PubMed Health⌕ Search

Biomedical subjects

H Tanke

Publications and source records attributed to H Tanke.

17 recordsLinked to original sources
PubMed2001

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR.

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype-phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNA(Lys) mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

Cell Line↗
PubMed2001

Use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: a rapid, sensitive DNA test to identify human papillomavirus type 16 infection.

BACKGROUND: A lateral-flow (LF) device using the new reporter up-converting phosphor technology (UPT) was applied to DNA (hybridization) assays for the detection of specific nucleic acid sequences, thereby aiming to perform the test outside well-equipped laboratories. The methodology reported here is sensitive and provides a rapid alternative for more elaborate gel electrophoresis and Southern blotting. In a preliminary study, it was applied to screen for the presence of human papillomavirus type 16 (HPV16) in a defined series of cervical carcinomas. METHODS: A LF assay was used to capture haptenized DNA molecules and hybrids, which were immunolabeled (before LF) with 400-nm UPT particles. These particles emit visible light after excitation with infrared in a process called up-conversion. Because up-conversion occurs in only the phosphor lattice, autofluorescence of other assay components is virtually nonexistent. RESULTS: The use of the UPT reporter in LF-DNA tests, as compared with colloidal gold, improved the detection limit at least 100-fold. UPT LF-DNA tests were successfully applied to detect (in a blind test) the presence of HPV16 in DNA extracts obtained from cervical carcinomas. Test results matched 100% with previous characterization of these carcinomas. CONCLUSIONS: The use of UPT in LF assays to detect specific nucleic acids provides low attamole-range sensitivity. Hybridization and consecutive detection of PCR-amplified HPV16 sequences were successful in a background of 10 microg of fish-sperm DNA. The sensitivity of UPT detection in these complex mixtures indicates that detection of viral infections without PCR or other amplification technique is achievable.

Antibodies, Monoclonal↗
PubMed2000

Individuals with abnormal phenotype and normal G-banding karyotype: improvement and limitations in the diagnosis by the use of 24-colour FISH.

The simultaneous identification, by fluorescence in situ hybridisation (FISH), of each chromosome in a distinct colour became feasible a few years ago. The key question in the application of this and many other developments in molecular cytogenetics to clinical situations is whether the results add significant further information that is relevant to the diagnosis. So far, limited data exist regarding how much improvement the technique brings to the diagnosis of phenotypically abnormal individuals in whom no abnormalities have been detected by conventional G-banding analysis. Because of the lack of a conclusive diagnosis, genetic counselling, estimation of recurrence risk and prenatal diagnosis of these individuals and their relatives is problematic. We report a study with 24-colour whole-chromosome painting of 10 familial and 11 isolated cases with abnormal phenotypes and normal G-banding karyotypes. Previously undetected unbalanced translocations were revealed in two cases. The value and current cost-effectiveness of multicolour FISH for cytogenetic diagnosis is discussed.

Adolescent↗
PubMed1999

Recurrent integration of human papillomaviruses 16, 45, and 67 near translocation breakpoints in new cervical cancer cell lines.

Progressive chromosomal changes and integration of human papillomavirus (HPV) sequences mark the development of invasive cervical cancer. Chromosomal localization of HPV integration is essential to the study of genomic regions involved in HPV-induced pathogenesis. Yet, the available information about HPV integration loci is still limited, especially with respect to different HPV types. We have established cell lines from five cervical cancers with HPV-16, HPV-45, and HPV-67. We have determined HPV integration sites and karyotype abnormalities by using the multicolor combined binary ratio-fluorescence in situ hybridization method (Tanke et al.) with 24 chromosome-specific paints in combination with full-length HPV DNA probes. All cell lines were cytogenetically abnormal, and exhibited numerical and structural chromosomal deviations. HPV sequences were integrated at various (segments of) chromosomes. Duplicate integration sites were seen in all multiploid cell lines, suggesting that viral integration had preceded chromosomal endoreduplication. HPV-16 was found near the t(3p14.1-14.3;14) breakpoint in cervical squamous cell carcinoma (CSCC)-7 and mainly in episomal form in CSCC-1. HPV-45 was integrated near 3q26-29 in cervical (adeno or adenosquamous) carcinoma (CC)-8 and near 1q21-23 as well as near the t(1q21;22q13) breakpoint in CC-10A and CC-10B variant lines. HPV-67 was localized near the breakpoint of t(3p23-26;13q22-31) in CC-11. Southern blot analysis showed that, except for CSCC-1, the physical state of HPV in the cell lines was the same as in the original tumor lesions. This set of six cervical cancer cell lines included three lines with HPV-45, a major non-Western high-risk HPV type, the first reported HPV-67-positive cell line, and two cell lines with integrated and episomal HPV-16 DNA, respectively. The novel combined binary ratio-fluorescence in situ hybridization technique enabled us to simultaneously map chromosomal rearrangements and HPV integration sites, thereby revealing recurrent integration near translocation junctions for all of these HPV types in the cell lines from three of the five primary tumors. The detection of multiple HPV integration sites at rearranged chromosomes at such high frequency in cervical cancer-derived cells may reflect events that are relevant to the development of cervical cancer.

