Immunoaffinity isolation of apolipoprotein E-containing lipoproteins

Discrete apolipoprotein E-containing lipoproteins can be identified when EDTA plasma is fractionated on columns of 4% agarose. The present study has demonstrated, by physical and metabolic criteria, that these apolipoprotein E-containing lipoprotein subclasses may be further isolated by immunoaffinity chromatography. Whole plasma was first bound to an anti-apolipoprotein E immunoadsorbent prior to gel filtration on 4% agarose. After elution from the affinity column and dialysis, the bound fraction was chromatographed on 4% agarose. Discrete subfractions of apolipoprotein E could be demonstrated within elution volumes similar to those observed in the original plasma. When whole plasma was first submitted to gel filtration and the apolipoprotein E-containing lipoproteins of either intermediate- or of high-density lipoprotein (HDL) size were subsequently bound to anti-apolipoprotein E columns, the bound eluted fractions maintained their size and physical properties as shown by electron microscopy and by rechromatography on columns of 4% agarose. The metabolic integrity of apolipoprotein E-containing very-low-density lipoproteins (VLDL) was examined by coinjection into a cynomolgus monkey of 125I-labeled apolipoprotein E-rich and 131I-labeled apolipoprotein E-deficient human VLDL which had been separated by immunoaffinity chromatography. The plasma specific activity time curves of the apolipoprotein B in VLDL, intermediate-density (IDL) and low-density (LDL) lipoproteins demonstrated rates of decay and precursor-product relationships similar to those obtained after injection of whole labeled VLDL, supporting the metabolic integrity of VLDL isolated by immunoaffinity chromatography.     

Chromosomal localization of several families of repetitive sequences by in situ hybridization.

Four recombinant DNA clones (H1, H7, H12, and H15) carrying low-repetitive human DNA were previously isolated from a human genomic library based on their specificity for chromosome 21 and were studied for their distribution as determined by in situ hybridization. Clone H7 hybridized to the satellite regions of chromosomes 13, 14, 15, 21, and 22 as well as to the centromere region of chromosome 1. Clone H12 hybridized strongly to chromosomes 11 and 17 and the centromere of the X. Clones H1 and H15 had a very widespread distribution throughout the genome. Clone H15 hybridized significantly more to the short arm of chromosome 18 than to any other chromosomal segment. Clone H1 hybridized strongly to the centromere of chromosome 19 and also showed random distribution on all the other human chromosomes. We conclude that these probes appear to represent four repetitive families that demonstrate in situ hybridization patterns that do not correspond with those of any other repetitive family. Further, the in situ hybridization patterns do not show the strong chromosome 21 specificity originally defined by Southern blot analysis. The nature and chromosomal localization of these repetitive families should be useful in regional mapping and evolutionary studies and give additional insight into chromosomal organization.     

Mechanisms underlying the asynchronous replacement of myosin light chain isoforms during stimulation-induced fibre-type transformation of skeletal muscle

During the fibre-type transformation induced by chronic electrical stimulation of rabbit fast-twitch muscle, replacement of the fast forms of the two classes of myosin light chain by their slow isoforms occurs asynchronously. Studies of total cellular myosin light chains and of the slow-to-fast transition now justify the conclusion that the asynchrony is due to switching between the expression of fast and slow genes for the two light chain classes at sequential stages of the transformation process.     

Effect of temperature on the circular dichroism spectra of beta 2-microglobulins

When the temperature was lowered from 25 to 5 degrees C dramatic changes were observed in the near-ultraviolet circular dichroism spectra of bovine and caprine but not human beta 2-microglobulin. Comparison of the protein sequences suggests that the conformational change occurs in the amino-terminal 24 residues and that a tyrosine residue located on a potential beta-turn acts as a reporter group. Because delta H degrees is small (-22 kcal X mol-1), such conformational changes, possibly not readily observed, may occur at low temperatures in other proteins having potential beta-turns in otherwise aperiodic regions of sequence.     

Myoglobin: cytochrome b5 interactions and the kinetic mechanism of metmyoglobin reductase

Metmyoglobin (metMb) reduction by metMb reductase from heart muscle requires cytochrome b5 as electron-transfer mediator. The existence of a metMb-ferrous cytochrome b5 complex is demonstrated by mutual perturbation of the proteins' respective electrophoretic titration curves between pH 4 and 7. The same technique shows a preferential binding of cytochrome b5 over metMb by the enzyme. The paramagnetic hyperfine shifts in the cytochrome b5 1H NMR spectrum are perturbed by metMb, indicating the formation of a specific bimolecular complex with a 1:1 stoichiometry and a binding constant estimated to be less than 10 microM. The resonances assigned to the cytochrome b5 heme 6-propionate methylene group exhibit the largest complexation shifts. Computer modeling implicates lysines 47, 50, and 98 of metMb as contact points with cytochrome b5 carboxylate residues 43, 44, 60, and heme 6-propionate. The mechanism of the enzymatic reduction establishes metMb reductase as an NADH-cytochrome b5 oxidoreductase. Cytochrome b5 is reduced at near diffusion-controlled rates by the enzyme with a turnover number of 1000 min-1 X Km for the cytochrome is 0.9 microM versus 100 microM reported for the erythrocyte enzyme. Ferrous cytochrome b5 then reduces metMb nonenzymatically with an apparent rate constant of 4.9 X 10(4) M-1 min-1 X Acetylation of metMb, which does not affect its oxygen affinity or chemical reduction, renders it a poor substrate for enzymatic reduction. This study suggests a function for the three exterior lysine residues conserved in all mammalian myoglobin sequences: they are contact points for complexation with cytochrome b5.     

