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Biomedical subjects

G Schuster

Publications and source records attributed to G Schuster.

At least 19 recordsLinked to original sources
PubMed2000

RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1.

The chloroplast ribosomal protein CS1, the homolog of the bacterial ribosomal protein S1, is believed to be involved in the process of ribosome binding to mRNA during translation. Since translation control is an important step in chloroplast gene expression, and in order to study initiation complex formation, we studied the RNA-binding properties of CS1 protein. We found that most of the CS1 protein in spinach chloroplast co-purified with the 30S ribosomal subunit. The relative binding affinity of RNA to CS1 was determined using the UV-crosslinking competition assay. CS1 protein binds the ribohomopolymer poly(U) with a relatively high binding affinity. Very low binding affinities were obtained for the other ribohomopolymers, poly(G), poly(A) and poly(C). In addition, no specific binding of CS1, either in the 30S complex or as a recombinant purified protein, was obtained to the 5'-untranslated region of the mRNA in comparison to the other parts. RNA-binding experiments, in which the N- and C-termini of the protein were analyzed, revealed that the RNA-binding site is located in the C-terminus half of the protein. These results suggest that CS1 does not direct the 30S complex to the initiation codon of the translation site by specific binding to the 5'-untranslated region. In bacteria, specific binding is derived by base pairing between 16S rRNA and the Shine-Dalagarno sequences. In the chloroplast, nuclear encoded and gene-specific translation factors may be involved in the determination of specific binding of the 30S subunit to the initiator codon.

5' Untranslated Regions↗
PubMed2000

Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence.

Polyadenylation of RNA molecules in bacteria and chloroplasts has been implicated as part of the RNA degradation pathway. The polyadenylation reaction is performed in Escherichia coli mainly by the enzyme poly(A) polymerase I (PAP I). In order to understand the molecular mechanism of RNA poly-adenylation in bacteria, we characterized the biochemical properties of this reaction in vitro using the purified enzyme. Unlike the PAP from yeast nucleus, which is specific for ATP, E.coli PAP I can use all four nucleotide triphosphates as substrates for addition of long ribohomopolymers to RNA. PAP I displays a high binding activity to poly(U), poly(C) and poly(A) ribohomopolymers, but not to poly(G). The 3'-ends of most of the mRNA molecules in bacteria are characterized by a stem-loop structure. We show here that in vitro PAP I activity is inhibited by a stem-loop structure. A tail of two to six nucleo-tides located 3' to the stem-loop structure is sufficient to overcome this inhibition. These results suggest that the stem-loop structure located in most of the mRNA 3'-ends may function as an inhibitor of poly-adenylation and degradation of the corresponding RNA molecule. However, RNA 3'-ends produced by endonucleolytic cleavage by RNase E in single-strand regions of mRNA molecules may serve as efficient substrates for polyadenylation that direct these molecules for rapid exonucleolytic degradation.

Binding Sites↗
PubMed2000

Organotypic cultures of gingival cells: an epithelial model to assess putative local effects of orthodontic plate and occlusal splint materials under more tissue-like conditions.

This article explores whether organotypic cultures of immortalized gingival keratinocytes constitute a suitable model for assessing the epithelial cell compatibility of two groups of dental resins, each of them representing one group used in orthodontics and temporo-mandibular disorders (TMD) therapy under conditions more closely resembling the actual tissue situation. The resins were tested with the agar diffusion assay (ADA) in conventional monolayer and organotypic cultures. Compared to the control exhibiting a neutral red destaining index of 3, the index of 4 obtained after exposure of monolayers to one soft permanent resin (Durabase) indicated the presence of a non-lytic but physiologically active substance. In contrast, the adaptation of the ADA to organotypic cultures revealed no apparent lesions at the epithelial surface by performing scanning electron microscopy, while histoarchitecture indicated the development of stratified surface epithelia. This was substantiated by undamaged cells in the uppermost cell layers and by the preservation of cell-to-cell contacts. Furthermore, indirect immunofluorescence for Ki-67 and the cytokeratins ck 14 and ck4 revealed that cell proliferation and epithelial structure were maintained, while differentiation was enhanced, possibly increasing epithelial resistance. The results obtained from the organotypic cultures suggest that (i) cell-affecting effects of materials visible in monolayer cultures may not be seen in epithelia resembling that in vivo and that (ii) enhanced differentiation may be associated with increased stability of the epithelial cells. Thus, organotypic cultures of gingival cells constitute a tissue model allowing short-term tissue compatibility studies of dental materials and rendering a potential candidate also for long-term studies.

