Biomedical subjects
Publications and source records attributed to D Fischer.
The authors report a mutation in exon 5 of GCH1 in a patient with adult-onset oromandibular dystonia and no obvious family history of dystonia. The patient responded positively to treatment with L-dopa. These findings demonstrate that GCH1 mutations must be considered even in patients with dystonic symptoms not typical of dopa-responsive dystonia.
Endoglin is a component of the transforming growth factor-beta receptor complex whose expression is limited to a small number of cell types, including endothelial cells (ECs), activated monocytes, tissue macrophages and erythroid precursors. Of particular interest is its preferential expression in the vasculature of many malignant tumors, especially in view of potential therapeutic applications. We have cloned the human endoglin promoter, analyzed its structure and demonstrate that the isolated genomic fragment shows strong promoter activity in ECs (compared to other known EC-selective promoters), but not in epithelial cells and fibroblasts. These findings suggest that the endoglin promoter may prove a useful tool for the transcriptional targeting of ECs by gene therapy. We also determined the domains that are responsible for both efficient transcription and the observed preferential activity in ECs. The region around the major site of transcription initiation was found to be essential for transcription in both ECs and non-ECs. In contrast, cell type specificity does not appear to be governed by a single mechanism, but rather seems to be due to functionally distinct regulatory mechanisms acting on different upstream sequences.
The results of the first Critical Assessment of Fully Automated Structure Prediction (CAFASP-1) are presented. The objective was to evaluate the success rates of fully automatic web servers for fold recognition which are available to the community. This study was based on the targets used in the third meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP-3). However, unlike CASP-3, the study was not a blind trial, as it was held after the structures of the targets were known. The aim was to assess the performance of methods without the user intervention that several groups used in their CASP-3 submissions. Although it is clear that "human plus machine" predictions are superior to automated ones, this CAFASP-1 experiment is extremely valuable for users of our methods; it provides an indication of the performance of the methods alone, and not of the "human plus machine" performance assessed in CASP. This information may aid users in choosing which programs they wish to use and in evaluating the reliability of the programs when applied to their specific prediction targets. In addition, evaluation of fully automated methods is particularly important to assess their applicability at genomic scales. For each target, groups submitted the top-ranking folds generated from their servers. In CAFASP-1 we concentrated on fold-recognition web servers only and evaluated only recognition of the correct fold, and not, as in CASP-3, alignment accuracy. Although some performance differences appeared within each of the four target categories used here, overall, no single server has proved markedly superior to the others. The results showed that current fully automated fold recognition servers can often identify remote similarities when pairwise sequence search methods fail. Nevertheless, in only a few cases outside the family-level targets has the score of the top-ranking fold been significant enough to allow for a confident fully automated prediction. Because the goals, rules, and procedures of CAFASP-1 were different from those used at CASP-3, the results reported here are not comparable with those reported in CASP-3. Nevertheless, it is clear that current automated fold recognition methods can not yet compete with "human-expert plus machine" predictions. Finally, CAFASP-1 has been useful in identifying the requirements for a future blind trial of automated served-based protein structure prediction.
The purpose of this study was to determine, using random peptide library (RPL) technologies, the minimal epitope requirements of the mouse monoclonal anti-interphotoreceptor-retinoid-binding protein antibody, H3B5. This previously characterized antibody is used as an example to examine whether RPL's offer a relatively easy and rapid route to obtaining detailed epitope mapping data.A pentadecamer random peptide library (RPL) displayed on the major coat protein (gene 8) of filamentous bacteriophage (F88-4-15) was used as a target for selection by the anti-IRBP monoclonal antibody, H3B5. Three rounds of library selection were performed, and 90 of the resultant RPL clones were examined for affinity to H3B5 by enzyme-linked immunosorbent assay (ELISA). DNA sequencing of ELISA positive clones provided sequence of the region encoding the random peptide. After three rounds of selection of the RPL, 76.7% of clones examined interacted with H3B5, 17.7% did not show significant binding and 6.6% bound to control antibody also. The essential elements of the peptide epitope were determined by sequence comparison of 24 clones to be the four amino-acid sequence (Aspartic or glutamic acid)-Proline-Arginine-(Leucine, Isoleucine or Valine). This motif [(D/E) PR (L/I/V)] is in agreement, but at greater resolution, than previous synthetic peptide studies where the motif AASEDPRL was identified. Other motifs were found which bound to H3B5 but did not share primary structure similarities (peptidomimetics). Selection from a RPL has rapidly defined the minimal requirements for the H3B5 epitope in fine detail. Such a process offers great potential for investigating antibody-antigen interactions and core sequences of an epitope, and enables the identification of motifs in other proteins which may be recognized by the antibody, providing information on possible cross-reactivity.