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two- to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate. Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.     

Site-specific maturation of enveloped viruses in L cells treated with cytochalasin B

Treatment of infected L cells with 10 micrograms/ml cytochalasin B (CB) was found to promote a rapid relocalization of viral glycoproteins on the cell surface. Whereas the vesicular stomatitis virus G protein and the influenza virus hemagglutinin were uniformly distributed on the surface of untreated cells, in CB-treated cells, they were strikingly concentrated at cell extremities in the regions of clustered blebs. Glycoprotein concentration at cell extremities was accompanied by preferential maturation of virus particles from the same sites; both vesicular stomatitis and influenza viruses budded predominantly from the vicinity of clustered blebs. This effect of CB was completely reversible. Removal of CB from the cell growth medium resulted in a return of viral glycoproteins to the uniform distribution characteristic of untreated cells and to uniform virus budding. The results of this study are interpreted in terms of a model that suggests that preferential budding of viruses from the regions of bleb clusters is due to the concentration of viral glycoproteins at these sites.     

Effect of oral contraceptives and some psychological factors on the menstrual experience

This study attempts to clarify the etiology of menstrual distress by using objective measures of menstration, enlightened statistical treatment and standardized measures of psychological factors. In addition to observing the traditionally associated psychological factors, measures of health locus of control, preventive health behavior and menstrual socialization (e,g., age at menarche) are included in order to assess the relevance of attitudes towards health. 57 women (mean age=23.5 years), 1/2 of whom (29) used an oral contraceptive, completed Moos' menstrual distress questionnaire at each of the 3 menstrual phases. In addition they kept menstrual diary cards for 50 days, recording days on which menstrual blood loss occurred. During an intermenstrual phase, they completed a general information questionnaire with questions on menstrual socialization and demographic variables; Eysenck's personality inventory; the multidimensional health locus of control scale; the Bem sex role inventory; and a measure of preventive health behavior. Analyses investigating the effects of pill use and psychological factors on the incidence and intensity of menstrual distress found few significant associations between these measures, especially when symptom changes over the menstrual cycle were the dependent variables. The results generally support the notion that menstruation is a negative event for most women (2/3 of the sample). Neuroticism was found to correlate with all the premenstrual MDQ scores except the positive aspect of increased arousal, with negative affect at both menstrual and intermenstrual phases, with menstrual pain and with intermenstrual concentration. The regression analyses indicate that changes in symptoms of menstrual distress over the menstrual cycle are not well predicted by the measures investigated in this study. Of the few significant associations noted, most are explicable in common sense terms. The more objective approach adopted in this study gives little support for a psychological etiology of distres. However, it also queries the appropriateness of a physiological explanation because of the limited differences found between pill users and nonusers. The inability of locus of control scores, menstrual socialization measures and a preventive health behavior measure to contribute to the definition of a woman at risk, suggests that distress is not related to an individual's general health concepts nor perceived control. 1 aspect not investigated in this study and a topic for future research is the role of a woman's expectations on her experience.     

Bulinus tropicus, a natural intermediate host for Schistosoma margrebowiei in Lochinvar National Park, Zambia

Snails, provisionally identified as Bulinus tropicus, on the basis of chromosome number, egg protein profile, AcP and HBDH enzymes of the digestive gland, and radular morphology, from Lochinvar National Park, Zambia have been demonstrated to transmit Schistosoma margrebowiei naturally. The identification of the unpaired male schistosomes was confirmed by PGM and AcP analyses. The observations confirm earlier epidemiological predictions, and add another species of mollusc to the two, B. forskalii and B. scalaris, known to be natural intermediate hosts of S. margrebowiei.     

Biological and biochemical characterization of bovine interleukin 2. Studies with cloned bovine T cells

Bovine peripheral blood lymphocytes stimulated with the T cell mitogen concanavalin A (Con A) secrete a lymphokine with biological properties similar to T cell growth factor (TCGF) or interleukin 2 (IL 2) from other species. The material supports proliferation of Con A-derived T cell blasts, limiting dilution cloning of T cell blasts, and continuous growth of T cell clones for over 6 mo in vitro. A quantitative microassay with the use of TCGF-dependent, Con A-unresponsive cloned T cells was used to determine the biological activity during purification of IL 2. A single peak of activity with an apparent m.w. of 25,000 to 28,000 was recovered after gel filtration. This material eluted from DEAE-Sephacryl between 135 and 165 mM NaCl. After isoelectric focusing, high pressure liquid chromatography, and gel electrophoresis under reducing conditions, peak IL 2 activity was associated with proteins having m.w. of 20,000 and 23,000.