The lipoxins. Stereochemical identification and determination of their biosynthesis

The stereochemistry and double bond geometry of a novel series of leukocyte-derived arachidonic acid metabolites, the lipoxins, was determined by comparison to pure unambiguous synthetic standards. The lipoxins were found to be a mixture of four lipoxin A isomers and two lipoxin B isomers. In determining the biosynthesis of these compounds, they were shown to be formed via a tetraene epoxide. In addition, it was shown that all of the lipoxin isomers formed by the incubation of 15-hydroperoxyeicosatetraenoic acid with human leukocytes were also formed by nonenzymatic hydrolysis of this tetraene epoxide.     

The biosynthesis of galactofuranosyl residues in galactocarolose

1. Cell-free extracts of Penicillium charlesii G. Smith were used in a study of the biosynthesis of the galactofuranose polymer, galactocarolose. 2. UDP-glucose and UDP-galactopyranose were precursors of galactocarolose and it was shown that the galactofuranose residues in the polymer were formed from glucose without fission of the hexose carbon chain. 3. A new nucleotide, UDP-alpha-d-galactofuranose, was formed by the system and was a major product when polymer synthesis was inhibited by F(-) or Zn(2+); the nucleotide was isolated and its structure determined. 4. UDP-alpha-d-galactofuranose was efficiently utilized for polymer synthesis and shown to be formed from the pyranose nucleotides. 5. A route for the biosynthesis of galactocarolose, involving a novel ring contraction of the hexose residue while still attached to the nucleotide, is proposed.     

Limited proteolysis of human erythrocyte Ca2+-ATPase in membrane-bound form. Identification of calmodulin-binding fragments

Water-soluble and membrane-bound calmodulin-binding polypeptides formed upon limited proteolysis of erythrocyte ghosts were isolated by means of affinity chromatography. Immune blotting revealed that all isolated fragments originated from Ca2+-ATPase. Among the fragments obtained those having formed an acylphosphate intermediate were identified. The N-terminal residue of purified intact Ca2+-ATPase was shown to be blocked (probably acylated).     

Deamination of protein lysyl ε-amino groups by peroxidase in vitro

Peroxidase, catechol, and hydrogen peroxide were shown to react with proteins, causing a decrease in lysine detectable after acid hydrolysis. The loss of lysine did not occur in the presence of benzenesulfinic acid which suggested that the quinones formed by peroxidase had oxidized some lysyl residues to lysyl aldehyde that formed a cyclic ninhydrin negative Shiff's base. When peroxidase treated protein was oxidized with performic acid prior to hydrolysis a new ninhydrin positive compound was found, which was shown by cochromatography and mass spectroscopy to be α-aminoadipic acid. The α-aminoadipic acid recovered accounted for (20–40)% of the lysine lost.     

Formation by bifunctional furocoumarins of DNA crosslinks and their repair in cultured mouse embryo fibroblasts

Formation of crosslinks in DNA by three bifunctional psoralen derivatives plus UVA light in mouse embryo fibroblasts was evaluated by a NaI density gradient centrifugation method. Psoralen was shown to be a more active cross-linking agent than 8-methoxypsoralen. As for 4,5',8-trimethylpsoralen, it needed much lower concentrations and much less 365 nm light fluence to yield high percentages of crosslinked DNA. Repair of adducts formed by these psoralen derivatives was studied by splitting the irradiation dose into two equal parts separated by variously long dark repair periods. It was shown that essentially only monoadducts formed during the first irradiation period were repaired. These mouse embryo fibroblasts seem unable to repair interstrand DNA crosslinks.     

Polymerization of β-like actin from scallop adductor muscle

Scallop adductor muscle β-like isoactin differs from rabbit skeletal muscle α-actin in the rate, extent and critical concentration of polymerization. The difference is temperature- and [KCl]-dependent. In the presence of DNase I scallop actin was shown to be depolymerized more rapidly than rabbit actin. It was suggested that the polymers formed by β-actin are less stable than those formed by α-actin.     

