Isolation of high molecular weight DNA from wholeEuglena cells

Summary A method for isolating high quality DNA from wholeEuglena cells is described. The procedure consists in: the weakening of the cell pellicle in glycerol avoiding the mechanical disruption of cells and shearing damage in DNA molecules; the decondensation ofEuglena compact chromatin directly inside the cells; the complete dissociation of cells and nucleoproteins in sarkosyl detergent; the optional digestion of proteins and RNA with DNase-free enzymes and the final purification of DNA by isopycnic banding in CsCl gradients. Degradation of DNA is prevented all along the extraction procedure by glycerol, antioxydants, EDTA and sarkosyl detergent. Using the enzymatic digestion step, DNA containing few single-stranded nicks is obtained with a yield approaching 100%. DNA with no single-stranded nick could be obtained with a 35% yield when the enzymatic digestion step was omitted. In both cases, the double-stranded DNA has an average molecular weight equal or greater than 6×10⁷. It is free of contaminants and could be easily digested with restriction enzymes. After digestion with Eco RI and size-fractionation in agarose gel this DNA has permitted specific hybridization of the rDNA sequences with a radioactive rRNA probe.Abbreviations Kbp kilobasepairs - Kb kilobases     

Biosynthesis of endothelium-derived relaxing factor: a cytosolic enzyme in porcine aortic endothelial cells Ca2+-dependently converts L-arginine into an activator of soluble guanylyl cyclase

In the presence of porcine aortic endothelial cytosol, soluble guanylyl cyclase purified from bovine lung was activated by L-arginine up to 2.5-fold, with an EC50 of about 6 microM. This activation was dependent on NADPH and Ca2+. The EC50 for Ca2+ was about 60 nM. No effect of L-arginine on guanylyl cyclase was observed when the cytosolic proteins were heat-denaturated. The effect of L-arginine was inhibited by NG-monomethyl-L-arginine and hemoglobin. These results indicate that endothelial cells contain a cytosolic enzyme which is directly or indirectly regulated by Ca2+ and converts L-arginine into a compound which in stimulating soluble guanylyl cyclase behaves similar to endothelium-derived relaxing factor.     

Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase, located in mitochondiral outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.     

SIMS microscopy in the biomedical field.

We attempted to indicate the requirements for biomedical applications of SIMS microscopy. Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue. Furthermore, it is often necessary to correlate ionic and light microscope images. This implies a common methodological approach to sample preparation for both microscopes. The use of low or high mass resolution depends on the elements studied and their concentrations. To improve the acquisition and processing of images, digital imaging systems have to be designed and require both ionic and optical image superimposition. However, the images do not accurately reflect element concentration; a relative quantitative approach is possible by measuring secondary ion beam intensity. Using an internal reference element (carbon) and standard curves the results are expressed in micrograms/mg of tissue. Despite their limited lateral resolution (0.5 microns) the actual SIMS microscopes are very suitable for the resolution of biomedical problems posed by action modes and drug localization in human pathology. SIMS microscopy should provide a new tool for metabolic radiotherapy by facilitating dose evaluation. The advent of high lateral resolution SIMS imaging (less than 0.1 microns) should open up new fields in biomedical investigation.     

Scanning electron microscopic study of the oesophageal mucous layer in the eel, Anguilla anguilla L.

The structure of the mucous layer covering the oesophageal epithelium was analysed by scanning electron microscopy in the eel, Anguilla anguilla . Different fixation procedures were used to conserve the mucus in situ. The mucous layer changes progressively down the oesophagus from a thick dense layer to a very thin fibrous network. The possible roles of these mucous structures in ion absorption are discussed.     

Abnormal electrophoretic mobility of spectrin tetramers in hereditary elliptocytosis

Summary Hereditary elliptocytosis (HE) is a genetically determined disorder of the red cell membrane. The main protein which composes the proteinaceous skeleton of the membrane is an elongated molecule named spectrin which is a heterodimer composed of two chains, and . In the membrane spectrin dimers are associated head-to-head to form tetrameric structures. We and other authors have reported that spectrin studied from many HE patients exhibited a dimer self-association defect (type I HE). A mutation in the head of the spectrin chain was mostly found in type I HE. We have previously described one of the three known spectrin pathological variants shown on mild tryptic digest pattern. This variant was characterized by the appearance of an abnormal 65,000-dalton peptide (Sp ^I/65). Using nondenaturating gel electrophoresis, we describe in this paper a triplicated pattern of the spectrin tetramer bands which is found in heterozygous HE cases displaying the 65,000-dalton variant. Study of a homozygous case allowed us to characterize the electrophoretic mobility of the abnormal symmetrical spectrin tetramer (₂ ^I/65-₂) and to study the correlation between the fraction of this abnormal symmetrical tetramer found in heterozygous patients and the amount of the 65,000-dalton peptide observed in spectrin tryptic digests.     