Assigning three-dimensional protein folds to genome sequences is essential to understanding protein function. Although experimental three-dimensional structures are currently available for only a very small fraction of these sequences, computational fold assignment is able to assign folds to 20-30% of the sequences in various genomes. This percentage varies depending on the particular organism under analysis, on the sensitivities of the methods used and on the number of experimental structures available at the time the assignment is carried out. The fraction of assignable sequences is currently increasing at an annual rate of roughly 18%. If this rate is sustained throughout the coming years, three-dimensional computational models for more than half of the genome sequences may be available by the year 2003.
PURPOSE: Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency. METHODS: The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and 13C-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promotor as reporter gene system. RESULTS: LMW-PEI (Mw 11'900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw 1'616'000 D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition. CONCLUSIONS: The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.
Several therapeutic approaches to sepsis and disseminated intravascular coagulation (DIC) have shown promising results in animal models. Large controlled trials in humans, however, have failed to show a clearly beneficial effect of a single drug or substance on outcome and survival so that treatment remains uncertain. As one researcher stated: ". . . sepsis is a classical example of a disease greater than the sum of its parts; it is a complex process in which intervention in one area might have only a modest effect on the final outcome." We believe that the complex pathophysiological setting of septic shock will undoubtedly require a multifaceted approach. Consequently, we attempt to arrest DIC and restore adequate tissue perfusion by intervention with heparin, AT and if possible protein C (PC) in the earliest stage of the disease, with the aim of blocking ongoing microthrombus formation and to support fibrinolysis. Growing understanding of the basic underlying mechanisms teaches us how to successfully stabilise the individual decompensated sub-systems like coagulation in septic patients. We should learn to accept these steps to reach the goal of a better outcome in terms of survival in this devastating illness.
BACKGROUND: although several reports indicate that qualitative and quantitative assessments of parotid salivary function are age-stable in healthy adults, there have been no studies of the influence of age on the variability of repeated parotid flow rates. OBJECTIVE: to examine the influence of age on the variability of repeated parotid flow rates in unmedicated, healthy adults. MATERIALS AND METHODS: we assessed unilateral unstimulated and 2% citrate-stimulated parotid flow rates and collected responses to a five-item subjective xerostomia questionnaire in 14 subjects aged 20-40 years and 14 subjects aged 60-80 years. All subjects were healthy and unmedicated. We collected saliva and asked subjects to complete the questionnaire at baseline, 1 h and 2 h. RESULTS: unstimulated and stimulated parotid flow rates were similar at each time point in young and older subjects, and remained stable over the collection period. There were no differences in the standard deviations of the three collections of unstimulated and stimulated flow rates between young and older subjects. There were no differences between questionnaire responses between the two groups, and no change in response over time. CONCLUSIONS: there is no increased age-related variability in parotid salivary flow rates over repeated measures. Stimulated parotid flow rates are stable over 2 h in healthy people, and are similar in young and older individuals. These results suggest that parotid glands have a large secretory reserve.
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Heregulins are members of the protein family of EGF-like growth and differentiation factors. The primary cell-surface targets of heregulins are heterodimers of the EGF-receptor homolog HER2 with either HER3 or HER4. We used a weighted evolutionary trace analysis to identify structural features that distinguish the EGF-like domain (hrg) of heregulins from other members of the EGF family. In this analysis, each amino acid sequence is weighted according to its uniqueness and the variability in each position is assigned by an amino acid substitution matrix. Conserved residues in heregulin that are variable in other EGF-like domains are considered possible specificity-conferring residues. This analysis identifies two clusters of residues at the foot of the boot-shaped hrg domain. The residues in one cluster are recruited from the N-terminus; those in the other are from the ohm-loop region and show a weak sequence similarity to the N-terminal residues at the opposite side of the boot. The remaining residues with high conservation scores distribute themselves into these two distinct surfaces on hrg. This pseudo-twofold symmetry and the presence of two distinct interfaces may reflect the preference of hrg for heterodimeric versus homodimeric HER complexes.
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Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the Yersinia fep-fes gene cluster orthologous to the Escherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by fepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD, fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD or fes genes abrogates enterochelin-supported growth of Y. enterocolitica on iron-chelated media. In contrast to E. coli, the fep-fes gene cluster in Y. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, and Yersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the family Enterobacteriaceae.
