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T Wolff

Publications and source records attributed to T Wolff.

At least 73 records · Page 4Linked to original sources
PubMed1994

Splicing function of mammalian U6 small nuclear RNA: conserved positions in central domain and helix I are essential during the first and second step of pre-mRNA splicing.

On the basis of mutational analyses in yeast, the highly conserved ACAGAGA sequence of U6 small nuclear RNA (snRNA) and the adjacent U6-U2 helix I have been proposed to be part of the active center of the spliceosome. We report here a detailed analysis of the human U6 snRNA sequence requirements during the first and second step of splicing, using a mammalian in vitro splicing-complementation system and a mutational approach. Positions A53G54C55 (helix Ib) were identified as important specifically for the first step, but not for spliceosome assembly. A45 of the ACAGAGA sequence and U52 of helix Ia function during the second step; in addition, the bulge separating helices Ia and Ib appears critical for the second step. In contrast, no splicing-essential sequences could be identified in the central domain upstream of the ACAGAGA sequence. In sum, our data demonstrate for the mammalian splicing system that discrete positions within the ACAGAGA sequence and helix I of U6 snRNA function during the first and second step of splicing, suggesting that these two sequence elements are closely associated with the catalytic center of the spliceosome. Comparison with previous results in yeast indicates a fundamental conservation of the U6 snRNA function in the pre-mRNA splicing mechanism.

Base Sequence↗
PubMed1994

Regional anaesthesia and subsequent long-term pain.

The incidence of long-term pain (between 1 and 48 weeks and at 2 year follow-up) unrelated to the surgical site following either regional brachial plexus or general anaesthesia was determined. In 834 patients with regional anaesthesia, the incidence (11.1%) was significantly higher than in the 86 patients with general anaesthesia (3.6%; P = 0.03). The incidence of pain was not significantly different among four common techniques of positioning the needle tip in the axillary sheath (9.9 to 11.1%). Parascalene blocks had a slightly but not significantly higher rate (16.3%). A regional re-block was not associated with a higher incidence when compared to those blocked only once. A more distal local re-block was associated with a higher incidence of pain (23%). 2 years post-operatively, 0.5% of patients had pain related to the regional block. A significant proportion of patients developed some long-lasting post-operative pain following regional brachial plexus anaesthesia, although ultimate morbidity was minimal.

Anesthesia, Conduction↗
PubMed1994

Expression of baculovirus P35 prevents cell death in Drosophila.

The baculovirus P35 protein functions to prevent apoptotic death of infected cells. We have expressed P35 in the developing embryo and eye of the fly Drosophila melanogaster. P35 eliminates most, if not all, normally occurring cell death in these tissues, as well as X-irradiation-induced death. Excess pupal eye cells that are normally eliminated by apoptosis develop into pigment cells when their death is prevented by P35 expression. Our results suggest that one mechanism by which viruses prevent the death of the host cell is to block a cell death pathway that mediates normally occurring cell death. Identification of molecules that interact biochemically or genetically with P35 in Drosophila should provide important insights into how cell death is regulated.

Animals↗
PubMed1993

The TGF beta homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophila retina.

Development of the Drosophila retina occurs asynchronously; differentiation, its front marked by the morphogenetic furrow, progresses across the eye disc epithelium over a 2 day period. We have investigated the mechanism by which this front advances, and our results suggest that developing retinal cells drive the progression of morphogenesis utilizing the products of the hedgehog (hh) and decapentaplegic (dpp) genes. Analysis of hh and dpp genetic mosaics indicates that the products of these genes act as diffusible signals in this process. Expression of dpp in the morphogenetic furrow is closely correlated with the progression of the furrow under a variety of conditions. We show that hh, synthesized by differentiating cells, induces the expression of dpp, which appears to be a primary mediator of furrow movement.

Animals↗
PubMed1993

Development of a pharmacophore for inhibition of human liver cytochrome P-450 2D6: molecular modeling and inhibition studies.