Localisation,structure et genèse des concrétions minérales dans le mésentéron des Collemboles Tomoceridae (Insecta,Collembola)

Résumé Le mésentéron des Collemboles est riche en inclusions minérales appelées sphérites. L'étude de leur genèse nous montre qu'ils se forment dans les citernes du réticulum endoplasmique. Leur croissance et leur évolution se poursuivent dans les mêmes citernes par apport de matériel divers. Les sphérites sont formés de nucléi opaques aux électrons entourés de couches concentriques alternativement claires et sombres. Les critaux âgés sont souvent associés à des enroulements membranaires. Le rôle physiologique est discuté. Ces concrétions apparaissant dès l'éclosion, peuvent s'éliminer de differentes manières: par extrusion apocrine, par dégénérescence cellulaire et surtout par le renouvellement périodique de l'épithélium intestinal qui se fait à chaque mue. Ces sphérites peuvent représenter une voie d'excrétion par accumulation et semblent jouer le rôle des tubes de Malpighi qui font défaut chez les Collemboles.

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Summary The mesenteron of Collembola is rich in mineral inclusions called spherites. The study of their genesis shows us that they are produced in the cisternae of the endoplasmic reticulum. Their growing and evolution occur in the same cisternae by adduction of diverse material. The spherites are composed of dark nuclei surrounded by alternatively clear and dark concentric strata. The aged cristals are often connected with membraneous windings. The physiological role is discussed. These concretions which appear already after hatching, cancel out by apocrine extrusion, by cellular degeneration and especially by periodic renovation of the intestinal epithelium which occurs at each moulting. These spherites could reflect a process of excretion by accumulation and seem to have the same part as the Malpighian tubules which lack in Collembola.

     

Defective gamma subunit of ATP synthase (F1F0) from Escherichia coli leads to resistance to aminoglycoside antibiotics.

A strain of Escherichia coli which was derived from a gentamicin-resistant clinical isolate was found to be cross-resistant to neomycin and streptomycin. The molecular nature of the genetic defect was found to be an insertion of two GC base pairs in the uncG gene of the mutant. The insertion led to the production of a truncated gamma subunit of 247 amino acids in length instead of the 286 amino acids that are present in the normal gamma subunit. A plasmid which carried the ATP synthase genes from the mutant produced resistance to aminoglycoside antibiotics when it was introduced into a strain with a chromosomal deletion of the ATP synthase genes. Removal of the genes coding for the beta and epsilon subunits abolished antibiotic resistance coded by the mutant plasmid. The relationship between antibiotic resistance and the gamma subunit was investigated by testing the antibiotic resistance of plasmids carrying various combinations of unc genes. The presence of genes for the F0 portion of the ATP synthase in the presence or absence of genes for the gamma subunit was not sufficient to cause antibiotic resistance. alpha, beta, and truncated gamma subunits were detected on washed membranes of the mutant by immunoblotting. The first 247 amino acid residues of the gamma subunit may be sufficient to allow its association with other F1 subunits in such a way that the proton gate of F0 is held open by the mutant F1.     

Mouse B lymphocyte specific endocytosis and recycling of MHC class II molecules

In B lymphocytes, the processing of exogenous proteins and the subsequent binding of antigenic peptides to class II molecules encoded by the major histocompatibility complex (MHC) occurs most likely within endocytic compartments. To examine the endocytic transport of MHC class II molecules, we used (i) surface iodination followed by internalization, pronase treatment and immunoprecipitation, (ii) in situ iodination of endosomal compartments, and (iii) confocal microscopy to visualize the fate of fluorescence coupled Fab fragments. In murine I-Ak, I-Ek positive B lymphoma cells, cell surface MHC class II molecules are partially protected from pronase digestion after 15 min at 37 degrees C and recycled back to the cell surface within the next 30 min. The fluorescence coupled Fab fragments are delivered to juxtanuclear endocytic compartments in 15 min. In contrast to the murine B cells, L fibroblasts transfected with either I-A alpha beta k or I-E alpha k beta k,d fail to internalize their surface class II molecules. A fraction of class II molecules, however, is still present in endosomal compartments as detected after in situ iodination in L fibroblasts. We conclude that the recipient L fibroblasts lack one or several factors needed for the transport of MHC class II molecules from the cell surface to the endosomes. We suggest that in murine B lymphoma cells, antigenic peptides can gain access to a pool of recycling class II molecules whereas in L cells they meet newly synthesized class II molecules targeted to the endosomal compartments.