AIMS: To determine whether the infiltrating cells in the vitreous and subretinal fluid of patients with proliferative vitreoretinopathy (PVR) express messenger RNA for various cytokines found in this condition. METHODS: The presence of mRNA coding for HPRT, IL-6, IL-1beta, IL-8, and TNFalpha was investigated in 20 vitreous and subretinal fluid (SRF) samples from patients with PVR by reverse transcriptase polymerase chain reaction (RT-PCR). 16 samples from patients with retinal detachment and macular holes were used as controls. RESULTS: HPRT was detected in all samples of PVR and in 11 (69%) control cases. Patients with PVR demonstrated mRNA for the cytokines tested more often than controls. The difference was statistically significant. CONCLUSION: The presence of mRNA encoding for IL-6, IL-1beta, IL-8, and TNFalpha is significantly detected by RT-PCR in vitreous and SRF samples of patients with PVR, indicating local production of these cytokines by vitreous and SRF cells.
Recent studies suggest that sepsis stimulates ubiquitin-dependent protein breakdown in skeletal muscle. In this proteolytic pathway, ubiquitinated proteins are recognized, unfolded, and degraded by the multicatalytic 26S protease complex. The 20S proteasome is the catalytic core of the 26S protease complex. The role of the 20S proteasome in the regulation of sepsis-induced muscle proteolysis is not known. We tested the hypothesis that sepsis increases 20S proteasome activity and the expression of mRNA for various subunits of this complex. Proteolytic activity of isolated 20S proteasomes, assessed as activity against fluorogenic peptide substrates, was increased in extensor digitorum longus muscles from septic rats. The proteolytic activity was inhibited by specific proteasome blockers. Northern blot analysis revealed an approximately twofold increase in the relative abundance of mRNA for the 20S alpha-subunits RC3 and RC9 and the beta-subunit RC7. However, Western blot analysis did not show any difference in RC9 protein content between sham-operated and septic rats. The increased activity and expression of the 20S proteasome in muscles from septic rats lend further support for a role of the ubiquitin-proteasome-pathway in the regulation of sepsis-induced muscle proteolysis.
OBJECTIVES: To evaluate the safety and immunogenicity of a two-component acellular pertussis vaccine in preterm infants. STUDY DESIGN: Fifty preterm infants (25-35 weeks of gestation; mean, 30.8 weeks) and 50 term infants as a control group received a two-component acellular pertussis vaccine irrespective of their biological age and actual weight. Adverse reactions were registered by parents on a diary card and reviewed on each visit. Antibodies against pertussis toxoid (PT) and filamentous hemagglutinin (FHA) were determined with an enzyme-linked immunosorbent assay before the first and after the third vaccination. RESULTS: The infants of both groups showed an increase in geometric mean titers (GMT) against PT and FHA after vaccination (3 doses). There was a significant difference of antibody concentration between the preterm and the control group. The GMT for PT antibody of the preterm infants was 64. 16 U/L, and for the term infants it was 98.96 U/L. The GMT for FHA was 50.92 U/L in preterm versus 86.02 U/L in the control group. Efficacy of the immunization (more than a fourfold increase of antibody concentration in each infant) was 93.5% in the preterm group with respect to PT and 82.6% with respect to FHA. The incidence of adverse reactions was low and comparable in both study groups. CONCLUSION: Immunization with an acellular pertussis vaccine is safe for preterm infants. The immune response is significantly lower compared with a control group of term infants, but efficacy is high.
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Homology or comparative modeling is aimed at modeling the three-dimensional structure of a target sequence of unknown structure using the framework of an already known fold. Traditionally, homology modeling has been applied to targets with clear sequence similarity to proteins of known structure. Because methods to identify increasingly distant relationships have been developed, homology models can now be built for a wider range of targets. The first challenge in homology modeling is to obtain an initial, accurate, sequence-structure alignment with the most compatible fold. In CASP3, the abilities of fold-recognition methods to fulfill this challenge were evaluated with a number of target sequences of unknown structure. Sequence-structure alignments for 33 of the CASP3 targets using the fold-recognition method SDP were submitted (Fischer and Eisenberg, Protein Sci 1996; 5:947-955). After the three-dimensional structures of the sequences were subsequently released, the quality of the predictions were evaluated. Here I describe three of the predictions for targets with little sequence similarity to proteins of known structure that were judged by the assessors to be of higher quality. For two of these predictions, the sequence-structure alignment corresponded perfectly to the structural alignment (zero average shift), and for the third, the average shift was 0.1. This alignment accuracy entails an ideal starting point for homology modeling.