[Why should the ophthalmologic part of medical education be reduced?].
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Humans↗
Biomedical subjects
K Wilke
Publications and source records attributed to K Wilke.
Diagnosis of haploidy and triploidy based on measurement of gene copy number by real-time PCR.
We report the development of a method for diagnosis of heterozygous deletions or duplications based on measurement of gene copy number. The method involves amplifications of a test locus with unknown copy number and a reference locus with known copy number using real-time PCR. Progress of the PCR reactions is monitored using fluorigenic probes and a real-time fluorescence detection system. For each reaction, the number of cycles is measured at which a defined threshold fluorescence emission is reached. Using standard curves, the copy number of the test DNA relative to a common standard DNA is determined for each locus. From the ratio of the relative copy numbers, the genomic copy number of the test locus is determined. In order to demonstrate the accuracy and reliability of the method for genetic testing, we analyzed 43 patients with hereditary neuropathy with liability to pressure palsies (HNPP), containing a heterozygous deletion of a 1.5 Mb region on chromosome 17p11.2-p12, eight patients with Charcot-Marie-Tooth disease, containing a heterozygous duplication of the same genomic region, and 50 normal control individuals. As a test locus we analyzed the PMP22 gene located within the 1.5 Mb region. The genomic copy number of the test locus was precisely measured, and the presence or absence of the genomic deletion or duplication was unambiguously diagnosed in all individuals.
Charcot-Marie-Tooth Disease↗
MHC expression in fragment and full-thickness allogeneic embryonic retinal transplants.
BACKGROUND: The study was carried out to evaluate the expression of major histocompatibility complex (MHC) molecules in retinal transplants with different tissue integrity. METHODS: Twelve adult rabbits received an allogeneic subretinal neuroretinal transplant, in the form of either fragmented embryonic cells or a complete full-thickness embryonic retina. A controlled transvitreal approach was used for both transplantation types. The grafts were examined histologically after 31 or 49 days with hematoxylin and eosin staining and immunohistochemical analysis of MHC class I and class II expression. RESULTS: All five fragment transplants developed into rosettes. Two of them displayed MHC class I-labeled cells, and four MHC class II-labeled cells. The cells were concentrated on the scleral side of the graft, and there was also a marked increase of labeled cells in the choroid. MHC labeling was often associated with defects in the retinal pigment epithelium. Six of the seven full-thickness grafts displayed a laminated morphology with well-developed retinal layers. The seventh consisted of rosettes. None of these grafts displayed MHC class I- or class II-labeled cells. CONCLUSIONS: The findings suggest that host immune response against fragmented and intact neuroretinal grafts is different, indicating tissue integrity as one factor affecting graft-host immune interactions. The absence of immune response in full-thickness grafts is encouraging and important in the struggle to find therapies for retinal degenerative disease.
Animals↗
Beta-thalassemia in the German population: mediterranean, Asian and novel mutations. Mutations in brief no.228. Online.
The beta-thalassemia mutations of 13 unrelated heterozygous Germans who remained unidentified in a previous study of 40 subjects were investigated at the DNA level. Two Mediterranean, one Asian and three novel mutations (CD6 -G, CDs 108 /112-12nt, CDs 130/131 + GCCT) were identified. Altogether, in 30 of the 35 subjects (86%) in which a mutation in the beta-globin gene was identified, the mutation was of Mediterranean origin. The geographical distribution suggests recent migration from the Mediterranean region as cause of the high proportion of frequent Mediterranean beta-thalassemia mutations in the German population. Our results support the notion that the majority of beta-thalassemia genes in the western and central European population are of Mediterranean origin.
DNA Mutational Analysis↗
Different ocular abnormalities in individuals of a three-generation family caused by a new nonsense mutation in the PST domain of the PAX6 gene. Mutations in brief no. 189. Online.
PAX6 is a candidate gene for familial aniridia. We have carried out a mutational analysis of the PAX6 gene in a three-generation family from Germany, containing 5 individuals affected with ocular abnormalities. In all affected individuals, a heterozygous mutation was detected in the PAX6 gene, exchanging tyrosine 369 by a stop codon. The mutation is located in the 3' moiety of the PST domain, at the C terminus of the PAX6 protein. In the affected family members, the same heterozygous mutation leads to distinct phenotypes of varying severity. Most notably, no aniridia was observed in one of the family members carrying the mutation, although other ocular abnormalities (underdeveloped iris and cataracts) were present. We discuss the possibility that small C terminal truncations of the PAX6 protein might lead to less severe or more divergent phenotypes than trancations at internal positions.
Aniridia↗
Molecular diagnostics of 15 hemophilia A patients: characterization of eight novel mutations in the factor VIII gene, two of which result in exon skipping.