Acrylic Resins↗
PubMed2000

Adapting the waterproof BMS-96 diode array for isodose determination of dynamic beams.

We report the application of the Schuster BMS-96 waterproof linear diode array for isodose determination of dynamic beams. The array recorded beam profiles correctly, while depth dose distributions of dynamic beams with large variations in dose rate were registered erroneously. The deviations could be eliminated by appropriate software modifications. Until the software is revised, true isodoses can be obtained by rescaling each individual profile to the depth dose curve as measured with a single ionization chamber. After the corrections presented in the paper, isodoses interpolated from these corrected data sets agreed with ionization chamber measurements within 1-2%.

Radiation Dosage↗
PubMed2000

Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhadtii.

Three classes of RNA, represented by atpB and petD mRNAs, Arg and Glu tRNAs, and 5S rRNA, were found to exist in polyadenylated form in Chlamydomonas reinhardtii chloroplasts. Sequence analysis of cDNA clones derived from reverse transcriptase-polymerase chain reaction protocols used to select polyadenylated RNAs revealed that, at least for the mRNAs and tRNAs, there are three apparent types of polyadenylation. In the first case, the poly(A) tail is added at or near the mature 3' end, even when this follows a strong secondary structure. In the second case, the tail is added to pre-mRNA or pre-tRNA, suggesting a possible competition between polyadenylation and RNA-processing pathways. Finally, in all cases, the poly(A) tail can be added internally, possibly as a part of an RNA-decay pathway. The tails found in Chlamydomonas chloroplasts differ from those of spinach chloroplasts in adenine content, being nearly homopolymeric (>98% adenine) versus 70% in spinach, and are similar in length to those of Escherichia coli, being mostly between 20 and 50 nt. In vitro assays using a Chlamydomonas chloroplast protein extract showed that a 3' end A25 tail was sufficient to stimulate rapid degradation of atpB RNA in vitro, with a lesser effect for petD, and only minor effects on trnE. We therefore propose that polyadenylation contributes to mRNA degradation in Chlamydomonas chloroplasts, but that its effect may vary.

Animals↗
PubMed2000

[Mesial temporal sclerosis syndrome in adult patients].

Magnetic resonance imaging (MRI) has become an essential tool in the work-up of epilepsy. Since its appearance it has been possible to identify pathologies, such as hippocampal sclerosis (HS), that had previously only been detected by histopathological assays. The aim of this study was to analyze the clinical manifestations, EEG and the outcome of patients with HS as shown by MRI. We revised the clinical histories of 384 outpatients from the Epilepsy Center, Ramos Mejía Hospital, who had been studied by MRI. Thirty five of them (15.5%) had a diagnosis of HS, based on the structural changes observed on the images. Six patients were excluded because of incomplete clinical data. Therefore, we studied 29 patients including 15 men. The mean age was 32.7 +/- 10.2 years (range: 19-58). All of them had partial seizures. Ten subjects had had febrile convulsions (34.5%) in childhood. Neurological examination was normal in all subjects. Interictal EEG showed focal abnormalities that were coincident in their location with the MRI abnormalities in 16 patients (55.1%). Fourteen patients (48.3%) showed right side hippocampal lesions on MRI, thirteen on the left side (44.9%) and 2 bilateral HS (6.8%). Twenty-seven patients (93.1%) had intractable epilepsy. Anterior temporal lobectomy was performed in 3 subjects with good outcome. The identification of these patients who present certain clinical and MRI characteristics, provides an opportunity to define the mesial temporal sclerosis syndrome. This could benefit patients in their prognosis and for specific treatments.

