Native Molecular Forms of Head Acetylcholinesterase from Adult Drosophila melanogaster: Quaternary Structure and Hydrophobic Character

The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100, 10-oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt-soluble fraction contained the same molecular forms as the low salt-soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.     

Properties of the 12-O-Tetradecanoylphorbol-13-Acetate-Stimulated S6 Kinase from Rat Astroglial Cells

The S6 kinase activity of astroglial cells in primary culture stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) has been studied. This activity was eluted as a single peak at 0.15 M NaCl from a DEAE-Sephacel column. The chromatography of this peak on phosphocellulose revealed an activity eluted at 0.15 M NaCl. This partially purified enzyme had a sedimentation coefficient of 3.7S; Km values were 2 X 10(-5) M for ATP and 10(-6) M for 40S ribosomal subunits. The optimal Mg2+ concentration requirement was 2-3 mM. Mn2+ and Co2+ could substitute for Mg2+ (optimum concentrations 1.5 and 0.8 mM, respectively), but these cations were strong inhibitors in the presence of Mg2+. The enzyme was inhibited by N-ethylmaleimide, indicating that it contained thiol groups. This S6 kinase used ATP, but not GTP, as a phosphate donor, and exhibited great specificity for S6 as phosphate acceptor. Whole histones and protamine were slightly phosphorylated whereas phosvitin, histone H1, and surprisingly the peptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala were not phosphorylated. The TPA-stimulated S6 kinase resembles the insulin-, fibroblast growth factor- and cyclic AMP-stimulated enzymes, suggesting that several pathways might activate the same entity.     

The complete nucleotide sequence of the colicinogenic plasmid ColA. High extent of homology with ColE1

Summary The complete nucleotide sequence of the colicinogenic plasmid ColA has been determined. The plasmid DNA consists of 6720 bp (molecular weight 4.48×10⁶). Fifteen putative biological functions have been identified using the functional map previously determined. These include 11 genes and 3 DNA sites. Nine genes encode proteins of which 3 have been fully characterized. The replication region of ColA coding for RNAI and RNAII is highly homologous to that of ColE1 andClo DF13. The same holds true for the site-specific recombination region containing palindromic symmetry and involved in stable maintenance of the plasmids. A high percentage of homology has been detected for putative mobility proteins encoded by ColA and ColE1. The exclusion proteins are also highly homologous.     

Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

Genomic organization and nucleotide sequence of a long mosaic repetitive DNA in the mouse genome

A long mosaic repetitive sequence (LMRS) was isolated from a mouse liver genome library using a mouse repetitive DNA as a probe. LMRS exhibits the following features: (1) it is almost 15 kb in length; (2) it is partly organized in tandem array and frequently interrupted by other repeated sequences; and (3) it is located predominantly on the A3 band of the mouse X Chromosome (Chr). One fragment of LMRS (B6) shows restriction fragment length polymorphism (RFLP) between different mouse strains, and is thus potentially useful for mapping studies. The nucleotide sequence confirms a mosaic organization of LMRS which includes three repeats in the 5 part, showing similarity with the 5 end of L1Md-A2, and seven long A+T rich segments in the central part of the element. Our findings suggest that this sequence may have arisen from the duplication of an ancestral motif and has expanded by successive waves of amplification and invasion by foreign sequences.The nucleotide sequence data reported in this paper have been submitted to EMBL and have been assigned the accession number X55036.     

Correspondence of the monocyte antigen HMA-1 to the non-HLA antigen 9a

Monocyte-specific antibodies are detrimental to bone marrow and renal transplantation. By using human antimonocyte sera we were able to identify two monocyte-specific antigens, human monocyte antigen 1 and 2 (HMA-1 and HMA-2). The presence of HMA-1 and HMA-2 was compared with the presence of several non-HLA antigens. In panel and inhibition studies, HMA-1 corresponded to the previously described non-HLA granulocyte antigen 9a. Absorption studies showed that HMA-1 and 9a were both present on granulocytes and monocytes. The clinical relevance of these antigens is discussed.     

