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R Schroeder

Publications and source records attributed to R Schroeder.

At least 19 recordsLinked to original sources
PubMed1996

The environment of two metal ions surrounding the splice site of a group I intron.

Several divalent metal ions (Ca2+, Sr2+ and Pb2+) do not promote splicing, but instead induce cleavage at a single site in the conserved group I intron core in the absence of the guanosine cofactor at elevated pH, generating products with 5'-OH and 3'-phosphate ends. The reaction is competed by Mg2+, which does not cleave at this position, but hydrolyses the splice sites producing 3'-OH and 5'-phosphate ends. Mn2+ promotes both core cleavage and splice site hydrolysis under identical conditions, suggesting that two different metal atoms are involved, each responsible for one type of cleavage, and with different chemical and geometric requirements. Based on the core cleavage position and on the previously proposed coordination sites for Mg2+, we propose a structural location for two metal ions surrounding the splice site in the Michel-Westhof three-dimensional model of the group I intron core. The proposed location was strengthened by a first mutational analysis which supported the suggested interaction between one of the metal ions and the bulged residue in P7.

Bacteriophage T4↗
PubMed1996

Antibiotic-induced oligomerisation of group I intron RNA.

Antibiotics act as inhibitors of various biological processes. Here we demonstrate that some tuberactinomycins, hitherto known as inhibitors of prokaryotic protein synthesis and of group I intron self-splicing, have a modulatory effect on group I intron RNAs. The linear intron, which is excised during the self-splicing process, is still an active molecular capable of performing an intramolecular transesterification resulting in a circular molecule. However, in the presence of sub-inhibitory concentrations of tuberactinomycins, the intron reacts intermolecularly leading to the formation of linear head-to-tail intron-oligomers. The antibiotic stimulates the intron to react in trans instead of in cis. The phage T4-derived td intron uses the same sites for oligomerisation as for circularisation. Gel- retardation experiments demonstrate that the intron RNA forms non-covalent complexes in the presence of the antibiotic. It might be envisaged that the role of these peptide antibiotics is to bridge RNA molecules mediating RNA-RNA interactions and thus enabling their reaction. The tuberactinomycins are further able to induce the interaction of heterologous introns. The ligation of the T4 phage-derived td intron with the Tetrahymena rRNA intron is very efficient, resulting in molecules composed of two introns derived from different species. The td intron attacks the Tetrahymena intron at various sites, which are located within double-stranded regions. These observations suggest that small molecules like these basic peptide antibiotics could have mediated RNA-RNA interactions in a pre-protein era.

Animals↗
PubMed1996

Transforming growth factor beta mediates the angiotensin-II-induced stimulation of collagen type IV synthesis in cultured murine proximal tubular cells.

BACKGROUND: Angiotensin II (Ang II) stimulates synthesis of type IV collagen in a cultured murine proximal tubular cell line (MCT cells). In addition, Ang II also induces the expression of TGF-beta1 in these cells. Since TGF-beta has well-known stimulatory effects on the transcription of various collagens, we tested whether the Ang-II-mediated stimulation of type IV collagen is due to induction of endogenous TGF-beta1 synthesis in MCT cells. RESULTS: A neutralizing monoclonal anti-TGF-beta1-3 antibody abolished the Ang II-stimulated release of type IV collagen in culture supernatants. The anti-TGF-beta1-3 antibody also partly blocked Ang-II-mediated incorporation of 3[H]proline into de novo synthesized collagens. Moreover, 5 microM TGF-beta1 antisense oligonucleotides, but not the same concentration of sense oligonucleotides, completely blocked Ang-II-stimulated 3[H]proline incorporation. MCT cells incubated with TGF-beta1 antisense phosphorothioate-modified oligonucleotides failed to synthesize TGF-beta1 protein after Ang II treatment as measured by a sandwich ELISA in culture supernatants. SDS-polyacrylamide electrophoresis of 3[H]proline-labelled collagens and comparison with standard collagens also demonstrated that the neutralizing anti-TGF-1-3 antibody abolished the Ang-II-mediated stimulation in type IV collagen. Semiquantitative cDNA amplification for collagen type alpha1 (IV) transcripts revealed that the anti-TGF-beta1-3 antibody abrogates the increase in mRNA after Ang II treatment. Transient transfection studies in MCT cells using murine collagen alpha1 (IV) enhancer/promoter constructs also demonstrated the suppressive effect of the neutralizing antibody on Ang-II-stimulated gene transcription. CONCLUSIONS: Our data collectively suggest that the Ang-II-mediated increase in type IV collagen in MCT cells is mediated by endogenous synthesis and autocrine action of TGF-beta1. These findings may be important in changes of the tubulointerstitial architecture during the progression of renal disease.

