Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.     

ADP-ribosylation of actin from the green algaChara corallina byClostridium botulinum C2 toxin andClostridium perfringens iota toxin

Summary The ability of two bacterial toxins to modify a plant actin by covalent ADP-ribosylation was tested in the green algaChara corallina. Using [³²P]NAD, bothClostridium botulinum C2 toxin andClostridium perfringens iota toxin labelled a protein of M_r 42 kDa which comigrated with actin and was immunoprecipitated by a monoclonal anti-actin antibody. ADP-ribosylation ofChara actin was more efficient with iota toxin than with C2 toxin. The actin bundles in perfusedChara cells were not affected by toxin-containing media competent for ADP-ribosylation. The data indicate that monomeric plant actin is substrate for ADP-ribosylation by the bacterial toxins.Abbreviations ADP adenosine-diphosphate - EGTA ethyleneglycol-bis-(-aminoethyl)N,N,N,N-tetraacetic acid - NAD nicotinamide dinucleotide - pCA -log [Ca²⁺] - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Immunocytochemical localization of Na+/K+-ATPase and V-H+-ATPase in the salivary glands of the cockroach,Periplaneta americana

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular, intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na⁺/K⁺-ATPase (sodium pump) and a vacuolar-type H⁺-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na⁺/K⁺-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H⁺-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electrondense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na⁺/K⁺-ATPase in the peripheral cells is probably directly involved in the formation of the Na⁺-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H⁺-ATPase.     

The F3 Neuronal Glycosylphosphatidylinositol-Linked Molecule Is Localized to Glycolipid-Enriched Membrane Subdomains and Interacts with L1 and Fyn Kinase in Cerebellum

Abstract: The F3 molecule is a member of the immunoglobulin superfamily anchored to plasma membranes by a glycosylphosphatidylinositol group. In adult mouse cerebellum, F3 is predominantly expressed on a subset of axons, the parallel fibers, and at their synapses. In vitro studies established that it is a plurifunctional molecule that, depending on the cellular context and the ligand with which it interacts, either mediates repulsive interactions or promotes neurite outgrowth. In the present study, we report the isolation of two fractions of F3-containing microdomains from adult cerebellum on the basis of their resistance to solubilization by Triton X-100 at 4°C. Both fractions were composed of vesicles, ranging from 100 to 200 nm in diameter. Lipid composition analysis indicated that the lighter fraction was enriched in cerebrosides and sulfatides. F3 sensitivity to phosphatidylinositol phospholipase C differed between the two fractions, possibly reflecting structural differences in the lipid anchor of the F3 molecule. Both fractions were highly enriched in other glycosylphosphatidylinositol-anchored proteins such as NCAM 120 and Thy-1. It is interesting that these vesicles were devoid of the transmembrane forms (NCAM 180 and NCAM 140), which were recovered in Triton X-100-soluble fractions, but contained the L1 transmembrane adhesion molecule that is coexpressed with F3 on parallel fibers and the fyn tyrosine kinase. Immunoprecipitation experiments indicated that F3, but not NCAM 120 or Thy-1, was physically associated in a complex with both L1 and fyn tyrosine kinase. This strongly suggests that the interaction between L1 and F3, already described to occur with isolated molecules, is present in neural tissue. More important is that our study provides information on the molecular machinery likely to be involved in F3 signaling.     

Structural basis for the RNA binding selectivity of oligonucleotide analogues containing alkylsulfide internucleoside linkages and 2'-substituted 3'-deoxyribonucleosides.

In this report we describe the synthesis of oligonucleotides containing sulfide-linked dinucleoside units, namely rT(2'OH)sdT, rT(2'OMe)sdT, dTsrU(2'OMe) and dT(2'OMe)srU(2'OMe). We also describe the interactions of such oligomers with complementary DNA and RNA targets, and provide the structural basis for their remarkable RNA binding selectivity. In all cases, the Tm values of the S/P-chimera duplexes were lower than those of the corresponding unmodified duplexes. We attribute this to steric interactions between the 5'sulfur and the atoms of the nearby base/sugar residues. The 2'-substituents (i.e., 2'OH or 2'OMe) vicinal to the alkylsulfide internucleoside linkage significantly perturb the structure and stability of the duplexes formed with DNA, and more so than with RNA. The introduction of three rT(2'OH)sdTp (or rT(2'OMe)sdTp) units into an oligodeoxynucleotide sequence was sufficient to abolish binding to complementary DNA but not RNA. The same three substitutions with dTsrU(2'OMe)p and dT(2'OMe)srU(2'OMe)p did not abolish binding to DNA but the resulting complexes had poor thermal stability. The RNA-binding 'selectivity' exhibited by these oligomers is attributed to the tendency of the 2'-substituted (branched) furanoses to adopt the C3'-endo pucker, a conformation that is inconsistent with the B-form structure of helical DNA. The preference of these sugars to exist often exclusively in the C3'-endo form is attributed to stereoelectronic effects, namely gauche and anomeric effects. Our findings support the hypothesis that nucleoside analogues puckered exclusively in the C3'-endo form may result in them being especially good binders of targeted mRNA [S.H. Kawai (1991), Ph.D. Thesis, McGill University; Kawasaki et al. (1993) J. Med. Chem. 36, 831-841].     