The involvement of endogenous dolichol in the formation of lipid-linked precursors of glycoprotein in rat liver

Endogenous dolichol was shown to function as a natural acceptor of mannose residues by using regenerating rat liver containing [(3)H]dolichol. When subcellular fractions from this liver were incubated with GDP-[(14)C]mannose a double-labelled lipid, which represented 30% of the total [(14)C]mannolipid, could be isolated. This lipid was shown to be identical with the dolichol phosphate mannose formed from exogenous dolichol phosphate, by chromatography, stability to alkali and by chemical cleavage to mannose and dolichol derivatives. It was formed by the rough endoplasmic reticulum and mitochondria. If it is concerned in glycoprotein synthesis this would suggest that it functions in the formation of both secreted and mitochondrial glycoproteins. When both the dolichol and retinol of rat tissue were radioactive they made similar contributions to the synthesis of the lipid by liver microsomal fractions and intestinal epithelial cells.     

Effect of low temperatures on the phospholipase A2 activity of rat liver mitochondria

Phospholipase A2 is shown to be activated by freezing-thawing, possibly due to changes in the state of lipid bilayer under the effect of both the temperatures themselves and physicochemical factors formed in the low-temperature range.     

Metabolism of aflatoxin B1 by Petroselinum crispum (parsley)

On administration of aflatoxin B₁ to whole parsley (Petroselinum crispum) plants, a derivative was formed, which was shown to be aflatoxicol by its chromatographic properties and mass spectrometry. Optimum conditions for the production of the derivative was on the second day after administration of the toxin to the plants, which were 90 days old after germination. Cell-free preparations of parsley were found not to produce aflatoxicol A from added aflatoxin B₁; instead they formed two new derivatives, which from chromatographic properties, were shown to be more polar than either aflatoxin B₁ or aflatoxicol A.     

The development of the pouch-like structures at the angles of the mouth in the ontogeny of the hamster Phodopus campbelli]

The development of mouth angle sacciform structure was studied in the ontogenesis of hamster Phodopus campbelli. It was found that the true sacciform structure with all components characteristic for adult animals was formed by the 20th day of postnatal development. Histological data have shown than the sacciform structure is formed in ontogenesis as a result of a complex epidermal transformation involving muscular and connective tissues which comprise the external coat. The structure studied is not formed on the basis of sebaceous glands and cannot be assigned to the class of gland as considered before.     

Biochemical and genetic studies of recombination proficiency in Escherichia coli K12

Molecular Genetics and Genomics - Residual genetic recombination is carried out by recB - recC - mutants of E. coli. Recombinants (for one gene) formed by a recB - recC - parent were shown to be as...     

Spleen colony formation by mouse embryo liver cells of different age after administration of thymocytes

The number of spleen colonies produced by fetal liver cells of different gestational ages were compared after injection of the thymus cells into the irradiated recipients. It has been shown that thymocytes that lack influence on spleen colony formation by normal born marrow can increase the number of spleen colonies formed by 12-16 day fetal liver CFU-S. It can be concluded that the population of accessory T cells which have a role in spleen colony formation have been formed to the end of pregnancy.     

Functional reconstitution of the integral membrane proteins of influenza virus into phospholipid liposomes

The integral membrane proteins of influenza virus, a hemagglutinin and a neuraminidase, have been incorporated into liposomes composed of either phosphatidylcholine or a mixture of phosphatidylcholine and phosphatidylethanolamine (2:1 w/w) using detergent dialysis. The virus spike glycoproteins for reconstitution were selectively solubilized by using cetyltrimethylammonium bromide to leave a "core particle", which lacked a lipid bilayer but possessed quaternary structure as observed by electron microscopy. The viral spike proteins were combined with exogenous phospholipid in excess sodium cholate followed by exhaustive dialysis for 150 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that only the viral glycoproteins were associated with all the complexes formed. The level of sodium cholate remaining after dialysis was shown to be reduced to less than 1 molecule per 80 protein molecules. Viral proteins reconstituted into dimyristoylphosphatidylcholine liposomes were shown to have retained hemagglutination, low-pH-dependent hemolysis, and neuraminidase activities and were associated with a lipid bilayer in two types of complexes with average lipid to protein mole ratios after sucrose density gradient purification of either 590:1 or 970:1. The bilayer vesicles formed were of similar sizes and were shown by negative-stain electron microscopy to be 150-300 nm in diameter with well-defined spikes on their surface. Reconstituted liposomes of dimyristoylphosphatidylcholine were found to be unstable with respect to their trapped volume and therefore were unsuitable for fusion studies, unlike complexes formed with phosphatidylcholine or a mixture of phosphatidylcholine/phosphatidylethanolamine derived from hen eggs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane separation of protein precipitates: unstirred batch studies

