Detection of complex alleles by direct analysis of DNA heteroduplexes

DNA molecules derived from three alleles of the HLA-DRB3 locus and differing from each other at several nucleotide sites were denatured and cross-hybridized. Each allelic combination was found to generate a pair of heteroduplexes of different mobility. Their retardation as compared to homoduplexes was proportional to the number of mismatches. In each heteroduplexes pair the component possessing the highest number of Pyr-Pyr oppositions was the most retarded. The results are those predicted by a theoretical model implying a correlation between base-pair opening and bending of the DNA double helix. These observations introduce a new HLA typing method at the genomic level and indicate an experimental approach to the analysis of the superhelical DNA conformation as related to different types of base oppositions.     

Saccharification and protein enrichment of sugar beet pulp byPleurotus florida

Summary Pleurotus florida was grown in submerged culture with different concentrations of sugar beet pulp with a view to its nutritional upgrading. Micelial growth, protein enrichment and dry weight loss of the solid residues of cultures, as well as the evolution of reducing sugars in the liquid medium, were followed for 14 days. A product with 45% (w/w) protein and a saccharification extent as high as 35% (w/w) were obtained after 7 days with 1% (w/v) sugar beet pulp.     

Human erythrocyte glucose-6-phosphate dehydrogenase. Identification of a reactive lysyl residue labelled with pyridoxal 5'-phosphate

Human erythrocyte glucose-6-phosphate dehydrogenase contains a reactive lysyl residue, which can be labelled with pyridoxal 5'-phosphate. The binding of one mole of pyridoxal 5'-phosphate per mole of enzyme subunit produces substantial inactivation. The substrate glucose-6-phosphate prevents the loss of activity, suggesting that the reaction site is close to the substrate-binding site. A tryptic peptide containing the pyridoxal-5'-phosphate-binding lysyl residue has been isolated and characterised. The reactive lysyl residue has been identified in the glucose-6-phosphate dehydrogenase amino acid sequence. Comparison with glucose-6-phosphate dehydrogenase from other sources shows a high homology with a peptide containing a reactive lysyl residue, isolated from the enzyme from Saccharomyces cerevisiae; glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides also contains a region highly homologous with the sequence around the reactive lysyl residue in the human enzyme. The results of this communication provide the first direct evidence for the association of an essential catalytic function with a specific region of the molecule of human erythrocyte glucose-6-phosphate dehydrogenase.     

IFN-gamma facilitates NGF-induced neuronal differentiation in PC12 cells

Natural or recombinant murine interferon-gamma causes a reversible arrest of proliferation of PC12 cells. Treatment with other antimitotics (AraC, colchicine, mitomycin C, hydroxyurea) or removal of serum, on the contrary, leads to mitotic arrest followed by cell death. IFN-gamma-treated PC12 cells respond more rapidly to NGF in terms of speed of neuronal outgrowth. On the other hand, NGF potentiates the action of IFN-gamma in stimulating the enzyme 2',5'-A synthetase which shifts from an average of 4.4-fold stimulation at 48 h with IFN-gamma alone to increments varying between 5- and 18-fold when PC12 cells are treated for 48 h with IFN-gamma and NGF. NGF alone, on the contrary, does not exert any detectable effect on this enzyme. From the findings we propose the use of a combined treatment of PC12 cells with NGF and IFN-gamma for a more rapid induction of neuronal differentiation.     

Surface spreading of synaptonemal complexes in three isopod crustacean species

The technique of whole mount spreading is used to investigate the SC of three species of Asellidae (isopod crustaceans), Asellus aquaticus, Proasellus coxalis and Proasellus meridianus, which display considerable differences in genomic DNA content.The three species, originally considered to belong to the same genus Asellus, were subsequently assigned to two separate genera: Asellus and Proasellus. The SCs of the three species differ in morphological details related to the shape of the centromere region, the attachments to the nuclear envelope, the width of the central region and the presence of twists of the lateral elements. Furthermore, they display some differences in the degree of compaction of genomic DNA in the mitotic chromosomes. The greatest differences are found between A. aquaticus and P. coxalis, while P. meridianus has several features in common with either species.     

pp60c-src kinase activity in bovine coronary extracts is stimulated by ATP

pp60c-src kinase is believed to participate in regulating key cellular mechanisms including signal transduction and differentiation of smooth muscle during early embryogenesis. In this study, pp60c-src kinase activity was demonstrated in extracts from adult bovine coronary arterial smooth muscle. Activity, reflected by autophosphorylation of pp60c-src, phosphorylation of exogenous substrates, and phosphorylation of several endogenous substrates, was enhanced about 2 fold when added Mg2+ was replaced by Mn2+. Unexpectedly, activity was dramatically stimulated 20-50 fold by prior incubation with ATP. Such stimulation appears to be mediated through a novel mechanism which is independent of ATP-induced phosphorylation of reaction components. These new observations strongly suggest that a unique mechanism exists for regulation of coronary arterial pp60c-src kinase activity. Conceivably, this mechanism may serve important roles in modulating signal transduction and contractility of vascular smooth muscle.     

On the transposition of copia-like nomadic elements in cultured Drosophila cells

We studied the stability of the genomic distribution of six retrotransposon families in long-term and short-term cultures of Drosophila cells. In a subclone derived from Kc cells, no significant rearrangements were detected over an 8 year period. On the contrary, extensive reshuffling and amplification of transposon families were observed in recently established cell lines. These results show that in cultured Drosophila cells transposition appears to be restricted to the transition from the embryo to continuous cell lines.     

Purification and characterization of aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus, a thermoacidophilic organism isolated from an acidic hot spring (optimal growth conditions: 87 degrees C, pH 3.5) was purified to homogeneity. The enzyme is a dimer (Mr subunit = 53,000) showing microheterogeneity when submitted to chromatofocusing and/or isoelectric focusing analysis (two main bands having pI = 6.8 and 6.3 were observed). The N-terminal sequence (22 residues) does not show any homology with any stretch of known sequence of aspartate aminotransferases from animal and bacterial sources. The apoenzyme can be reconstituted with pyridoxamine 5'-phosphate and/or pyridoxal 5'-phosphate, each subunit binding 1 mol of coenzyme. The absorption maxima of the pyridoxamine and pyridoxal form are centered at 325 and 335 nm, respectively; the shape of the pyridoxal form band does not change with pH. The enzyme has an optimum temperature higher than 95 degrees C, and at 100 degrees C shows a half-inactivation time of 2 h. The above properties seem to be unique even for enzymes from extreme thermophiles (Daniel, R. M. (1986) in Protein Structure, Folding, and Design (Oxender, D. L., ed) pp. 291-296, Alan R. Liss, Inc., New York) and lead to the conclusion that aspartate aminotransferase from S. solfataricus is one of the most thermophilic and thermostable enzymes so far known.     

Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow

Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).