Adult↗
PubMed1999

The tooth-width relation of the incisors in closed bite.

On the basis of various opinions expressed in the literature, "closed bite" is associated in particular with an impaired front-tooth relation. Applying Tonn's index, evaluation of the records (models and cephalograms) of 1,100 patients, however, yielded no evidence of changes in the width ratio of the front teeth with regard to Angle classes, growth type, axial inclination of the central upper incisors, interincisal angle, or overbite.

Cephalometry↗
PubMed1999

Altering the 3 UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3-end maturation in vitro but not in vivo.

The 3' ends of chloroplast mRNAs are produced by the processing of longer precursors. The 3' ends of most plastid mRNAs are located at, or several nucleotides downstream of, stem-loop structures, which act as 3'-end-processing signals and RNA stability elements. In chloroplasts of the green alga Chlamydomonas reinhardtii, 3'-end maturation of atpB mRNA involves endonucleolytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotides downstream of the stem-loop structure. This cleavage is followed by exonucleolytic resection to generate the mature 3' end. In order to define critical nucleotides of the endonucleolytic cleavage site, we mutated its sequence. Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3'-end-processed products. However, in two cases where the AU-rich sequence of this site was replaced with a GC-rich one, the 3' end of the stable processing product differed from that of the wild-type product. To examine whether these mutations affected atpB mRNA processing or accumulation in vivo, the endogenous 3' UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts. Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3' end was generated. These results imply that Chlamydomonas atpB 3' processing parallels the situation with other endonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome.

3' Untranslated Regions↗
PubMed1999

Preferential degradation of polyadenylated and polyuridinylated RNAs by the bacterial exoribonuclease polynucleotide phosphorylase.

Polyadenylation of mRNA has been shown to target the RNA molecule for rapid exonucleolytic degradation in bacteria. To elucidate the molecular mechanism governing this effect, we determined whether the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated PNPase degraded polyadenylated and non-polyadenylated RNAs at similar rates. However, when the two molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. The same phenomenon was observed with polyuridinylated RNA. The poly(A) tail has to be located at the 3' end of the RNA, as the addition of several other nucleotides at the 3' end prevented competition for polyadenylated RNA. In RNA-binding experiments, E. coli PNPase bound to poly(A) and poly(U) sequences with much higher affinity than to poly(C) and poly(G). This high binding affinity defines poly(A) and poly(U) RNAs as preferential substrates for this enzyme. The high affinity of PNPase for polyadenylated RNA molecules may be part of the molecular mechanism by which polyadenylated RNA is preferentially degraded in bacterial cells.

Escherichia coli↗
PubMed1999

Genital incarceration with metal rings: their safe removal.

Strangulation of the genitalia with constricting metal bands presents a difficult problem with removal. We report the easy and safe removal by cutting the metal bands in two places using a hand-held Dremel Moto-Tool [corrected] with a metal cutting disk.

Adult↗
PubMed1998

YAC rescue of downless locus mutations in mice.

Mice with mutations at the downless (dl) locus have defects in hair follicle, tooth, sweat gland, preputial gland, Meibomian gland, and tail development. The dl phenotype is analogous to the human genetic disorder termed autosomal hypohidrotic (or anhidrotic) ectodermal dysplasia (HED). On the basis of the identification of two related transgenic insertional mutations in the downless gene, yeast artificial chromosomes (YACs) were identified that map to the critical region of mouse Chromosome (Chr) 10. To determine which of the YACs contain the dl gene, we generated YAC transgenic mice by mouse embryo microinjections. The 200-kb YAC B25.D9 was found to rescue all of the downless defects. In addition, the transgenic YAC rescued the dominant Sleek (Dlslk) allele. Since the sequences within the YAC are entirely deleted in one of the transgenic mutants, our results establish that Sleek encodes a dominant-negative protein whose effects can be reversed by expression of extra copies of the wild-type locus.