The study examines the use of ultrafiltration and microfiltration membranes for concentrating isoelectric soya protein. Experiments with an unstirred batch cell indicate that the flux is limited by the protein which remains in solution after precipitation of the major proportion. The porosityof the precipitate cake formed is shown to be a second important factor. A significant improvement in flux can be obtained by using membranes which permit passage of the soluble protein and by increasing the precipitate particle size. The results are shown to be within the range predicted theoretically by the two limiting cases of a particulate model and a soluble protein model.     

Formation of hydroxyapatite provides a tunable protein reservoir within porous polyester membranes by an improved soaking process

Biomineralization on porous polyester membranes was examined using an improved alternate soaking process (ASP). The effect of ion migration for the formation of hydroxyapatite (HAp) was shown to be crucial. Ion migration was improved by reducing the surface tension by mixing ethanol into an aqueous solution. The resulting hybrid materials were evaluated in terms of calcium content; structure using scanning electron microscopy (SEM), X-ray diffraction (XRD), and infrared spectroscopy (IR); and protein adsorption. The amount of formed HAp was controlled by the number of ASP cycles and also through the ethanol content of the mixed solvent. As the formation of HAp increased, the formed structure could be verified using SEM, IR, and XRD. Protein adsorption was investigated using albumin, gamma-globulin, and fibrinogen, and the amount of adsorbed protein was well-correlated with that of the formed HAp. This result shows that the total amount of the adsorbed proteins can be regulated by the HAp content. In summary, a tunable protein reservoir was formed on a porous polyester membrane.     

L-3-Phosphoglyceric acid,formed by ribulose-1,5-bisphosphate carboxylase,is the primary substrate for photorespiration: Experimental test of a hypothesis

Preparations of RuBP carboxylase are shown to carry out an oxygen dependent decarboxylation of L-3-phosphoglyceric acid. The product of this reaction is probably phosphoglycollate. L-3-phosphoglyceric acid, formed by RuBP carboxylase is therefore proposed to be the primary substrate for photorespiration.     

Biotransformation of tabersonine in cell suspension cultures of Catharanthus roseus.

To investigate the reactions involved in the biosynthesis of vindoline from tabersonine, the bioconversion products formed when the latter compound was fed to cell suspension cultures of Catharanthus roseus were isolated and characterized. Two biotransformation products of tabersonine were isolated and shown to be lochnericine, which is formed by epoxidation of tabersonine at positions 14, 15, and lochnerinine, the 11-methoxylation product of lochnericine. The bioconversion ratio of the main biotransformation product, lochnericine, reached a value of 80.6% within three days.     

Protonation of cyclopropene complexes of platinum(0) and the reduction of cyclopropene by nitrogenases

The reactions of a series of platinum(0) cyclopropene complexes with HCl have been studied, and it is shown that the products include both ring-intact (cyclopropane-type) and ring-opened (propene-type) materials. All products are believed to be formed by hydrocarbon insertions into transient platinum-hydrogen bonds formed by addition of HCl to platinum. Mechanistic interpretation is complicated by group migrations, and double-bond shifts. Oxidative addition of a σ-cyclopropys to platinum plays a part in ring-opening reactions, whereas the cyclopropanesare believed to be produced by further hydrocarbon insertions into transient platinum-hydrogen bonds. The relevance of these models to the mechanism of reduction of cyclopropene by nitrogenases is discussed.     

Endopeptidase activity in jackbeans and its effect on Concanavalin A

Endopeptidase activity in mature jackbeans has been characterised. One major activity is present, that of a neutral metallo-endopeptidase. Using specific inhibitors it can be shown that the Concanavalin A-associated fragments are not formed by proteolytic degradation of the intact subunit on hydration of the tissue. The intact subunit of the lectin is also resistant to the degradation by the endogenous endopeptidase activity during a prolonged incubation in vitro. These results suggest that the lectin fragments have been formed during seed maturation and no further limited proteolysis of Concanavalin A occurs during the early stages of germination.