Adult↗
PubMed1999

Chromosomal gains and losses in testicular germ cell tumors of adolescents and adults investigated by a modified comparative genomic hybridization approach.

Testicular germ cell tumors of adolescents and adults, both seminomas (SE) and nonseminomas (NS), are aneuploid, and classical karyotyping demonstrated a specific pattern of gains and losses. More recently, these data have been supported by in situ hybridization and comparative genomic hybridization (CGH) on a limited number of samples. Interpretation of CGH results is complicated by the intermediate ploidy of these tumors (3-4 n for SE and 2-3 n for NS). To circumvent this problem, this particular study was undertaken. CGH was performed on 8 SE and 10 NS, after which two single chromosome normalizations were applied, one for chromosome 4 (found to be associated to the lower ploidy level of the tumor) and one for chromosome 8 (found to be associated with the higher ploidy level of the tumor) Using this modified CGH interpretation method, chromosomal regions with a similar copy number of chromosome 4 and 8 were identified as belonging to the lower and higher ploidy level, and the regions below chromosome 4 and above chromosome 8 were identified as lost or gained outside the ploidy range of the tumor, respectively. Our results are in accordance with earlier findings, however they add novel data, including comparison of SE and NS. This approach reveals relevant information about the chromosomal constitution of testicular germ cell tumors, leading to a better understanding of the pathogenesis of these tumors.

Adolescent↗
PubMed1998

Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells.

The influence of fixation, air-drying and liquid-imaging on the morphology as well as on the viscoelasticity of malignant mesothelioma cells was studied by atomic force microscopy. In this study, dehydrated cells were more easily scanned and offered faster data recording than hydrated cells. However, the influence of fixation strength was more noticeable. Strong fixation induced flattening of the cytoplasm and loss of nuclear structure, resulting in a clearly visible cytoskeleton which could be easily seen as fibres orientated in the direction of the cell growth. By contrast, the morphology of hydrated cells was influenced to a lesser degree on fixation and showed an overall 'rounding' of the surface with vague, ill-defined structures. Nuclear areas of these samples were difficult to image. Viscoelasticity measurements also exhibited large differences. Dehydrated cells were much harder and showed a uniform indentation profile over the whole cell that was independent of fixation. Indentation on hydrated cells was large and depended on the height of the measuring spot, the submembranous structure and, to a lesser extent, on fixation. To calculate an overall 'cellular' viscoelasticity, different methods were tested on these samples. Indentations of multiple, randomly chosen points, covering the whole cell, were measured and averaged to yield a mean indentation score. We avoided the thin and shadowed areas since it was shown that these regions were less suited for measuring. Using this design, large viscoelasticity differences were found, on which the influence of the external parameters could be shown. In another set-up, layered imaging was tried. However, long data acquisition times caused cellular activation and rearrangement, making this scanning mode unsatisfactory.

Cells, Cultured↗
PubMed1998

Chromosomal constitution of human spermatocytic seminomas: comparative genomic hybridization supported by conventional and interphase cytogenetics.

No data on the chromosomal constitution of spermatocytic seminomas are available thus far because of their rarity. Ploidy analysis performed on paraffin-embedded cases showed varying results from (near-) diploid to aneuploid. We applied comparative genomic hybridization on four snap-frozen primary spermatocytic seminomas of three different patients. Conventional cytogenetic analysis was successful in one, and "interphase cytogenetics" with centromeric region-specific probes was applied to another. The results from comparative genomic hybridization showed almost exclusively numerical chromosomal aberrations, in agreement with the data from karyotyping. Despite the limited number of cases studied, a nonrandom pattern of chromosome imbalances was detected: chromosome 9 was gained in all spermatocytic seminomas. This suggests that that this aberration plays a role in the development of this cancer. Interphase cytogenetics shows that the copy number of most chromosomes ranges from two to four, with an average of near trisomic. This constitutes the first report on the chromosomal constitution of spermatocytic seminomas.

