Cystic fibrosis typing with DNA probes and screening for ΔF508 deletion in families from Southern France

Summary A sample of 235 individuals from 49 French cystic fibrosis (CF) families with at least one living affected child was typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, and was screened for the ΔF508 mutation. Using a combination of six probes, 44 out of the 49 families were sufficiently informative to enable prenatal diagnosis or carrier determination. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV2c: allele 1; KM19: allele 2), which accounts for about 78% of CF chromosomes in our families. The ΔF508 deletion was present in 64.3% of CF chromosomes.     

Somatic hybrid plants produced by electrofusion between dihaploid potatoes: BF15 (H1), Aminca (H6) and Cardinal (H3)

In order to regenerate somatic hybrids, mesophyll protoplasts from a dihaploid potato, BF15 (H1), were electrofused with those from two other dihaploid clones, Aminca (H6) and Cardinal (H3). Determination of the ploidy level by flow cytometry showed that 10% of plants regenerated from the fusion experiment with BF15 + Aminca were diploids, 14% triploids, 63% tetraploids and very few were mixoploids or had a higher ploidy level. Using morphological markers and vigour in plant growth, we were able to recover a total of 24 somatic hybrid plants, respectively 20 and 4 hybrids (accounting for 12% and 13% of regenerants) from the fusions BF15 + Aminca and BF15 + Cardinal. Most of the somatic hybrids were at the expected tetraploid level (2n=4x=48). The hybrid nature was confirmed by examining isoenzyme patterns for malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD).     

Assignment of 12 loci to rat chromosome 5: evidence that this chromosome is homologous to mouse chromosome 4 and to human chromosomes 9 and 1 (1p arm)

Twelve loci have been assigned to rat chromosome 5: aldolase B (ALDOB), atrial natriuretic factor (ANF = pronatriodilatin, PND), D4RP1, DSI1, galactosyltransferase (GGTB2), glucose transporter (GLUT1), interferon alpha 1 and related interferon alpha (INFA), interferon beta (INFB), lymphocyte-specific protein-tyrosine kinase (LCK), oncogene MOS, alpha 2U-globulin (major urinary protein, MUP), and orosomucoid (ORM, also called alpha 1-acid glycoprotein, AGP). Among these, the interferon alpha and beta genes map in the q22-23 region, which also contains a transformation suppressor gene (SAI1). The other loci reside outside this region. This study also indicated that the rat genome contains 2 LCK genes, unlike the human and murine genomes. These new assignments on rat chromosome 5 demonstrate that this chromosome is highly homologous to mouse chromosome 4 and carries synteny groups conserved on human chromosome 9 (interferon alpha and beta, galactosyltransferase, orosomucoid, and aldolase B genes) and on the short arm of human chromosome 1 (MYCL, glucose transporter, protein kinase LCK, and atrial natriuretic factor genes).     

Basal autophosphorylation of insulin receptor occurs preferentially on the receptor conformation exhibiting high affinity for insulin and stabilizes this conformation

Insulin signal transmission through the plasma membrane was studied in terms of relationship between basal autophosphorylation of the β-subunit and the ability by bind insulin by the -subunit of the insulin receptor. In a cell free system, receptors phosphorylated on tyrosine residues in the absence of insulin were separated from non-phosphorylated receptors using antiphosphotyrosine antibodies. Insulin binding assays were then performed on basally autophosphorylated and on non-phosphorylated receptors. We found that the tyrosine phosphorylated receptors, which corresponded to 25% of the total number of receptors, were accountable for 60–80% of insulin binding. Scatchard representation of binding data has shown that the plot corresponding to tyrosine phosphorylated receptors was localized above, and was steeper than the plot corresponding to non-phosphorylated receptors. These data make it likely that the conformation of -subunit which favours ligand binding is connected to the conformation of β-subunit which favours phosphate reception on tyrosine residues. Reciprocally, the high-affinity conformation of insulin receptor seems to become stabilized by basal autophosphorylation.     