The X-linked bleeding disorder hemophilia A is caused by mutations in the coagulation factor VIII gene. A high frequency of de novo mutations and the large size of this gene complicate the molecular diagnostic of hemophilia A. Characterization of mutations, however, may help identify amino acids or regions with essential functional or structural properties and thereby clarify the mechanism of pathogenesis. In the present study, we describe the identification of 15 mutations in the factor VIII gene, of which eight are novel. Among the patients with severe hemophilia A, two splice mutations (IVS5-3 and IVS19-2), a 4-bp deletion ((TACA) at codon 1215, and a missense mutation G1850V have been characterized. The missense mutations G479R, R531C, V537D, N2129S and I2190N were found for five patients with a moderate course of hemophilia A disease. A silent mutation resulting in activation of a cryptic acceptor splice site within exon 11 and four other missense mutations Y114C, R1689H, R2150H (2x), M2164V have been identified for six patients with mild hemophilia A.
Animals↗
A gene in human chromosome band Xq28 (GABRE) defines a putative new subunit class of the GABAA neurotransmitter receptor.
We have isolated and sequenced a novel human gene (GABRE) of the GABAA neurotransmitter receptor family. A cDNA sequence of the gene coding for a 506 amino acid protein was identified, representing a member of a putative new class (epsilon) of the GABAA receptor. The gene is transcribed at least at low level in several different tissues, with the highest levels being detected in adult heart and placenta. Alternative splicing of GABRE transcripts isolated from different tissues was observed at multiple positions of the gene, yielding an unusually complex variety of cDNA variants. The structure of the 5' region of most cDNAs is compatible with expression of protein sequence epsilon only in adult brain, whereas in other tissues, the majority of transcripts code for truncated protein sequences. The GABRE gene extends over 14 kb and is clustered together with the alpha 3 and the putative beta 4 GABAA receptor subunit genes in an approximately 0.8-Mb interval in chromosome band Xq28, located in the candidate regions of two different neurologic diseases. Based on features of conservation of protein sequences, gene structure, and genomic organization of GABAA receptor gene clusters, we propose that the epsilon and gamma subunit genes have a common ancestor and that GABAA receptor gene clusters in the human genome have diverged by multiple duplication events of an ancestral gene cluster containing one each alpha, beta, and gamma/epsilon precursor gene.
Adult↗
Isolation of human and mouse HMG2a cDNAs: evidence for an HMG2a-specific 3' untranslated region.
We have isolated cDNAs of the human gene for high mobility group protein HMG2a, using the method of direct cDNA selection. The gene maps to chromosome band Xq28, and is located within 40 kb from marker DXS1684, at a distance of 5.4 Mb from the telomere. The deduced human HMG2a protein sequence has a length of 199 amino acids and is 97% identical to the sequence of chicken HMG2a. The 3' untranslated regions of the HMG2a gene in both species are highly homologous (87% identical nucleotides), and are even more conserved than the coding sequences (84% identical nucleotides). In addition, a partial cDNA sequence of the putative HMG2a gene from mouse was identified. The 3' untranslated regions from human and mouse are 90% identical. We conclude that the 3' untranslated sequences have been under strong selective pressure during evolution. Whereas expression of the chicken HMG2a gene has previously been demonstrated in liver of newly hatched chicken, the human HMG2a gene is transcribed mainly in placenta.
Amino Acid Sequence↗
The melanoma antigen gene (MAGE) family is clustered in the chromosomal band Xq28.
The melanoma antigen gene (MAGE) family comprises 12 known genes, of which 6 are expressed in tumors. In the course of a systematic analysis of transcripts in Xq28, we have identified cDNAs related to different MAGE genes. Analysis of cell hybrids, ordered YACs, and cosmids showed that all MAGE genes are located in Xq28 and are clustered in three main intervals within 3.5 Mb. The six genes expressed in tumors are contained in the two intervals closest to the telomere and are highly homologous to each other. Analysis of different species suggests that human MAGE sequences are conserved in primates, but less well conserved in other vertebrate species.
Animals↗
Reversed ratio of color-specific cones in rabbit retinal cell transplants.