Aneuploidy↗
PubMed1996

Comparative scanning, transmission and atomic force microscopy of the microtubular cytoskeleton in fenestrated liver endothelial cells.

Endothelial fenestrae control the exchange of fluids, solutes and particles between the sinusoidal lumen and the microvillous surface of the parenchymal cells. Fenestrae have a critical dimension in the order of 150-200 nm, making it necessary to use microscopes with a resolution better than the light microscope. Comparative whole-mount preparations of isolated, purified and cultured rat liver sinusoidal endothelial cells (LEC) were studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). Examination of detergent-extracted LEC by SEM and TEM shows an integral cytoskeleton: sieve plates are delineated by a sieve plate-associated cytoskeleton ring and fenestrae by a fenestrae-associated cytoskeleton ring. By using microtubule altering agents we could demonstrate: (1) the architectural role of microtubules in arranging fenestrae, (2) the existence of a population of microtubules resistant against low temperature and colchicine, (3) the ability of LEC to shift the microtubule assembly-disassembly steady state under various conditions, (4) and the necessity of an intact microtubular cytoskeleton to support the increase in the number of fenestrae after cytochalasin B. Topographical examinations of AFM images revealed that sieve plates are delineated by elevated borders, probably projections of the underlying tubular cytoskeleton.

Animals↗
PubMed1992

HOME: highly optimized microscope environment.

The Highly Optimized Microscope Environment (HOME) is a computerized microscope designed to assist pathologists and cytotechnicians in clinical routine tasks. The prototype system consists of a IBM-PC compatible computer and a light microscope in which a built-in high-resolution computer display image is superimposed on the optical image of the specimen. Also, a manually operated encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on a slide to be uniquely defined and makes it possible to measure interactively lengths and areas larger than the size of the microscope field. Software, written in the C language and operating under the MS-DOS/MS-Windows environment, is controlled by means of a mouse-driven cursor moving over menu light-buttons displayed on the microscope image. The HOME microscope workstation is potentially useful in a wide range of applications such as i) tagging information on particular cells and tissue structures that can thus be accurately located and relocated, ii) performing morphometric measurement, differential counting, and stereological assessment of biological specimens, and iii) training and educating laboratory personnel. Finally, HOME will offer in the near future a user-friendly interface for automatic image processing of cells and tissue entities in interactively selected specimen areas.

Animals↗
PubMed1990

Early replication signals in nuclei of Chinese hamster ovary cells.

DNA replication sites generally known as replicon domains were resolved as individual replication signals in interphase nuclei of permeabilized Chinese hamster ovary cells by immunofluorescent microscopy. Biotin-11-dUTP was utilized as a tool to label newly replicated DNA in permeable cells and to study the distribution of nascent DNA in pulselabel and in pulsechase experiments. Active sites of DNA replication were visualized in exponentially growing cells and in synchronized cultures throughout the S phase. Fluorescent images of replication sites were analyzed by standard fluorescence microscopy and in three dimensions by confocal laser scanning microscopy. The rapid increase in number of discrete foci of newly replicated DNA is an indication that DNA synthesis starts at limited number of sites in mammalian nuclei rather than at thousands of foci at the same time.

Animals↗
PubMed1990

[The modular image analysis computer for assessment of prognosis in testicular tumors].

Since 1981 a total of 107 patients with non-seminomatous germ cell tumors of the testis (NSGCT) in clinical stage I were assigned to a "wait and see" protocol. At a median follow-up of 40 months (4-100) thirty seven pts. (35%) relapsed with 84% of these within the first year. Employing the Hedley-technique out of 50 primary tumor-tissues available nuclear suspensions were obtained, staining according to the DNA-Feulgen-procedure and evaluated by the modular image analysis computer (MIAC, Leitz, FRG). In 67% hyperpentaploidy was found in the cases with progression while only 23% exhibited a greater than 5c-rate in the NED-group. With logistic regression achieving a p-value of 0.0296 hyperpentaploidy has to be considered a significant prognosticator in NSGCT/CS I.

Dysgerminoma↗
PubMed1978

Identification of rosette-forming cells in permanent preparations.

A method is described for the identification of leucocytes in permanent cytocentrifuge rosette preparations made with the fluorescent methyl green pyronin-SITS (MPS) staining technique. The percentages of rosettes in stained slides agree well with the results obtained with the conventional haemocytometer method. Application of the MPS staining to cytocentrifuge preparations permits reliable morphological differentiation between free and rosetting leucocytes, and should be particularly valuable in studies on lymphocyte subpopulations in patients.

Centrifugation↗