Separation of macromolecules in isotachophoresis systems involving single or multiple counterions

The isotachcophoresis principle provides unique opportunities for rational designs of fractionation procedures involving charged molecules. Theoretically any two charged molecules that are soluble under the experimental conditions involved can be physically separated if their electrophoretic net mobilities differ only slightly in the electrophoresis medium used. A theoretical and practical outline is presented that enables the reader to set up this fractionation system and on a rational basis develop fractionation procedures for a given set of charged macromolecules by isotachophoresis with simple and well characterized ampholytes as spacer substances. The planning of preparative experiments in this approach is based on results obtained from rapid analytical screens on a microgram scale. The report includes an appendix containing the theoretical basis for computation of buffer compositions in the isotachophoretic steady state with mono/polyvalent constituents in systems involving one or more counterions and controlled amounts of interferiong ions.     

Lack of ornithine decarboxylase activity in isolated rat liver parenchymal cells

Polyamines are associated with fundamental metabolic and functional steps in cell metabolism. The activity of ornithine decarboxylase, the key enzyme in polyamine metabolism, was followed during the preparation of rat liver parenchymal cells and in the isolated cells during incubation. In experiments in which ornithine decarboxylase was not induced in vivo, enzyme activity dropped to barely measurable values during the preparation. An even more drastic loss of enzyme activity was noted in livers in which ornithine decarboxylase activity was stimulated in vivo 20-40fold by previous injection of bovine growth hormone, or thioacetamide or elevated because of circadian rhythmical changes of the enzyme activity. Within the first 20 min of liver perfusion to disintegrate the tissue, ornithine decarboxylase activity decreased by up to 80%. The presence of bovine growth hormone during cell preparation cannot prevent the loss of enzyme activity. Incubation of the isolated cells for periods of up to 240 min did not restore the enzyme activity. Furthermore, incubation of the cells with bovine growth hormone did not induce ornithine decarboxylase, even though the medium was supplemented with amino acids in physiological concentrations. During normal liver perfusion and in contrast to the situation with isolated cells, there is no loss of enzyme activity but a small rise. Following pretreatment of the animals with bovine growth hormone or thioacetamide the highly stimulated activity of ornithine decarboxylase declined slowly during liver perfusion, but never dropped to values lower than normal for perfusion periods of up to 240 min. Moreover, in the intact perfused organ ornithine decarboxylase remains responsive to bovine growth hormone. The experiments demonstrate that enzymatic tissue dispersion by collagenase in particular or the preparation of isolated cells in general drastically alters the metabolic and functional state of rat liver parenchymal cells.     

Autoregulatory control of actin synthesis in cultured rat hepatocytes.

ADP-ribosylation of actin by Clostridium botulinum C2 toxin resulted in a depolymerization of filamentous F-actin and an increase of monomeric G-actin in cultured hepatocytes. Simultaneously the de novo synthesis of actin was largely reduced, while the synthesis of albumin and of other proteins was not significantly impaired. The specific decrease of actin mRNA to 30% of the control indicates a down-regulation of actin synthesis at a pretranslational level. On the other hand, treatment with the mycotoxin phalloidin resulted in an increase of F-actin and a decrease of monomeric G-actin. Under this condition the de novo synthesis of actin was specifically enhanced and the level of actin mRNA was increased to 600% of the control. The data suggest an autoregulatory control of the actin synthesis.     

Characterization of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin

Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin belong to a novel family of actin ADP-ribosylating toxins. ADP-ribosylation of actin inhibits actin polymerization and G-actin-associated ATPase activity. The ADP-form of actin is ADP-ribosylated at a higher rate than actin with bound ATP. ADP-ribosylation of actin is reversible, a reaction, which is accompanied by reconstitution of actin ATPase activity.