Ultrastructural identification of corticotropes of the fetal rat

Corticotropes of rat fetuses aged 16, 18 and 21 days were localized by the indirect antibody-enzyme method on semithin sections of the pituitary. The development of the ultrastructure of these cells was observed on consecutive ultrathin sections. In comparison with previous data our present results show that identification of a fetal cell type cannot be based entirely on morphological criteria. The structural peculiarities of corticotropes obtained from studies in vivo are compared with those observed in cells maintained in vitro.     

Extra terrestrial abiogenic organization of organic matter: The hollow spheres of the Orgueil meteorite

Fragments of the Orgueil meteorite were macerated in mineral acids (HNO₃-HF-HNO₃) to dissolve the mineral matrix and separate the acid-resistant organic residues; a routine procedure in the extraction of pollen and spores from terrestrial sediments. Numerous spherical hollow objects were found, optically resembling the brown amorphous residual organic matrix of the meteorite. Their morphology, size-distribution, and chemical composition, revealed by electron microprobe with reference to carbon and phosphorus, are described, and evaluated in connection with criteria of biogenicity. The intrinsic criteria are satisfactorily met, but the extrinsic requirement of a sedimentary environment is not met. A review of the literature concerning the meteoritic environment suggests an explanation of these spheres based on the environment of their formation. It is proposed that they are organic coatings on olivine microchondrules, magnetite and glass globules, the mineral component of which has been dissolved by the acid maceration. They could have initially resulted from the polymerization of dispersed small organic molecules condensing on the surface of the microchrondrules. The latter were injected from a volcanic nuée ardente into the dispersed cold primordial cosmic dust of hydrated silicates and organic molecules, around the meteorite parent-body. This presumably occurred before the cosmic dust accreted as the carbonaceous chondritic outer layer of the parent-body. Upsurging reducing hot gases from the nuée ardente would polymerize part of the dispersed organic matter as the insoluble brown amorphous matrix, possibly the sticking agent when the cosmic dust accreted. The spiraled form of several of the organic structures described here are suggestive of atmospheric heat microturbulences. Organic membranes and comet-form tails of spherical coatings suggest polymerization in the wake of injected microchondrules. These diverse organic structures would result in our view from the abiogenic thermal organization of organic matter in an extraterrestrial gas-solid system.

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Résumé Plusieurs fragments de la météorite d'Orgueil ont été macérés dans des acides minéraux (HNO₃, HF, HNO₃), afin de dissoudre la fraction minérale et isoler la fraction résiduelle résistant aux acides. C'est là un procédé utilisé couramment en palynologie pour extraire les grains de pollen et les spores des sédiments terrestres.De nombreux objets microscopiques, sphériques et creux, ont été mis en évidence. Ils sont optiquement similaires au résidu organique brun, amorphe, dans lequel ils sont enrobés. Leur morphologie, leur répartition en fonction de leur taille, et leur composition chimique élémentaire, analysée par la microsonde électronique, qui révèle la présence de carbone et de phosphore, sont décrites, puis évaluées en fonction des critères disponibles d'une éventuelle origine biologique. Les critères intrinsèques aux objets sont bien satisfaits, mais non le critère extrinsèque d'un environnement sédimentaire convenable.L'analyse des hypothèses qui ont été avancées pour décrire l'environnement originel de la météorite, permet de suggérer une explication de ces sphères creuses organiques, qui repose entièrement sur cet environnement à l'époque de leur formation. Ce sont des revêtements organiques à la surface de microchondrules d'olivine, de globules de verre et de magnétite, minéraux de haute température appartenant à la fraction minérale de la météorite qui a été dissoute par la macération acide.Ces coques organiques résulteraient de la polymérisation de petites molécules organiques dispersées, qui se seraient condensées à la surface de gouttelettes minérales en fusion. Ces dernières ont pu être éjectées par une nuée ardente volcanique issue du corps parent de la météorite, et projetées dans la poussière cosmique primitive froide en suspension autour de ce corps parent, composée de silicates hydratés et de petites molécules organiques.C'est ensuite seulement que cette suspension de poussière primitive aurait subi l'accrétion pour former finalement la couche extérieure froide de matière étéoritique carbonée du corps parent. En outre, des gaz réducteurs à haute température, s'élevant de la nuée ardente, ont pu polymériser en partie la matière organique en suspension, pour former la matière météoritique organique amorphe, résistant aux acides, qui a peut-être été l'agent agglomérant lors de l'accrétion.Les formes spiralées de plusieurs des structures organiques décrites ici suggèrent des microturbulences atmosphériques dûes à la chaleur. Des membranes organiques, et l'appendice en forme de queue de comète d'une sphérule, suggèrent une polymérisation organique dans le sillage de la trajectoire de microchondrules. Selon notre opinion, ces diverses structures organiques résultent donc de l'organisation abiogénique sous l'effet de la température, de matière organique préexistante, plus simple, dans un système solide-gaz extraterrestre.