Angiotensin II↗
PubMed1995

Evaluation of physicochemical parameters important to the oral bioavailability of peptide-like compounds: implications for the synthesis of renin inhibitors.

A series of radiolabeled compounds related to renin inhibitor structures was synthesized to represent a range of physicochemical properties. These compounds were tested in assays for intestinal absorption and hepatic clearance in order to define parameters conducive to optimizing bioavailability. In general, compounds with higher lipophilicity were better absorbed from the intestine. Absorption may also be dependent on molecular charge, as compounds with ionizable functionality were less well-absorbed than neutral compounds. Neutral compounds showed some dependency on molecular weight, with smaller compounds exhibiting better absorption. While uptake into hepatic cells was rapid regardless of partition coefficient or molecular weight, rate of appearance in bile was dependent on the molecular weight of the compounds.

Administration, Oral↗
PubMed1995

ANG II is a mitogen for a murine cell line isolated from medullary thick ascending limb of Henle's loop.

A murine SV40-transformed renal epithelial cell line derived from medullary thick ascending limb of Henle's loop (MTAL) was established and characterized by morphology, antigen expression, and biochemical criteria. These MTAL cells express a single class of high-affinity receptors for angiotensin II (ANG II) and transcripts for the AT1 subtype of ANG II receptors. ANG II, in a dose-dependent manner, induced proliferation of MTAL cells. This observation is in striking contrast to syngeneic proximal tubular cells in which it was previously shown that the peptide induced cellular hypertrophy and slightly inhibited proliferation [G. Wolf and E. G. Neilson. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28: F768-F777, 1990]. The AT1-receptor antagonist losartan (10(-6) M), but not an AT2-receptor antagonist, blocked the mitogenic effects of ANG II in MTAL cells. Coincubation of quiescent MTAL cells with ANG II and 5% fetal calf serum further increased proliferation compared with cells grown only in serum. In contrast to proximal tubular cells, ANG II failed to induce transforming growth factor-beta 1 mRNA and protein synthesis in MTAL cells. Our data collectively suggest that ANG II is a mitogen for MTAL cells in vitro. Therefore, epithelial cells derived from different parts of the nephron, even when transformed with SV40 virus and while under cell culture conditions, exhibit a distinct pattern of growth behavior after stimulation with ANG II.

Angiotensin II↗
PubMed1994

Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior.

Proteins anchored by GPI are poorly solubilized from cell membranes by cold nonionic detergents because they associate with detergent-resistant membranes rich in cholesterol and sphingolipids. In this study, we demonstrated that cholesterol and sphingolipid-rich liposomes were incompletely solubilized by Triton X-100. GPI-anchored placental alkaline phosphatase incorporated in these liposomes was also not solubilized by cold Triton X-100. As sphingolipids have much higher melting temperatures (Tm) than cellular phospholipids, a property correlated with Tm might cause detergent inextractability. In support of this idea, we found that the low-Tm lipid dioleoyl phosphatidylcholine (DOPC) was efficiently extracted from detergent-resistant liposomes by Triton X-100, whereas the high-Tm lipid dipalmitoyl phosphatidylcholine (DPPC) was not. The fluorescence polarization of liposome-incorporated diphenylhexatriene was measured to determine the "fluidity" of the detergent-resistant liposomes. We found that these liposomes were about as fluid as DPPC/cholesterol liposomes, which were present in the liquid-ordered phase, and much less fluid than DOPC or DOPC/cholesterol liposomes. These findings may explain the behavior of GPI-anchored proteins, which often have saturated fatty acyl chains and should prefer a less-fluid membrane. Therefore, we propose that acyl chain interactions can influence the association of GPI-anchored proteins with detergent-resistant membrane lipids. The affinity of GPI-anchored proteins for a sphingolipid-rich membrane phase that is not in the liquid crystalline state may be important in determining their cellular localization.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Peptide antibiotics of the tuberactinomycin family as inhibitors of group I intron RNA splicing.