To gain insight into the specificity of cytochrome P-450 2D6 toward inhibitors, a preliminary pharmacophore model was built up using strong competitive inhibitors. Ajmalicine (1), the strongest inhibitor known (Ki = 3 nM) was selected as template because of its rigid structure. The preliminary pharmacophore model was validated by performing inhibition studies with derivatives of ajmalicine (1) and quinidine (9). Bufuralol (18) was chosen as substrate and the metabolite 1'-hydroxybufuralol (19) was separated by high performance liquid chromatography. All incubations were carried out using human liver microsomes after demonstration that the Ki values obtained with microsomes were in accordance with those obtained with a reconstituted monooxygenase system containing purified cytochrome P-450 2D6. Large differences of Ki values ranging between 0.005 and 100 microM were observed. Low-energy conformers of tested compounds were fit within the preliminary pharmacophore model. The analysis of steric and electronic properties of these compounds led to the definition of a final pharmacophore model. Characteristic properties are a positive charge on a nitrogen atom and a flat hydrophobic region, the plane of which is almost perpendicular to the N-H axis and maximally extends up to a distance of 7.5 A from the nitrogen atom. Compounds with high inhibitory potency had additional functional groups with negative molecular electrostatic potential and hydrogen bond acceptor properties on the opposite side at respective distances of 4.8-5.5 A and 6.6-7.5 A from the nitrogen atom. The superposition of strong and weak inhibitors led to the definition of an excluded volume map. Compounds that required additional space were not inhibitors. This is apparently the first pharmacophore model for inhibitors of a cytochrome P-450 enzyme and offers the opportunity to classify compounds according to their potency of inhibition. Adverse drug interactions which occur when both substrates and inhibitors of cytochrome P-450 2D6 are applied may be predicted.

Alkaloids↗
PubMed1993

Evidence for agonist-induced export of intracellular Ca2+ in epithelial cells.

There is increasing evidence that some agonists not only induce intracellular Ca2+ increases, due to store release and transmembranous influx, but also that they stimulate Ca2+ efflux. We have investigated the agonist-stimulated response on the intracellular Ca2+ activity ([Ca2+]i) in the presence of thapsigargin (10(-8) mol/l, TG) in HT29 and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10(-7)-10(-3) mol/l, n = 9), carbachol (10(-6)-10(-3) mol/l, n = 5) and neurotensin (10(-10)-10(-7) mol/l, n = 6) all induced a concentration-dependent decrease in [Ca2+]i in the presence of TG. Similar results were obtained with HT29 cells. This decrease of [Ca2+]i could be caused by a reduced Ca2+ influx, either due to a reduced driving force for Ca2+ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca2+ permeability. Using the fura-2 Mn2+ quenching technique we demonstrated that ATP did not slow the TG-induced Mn2+ quench. This indicates that the agonist-induced [Ca2+]i decrease in the presence of TG was not due to a reduced influx of Ca2+ into the cell, but rather due to stimulation of Ca2+ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of -49 +/- 3.6 mV (n = 9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

Cyproterone acetate generates DNA adducts in rat liver and in primary rat hepatocyte cultures.

Cyproterone acetate (CPA), an active component of certain contraceptive and antiandrogenic drugs, has been shown recently to induce DNA repair synthesis in rat hepatocytes in vitro. In the present study we examined whether CPA can cause the formation of DNA adducts detectable by the 32P-postlabeling technique in hepatic cells in vitro and in vivo. Incubation of primary cultures of hepatocytes from male Wistar rats with CPA resulted in the occurrence of radioactive spots in the radiochromatograms of 32P-postlabeled DNA digests indicating the formation of two DNA adducts ('A' and 'B'). At 30 microM CPA, the highest concentration tested, approximately 50 'A' adducts and five 'B' adducts were found per 10(9) nucleotides. DNA of hepatocyte cultures from female rats was found to contain adduct A and a minor adduct termed 'D', but adduct B was not observed. Between 1 and 10 microM CPA, the relative level of adduct A was approximately 20-fold higher than the level observed in male hepatocytes. In vivo DNA adducts were detected almost exclusively in hepatic DNA. Hepatic DNA from male Wistar rats treated with single doses of CPA (1-100 mg/kg) by gavage, showed the major adducts A and B and two further spots of minor intensity ('C' and 'D') in the radiochromatograms. No adducts were detectable in extrahepatic tissues. The adduct pattern of liver DNA from females exposed to single oral doses between 0.1 and 30 mg CPA/kg body wt was similar to that observed in males; however, the relative levels for adducts A and D were approximately 100-fold higher. In females, linear relationships between dose and adduct levels were observed for all four adducts. The present findings show that CPA causes damage to hepatic DNA not only in vitro, but also in vivo. Thus it appears possible that DNA adduct formation is involved in the formation of hepatic tumors during long-term treatment of rats with the synthetic steroid.

