2-Bromoethyl glycosides in glycoside synthesis: preparation of glycoproteins containing alpha-L-Fuc-(1----2)-D-Gal and beta-D-Gal-(1----4)-D-GlcNAc

The applicability of 2-bromoethyl glycosides in carbohydrate synthesis is demonstrated by the synthesis of glycosides of alpha-L-Fuc-(1----2)-D-Gal and beta-D-Gal-(1----4)-D-GlcNAc. The bromoethyl aglycon was transformed into the methoxycarbonylethylthioethyl spacer, which allowed coupling of the sugars to proteins (BSA and KLH).     

Phosphorus nutrition of barley, buckwheat and rape seedlings. II. Influx and efflux of phosphorous by intact roots of different P status

Seedlings of barley (Hordeum vulgare L. cvs Salka and Zita), buckwheat (Fagopyrum esculentum Moench) and rape (Brassica napus L. ssp. napus cv. Line) were raised at 8 or 10 different extenral P concentrations in the range 0–2000 μM. Apart from P, the nutrient solutions were complete. Phosphate influx in roots of different P status was determined by use of a nutrient solution containing 0.1 mM³²P-labelled phosphate. A double labelling technique was used for simultaneous determination of influx (³³P) and efflux (³²P) of phosphorus by roots of barley and rape with three selected P levels. Flux determinations were also done in presence of a metabolic uncoupler (2,4-dinitrophenol) and a protein synthesis inhibitor (cycloheximide). Influx of phosphate was maximal at a certin optimal P level of the roots and decreased at both lower and higher P levels. Maximum phosphate influex [μmol (g root)-^?1 h^?1] were: rape 4,4, buckwheat 2.2, barley cv. Salka 1.6, barley cv. Zita 1.5. Both Hill plots and plots of the untransformed decreasing phosphate influx vs root P concentrations above the optimal were linear and had high correlation coefficients. The Hill coefficient varied between -3.1 and -4.2. The decrease of phosphate influx from the maximum to the lowest value at the highest P concentration of the root was 60–70%. Hence, phosphate influex appeared to be regulated through negative feedback by the internal level of phosphorous in the roots. The regulation mechanism seems bascially similar for the three species and may be of an allosteric type. P efflux from roots of low and optimal (with regard to P influx) P status was 15–20% of the simultaneous P influx. Contary to P influx, P efflux increased at high P status and almost eliminated (barley) or halved (rape) net P uptake. 2,4-Dinitrophenol reduced both P influx and P efflux by low P roots and gave linearly increasing P efflux with increasing root P status. This indicates that P efflux partly occurred by counter transport and ion exchange at the uptake sites, partly by passive P efflux along an electrochemical potential gradient. Phosphate influx was not affected by inhibition of barley root growth with cycloheximide, but P efflux increased considerably.     

Direct plasmid transfer from replica-plated E. coli colonies to competent B. subtilis cells. Identification of an E. coli clone carrying the hisH and tyrA genes of B. subtilis

Summary We have cloned the hisH tyrA wild-type genes of Bacillus subtilis with the aid of the chimeric plasmid pBJ194, which replicates both in B. subtilis and Escherichia coli. Primary cloning was done in E. coli. The original E. coli clone, carrying the recombinant plasmid (pGR1) which complements hisH tyrA mutants of B. subtilis, was selected directly from a mixture of plated E. coli clones by replicaplating these clones onto minimal agar plates without tyrosine spread just before with competent B. subtilis cells. After overnight incubation clusters of small colonies had developed exclusively in the E. coli [pGR1] colony prints.The Tyr⁺ minicolonies were shown to be B. subtilis carrying pGR1 because (i) their appearance depended linearly on the number of B. subtilis cells plated, (ii) they produced extracellular protease and amylase and (iii) plasmids could be reisolated from the minicolonies and used to transform B. subtilis recE4 tyrA1 both to Cm^r and Tyr⁺.Plasmid pGR1 transfer through replica plating was compared with plasmid transfer in liquid. Both systems depended on transformable B. subtilis strains and were sensitive to DNAseI. However, whereas integration of the tyrA ⁺ gene into the chromosome and concomittant loss of plasmids occurred frequently during regular plasmid transformation of Rec⁺ B. subtilis, this was a rare event during plasmid transfer through replica plating.     