     

Characterization of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and-insensitive (IisCaP) nonmitochondrial Ca2+ pools in rat pancreatic acinar cells

Summary We have measured Ca²⁺ uptake and Ca²⁺ release in isolated permeabilized pancreatic acinar cells and in isolated membrane vesicles of endoplasmic reticulum prepared from these cells. Ca²⁺ uptake into cells was monitored with a Ca²⁺ electrode, whereas Ca²⁺ uptake into membrane vesicles was measured with⁴⁵Ca²⁺. Using inhibitors of known action, such as the H⁺ ATPase inhibitors NBD-Cl and NEM, the Ca²⁺ ATPase inhibitor vanadate as well as the second messenger inositol 1,4,5-trisphosphate (IP₃) and its analog inositol 1,4,5-trisphosphorothioate (IPS₃), we could functionally differentiate two non-mitochondrial Ca²⁺ pools. Ca²⁺ uptake into the IP₃-sensitive Ca²⁺ pool (IsCaP) occurs by a MgATP-dependent Ca²⁺ uptake mechanism that exchanges Ca²⁺ for H⁺ ions. In the absence of ATP Ca²⁺ uptake can occur to some extent at the expense of an H⁺ gradient that is established by a vacuolar-type MgATP-dependent H⁺ pump present in the same organelle. The other Ca²⁺ pool takes up Ca²⁺ by a vanadate-sensitive Ca²⁺ ATPase and is insensitive to IP₃ (IisCaP). The IsCaP is filled at higher Ca²⁺ concentrations (10^–6 mol/liter) which may occur during stimulation. The low steady-state [Ca²⁺] of 10^–7 mol/liter is adjusted by the IisCaP.It is speculated that both Ca²⁺ pools can communicate with each other, the possible mechanism of which, however, is at present unknown.     

Ferritin H gene polymorphism in idiopathic hemochromatosis

Summary We have analysed karyotypes and DNA from three patients with aniridia (congenital absence of irises) and Wilms' tumour. All three had constitutional deletions from the short arm of chromosome 11. The minimum region of overlap of the deletion involves a small region of band 11p13 presumed to contain the genetic loci responsible for both phenotypic abnormalities. Using cells from these patients, somatic cell hybrids with transformed mouse cells have been prepared. Individual subclones retaining either the deletion-11 chromosome or the normal chromosome 11, in addition to a variety of other human chromosomes, have been identified. The relative position of these breakpoints have been determined and the panel of hybrids has been used to map randomly-isolated 11p13 DNA sequences. The characterisation of these deletions has provided a useful panel of hybrids for random mapping strategies designed to identify the Wilms' and aniridia genes.