Animals↗
PubMed1993

Conformational changes of U6 RNA during the spliceosome cycle: an intramolecular helix is essential both for initiating the U4-U6 interaction and for the first step of slicing.

During each spliceosome cycle, U6 RNA undergoes several conformational changes, involving the formation and disruption of base-pairing interactions with U4 and U2 RNAs. By use of a mutational approach we have focused on the stem II region of U6, which can adopt alternative conformations: In the singular form of U6, it can form an intramolecular stem-loop structure; in the U4/U6 snRNP, the stem II region base-pairs with U4 RNA; in the active spliceosome, this region has been proposed to fold back into an intramolecular U6 helix in the context of a U6-U2 structure. Using chemical modification/interference assays and a mutational approach we found that the 3' terminal loop of the singular U6 (nucleotides 65-69) is essential for initiating the U4-U6 base-pairing interaction. A series of point mutations in the adjacent helix was designed to alter the stability of the intramolecular helix. Stabilizing mutations inhibited the formation of the U4/U6 snRNP. In contrast, mutant U6 RNAs with a destabilized intramolecular helix were still active in U4-U6 interaction and spliceosome assembly; however, their ability to support the first step of splicing was strongly reduced, suggesting that the intramolecular U6 helix has an important function in the first step of splicing. Affinity-purified U4 snRNP and U6 RNA did not assemble into a stable U4/U6 snRNP, unless complemented by nuclear extract, indicating that a protein factor (or factors) is necessary for the U4-U6 interaction. In sum, these data demonstrate that the stem II region of U6 functions both in U4-U6 interaction and in the first step of splicing; they also provide evidence that the balanced stability of different conformations of U6 RNA is critical for its function.

Base Sequence↗
PubMed1993

Biomonitoring of human exposure to carcinogenic nitroaromatic compounds: a pilot study on DNA adduct formation by 1,6-dinitropyrene in rats.

DNA adduct formation by 1,6-dinitropyrene (DNP) was examined in various rat tissues. Intraperitoneal administration of 0.2, 1.0 or 5.0 mg DNP/kg b.w. caused the formation of one major and, at the higher doses, two minor DNA adducts. The highest level of about 300 adducts/10(9) nucleotides was found in urinary bladder DNA. 5-10-fold lower adduct levels were found in the DNA of white blood cells (WBC), liver, lung and small intestinal mucosa. DNA adduct levels in the bladder were highest in Sprague-Dawley males followed by Sprague-Dawley females and Wistar males. Administration by gavage was less effective than intraperitoneal injection. Intratracheal instillation of microcrystalline DNA suspensions did not lead to any detectable adduct formation. The results indicate that WBC DNA may not be a reliable DNA source for biomonitoring human exposure to nitroarenes and that nitroarene-DNA adducts may only be detectable at high levels of exposure.

Animals↗
PubMed1992

Reconstituted mammalian U4/U6 snRNP complements splicing: a mutational analysis.

We have developed an in vitro complementation assay to analyse the functions of U6 small nuclear RNA (snRNA) in splicing and in the assembly of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. U6-specific, biotinylated 2'-OMe RNA oligonucleotides were used to deplete nuclear extract of the U4/U6 snRNP and to affinity purify functional U4 snRNP. The addition of affinity purified U4 snRNP together with U6 RNA efficiently restored splicing activity, spliceosome assembly and U4/U5/U6 multi-snRNP formation in the U4/U6-depleted extract. Through a mutational analysis we have obtained evidence for multiple sequence elements of U6 RNA functioning during U4/U5/U6 multi-snRNP formation, spliceosome assembly and splicing. Surprisingly, the entire 5' terminal domain of U6 RNA is dispensable for splicing function. In contrast, two regions in the central and 3' terminal domain are required for the assembly of a functional U4/U5/U6 multi-snRNP. Another sequence in the 3' terminal domain plays an essential role in spliceosome assembly; a model is strongly supported whereby base pairing between this sequence and U2 RNA plays an important role during assembly of a functional spliceosome.

