Effect of vitamin B-6 (pyridoxine) deficiency on lung elastin cross-linking in perinatal and weanling rat pups.

Weanling and perinatal rats were rendered vitamin B-6 (pyridoxine)-deficient. The rat pups were nursed from vitamin B-6-deficient or -sufficient dams and were killed at day 15 after parturition. The weanling rats were fed vitamin B-6-deficient or -sufficient diets and were killed after 5 weeks of treatment. Lung elastin from the groups of rats was then studied with respect to its content of lysine-derived cross-linking amino acids. Lung lysyl oxidase activity was also measured. B-6 deficiency decreased the number of lysine residues in elastin that were converted into the cross-linking amino acid precursor allysine. However, a more significant defect in cross-link formation was an apparent block in the condensation steps leading to the formation of desmosine. Desmosine was decreased, with an increase in the amounts of aldol condensation products (aldol CP) in elastin. It is proposed that the elevation in aldol CP results from the formation of thiazines, which are produced from the reaction between aldehyde and homocysteine. The concentration of homocysteine is significantly elevated in vitamin B-6-deficient rats.     

Mast cell products stimulate collagenase and prostaglandin E production by cultures of adherent rheumatoid synovial cells

Mast cells were purified from histologically-confirmed dog mastocytomas and extracted for whole mast cell products (MCP). When added to cultures of human adherent rheumatoid synovial cells MCP induced a 50-400 fold increase in prostaglandin E synthesis and a 10-50 fold stimulation of collagenase production. The mast cell stimulatory factor has not been identified and was not due to histamine, heparin or prostaglandin E. These results indicate a novel way in which mast cells might interact with synovial cells to promote the production of inflammatory mediators and proteolytic enzymes which might contribute to connective tissue degradation.     

Recognition of super-secondary structure in proteins

A procedure to recognize super-secondary structure in protein sequences is described. An idealized template, derived from known super-secondary structures, is used to locate probable sites by matching with secondary structure probability profiles. We applied the method to the identification of βαβ units in β/α type proteins with 75% accuracy. The location of super-secondary structure was then used to refine the original (Garnier et al., 1978) secondary structure prediction resulting in an 8.8% improvement, which correctly assigned 83% of secondary structure elements in 14 proteins. Slight modifications to the Garnier et al. method arc suggested, producing a more accurate identification of protein class and a better prediction for β/α. type proteins. A method for the incorporation of hydrophobic information into the prediction is also described.     

A method for incorporating macromolecules into adherent cells

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.     

Isolation and characterization of a 30,000-dalton calcium-sensitive actin cross-linking protein from Dictyostelium discoideum

A calcium-sensitive actin-binding protein having a subunit molecular mass of 30,000 daltons (30K protein) has been isolated from Dictyostelium discoideum. Structural, immunological, and functional analyses demonstrated that the 30K protein was distinct from other actin-binding proteins of D. discoideum. A native molecular mass of 31,700 daltons was determined by equilibrium sedimentation, indicating that the protein is monomeric. The Stokes radius was 30 A. The frictional coefficient calculated from these measurements was 1.44, indicating an asymmetric shape. The 30K protein induced an increase in the viscosity of a solution of F-actin. Bundles of actin filaments were observed in negatively stained mixtures of actin and the 30K protein. Both the formation of filament bundles and the increases in viscosity of actin induced by the 30K protein were observed in the presence of 1 X 10(-8) M but not 2 X 10(-6) M calcium. Variation of the pH from 6.6 to 7.8 had no effect on the activity of the 30K protein. Calcium induced neither a large change in quaternary structure of the 30K protein nor a restriction of the lengths of actin filaments by the 30K protein. The apparent affinity of the 30K protein for actin was decreased in the presence of calcium. Reversible cross-linking of actin filaments by the 30K protein may contribute to regulation of the consistency and contractility of cytoplasm in D. discoideum.     

Escherichia coli dnaJ- and dnaK-gene products: synthesis in minicells and membrane-affinity

Escherichia coli dnaJ- and dnaK-gene products have been identified in a system of minicells infected with the appropriate transducing lambda phages. The molecular weights of these polypeptides in dodecyl sulphate/acrylamide electrophoresis amounted to 39,000 and 77,000, respectively. Equilibrium sedimentation of minicell lysates in metrizamide density gradients has revealed that both these host proteins, indispensable for lambda DNA replication, are membrane-bound.     

Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus.

Analysis of duck hepatitis B viral DNA by gel electrophoresis, Southern blotting, and binding to benzoylated naphthoylated DEAE-cellulose showed that a protein is bound to the minus-strand virion DNA as well as to the full-length single strand, minus-strand species, and minus-strand DNA intermediates isolated from replicating complexes present in infected duck liver. By utilizing a modified dideoxynucleotidyl sequencing method, it was shown that the protein is covalently bound to the smallest detectable growing strands (ca. 30 bases) and that minus-strand synthesis begins at a unique site. These results support the notion that the protein may function as a primer for synthesis of the minus-strand DNA.     

Interaction of mycoplasmas and phagocytes

Aspects of the interaction of certain mycoplasmas with macrophages and neutrophils in vivo and in vitro have been studied using two systems, one involving M. pulmonis in mice and the other involving M. bovis with bovine leucocytes. Studies with M. pulmonis indicated that the disappearance of viable organisms from the peritoneal cavity was not enhanced in SPF mice in which a peritoneal exudate rich in neutrophils had been induced. However, viable M. pulmonis organisms disappeared more rapidly from the peritoneal cavities with exudates containing increased numbers of macrophages. Experiments in vitro studied the opsonic effect of bovine IgG isotypes for bovine neutrophils and alveolar macrophages. Both IgG1 and IgG2 promoted killing of M. bovis by alveolar macrophages but IgG2 was more effective than IgG1 at promoting mycoplasma killing by neutrophils. Further studies in vitro indicated that certain bovine mycoplasma could inhibit killing of Escherichia coli by bovine neutrophils.