Isolation and RNA-binding analysis of NAD+-isocitrate dehydrogenases from Kluyveromyces lactis and Schizosaccharomyces pombe

Krebs cycle NAD⁺-isocitrate dehydrogenase (Idh) binds to the 5-UTRs of all mitochondrial mRNAs in Saccharomyces cerevisiae. We hypothesize that this leader-binding activity plays a role in translational regulation, thereby linking mitochondrial biogenesis to the need for respiratory function. Analysis of effects of leader binding on mitochondrial translation is complicated by the involvement of the enzyme in mitochondrial metabolism. We have therefore searched for an Idh altered in RNA binding, but retaining full enzyme activity. Idh from Kluyveromyces lactis and Schizosaccharomyces pombe was partially purified and examined for the ability to bind Cox2 mRNA. Sch. pombe Idh, like the S. cerevisiae enzyme, has high affinity for both its own, K. lactis and S. cerevisiaeCOX2 leaders. In contrast, Idh purified from K. lactis shows only low affinity for all mRNAs tested. To determine what distinguishes K. lactis Idh from S. cerevisiae Idh, genes encoding the two subunits of Idh in K. lactis were cloned and sequenced. Sequence comparison revealed high levels of similarity throughout the proteins, in particular in regions involved in enzyme activity, co-factor and regulator binding. Non-conserved residues between the subunits from the two yeasts are candidates for involvement in the interaction with RNA. Received: 19 January 2000 / 24 March 2000     

Characterization of FMR1 proteins isolated from different tissues

FMR1 protein expression was studied in different tissues. Inhuman, monkey and murine tissues, high molecular mass FMR1 proteins(67–80 kDa) are found, as shown in lymphobiastoid celiiines. The identity of these proteins was confirmed by theirabsence in tissues from patients with the fragile X syndromeand a FMR1 knock-out mouse. An IIe367Asn substitution in theFMR1 protein did not aiter the transiation, processing and localizationof FMR1 proteins in lymphoblastoid cells from a patient carryingthis mutation. All the high molecular mass FMR1 proteins isolatedfrom normal lymphoblastoid cells and cells from the patientwith the IIe367Asn substitution were able to bind RNA. However,the FMR1 proteins of the patient had reduced affinity for RNAbinding at high salt concentrations. In some human, monkey andmurine tissues low molecular mass FMR1 proteins (39–41kDa) were found, which had the same N terminus as the 67–90kDa isoforms, but differ in their C terminus and are thereforemost likely the result of carboxy-terminal proteolytic cleavage.These low molecular mass FMR1 proteins did not bind RNA, incontrast with the high molecular mass FMR1 proteins. The significanceof these low molecular mass proteins remains to be studied.     

On the mechanism of anionic polymerization of maleic anhydride, 2

The triphenyl and tributyl phosphine initiated polymerization of maleic anhydride was investigated. The structure of the macro zwitterions, formed under the given reaction conditions, depended on the initiator employed. If the polymerization was initiated by triphenyl phosphine, the macromolecules contained succinic anhydride units and cyclopentanone derivatives, formed from further rearrangement. If, however, tributyl phosphine was employed as initiator, the polymers were mostly made up of conjugated ketoolefinic units. IR, NMR and mass spectrometric measurements were carried out to determine the structure of the polymers. Existence of the macro zwitterions was proved by high voltage electrophoresis. The semi-conductor properties of the polymers containing conjugated ketoolefinic units are discussed. Spectroscopic investigation of the kinetics of the triphenyl phosphine initiated polymerization of maleic anhydride showed that the initiation reaction of the ylide, formed with the monomer, proceeded about 90 times slower than the propagation reaction due to addition of the monomer to the growing chain-end.     

A-raf oncogene localizes on mouse X chromosome to region some 10–17 centimorgans proximal to hypoxanthine phosphoribosyltransferase gene

The localization of the A-rafcellular oncogene on the mouse X chromosome has been determined using Xbal-restricted DNAs prepared from progeny of an interspecies backcross between the B6.CBA.R1 and the Spe/Pas mouse strains. This localization to the proximal part of the mouse X chromosome has been confirmed by the use of somatic cell hybrids, carrying partially deleted X chromosomes and suggests that the A-raf oncogene localizes to a region lying some 10–17 centimorgans proximal to the hypoxanthine phosphoribosyltransferase (Hprt) gene between the locus DXPas4and the locus DXPas7defined by the cross-reacting human X chromosome-specific probe DXS32 (M2C). This localization on the mouse X chromosome is compatible with the presence of the A-rafoncogene on the short arm of the human X chromosome between the centromere and Xp21.     