Alleles↗
PubMed1998

The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability.

A general characteristic of the 3'-untranslated regions (3' UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops are RNA 3'-end processing signals and determinants of mRNA stability, not transcription terminators. Incubation of synthetic RNAs corresponding to the 3' UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products. Synthetic RNAs of the petA 3' UTR and the antisense strand of atpB 3' UTR were degraded in the extract. To examine 3' UTR function in vivo, the atpB 3' UTR was replaced with the 3' UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR plus trnR, rbcL, petA and E. coli thrA by biolistic transformation of Chlamydomonas chloroplasts. Each 3' UTR was inserted in both the sense and antisense orientations. The accumulation of both total atpB mRNA and ATPase beta-subunit protein in all transformants was increased compared to a strain in which the atpB 3' UTR had been deleted. However, the level of discrete atpB transcripts in transformants containing the antisense 3' UTR sequences was reduced to approximately one-half that of transformants containing the 3' UTRs in the sense orientation. These results imply that both the nucleotide sequences and the stem-loop structures of the 3' UTRs are important for transcript 3'-end processing, and for accumulation of the mature mRNAs.

Adenosine Triphosphatases↗
PubMed1998

Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse.

Visceral left-right asymmetry occurs in all vertebrates, but the inversion of embryo turning (inv) mouse, which resulted following a random transgene insertion, is the only model in which these asymmetries are consistently reversed. We report positional cloning of the gene underlying this recessive phenotype. Although transgene insertion was accompanied by neighbouring deletion and duplication events, our YAC phenotype rescue studies indicate that the mutant phenotype results from the deletion. After extensively characterizing the 47-kb deleted region and flanking sequences from the wild-type mouse genome, we found evidence for only one gene sequence in the deleted region. We determined the full-length 5.5-kb cDNA sequence and identified 16 exons, of which exons 3-11 were eliminated by the deletion, causing a frameshift. The novel gene specifies a 1062-aa product with tandem ankyrin-like repeat sequences. Characterization of complementing and non-complementing YAC transgenic families revealed that correction of the inv mutant phenotype was concordant with integration and intact expression of this novel gene, which we have named inversin (Invs).

Amino Acid Sequence↗
PubMed1998

3'-Processed mRNA is preferentially translated in Chlamydomonas reinhardtii chloroplasts.

3'-end processing of nucleus-encoded mRNAs includes the addition of a poly(A) tail that is important for translation initiation. Since the vast majority of chloroplast mRNAs acquire their 3' termini by processing yet are not polyadenylated, we asked whether 3' end maturation plays a role in chloroplast translation. A general characteristic of the 3' untranslated regions of chloroplast mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops and their flanking sequences serve as RNA 3'-end formation signals. Deletion of the Chlamydomonas chloroplast atpB 3' IR in strain Delta26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase beta-subunit, leading to weakly photosynthetic growth. Of the residual atpB mRNA in Delta26, approximately 1% accumulates as a discrete RNA of wild-type size, while the remainder is heterogeneous in length due to the lack of normal 3' end maturation. In this work, we have analyzed whether these unprocessed atpB transcripts are actively translated in vivo. We found that only the minority population of discrete transcripts of wild-type size is associated with polysomes and thus accounts for the ATPase beta-subunit which accumulates in Delta26. Analysis of chloroplast rbcL mRNA revealed that transcripts extending beyond the mature 3' end were not polysome associated. These results suggest that 3'-end processing of chloroplast mRNA is required for or strongly stimulates its translation.

Adenosine Triphosphatases↗
PubMed1997

The mechanism of preferential degradation of polyadenylated RNA in the chloroplast. The exoribonuclease 100RNP/polynucleotide phosphorylase displays high binding affinity for poly(A) sequence.

