Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.     

Production and characterization of KDO-specific monoclonal antibodies recognizing lipopolysaccharides from heptoseless mutants of Salmonella

Abstract Hybrid cell lines producing monoclonal antibodies with specificity for the lipopolysaccharide (LPS) from the deep rough mutant Salmonella minnesota R595 have been established. Spleen cells from BALB/c mice immunized with live R595 bacteria were fused with Sp 2/0 myeloma cells and three hybridomas producing antibodies specific for heptoseless LPS from Salmonella were selected. All three monoclonal antibodies were shown to bind only to heptoseless, but 3-deoxy- d -manno-octulosonic acid (KDO) containing LPS when tested in enzyme-linked immunosorbent assay (ELISA) against a set of structurally defined LPS and lipid A from Salmonella, Shigella and Escherichia coli . Synthetic KDO was an efficient inhibitor of the antibody-R595 LPS interaction defining that KDO is in an immunodeterminant position interacting with the monoclonal antibodies.     

Interaction of 1-propargyl-5-chloropyrimidin-2-one (a metahalone) with rat brain tubulin

The mitotic inhibitor 1-propargyl-5-chloropyrimidin-2-one (a metahalone) was found to bind to DEAE-cellulose purified rat brain tubulin. A decrease in the fluorescence of 1-propargyl-5-chloropyrimidin-2-one was seen when the drug was incubated in the presence of increasing tubulin concentrations. The decrease in metahalone fluorescence was not affected by the addition of GTP, indicating drug interaction at other portions of the tubulin molecule than the nucleotide binding sites. Scatchard plot analysis following incubation of tubulin with 1-propargyl-5-chloro-[2-14C]pyrimidin-2-one revealed that 1 mol of metahalone bound to 1 mol of tubulin dimer with a measured association constant of 8.0 X 10(3) M-1. Double reciprocal plots of vincristine and colchicine binding to tubulin in the presence of 1-propargyl-5-chloropyrimidin-2-one showed that the metahalone competitively inhibited colchicine binding to tubulin but had no influence on vincristine binding. This conclusion was supported by gel filtration chromatography where an increase in unbound colchicine was measured when 1-propargyl-5-chloropyrimidin-2-one was present in an incubation mixture containing colchicine and tubulin. In the presence of 5 mM 1-propargyl-5-chloropyrimidin-2-one, tubulin self-aggregated into crystalline structures. The binding of 1-propargyl-5-chloropyrimidin-2-one to tubulin at or near the colchicine binding site may be responsible for the metaphase arresting characteristics of this drug.     

Synthesis of mannose oligosaccharides via reversal of the α-mannosidase reaction

Summary Three naturally occurring isomers of the disaccharideO--d-mannosyl-d-mannoside were synthesized by reversing the hydrolytic activity of jack bean -mannosidase at 75°C in a very high concentration of mannose. Higher oligosaccharides were also obtained at the later stages of the reaction. The maximum total yield of disaccharides was 37% (w/w) based on the total amount of saccharides.     

Studies on the metabolism of steroids in the foetus. Biosynthesis of 6α-hydroxytestosterone in the human foetal liver

After incubation of testosterone with 105000g microsomes of human foetal liver, 6alpha-hydroxytestosterone was isolated and identified by t.l.c. and g.l.c.-mass spectrometry. This is the first example of 6alpha-hydroxylation of C(19) steroids in the human liver, and the finding is discussed in relation to earlier reports of 6-oxygenated C(19) and C(18) steroids in pregnant women.     

Phytanic acid alpha-oxidation and complementation analysis of classical Refsum and peroxisomal disorders

Summary We have measured the production of ¹⁴CO₂ from exogenous [1-¹⁴C] phytanic acid in fibroblast monolayers from patients with classical Refsum's disease and peroxisomal disorders. Activities in the different disorders were (percentage of control): classical Refsum's disease (5%), isolated peroxisomal acyl-CoA oxidase deficiency (75%), Zellweger syndrome (4%), neonatal adrenoleukodystrophy (5%), and rhizomelic chondrodysplasia punctate (3%). Absence of complementation was demonstrated between Zellweger syndrome and infantile Refsum's disease lines after polyethylene glycol fusion, with decreases of average activity of 11% relative to unfused cell mixtures. Classical Refsum's disease, rhizomelic chondrodysplasia punctata, and neonatal adrenoleukodystrophy lines all complemented one another, and Zellweger syndrome or infantile Refsum's disease lines, with average activity increases of 522%–772%. No intragenic complementation was observed within either group. Four complementation groups were detected suggesting that at least four genes are involved in phytanic acid -oxidation: one gene for the enzyme phytanic acid -hydroxylase (probably mitochondrial); one gene for a regulatory factor for the expression of phytanic acid -decarboxylation activity and two membrane-bound peroxisomal enzymes involved in the synthesis of plasmalogens; two genes for the assembly of functional peroxisomes and/or import of proteins into peroxisomes.     

Immunogenicity of the Shigella flexneri Serotype Y (SFL124) Vaccine Strain Expressing Cloned Glucosyl Transferase Gene of Converting Bacteriophage SfX

The glucosyl transferase gene (gtr) from bacteriophage phage X (SfX) caused partial conversion of serotype Y (group antigen 3, 4) to X (group antigen 7, 8) when introduced into a candidate vaccine strain of Shigella flexneri serotype Y (SFL124). The gtr gene caused conversion of O-antigens but did not eliminate the adsorption of the corresponding phage SfX. The hybrid strain expressing both group antigens 7, 8 and 3, 4 showed 75% protection when immunized guinea pigs were challenged with a wild-type S. flexneri serotype X strain. No protection was observed against serotype Y challenge, although group antigen 3, 4 was detected in the LPS of the hybrid strain. This suggests the importance of O-antigen immunity in the host defense against shigellosis.