New polymer syntheses, 3. Binding of nucleosides to basic polypeptides via isocyanato-isothiocyanates

New aliphatic α-isocyanato-ω-isothiocyanates of various chain lengths were synthesized from ω-isothiocyanatocarboxylic acid chlorides and trimethylsilyl azide. The 5′-OH group of 2′-O,3′-O-isopropylideneuridine and of N⁶-benzoyl-2′-O,3′-O-isopropylideneadenosine was added selectively onto the isocyanate group of aromatic and aliphatic α-isocyanato-ω-isothiocyanates. The isothiocyanate group of the resulting nucleoside derivatives was then reacted with the free amino groups of polylysine, isopolylysine, polyornithine and isopolyornithine. The resulting polypeptides with pending nucleosides were characterized by elemental analyses and 360 MHz ¹H NMR spectra. A new “one-pot-procedure” was developed to protect the primary amino group (N⁶) of 2′-O,3′-O-isopropylideneadenosine.     

The blocking effects of omega-conotoxin on Ca current in bovine chromaffin cells

The effects of omega-conotoxin (omega-CgTX) on voltage-sensitive Ca currents (ICa) were studied in cultured bovine adrenal chromaffin cells. A maximal block of ICa of 40-50% was obtained with omega-CgTX in the microM range, and was independent of the holding potential. The onset of block was both concentration- and time-dependent. In bovine chromaffin cells, Ca channels, both sensitive and insensitive to omega-CgTX, appear to be present.     

Correlation of light and electron micrographs of human metaphase chromosomes after incorporation of BUdR and staining with "33258 Hoechst" and Giemsa.

Stained (Giemsa, "33258 Hoechst"1) and "33258 Hoechst" + Giemsa) and unstained metaphase chromosomes from human peripheral lymphocytes, after two rounds of replication in the presence of 5-bromodeoxyuridine (BUdR), have been prepared for electron microscopy. There is a positive correlation between light and electron micrographs. The same differential contrast on electron micrographs has been obtained whether the preparations have been stained or not. We attribute this differential contrast primarily to the lesser condensation of the bifilarly substituted chromatid.     

Exploring cancer register data to find risk factors for recurrence of breast cancer – application of Canonical Correlation Analysis

Background  

A common approach in exploring register data is to find relationships between outcomes and predictors by using multiple regression analysis (MRA). If there is more than one outcome variable, the analysis must then be repeated, and the results combined in some arbitrary fashion. In contrast, Canonical Correlation Analysis (CCA) has the ability to analyze multiple outcomes at the same time.     

New insights into the pathogenesis of dilated cardiomyopathy: possible underlying autoimmune mechanisms and therapy

In the present study, autoimmune processes involved in the pathogenesis of dilated cardiomyopathy (DCM) are discussed. Genetic predisposition, persistent viral infection, and molecular mimicry have previously been described as the underlying mechanisms of the disease, and prevalence of autoantibodies (AABs) against several intra- and extracellular cardiotropic targets has been confirmed. These autoantibodies are able to disturb the normal physiological activity of the cardiomyocytes. They also could function as mediators in an activated immune system and direct a great deal of attention to injured tissue via (1) complement activation and (2) genesis of circulatory immunocomplexes (CICs) in association with self-antigens. The number as well as duration of accessible autoantigens or CICs seem to play an important role in activation of the antigen-presenting cells (APCs) and, consequently, promotion of autoimmunity. Since AABs play such a decisive role, their exclusion by immunoadsorption (IA) therapy has been discussed as a new approach in DCM treatment. Hitherto, all performed pilot studies using this approach have shown improvement in cardiac function and quality of life in the vast majority of treated DCM patients. The removal of circulating AABs may downregulate the autoimmune system, moderate the inflammatory signals, and hasten the recovery of the affected tissue.