Cystic fibrosis typing with DNA probes: experience of a screening laboratory

Summary A sample of 125 individuals from 37 British cystic fibrosis (CF) families with at least one living affected child were typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene. These probes were MetD, MetH, pJ3.11 and 7C22. Using this combination of probes, 30 out of the 37 families were sufficiently informative to enable prenatal diagnosis of the disease. Linkage analysis has also proved to be useful in excluding CF in two cases where diagnosis of the disease was equivocal in the sibling of an affected child.     

Pericentric inversion of chromosome 19 in three families

Summary Pericentric inversion of chromosome 19 has been found in several members of three unrelated families from a restricted geographical region. In one of the families, an additional pericentric inversion of chromosome 9 was observed. Reproductive problems, multiple abortions in two families and a neonatal death in the third, were present. A review of previously described cases is included, and the genetic risk connected with this type of rearrangement is also discussed.     

Effect of culture media and growth phase on the morphology of lactobacilli and on their ability to adhere to epithelial cells

Previous studies have demonstrated that the ability of lactobacilli to attach to and colonize uroepithelial surfaces is an important characteristic that enhances interference against uropathogenic bacteria. This adherence capacity was found to vary amongst lactobacillus strains and with the type of growth medium used to culture the organisms. The present study was undertaken to examine further the effect of culture media and growth phase on lactobacillus adherence to uroepithelial cells in vitro. In addition, a freeze substitution technique was developed to examine the morphology of strainsLactobacillus casei ssrhamnosus RC-17,L. casei GR-1, andL. acidophilus T-13 in relation to growth conditions and adhesion. A growth curve was plotted for strain GR-1, and adherence was found to be lowest for bacteria in early log phase (39 bacteria per uroepithelial cell) and highest in stationary phase (59 bacteria per uroepithelial cell). Strains RC-17 and GR-1 attached in high numbers to uroepithelial cells, whereas T-13 was poorly adherent. The latter formed a long, relatively dense, fibrous capsule after growth in brain heart infusion yeast extract agar, unlike strains GR-1 and RC-17, which formed a short, tightly bound, electron-dense capsule which surrounded the cells in a radial fashion. Growth of RC-17 in batch cultures of human urine, with and without addition of carbohydrates, resulted in formation of an irregular, fibrous extracellular matrix. These experiments illustrate that growth phase and culture conditions affect the extracellular structure of lactobacilli and also affect the adherence capacity of these bacteria. Structural changes mediated by availability of nutrients may partly explain why lactobacilli vary between species and between hosts in their colonization of the urogenital tract.     

Antigenic determinants expressed by human C4 allotypes; a study of 325 families provides evidence for the structural antigenic model

The antigenic determinants of human C4 have been defined by human IgG antisera, Rodgers (Rg) and Chido (Ch), in hemagglutination-inhibition assays (HAI). Eight (2 Rg and 6 Ch) are of high frequency, > 90% , and 1, WH, is of low frequency, 15 %. The phenotypic combinations are complex; generally, C4A expresses Rg, and C4B has Ch, but reverse antigenicities have been established both by HAI and by sequence data of selected C4 allotypes. A study of 325 families provides data on the antigenic expression of each C4 allotype and demonstrates strong associations. A structural model for the antigenic determinants of C4 proteins has been proposed and is completely supported by the family material. Of the 16 possible antigenic combinations for C4 proteins, only 3 are undetected. A new Ch combination has been recorded in two French families. The reported sequence variation within the C4d region can account for the antigenic determinants but leaves the location of electrophoretic variation in C4 still unclear.     

Synthesis of eicosanoids by gamma-interferon-differentiated U937 cells

Interferons induce morphological, biochemical and functional alterations in monocyte macrophage and myeloid cell lines. We studied the effect of 3 days incubation with gamma-interferon from human buffy coats on the global synthesis of arachidonic acid metabolites by U937 cells. Interferon-induced morphologic changes including cytoplasmic and nuclear changes and the appearance of multiple lysosomal-like granules consistent with cellular differentiation were observed by electron microscopy. The labeling of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine was increased and that of phosphatidylinositol, free fatty acids as 3H-arachidonic acid and neutral lipids reduced, when interferon-treated cells were incubated with 3H-arachidonic acid. Interferon caused qualitative and quantitative changes in the synthesis of cyclooxygenase and lipoxygenase products. A23187, a calcium ionophore, and the tumor promotor, phorbol myristate acetate, greatly increased the synthesis by interferon-differentiated cells of 2 cyclooxygenase products; synthesis of lipoxygenase products was reduced. In the presence of indomethacin, 'shunting' into putative lipoxygenase products occurred. The relationship between interferon-induced morphologic and functional changes, the development of altered phospholipid and eicosanoid metabolism and the identity of these metabolites are yet to be established.     

Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro.

Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli. The protein was soluble and could be purified by a simple scheme. It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites. The products of processing P1 were 1ABC and 1D (VP1). The activity was insensitive to Triton X-100. Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VP0), 1C (VP3), and 1D in the same assay system but were sensitive to detergent. 3C from cell extracts that was separated from its precursors resembled the recombinant proteinase in its activity. Recombinant 3C cleaved the peptide dansyl-Glu-Glu-Glu-Ala-Met-Glu-Gln-Gly-Ile-Thr-Asn-Lys-NH2 at the Gln-Gly bond. We conclude that 3C is merely the core of the Gln-Gly-cleaving activity which processes P1 in vivo and that there is probably a hydrophobic contact between a larger 3C precursor and its P1 substrate which allows the second processing reaction: 1ABC, 1D----1AB, 1C, 1D.     

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.     

Bleomycin and X-ray-hypersensitive Chinese hamster ovary cell mutants: genetic analysis and cross-resistance to neocarzinostatin

We have previously reported the isolation of 3 mutants of Chinese hamster ovary cells which exhibit hypersensitivity to bleomycin. 2 mutants were isolated on the basis of bleomycin-sensitivity [designated BLM-1 and BLM-2, Robson et al., Cancer Res., 45 (1985) 5304-5309] and 1 as adriamycin-sensitive [ADR-1, Robson et al., Cancer Res., 47 (1987) 1560-1565]. Because bleomycin generates DNA-strand breaks via a free-radical mechanism, we have studied the survival response of these mutants to a range of drugs which also generate free radicals and consequently DNA-strand breaks. The mutants are all hypersensitive to phleomycin, which differs from bleomycin in being unable to intercalate due to a modified bithiazole moiety. However, BLM-2 cells alone are hypersensitive to pepleomycin, a semi-synthetic bleomycin analogue. In contrast, BLM-1 cells are more sensitive than BLM-2 to streptonigrin (which operates via a hydroquinone intermediate). ADR-1 cells show wild-type resistance to streptonigrin. The results obtained with neocarzinostatin, an antibiotic requiring thiol activation, are unusual in that both BLM-1 and BLM-2 are approximately 3-fold more resistant than parental cells. However, the steady-state intracellular level of the major non-protein thiol, glutathione, is not altered in BLM-1 or BLM-2 cells. ADR-1 cells show essentially wild-type resistance to neocarzinostatin. Analysis of cell hybrids shows that BLM-1 and BLM-2 cells are phenotypically recessive in combination with parental CHO-K1 cells and represent different genetic complementation groups not only from one another, but also from the bleomycin-sensitive mutant xrs-6, isolated on the basis of X-ray sensitivity by Jeggo and Kemp [Mutation Res., 112 (1983) 313-319]. These results indicate that at least 3 gene products are involved in cellular protection against bleomycin toxicity in mammalian cells.     

Atrial granules contain an amino-terminal processing enzyme of atrial natriuretic factor

At least three enzymes have been identified in atrial tissue homogenates that are capable of processing pro-atrial natriuretic factor to active atrial peptides. The atrial peptides possess potent natriuretic, diuretic, vasorelaxant, and hemodynamic properties, and their existence has implicated the mammalian heart as an endocrine organ. We have purified and characterized a serine proteinase (Mr approximately equal to 70,000) associated with atrial granules that preferentially hydrolyzes the Arg-Ser bond in the synthetic substrates Gly-Pro-Arg-Ser-Leu-Arg, benzoyl-Gly-Pro-Arg-Ser-Leu-Arg, and benzoyl-Gly-Pro-Arg-Ser-Leu-Arg-Arg-2-naphthylamide, the Arg-2-naphthylamide bond in the substrate benzoyl-Gly-Pro-Arg-2-naphthylamide, and the Arg-Ser bond in a 31-residue substrate (Gly96-Tyr126 peptide) corresponding to residues Arg98-Ser99 in pro-atrial natriuretic factor. The Gly96-Tyr126 peptide contains the putative processing site in pro-atrial natriuretic factor and the sequence for the bioactive peptides. Our results indicate that the minimum processing site sequence is -Gly-Pro-Arg-Ser-Leu-Arg-Arg- and that the Ser99-Tyr126 natriuretic peptide is the predominant hydrolytic product. After prolonged incubation or at high enzyme concentrations, the Ser103-Tyr126 natriuretic peptide may also be formed. The Ser103-Arg125 natriuretic peptide was only a very minor product. The doublet of basic amino acids is not the primary processing site in pro-atrial natriuretic factor, but their presence may influence cleavage at the single Arg residue "upstream." Our findings are consistent with the idea that the pro-protein and the processing enzymes are packaged into the secretory granule and in response to the proper stimulus, the pro-protein is processed to the active peptides, probably during the process of secretion. The processing pathway of pro-atrial natriuretic factor is discussed.