Resistance to Disruption of Multilamellar Fragments of Central Nervous System Myelin

Abstract: Single-bilayer vesicles of myelin are desirable for studying myelin development and metabolism. Accordingly, our interest was drawn to a procedure for ves-iculating myelin (Steck et al., Biochim. Biophys. Acta 509, 397–408, 1978). We used X-ray diffraction analysis to examine these putative vesicle preparations because much larger amounts of material can be surveyed by this method than by electron microscopy. The sharpness (width) of the rings in the X-ray diffraction pattern varies inversely with the number of bilayers per multilayer structure. We therefore expected to see the diffuse diffraction pattern characteristic of single bilayers. Diffraction patterns were recorded from isolated rat brain myelin before and after the vesiculation procedure. Both patterns showed sharp rings, indicating numerous multilayered structures. Average values ranging from 7 to 10 bilayers per multilayer were calculated in both cases. This procedure did produce a small fraction of single-bilayer structures, which were isolated by differential centnfu gation; however, these accounted for only about 1% of the total myelin present. The diffraction pattern of this material showed the diffuse band typical of single-bilayer structures, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated it had the same protein composition as in normal myelin. Similar results were also obtained using either fresh or frozen bovine brain myelin. Variations of the published vesiculation procedure (incubation in 0.1 M NaCl or in buffers containing glycerol; disruption by sonication or use of a Tissumizer) also were not effective in breaking down the multilamellar fragments into thinner structures. We conclude that the multilamellar fragments of isolated CNS myelin resist disruption into single-bilayer structures.     

Boar sperm surface glycoproteins: Isolation,localization, and temporal expression during spermatogenesis

Boar sperm glycoprotein fractions were isolated by Lens culinaris hemagglutinin affinity chromatography of detergent-solubilized ejaculated spermatozoa, followed by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In order to develop methods for further investigations of the sperm proteins, we proceeded with two of the isolated glycoproteins. Antibodies were raised in female rabbits against each of the two sperm glycoproteins. By a combination of immunosorbent chromatography, using the antibodies obtained, and preparative SDS polyacrylamide gel electrophoresis, highly purified sperm proteins were isolated. The sperm proteins were immobilized on Sepharose gel columns and specific immunoglobulin Fab fragments were enriched by affinity chromatography. The specificity of the Fab fragments was ascertained by immunoprecipitation analysis. The Fab fragments were used in indirect immunofluorescence analysis to localize the corresponding antigens on the surface of boar spermatozoa. Both antigens were exclusively confined to the postacrosomal region. Immunohistochemical staining of boar testis sections revealed that both antigens are expressed from the spermatid stage. This technique also revealed that one of the antigens congregated at the Golgi complex-acrosome region during spermatogenesis.     

Improved clonal and nonclonal growth of human,rat and bovine adrenocortical cells in culture

Summary This report describes the development of a culture system for long-term growth and cloning of human fetal adrenocortical cells. Optimal conditions for stimulating clonal growth were determned by testing the efficacy of horse serum (HS), fetal bovine serum (FBS), fibroblast growth factor (FGF), epidermal growth factor (EGF), fibronectin, and a combination of growth factors, UltroSer G, in stimulating growth from low density. Optimal conditions for clonal growth were achieved using fibronectin-coated dishes and DME/F12 medium with 10% FEBS, 10% HS, 2% UltroSer G, and 100 ng/ml FGF or 100 pM EGF. Conditions for growth at clonal density were found to be optimal for growth of early passage, nonclonal cultures at higher densities. The improved growth conditions used for cloning were shown to allow continued long-term growth of nonclonal human adrenocortical cells without fibroblasts overgrowth. All cells in cultures grown in HS, FBS, and UltroSer G had morphologic characteristics of adrenocortical cells, whereas cells grown in FBS only rapidly became overgrown with fibroblasts. Clonal and nonclonal early passage human adrenocortical cells had smilar mitogenic responses to FGF and EGF. Whereas FGF, EGF, and UltroSer G showed similar stimulation of DNA synthesis and clonal growth in human adrenocortical cells and human adrenal gland fibroblasts, the tumor promoter 12-O-teradecanoylphorbol-13-acetate stimulated growth only in adrenocortical cells and was strongly inhibitory to growth in fibroblasts. In both cell types, forskolin inhibited DNA synthesis. Human adrenocortical cell cultures were functional and synthesized cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. The improved growth conditions for clonal growth of human adrenocortial cells also provided optimal conditions for long-term growth of cultured rat adrenocortical cells and ncreased the cloning efficiency of cultured bovine adrenocortical cells. This work was supported by Research grants AG-00936 and AG-06108 from the National Institute on Aging, Bethesda, MD.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opzonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B₄ and C₄. After 3–4 min, the levels of LTB₄ and LTC₄ were 93 and 35 pmol/310⁷ cells, respectively. These amounts were 2–4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC₄ were 8 and 20 times lower than those of LTB₄ after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.