The tuberactinomycins are a group of cyclic peptide antibiotics, which are potent inhibitors of prokaryotic protein synthesis. We report the inhibitory effect of viomycin, di-beta-lysyl-capreomycin IIA and tuberactinomycin A on group I intron self-splicing. They compete with the guanosine cofactor for the G-binding site located in the conserved core of the intron. They are 100-fold more active than all other competitive inhibitors described so far (dGTP, arginine or streptomycin), inhibiting splicing at concentrations between 10 and 50 microM. Mutation of the G-binding site leads to partial resistance, and the inhibitory effect of these drugs is dependent on Mg2+ concentration. This suggests that the tuberactinomycins have more than one contact site with the intron RNA: via the G-binding site and via additional contacts with the RNA backbone. Positioning the tuberactinomycins in the three-dimensional model of the td intron core suggests that the charged lysyl side-chain (R1) is in contact with the backbone of the P1 helix. Structure/function analyses with various tuberactinomycin analogues with different activities confirm the involvement of this sidechain in inhibition of group I self-splicing. The demonstration of a new class of splicing inhibitors, the peptide antibiotics, illustrates how antibiotics may interact with catalytic RNA.

Bacteriophage T4↗
PubMed1994

Behavioral effects of persistent lymphocytic choriomeningitis virus infection in mice.

Lymphocytic choriomeningitis virus (LCMV) is a nonlytic murine virus that provides a valuable model system for studying the behavioral correlates of CNS viral infection. Newborn or immunosuppressed mice infected with LCMV develop a persistent tolerant infection characterized by continuous viral production. Virus can be found in various body organs including lung, liver, kidney, and brain. In brain, neurons are the predominant CNS cells infected and the greatest number of persistently infected neurons are found in the cerebral cortex, hippocampus, other limbic structures and parts of the hypothalamus. Despite continuous infection throughout the animal's life, neurons show no structural injury or dropout. Mice from the DBA/2J strain were infected with LCMV (1000 plaque-forming units) within 18 h of birth and tested for behavioral function as adults. Plaque assays indicated persistent infection in virus-injected mice. Mice were tested for their ability to learn a Y-maze spatial discrimination to avoid the onset of a mild footshock (0.43 mA). The number of correct avoidance responses made during training was taken as a measure of acquisition performance. The virus-infected mice showed a deficit in acquisition of the Y-maze discrimination compared to that seen in vehicle-injected and noninjected controls. Following additional training to reach control levels of performance, the infected mice and the controls were injected with the cholinergic antagonist scopolamine. Scopolamine (2.0 mg/kg) disrupted the performance of the infected mice significantly more than control performance, suggesting that a cholinergic dysfunction accounted for some of the learning deficit. A separate group of virus-infected mice exhibited hypoactivity during the first exposure to a locomotor testing apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Induction and habituation of immediate early gene expression in rat brain by acute and repeated restraint stress.

Acute exposure to stress leads to activation of the pituitary-adrenal axis (PA-axis) while repeated exposure to a homotypic stressor generally results in habituation of this response. Previous studies suggested that such habituation is largely due to changes in afferents of the PA-axis. To examine where within these afferents habituation occurs, we studied the effect of acute and repeated exposure to 2 hr restraint stress on expression of c-fos mRNA, as a marker of altered neuronal activity, in brain regions previously shown to influence the activity of the PA-axis. Acute restraint stress increased expression of c-fos mRNA in cortex, hippocampus, hypothalamus, septum, and brainstem. In contrast, the effect of restraint stress on c-fos expression in the aforementioned brain regions was much smaller in animals restrained once daily for 4 d, and nonexistent in animals restrained once daily for 9 d. A similar pattern of induction and habituation of jun-B, but not zif-268, c-jun, or jun-D mRNA expression, was observed in the cortex of animals exposed to acute versus repeated restraint stress. The habituation of c-fos responses was stressor specific: exposure of restraint-adapted animals to a novel (20 min swim) stress produced an increase in levels of c-fos mRNA in every examined brain region comparable to that seen in animals exposed to this stressor for the first time. Adrenalectomy did not alter the pattern of c-fos expression induced by acute and repeated restraint stress. Therefore, activation and habituation of these c-fos responses are independent of changes in circulating levels of corticosterone.

Adrenalectomy↗
PubMed1994

[Status of portal perfusion in colorectal cancer. Swiss Study Group for Clinical Cancer Research].