A practical test for in vitro evaluation of photosensitization: Assessment with hematoporphyrin derivative (HPD)

A simple procedure is described for the determination of the photosensitizing potency of drugs, using three leukemic cell lines, two of lymphocytic origin, L1210 and P388 and one of erythroid type, Friend-745. The procedure allows one to investigate several aspects of the photosensitization properties of tested compounds such as cellular localization and direct (trypan blue exclusion) or delayed (clonogenicity) photomediated toxicities.The method was assessed using crude hematoporphyrin derivative (HPD) as well as dihematoporphyrin ether (DHE) or commercially available Photofrin II. Results were compared to those obtained with normal cells, e.g spleen lymphocytes and erythropoietic stem cells (CFU-e), and discussed in the light of the relative response of normal versus transformed cells.Abbreviations DHE Dihematoporphyrin Ether - FCS Fetal Calf Serum - HPD Hematoporphyrin Derivative - PDT Photodynamic Therapy     

C1q,a collagen-like complement subcomponent,in dermatosparactic cattle: its extracellular modification is not affected by lack of procollagen N-terminal proteinase (pN-proteinase)

C1q, a collagen-like complement protein, was purified from the serum of a ddermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum Clq from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No differdence, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and typtic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q.     

3-Amino-6-methoxy-9-(2-hydroxyethylamino) acridine: A new fluorescent dye to detect mycoplasma contamination in cell cultures

A new fluorescent acridine orange derivative, 3-amino-6-methoxy-9-(2-hydroxyethylamino) acridine (AMHA), has been applied to Hela cells in order to set up appropriate conditions for the detection of mycoplasma contaminations. Since AMHA staining reveals intensely fluorescent nuclei and slight fluorescent cytoplasm, we can visualize and localize mycoplasma contamination on each cell. In combination with a shortened Chen's staining method (1977), AMHA should allow a better detection of mycoplasma in animal cell cultures than the well established Hoechst dye.     

Basic nuclear proteins of transition during spermiogenesis in Scylliorhinus caniculus

During dog-fish spermiogenesis, 2 basic nuclear protein transitions occur: the first from histones to spermatid-specific proteins S1 and S2, the second leading to protamines. S1, the most abundant transition protein, is a polypeptide containing 87 residues (Mr = 11,179 Da) whereas S2, the minor transition protein, contains 80 residues (Mr = 9,726 Da). The 2 proteins are mainly characterized by an asymmetry of the molecule, a very high content of basic residues, a relatively high level of hydrophobic residues and a cluster of acidic residues in the carboxy-terminal quarter of the molecule. The 2 proteins are phosphorylated on serine residues and the degree of phosphorylation is relatively important in protein S1. The 2 transition proteins are structurally unrelated to testis histones or sperm protamines and cannot be considered either as their proteolytic degradation products or as their precursors.     

Controlled addition of small amounts of oxygen with membranes

Summary In some fermentations micro-aerobic conditions are desirable. The controlled addition of trace amounts of oxygen may be achieved by bubble free oxygen supply using silicon membrane tube, operated in dead-end mode. The small oxygen uptake rate is accurately determined with this self-constructable device.     

A survey of the kinetic parameters of class C beta-lactamases. Cephalosporins and other beta-lactam compounds.

Various cephalosporins, cefoxitin, moxalactam, imipenem and aztreonam were studied as substrates of six class C beta-lactamases. Nitrocefin, cephaloridine, cefazolin, cephalothin and cephalexin were good substrates, with kcat. values ranging from 27 to 5000 s-1. Cefuroxime, cefotaxime and cefoxitin exhibited low kcat. values (0.010-1.7 s-1) and low Km values, which suggested a rate-limiting deacylation. Imipenem and aztreonam were even poorer substrates (kcat. 2 x 10(-4)-3 x 10(-2) s-1) and, in the presence of a reporter substrate, behaved as transient inactivators. With moxalactam, biphasic kinetics were observed, indicating a possible rearrangement of the acyl-enzyme.