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Biomedical subjects

D Fischer

Publications and source records attributed to D Fischer.

At least 73 records · Page 4Linked to original sources
PubMed2000

Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture.

PURPOSE: To examine and quantify neuroprotective and neurite-promoting activity on retinal ganglion cells (RGCs) after injury of the lens. METHODS: In adult albino rats, penetrating lens injury was performed by intraocular injection. To test for injury-induced neuroprotective effects in vivo, fluorescence-prelabeled RGCs were axotomized by subsequent crush of the optic nerve (ON) with concomitant lens injury to cause cataract. The numbers of surviving RGCs were determined in retinal wholemounts and compared between the different experimental and control groups. To examine axonal regeneration in vivo, the ON was cut and replaced with an autologous piece of sciatic nerve (SN). Retinal ganglion cells with axons that had regenerated within the SN under lens injury or control conditions were retrogradely labeled with a fluorescent dye and counted on retinal wholemounts. Neurite regeneration was also studied in adult retinal explants obtained either after lens injury or without injury. The numbers of axons were determined after 1 and 2 days in culture. Putative neurotrophins (NTs) were studied within immunohistochemistry and Western blot analysis. RESULTS: Cataractogenic lens injury performed at the same time as ON crush resulted in highly significant rescue of 746 +/- 126 RGCs/mm(2) (mean +/- SD; approximately 39% of total RGCs) 14 days after injury compared with controls without injury or with injection of buffer into the vitreous body (30 +/- 18 RGCs/mm(2)). When lens injury was performed with a delay of 3 days after ON crush, 49% of RGCs survived, whereas delay of 5 days still rescued 45% of all RGCs. In the grafting paradigm virtually all surviving RGCs after lens injury appeared to have regenerated an axon within the SN graft (763 +/- 114 RGCs/mm(2) versus 79 +/- 17 RGCs/mm(2) in controls). This rate of regeneration corresponds to approximately 40% of all RGCs. In the regeneration paradigm in vitro preceding lens injury and ON crush 5 days previous resulted in a maximum of regeneration of 273 +/- 39 fibers/explant after 1 day and 574 +/- 38 fibers/explant after 2 days in vitro. In comparison, in control retinal pieces without lens injury 28 +/- 13 fibers/explant grew out at 1 day, and 97 +/- 37 fibers/explant grew out at 2 days in culture. Immunohistochemical and Western blot analysis of potential NTs in the injured lens revealed no expression of ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), NT-4, nerve growth factor (NGF), and basic fibroblast growth factor (bFGF). CONCLUSIONS: The findings indicate that the lens contains high neuroprotective and neuritogenic activity, which is not caused by NT. Compared with the data available in the literature, this neuroprotection is quantitatively among the highest ever reported within the adult rat visual system.

Animals↗
PubMed1999

Follow-up recommendations for patients with American Joint Committee on Cancer Stages I-III malignant melanoma.

BACKGROUND: Guidelines for follow-up of melanoma patients are not established. In 1987, a follow-up protocol was instituted at the Yale Melanoma Unit to improve upon the detection of disease recurrence in patients with American Joint Committee on Cancer Stage I-III cutaneous melanoma. The follow-up protocol consists of a patient education program and a surveillance schedule based on stage of disease. METHODS: The authors retrospectively reviewed the records of 373 patients who were seen and followed according to the surveillance protocol in the Yale Melanoma Unit between January 1988 and December 1994 to determine 1) the time interval between the initial visit and recurrence; 2) the most common method of detecting recurrences; 3) whether the surveillance schedule or the patient detects more recurrences, i.e., asymptomatic recurrences versus symptomatic recurrences; 4) whether there is any survival difference between asymptomatic and symptomatic recurrences. RESULTS: The 5-year overall survival rates for Stage I, II, and III patients were 95%, 72%, and 52%, respectively. Of the 78 recurrences, 44 (56%) were detected by physician-directed surveillance examinations and 34 (44%) by patients. Most recurrences were found within the first (47%) or second (32%) year of follow-up. The estimated 6-month hazard rates for death or recurrence were 0.0044, 0.0088, and 0.0278 for Stage I, II, and III patients, respectively. The group of asymptomatic patients with recurrence had a survival advantage over the symptomatic recurrence group. In addition, patients with locoregional recurrence had better survival than those with distant recurrence. CONCLUSIONS: Although many recurrences arise rapidly and are recognized early by patients, in this study more than half were found by surveillance examinations before symptoms were manifest. Based on the hazard ratio for recurrences, the authors recommend the following surveillance schedules in addition to the patient education program for detection of recurrences: 1) Stage I, annually; 2) Stage II, every 6 months for Years 1-2 and annually thereafter; 3) Stage III, every 3 months for Year 1, every 4 months for Year 2, and every 6 months for Years 3-5; 4) at Year 6 and beyond, all patients should have surveillance annually, due to the risk of late recurrence and/or metachronous multiple primaries.

Adolescent↗
PubMed1999

Characterization of the integration site of Yersinia high-pathogenicity island in Escherichia coli.

The high-pathogenicity island (HPI) of virulent Yersiniae consists of (i) a functional core encoding for biosynthesis and uptake of the siderophore yersiniabactin and (ii) a 5- to 13-kb AT-rich region of unknown function. This Yersinia HPI has been shown to be widely distributed among different pathotypes of Escherichia coli. In this study, the insertion site of the HPI was defined in three different E. coli strains: The enteroaggregative E. coli (EAggEC) strain 17-2, the uropathogenic (UPEC) E. coli strain 536, and the probiotic E. coli DSM6601. We demonstrated that in all three E. coli isolates the HPI is associated with the asnT tRNA (5'-extremity) and truncated in the AT-rich region (3'-extremity) since the 17-bp direct repeat (DR) of the asn tRNA that flanks the HPI in Yersinia is missing in E. coli. Moreover, in comparison to the HPI-negative E. coli K-12 strain, a uniform deletion must have taken place in the E. coli chromosome adjacent to the 3'-border of the HPI.

Escherichia coli↗
PubMed1999

GCH1 mutation in a patient with adult-onset oromandibular dystonia.

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.

Age of Onset↗
PubMed1999

Cell type specificity of the human endoglin promoter.

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.

3T3 Cells↗
PubMed1999

CAFASP-1: critical assessment of fully automated structure prediction methods.

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.

Algorithms↗
PubMed1999

Epitope discovery using bacteriophage display: the minimum epitope of an anti-IRBP antibody.

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.

Amino Acid Sequence↗
PubMed1999

Predicting structures for genome proteins.

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.

Amino Acid Sequence↗
PubMed1999

A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity.

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.

Animals↗
PubMed1999

Neonatal sepsis: a challenge in hemostaseology.

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.

Animals↗
PubMed1999

Effect of age on variability of parotid salivary gland flow rates over time.

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.

Adult↗
PubMed1999

Analysis of heregulin symmetry by weighted evolutionary tracing.

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.

Amino Acid Sequence↗