoprotein-2 to high density lipoprotein-3. Roles of neutral lipid exchange and triglyceride lipases

Cholesterol esters accumulating in human plasma high density lipoproteins (HDL) are important in conversion of HDL3 to larger HDL2. We studied whether mechanisms of removal of cholesterol esters from HDL might be important in a reverse direction, i.e. conversion of HDL2 to HDL3. Native HDL2 or HDL3 is incubated with very low density lipoproteins (VLDL) and lipoprotein-poor plasma (d greater than 1.21 g/ml) at 37 degrees C. After incubation, "modified" (M) VLDL, and HDL2 or HDL3 are reisolated by ultracentrifugation. In modified M-HDL2 or M-HDL3, triglyceride becomes the major core lipid as the triglyceride/cholesterol ester weight ratio increases 8-10-fold relative to native HDL. With only small changes in protein/phospholipid ratios in M-HDLs, the large decrease in cholesterol ester/protein ratios suggest net cholesterol ester loss from HDL. Quantitative recovery analyses prove that the cholesterol esters lost from HDL are transferred to M-VLDL, which is now richer in cholesterol ester and poorer in triglyceride. These substantial exchanges of HDL lipids are not associated by significant transfer of HDL apoproteins but are dependent on neutral lipid transfer factors present in human lipoprotein-poor plasma (d greater than 1.21 g/ml). Similar results are obtained when purified core lipid transfer protein replaces d greater than 1.21 g/ml plasma in these incubations. After depletion of cholesterol ester from HDL, most but not all, exchanged triglyceride can be removed by lipolysis with either hepatic or lipoprotein lipase, resulting in a post-lipolysis HDL2 with an increased triglyceride content relative to normal HDL. With successive incubations with VLDL, and core lipid transfer factors, HDL2 loses more than two-thirds of its cholesterol esters. After lipolysis of acquired triglyceride, HDL2 is remodeled, in both composition and flotation parameters, toward HDL3.     

Predictors of success in hypertensives treated with biofeedback-assisted relaxation

This paper describes differences in response in seventeen patients with essential hypertension who participated in a treatment program consisting of electromyograph biofeedback assisted relaxation training. Responders were found to have higher treatment values of urinary and plasma cortisol, Trait Anxiety and forehead muscle tension compared to treatment failures. Responders also sustained greater decreases in plasma, and urinary cortisol after treatment. These data are discussed in light of the ability to predict which hypertensive patients may be most benefitted by a relaxation based treatment.We would like to thank Dr. Charles Spielberger for his permission to use the State-Trait Anxiety Inventory. We thank Michael Robinson for assistance with statistical analysis.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): partial chemical and biological characterization

Bovine pituitary fibroblast growth factor has been purified 222,000-fold to homogeneity by a combination of differential salt extraction, gel filtration, and ion exchange chromatography on Mono S column. Pituitary FGF is a single-chain polypeptide with an apparent molecular mass of 15,800 and an isoelectric point of 9.6. It is highly active in triggering the proliferation of bovine and human vascular endothelial cell [half-maximal stimulation at 23-40 pg/ml (1.5-2.6 pM) and saturation between 140 and 280 pg/ml (9.3-18.6 pM)]. It displays a similar activity on bovine vascular smooth muscle cells, corneal endothelial cells, granulosa and adrenal cortex cells, and rabbit costal chondrocytes.     

Inhibition of chlorophyll synthesis inHordeum vulgare by 3-amino 2,3-dihydrobenzoic acid (gabaculin)

Gabaculin (3-amino 2,3-dihydrobenzoic acid) is shown to be a very potent inhibitor of chlorophyll formation inHordeum vulgate. Exposure of leaf segments to 30/M gabaculin results in an 80% inhibition of chlorophyll synthesis, and this is paralleled by a decrease in carotenoid. Dual-inhibitor studies with dioxoheptanoic acid, which is an inhibitor of inolaevulinic acid dehydratase, show that gabaculin inhibits an earlier step than dioxoheptanoic acid and affects -aminolaevulinic acid synthesis rather than its subsequent metabolism.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.     