Bacterial Proteins↗
PubMed1992

Endogenous and exogenous factors modifying the activity of human liver cytochrome P-450 enzymes.

Cytochrome P-450 dependent monooxygenases play a dual role for xenobiotic metabolism. On one hand they initiate the primary rate limiting step for the elimination of a bulk of drugs and organic chemicals. On the other hand they catalyze the formation of toxic metabolites from chemical carcinogens and many other toxic chemicals. Numerous studies have shown that their activity in animals is subject to the influence of various modifying factors, such as strain, species, sex, age, diurnal rhythm and the effect of enzyme inducers. Less is known about the influence of these factors on human cytochrome P-450 enzymes. Here we report the results of an extended study on human liver cytochrome P-450 performed with liver biopsies of 178 individuals taken for diagnostic purposes. The enzymatic activity was determined by the aldrin epoxidase assay indicating a variety of enzymes inducible by phenobarbital and by glucocorticoid and androgenic hormones. The frequency histogram of individual aldrin epoxidase activities showed a unimodal distribution and a variation factor of 100 between maximal and minimal activity. Individuals with severe liver diseases, such as cirrhosis and fatty liver, exhibited a 50% loss of enzyme activity. Age and sex did not significantly influence the enzyme activity. No significant correlation was observable between the rate of aldrin epoxidation and debrisoquine 4-hydroxylation, a prototype of a genetically controlled cytochrome P-450 reaction. We assume that the broad interindividual variation of epoxidase activities is more likely due to the influence of exogenous and endogenous inducers rather than to a genetic polymorphism.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.

Animals↗
PubMed1991

Cell death in normal and rough eye mutants of Drosophila.

The regular, reiterated cellular pattern of the Drosophila compound eye makes it a sensitive amplifier of defects in cell death. Quantitative and histological methods reveal a phase of cell death between 35 and 50 h of development which removes between 2 and 3 surplus cells per ommatidium. The timing of this epoch is consistent with cell death as the last fate to be specified in the progressive sequence of cell fates that build the ommatidium. An ultrastructural survey of cell death suggests dying cells in the fly eye have similarities as well as differences with standard descriptions of programmed cell death. A failure of cell death to remove surplus cells disorganizes the retinal lattice. A screen of rough eye mutants identifies two genes, roughest and echinus, required for the normal elimination of cells from the retinal epithelium. The use of an enhancer trap as a cell lineage marker shows that the cone cells, like other retinal cells, are not clonally related to each other or to their neighbors.

Animals↗
PubMed1991

The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.

Events in the morphogenetic furrow set the stage for all subsequent compound eye development in Drosophila. The periodic pattern of the adult eye begins in the furrow with the spaced initiation of ommatidial rudiments, the preclusters. A wave of mitosis closely follows the furrow. A cell-by-cell analysis reveals details of these events. Early stages of ommatidial assembly can be resolved using a lead sulfide stain. Overt ommatidial organization begins in the morphogenetic furrow as cells gather into periodically spaced concentric aggregates. A stereotyped sequence of cell rearrangements converts these aggregates into preclusters. In the furrow, new rows of ommatidia are initiated at the equator and grow as new clusters are added to the peripheral ends. Mitotic labeling using BrdU feeds shows that all cells not incorporated into a precluster divide. BrdU injections show that cells divide roughly simultaneously between two adjacent rows of ommatidia.

Animals↗
PubMed1990

Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes.

U6 snRNA sequences required for assembly of U4/U6 snRNP and splicing complexes were determined by in vitro reconstitution of snRNPs. Both mutagenesis and chemical modification/interference assays identify a U6 snRNA domain required for U4/U6 snRNP formation. The results support the existence of a U4/U6 snRNA interaction domain previously proposed on the basis of phylogenetic evidence. In addition, two short U6 snRNA regions flanking the U4/U6 interaction domain are essential to assemble the U4/U6 snRNP into splicing complexes. These two regions may represent binding sites for splicing factors or may facilitate the formation of an alternative U6 snRNA secondary structure during spliceosome assembly.

Base Sequence↗