Recently, we have reported on the emergence of various retinal cell types in embryonic rabbit retina transplanted to adult rabbits. When comparing the relative numbers of the spectrally different cone types in the transplants to those in the host or age-matched control retinas, a surprising shift was observed. While in the normal rabbit retina the middle-wavelength-sensitive (M) cones are considerably more abundant than the short-wave-sensitive (S) cones, the S/M cone ratio was found to be the opposite in the graft. The number of rosettes containing only S-cones in high density was found to be considerably higher than that of M-cone rich rosettes. The number of S-cones also exceeded that of the M-cones in each rosette that contained both cell types. Our results were obtained from the systematic immunocytochemical analysis of 15 different transplants derived from transplantations of embryonic rabbit retinas into adult hosts of the same species. The emergence and proportion of the two cone types were followed between 14 and 63 days after transplantation (between 29 and 78 postconceptional days of the donor tissue). Sections from various parts of the transplants were reacted with the monoclonal antibodies COS-1 and OS-2, specific for the middle- and short-wavelength-sensitive cones, respectively. The explanation for the reverse cone ratio in these transplants is not known yet, however, the observed phenomenon may indicate differences between the specification of the two basic cone types.
Animals↗
Porcine (GT)n sequences: structure and association with dispersed and tandem repeats.
Loci containing (GT)n repeats were isolated from three different plasmid libraries with inserts of porcine genomic DNA between 140 and 200, 200 and 300, and 350 and 400 bp. Sequencing showed that the average repeat length and the fraction of perfect repeats were increased in the libraries containing longer inserts (> or = 200 bp). The polymorphism of (GT)n loci containing at least 10 repeat units was analyzed using the polymerase chain reaction and an automated DNA sequencer. Nearly all tested loci are polymorphic and can therefore be used as marker loci for gene mapping and for other applications. The (GT)n loci were categorized into three classes: (1) loci containing the (GT)n repeats associated with a SINE element, (2) loci containing the (GT)n repeats associated with one or more other simple repeats, and (3) loci containing (GT)n as the only detected repetitive element. At most loci of the first class, the (GT)n repeat was in a fixed configuration adjacent to the 3' end of the SINE. The findings support the notion of clustering of different repeat types in the mammalian genome.
Animals↗
Anti-endothelial antibodies in Takayasu arteritis.
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Autoantibodies↗
Development of cell markers in subretinal rabbit retinal transplants.
Retinas from embryonic rabbits at day E15 were transplanted to the subretinal space in adult rabbits. After survival times between 7 and 193 days, the rabbits were killed, and the transplants were processed for immunohistochemistry. The results show that subretinal transplants from embryonic rabbit retinas develop many, if not all, retinal neuronal types. The cells show approximately normal morphology and express a variety of cell-type-specific markers: photoreceptor cells express visual pigment proteins as identified by antibodies against rhodopsin (R2-15), color-specific cone pigments (COS-1, OS-2) and the cone specific antigen 50-1B11, rod bipolar cells express PKC, horizontal cells HPC-1 antigen and neurofilament 160 kDa, amacrine cells HPC-1 antigen, GABA and neurofilament 160 kDa, and glial cells express vimentin and glial fibrillary acidic protein. The high degree of rosette formation seen in many young grafts, diminishes with time; many transplant cells disappear, and the remaining cells present a less prominent formation of rosettes.
Animals↗
Interphotoreceptor matrix components in retinal cell transplants.
To further investigate the functional potential of retinal transplants we have used immunocytochemistry to study the distribution of four different interphotoreceptor matrix (IPM)-specific components in rabbit retinal transplants. The different components were: interphotoreceptor retinoid-binding protein (IRBP), chondroitin-6-sulfate, F22 antigen and peanut agglutinin (PNA) binding structures. IRBP acts as a retinoid-transport protein between the neural retina and the retinal pigment epithelium. Chondroitin-6-sulfate is a glycosaminoglycan and a part of the insoluble IPM skeleton. The identity and role of the F22 antigen is not known. However, it is a 250 kDa protein localized to specific extracellular compartments such as teh IPM. PNA is a lectin with a high binding affinity for D-galactose-beta (1-3) N-acetyl-D-galactosamine disaccharide linkages and binds to IPM domains surrounding cones, but not rods. The transplants (15-day-old embryonic rabbit retina) were placed between the neural retina and retinal pigment epithelium in adult hosts. The transplants developed the typical rosette formations with photoreceptors toward the center. IRBP labeling was distinct in the IPM in the host retina. However, no IRBP labeling could be detected in the transplants. The chondroitin-6-sulfate and F22 antibodies strongly labeled the IPM in the host retina and corresponding structures in the center of rosettes. A cone-specific labeling with PNA could be seen in the host retina. In the transplants, however, PNA labeling appeared in association with many more photoreceptors than in the host retina. There is no previous study available on the IPM in retinal cell transplants.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
A YAC clone map spanning 7.5 megabases of human chromosome band Xq28.