Binding of simple carbohydrates and some of their chromophoric derivatives to soybean agglutinin as followed by titrimetric procedures and stopped flow kinetics

The number of carbohydrate-binding sites of the GalNAc-specific lectin is four per tetramer. The binding parameters of N-acetyl-D-galactosamine and methyl-N-acetyl-alpha-D- galactosaminide , were determined by titrating the perturbation in the absorption spectrum of the protein. For D-galactosides, it was necessary to use p-nitrophenyl-N-acetyl-beta-D- galactosaminide as an indicator in substitution titrations. The association constants K were determined at several temperatures yielding 2.4 X 10(4) M-1 at 25 degrees C with delta H degree' = -45 kJ mol-1 and delta S degree' = -67 J X K-1 mol-1 for methyl-N-acetyl-alpha-D- galactosaminide and 1.0 X 10(3) M-1 at 25 degrees C, delta H degree' = -38 kJ mol-1 and delta S degree' = -69 J X K-1 mol-1 for methyl-alpha-D-galactoside. The increase in K by a factor of 25 caused by the acetamido group is largely enthalpic . Whenever different methods were used to determine the association constant of a given compound, the agreement was excellent. The observed changes in absorption or fluorescence of all chromophoric carbohydrate derivatives used are specific for the binding of carbohydrates. For large aromatic beta- aglycons such as p-nitrophenyl or 4-methylumbelliferyl groups, the increase in K of the N-acetyl-D- galactosaminide moiety is by a factor of 2 or less, but for a large N-5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group this factor is about 20 as compared with the acetyl group. The concomitant 10-fold increase in dansyl fluorescence, also observed with four other GalNAc-binding lectins together with a favorable and large delta S degree' = +60 J X K-1 mol-1 strongly point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The results of stopped flow kinetics with 4-methylumbelliferyl-N-acetyl-beta-D- galactosaminide and the lectin are consistent with a simple mechanism for which k+ = 1.1 X 10(4) M-1 S-1 and k- = 0.4 S-1 at 25 degrees C. This k- is slower than for any monosaccharide-lectin complex reported so far.     

A low-cost antifoam additive for agar-based culture media

A highly effective, low-cost silicone-based antifoam emulsion is described which, at a final concentration of 100 ppm, prevents bubble formation during the preparation and dispensing of agar media. The compound is heat-stable, has a long shelf-life and offers considerable savings in cost by reduction in wastage of time and materials. It has no demonstrable deleterious effect on the growth of a wide range of pathogenic and commensal bacteria, or on antibiotic disc susceptibility testing, when examined using a range of different media.     

DQ α and β RFLP reveals the composition of the DQ molecule recognized by T-cell clones

Pst I RFLP, revealed with DQ and DQ probes, was compared with Taq I RFLP using a panel of DR-homozygous cell lines and HLA-typed family members. Taq I patterns, characteristic for each DR-associated DQ and allelic forms, were recognized in the homozygous state and then proven to segregate in the heterozygous members of informative families. The presence of both specific and chains was found to be necessary to form the type of DQ molecule specifically recognized by two alloreactive T-cell clones. Particular and associations also seem to be responsible for some Dw splits of the DRw6-positive cells. Taq I RFLP analysis may be more complex than the Pst I analysis, but is certainly more informative and complete, considering the type of information we were seeking by performing these types of experiments.Abbreviations used in this paper BSA bovine serum albumin - GLO glyoxalase - kb kilobase(s) - LCL lymphoblastoid cell line - MHC major histocompatibility complex - PBL peripheral blood lymphocyte - PLT primed lymphocyte test - RFLP restriction fragment length polymorphism - SDS sodium dodecyl sulfate - SSC standard sodium citrate - SSCP sodium, sodium citrate, sodium phosphate - TBE Tris-borate, boric acid, ethylenediaminetetraacetate (EDTA) - TCGF T-cell growth factor     

pHG165: A pBR322 copy number derivative of pUC8 for cloning and expression

During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted. This has resulted in an elevated copy number for the pUC plasmids which makes the expression of beta-galactosidase activity constitutive in a host containing the Iqtss lac repressor. We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered. In addition, the lac alpha/lac promoter expression region has been inserted into a HpaI cassette. This facilitates the movement of recombinant DNA clones within the MCS. It also increases the complementation activity of the lac alpha peptide by an order of magnitude, allowing selection of recombinants by their Lac- phenotype on MacConkey agar.