533 patients with diagnosis of operable colorectal carcinoma were randomized to receive either a single course of portal infusion with Mitomycin-C (MMC) and 5-Fluorouracil (5-FU) starting immediately after operation, or no adjuvant treatment. Of these, 505 (94%) were evaluable. Over the median follow-up of 8 years, the adjuvant therapy reduced the risk of recurrence by 22% (Hazard ratio = 0.78%, 95% CI 0.61-0.99; P = 0.045). The relative reduction of relapse on death was similar in all subgroups (i.e. nodal status, localization). However, adjuvant portal chemotherapy proved to be most efficient in the subgroups of patients with tumor involvement of the regional lymph nodes (Dukes C) and of patients with colon cancer. Analysis of the pattern of relapse showed that most of the difference in overall and disease-free survival is to be attributed to a consistent reduction of all kinds of tumor recurrences (i.e. local relapses, liver metastases and/or other distant metastases) in the treated group, rather than to liver relapses alone. We conclude therefore, that part of significant benefit obtained for patients with operable colorectal carcinoma treated with a single course of adjuvant chemotherapy via the portal vein might be due to the additional systemic effects of the portal chemotherapy and further study of perioperative treatment with and without prolonged chemotherapy appears worthwhile.

Adenocarcinoma↗
PubMed1993

Lead cleavage sites in the core structure of group I intron-RNA.

Self-splicing of group I introns requires divalent metal ions to promote catalysis as well as for the correct folding of the RNA. Lead cleavage has been used to probe the intron RNA for divalent metal ion binding sites. In the conserved core of the intron, only two sites of Pb2+ cleavage have been detected, which are located close to the substrate binding sites in the junction J8/7 and at the bulged nucleotide in the P7 stem. Both lead cleavages can be inhibited by high concentrations of Mg2+ and Mn2+ ions, suggesting that they displace Pb2+ ions from the binding sites. The RNA is protected from lead cleavage by 2'-deoxyGTP, a competitive inhibitor of splicing. The two major lead induced cleavages are both located in the conserved core of the intron and at phosphates, which had independently been demonstrated to interact with magnesium ions and to be essential for splicing. Thus, we suggest that the conditions required for lead cleavage occur mainly at those sites, where divalent ions bind that are functionally involved in catalysis. We propose lead cleavage analysis of functional RNA to be a useful tool for mapping functional magnesium ion binding sites.

Base Sequence↗
PubMed1993

RNA as a catalyst: natural and designed ribozymes.

RNA can catalyse chemical reactions through its ability to fold into complex three-dimensional structures and to specifically bind small molecules and divalent metal ions. The 2'-hydroxyl groups of the ribose moieties contribute to this exceptional reactivity of RNA, compared to DNA. RNA is not only able to catalyse phosphate ester transfer reactions in ribonucleic acids, but can also show amino-acyl esterase activity, and is probably able to promote peptide bond formation. Bearing its potential for functioning both as a genome and as a gene product, RNA is suitable for in vitro evolution experiments enabling the selection of molecules with new properties. The growing repertoire of RNA catalysed reactions will establish RNA as a primordial molecule in the evolution of life.

Base Sequence↗
PubMed1993

U1 and U2 snRNA are localized in the sperm nucleus.

Analysis of epididymal rat sperm RNA strongly suggested the presence of U snRNAs, especially U1 and U2 snRNA. By Northern blot analysis with radiolabeled oligodeoxynucleotide probes, the presence of U1 and U2 snRNA in rat sperm was confirmed. To precisely localize these RNAs, in situ hybridization with antisense and sense oligo probes labeled with digoxigenin was carried out. The results indicate that U1 as well as U2 snRNA are confined to the sperm nucleus.

Animals↗
PubMed1992

Non-competitive inhibition of group I intron RNA self-splicing by aminoglycoside antibiotics.

Aminoglycoside antibiotics inhibit self-splicing of group I intron RNA in vitro at concentrations as low as 10(-6) M. The sites of interaction and the mechanism of inhibition have yet to be determined. A comparative study of inhibition by different 2-deoxystreptamine analogues reveals structural features of the aminoglycoside antibiotics required for their interaction and effect on group I introns. Complete antibiotic inhibition of the two steps of splicing was not reversed at high concentrations of guanosine, indicating a non-competitive inhibition. A mutant group I intron in which the conserved guanosine nucleotide of the G-binding site had been replaced by an adenosine, was sensitive to the antibiotics providing direct evidence that the antibiotics do not interact with the G-binding site in the same way as the guanine base. In addition kinetic analyses of the self-splicing process in the presence of antibiotic inhibitors supported a non-competitive mechanism of the mixed type for inhibition of the ribozyme.

Aminoglycosides↗