Polyadenylation of mRNA in the chloroplast has recently been shown to target the RNA molecule for rapid exonucleolytic degradation. A model has been suggested in which the degradation of chloroplast mRNA is initiated by endonucleolytic cleavage(s) followed by the addition of poly(A)-rich sequences and rapid exonucleolytic degradation. When in vitro transcribed RNAs were incubated with chloroplast protein extract, competition between polyadenylated and non-polyadenylated RNAs for degradation resulted in the rapid degradation of the polyadenylated molecules and stabilization of their non-polyadenylated counterparts. To elucidate the molecular mechanism governing this effect, we determined whether the chloroplast exoribonuclease 100RNP/polynucleotide phosphorylase (PNPase) preferably degrades polyadenylated RNA. When separately incubated with each molecule, isolated 100RNP/PNPase degraded polyadenylated and non-polyadenylated RNAs at the same rate. However, when both molecules were mixed together, the polyadenylated RNA was degraded, whereas the non-polyadenylated RNA was stabilized. In RNA binding experiments, 100RNP/PNPase bound the poly(A) sequence with much higher affinity than other RNA molecules, thereby defining the poly(A)-rich RNA as a preferential substrate for the enzyme. 100RNP/PNPase may therefore be involved in a mechanism in which post-transcriptional addition of poly(A)-rich sequence targets the chloroplast RNA for rapid exonucleolytic degradation.

Binding Sites↗
PubMed1997

Antiparkinsonian and other motor effects of flupirtine alone and in combination with dopaminergic drugs.

In this study we attempted to specify the behavioural profile of the analgesic flupirtine (1, 10 and 20 mg/kg p.o.) in the rat with respect to (i) its antiparkinsonian potential alone and as an adjunct to L-dihydroxyphenylalanine (L-DOPA) in the haloperidol-induced catalepsy (0.5 mg/kg haloperidol i.p.), (ii) locomotion and exploratory behaviour in the open field with holeboard, and (iii) possible psychomotor stimulating effects in the experimental chamber. In the two latter tests, behaviour was additionally challenged by D-amphetamine (2 mg/kg i.p.). In the catalepsy tests (horizontal bar, podium, grid) flupirtine alone was anticataleptic at doses of 10 and 20 mg/kg p.o., and the antiparkinsonian potential of a subthreshold dose of L-DOPA (50 mg/kg p.o.) was potentiated by 1 and 10 mg/kg p.o. flupirtine. On spontaneous forward locomotion in the open field with holeboard, flupirtine (1 and 10 mg/kg p.o.) had no marked effect but increased the frequency and duration of head dips, indicative for augmenting exploratory behaviour. Spontaneous rearing was reduced and D-amphetamine-induced rearing was enhanced by 1 mg/kg p.o. flupirtine. Grooming was reduced by 1 and 10 mg/kg p.o. flupirtine. In contrast, turning and grooming behaviour (spontaneous as well as D-amphetamine-induced) was not markedly influenced by flupirtine in the experimental chamber. Sniffing was increased in this test by 1 mg/kg p.o. flupirtine but not by the higher dose. Flupirtine is highly effective in antagonising neuroleptic-induced catalepsy as well as in potentiating L-DOPA treatment in the rat, suggesting it is a prospective new candidate for the therapy of Parkinson's disease.

Aminopyridines↗
PubMed1997

Comparison of the behavioral and morphological effects of colchicine- or neutral fluid-induced destruction of granule cells in the dentate gyrus of the rat.

Virtually complete destruction of dentate gyrus granule cells by colchicine injections produced a persistent incapacity to solve spatial problems in rats. A topographically more selective but only locally complete destruction of granule cells using injections of neutral fluid (NFL) impaired acquisition during the initial stages of Morris water maze testing, thus indicating that limited degeneration of granule cells may weakly but significantly alter spatial learning capabilities. A subamnestic dose (0.08 mg/kg ip) of the NMDA antagonist MK-801 worsened radial maze performance only in NFL-treated rats, suggesting that there may be a synergistic interaction between NMDA blockade and limited granule cell degeneration.

Animals↗