Xq28 has been of special interest in human genetics because a large number of diseases map to this region. As a step in the molecular analysis of the as yet uncloned disease genes, and as a test for the detailed analysis of larger regions of the genome, we have constructed YAC clone contigs covering the 7.5 Mb region between IDS to the telomere on the long arm of the human X chromosome. Contigs were assembled and verified by an integrated hybridization-based strategy. Data was combined from the physical map, from YAC and cosmid mapping experiments, and from the localization of specific transcripts in the region. Two gaps in the YAC map of 250 and 100 kb were covered in part by the aid of cosmid clones, but small gaps of 50 kb each remain. The cloned region is expected to contain yet unidentified genes for at least ten genetic diseases. The construction of ordered YAC clone contigs of Xq28 represents an important step in the molecular identification of these genes, and the further analysis of one of the genetically most interesting regions of the human genome.
Chromosomes, Artificial, Yeast↗
[Bacteria-reducing properties of copper-containing and non-copper-containing materials. II. Relationship between microbiocide effect of copper-containing materials and copper ion concentration after contamination with moist and dry hands].
The bactericide quality of different materials containing copper (copper, brass, brass-sheet) was tested and particularly the effect of humidity and copper-concentration was examined. In first examinations (I. communication) (19) the materials were contaminated by hands, which were dipped into a suspension of different microorganisms (Micrococcus luteus, Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa). The survival rate of the microorganisms on the surfaces was determined after defined exposure times (0, 5, 30, 60, 120 min). The following ways of contaminating the test materials were chosen: single contamination with wet hands, repeated contamination with wet and dry hands. In this examination the results were tested for statistical significance and in parallel experiments the copper-concentration on the surfaces of the materials was determined. Materials containing copper contaminated with wet hands show a significant bactericide effect after an exposure time of 120 min. The bactericide effect on Micrococcus luteus and Pseudomonas aeruginosa is inhibited after contamination with dry hands. This results can not be explained by a different copper-concentration on the surfaces of the materials, because a statistically significant difference of copper-ion concentrations between contamination with wet and dry hands could not be obtained.
Bacteria↗
[Bacterial reducing qualities of copper-containing and non-copper-containing materials. I. Contamination and sedimentation in humid and dry conditions].
In this examination the bactericide qualities of different materials were tested. Particularly the effect of humidity was examined. 3 materials containing copper ("copper", "brass" and "brass sheet") and 2 materials not containing copper ("polyamide B" and "high-grade steel") were tested. The surfaces of the 5 types of material were contaminated with a suspension of 2 different bacteria species (Staphylococcus aureus and Micrococcus luteus). After defined exposure times the survival rate of the microorganisms on the surfaces was determined by the standardized method of the "Guidelines of the German Society of Hygiene and Microbiology". Additionally the sedimentation of airborne microorganisms on the materials was tested by determining the number of colony-forming-units after defined exposure times. In further examinations the materials were contaminated by hands, which were dipped into a suspension of different microorganisms (Micrococcus luteus, Staphylococcus aureus, Enterococcus faecium, Escherichia coli, Enterobacter cloacae and Pseudomonas aeruginosa). The following ways of contaminating the test materials were chosen: single contamination with wet hands, repeated contamination with wet and dry hands. After defined exposure times the survival rate of the microorganisms on the surfaces was determined. The results show, a bactericide quality of copper depends on the percent of copper-content of the tested materials, exposure time, humidity, frequency of contamination and bacteria species. Humidity and only single contamination cause a reduction of bacteria. Reduction of gram-negative bacteria is more easily than reduction of gram-positive bacteria. Enterococcus faecium shows the highest, Pseudomonas aeruginosa the lowest reduction rate of bacteria in the following sequence: Enterococcus faecium, Escherichia coli, Enterobacter cloacae, Micrococcus luteus, Staphylococcus aureus and Pseudomonas aeruginosa.
Air Microbiology↗
Transplantation of embryonic retina to the subretinal space in rabbits.
Embryonic rabbit retina can be transplanted to the subretinal space of adult rabbit with a new method, which gives a high rate of successful short-term transplants. Embryonic (stage E 15) neural retina cells were injected through an incision just behind the sclerocorneal border with a thin (inner diameter 0.15-0.4 mm, outer diameter 0.3-0.5 mm) plastic tube attached to a specially designed instrument, by which the length of the protruding plastic tip could be controlled. The retina was penetrated from the vitreous side and the donor tissue was injected into the subretinal space. The cells survived in the host for at least 5 months, although the long-term survival rate tended to decrease. The transplanted cells matured and differentiated, forming an approximation of the layered, retinal structure with some anomalies (e.g. rosettes). The subretinal location offers an interesting and convenient way of studying the development of retinal cell transplants in rabbits. Large transplants can be produced, and the risk for failures due to erroneous vitreous placement is small.
Animals↗