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.     

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.     

Biosynthesis of glycoconjugates in mitochondrial outer membranes

The results reported in this paper show two distinct ways for the incorporation ofN-acetylglucosamine into mitochondrial outer membranes. The first one is the glycosylation of dolichol acceptors, which is indicated by the inhibition of the synthesis of these products by the inhibitors of the dolichol intermediates (tunicamycin and GDP). The second one is the incorporation ofN-acetylglucosamine into protein acceptors directly from UDP-N-acetylglucosamine. This second way of glycosylation is only localized in mitochondria outer membranes.The existence of a direct route forN-glycoprotein biosynthesis has been based on the following evidence. First, the synthesis of theN-acetylglucosaminylated protein acceptors was not inhibited by tunicamycin or GDP. Second, the addition of exogenous dolichol-phosphate did not change the rate of biosynthesis of glycosylated protein material. Third, the sequential incorporation ofN-acetylglucosamine and mannose from their nucleotide derivatives in the presence of GDP and tunicamycin led to the synthesis of glycosylated protein material which entirely bound to Concanavalin A-Sepharose. The oligosaccharide moiety of the glycosylated protein material resulting from the direct transfer of sugars from their nucleotide derivatives to the protein acceptor is of theN-glycan type. On sodium dodecylsulphate polyacrylamide gel electrophoresis, this glycosylated material migrated as a marker protein with a molecular weight between 45 000 and 63 000. HPLC chromatofocusing analysis revealed that the fraction studied was anionic. The oligosaccharide moiety of the glycoprotein material can only be elongated by the incorporation ofN-acetylglucosamine and galactose from their nucleotide derivatives.     

Ultrastructural study of cholecystokinin-like immunoreactivity in endocrine cells of the insect midgut

Summary Electron-microscopic immunocytochemistry for the demonstration of CCK-like material in basal endocrine cells of the midgut of Aeschna cyanea, Locusta migratoria, Carausius morosus and Periplaneta americana was performed by use of the peroxidase-antiperoxidase procedure and the colloidal gold method. Immunoreactive cells appeared scattered among digestive and regenerative cells of the epithelium. Immunoreactivity was specifically detected over round to oval electron-dense granules whose size appeared rather different from species to species. Thus, the average size of 30% (d30) of the largest granules ranged from 312 nm in Periplaneta americana to 159 nm in Carausius morosus with intermediate values in Aeschna cyanea (d30=195 nm) and Locusta migratoria (d30=225 nm).     

Morphometrical analysis of the gap-junctional area in parenchymal cells of the rat liver after administration of dibutyryl cAMP and aminophylline

Summary In view of the presumed involvement of gap junctions in the coordination of metabolic activities, the influence of cAMP as a regulatory signal of cell metabolism on gap junctions of hepatocytes has been examined. Male rats received two intraperitoneal doses of 10 mg dibutyryl cAMP/100 g body weight with a time interval of 2.5 h and were decapitated 2.5 h later. After this 5-h interval, analysis of freeze-fracture replicas of fixed liver tissue revealed an increase in the mean (± SEM) gap-junctional membrane portion on the lateral hepatocyte membranes from 0.049 + 0.003 (n = 66) in controls to 0.061 ± 0.003 (n = 70) in treated rats, while the configuration of the connexons appeared unaltered. This effect could not be reinforced by prior administration of aminophylline: the relative gapjunctional area is similarly extended from 0.054 ± 0.003 (n = 126) in the control group to 0.065 ± 0.004 (n = 105) in the experimental animals. Probing for the time course of the junctional response, a group of rats was sacrificed 3 h after the onset of treatment. Already within this time, the gapjunctional area is augmented from 0.042 ± 0.004 (n = 63) in the concurrent controls to 0.069 ± 0.006 (n = 42) in the treated rats. These statistically significant increases in area may suggest a stimulating effect of cAMP on gap junctions of hepatocytes in vivo.This investigation was supported by grant No. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium)     

Prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda

Summary The prevalence of the 281 (GlyGlu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.