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M Werner

Publications and source records attributed to M Werner.

At least 217 records · Page 12Linked to original sources
PubMed1996

Microwave pretreatment improves RNA-ISH in various formalin-fixed tissues using a uniform protocol.

RNA is situ hybridization technique (RNA-ISH) is hampered by formalin fixation of tissues. This necessitates a pretreatment step of enzymatic digestion. However, to achieve the best results, digestion times and concentrations of enzymes have to be adapted for every specimen in a cumbersome procedure. Microwave pretreatment (MP) of formalin-fixed tissues has developed to become a powerful tool in immunohistochemistry (IHC) in recent years. To evaluate whether MP could also be helpful for RNA-ISH, we compared MP and conventional enzymatic pretreatment systematically using different tissues and various fixation times. As a model for RNA-ISH we chose the detection of kappa/lambda light chain mRNA in tonsils and lymph nodes with follicular hyperplasia and lymph nodes of patients with lymphocyte predominant Hodgkin's Disease (LPHD). Signal intensity obtained after MP was at least as good or dramatically enhanced as that obtained with optimized, single case adapted conventional pretreatment, the morphology being much better preserved after MP. These results confirm MP as a suitable method to unify the staining protocols in RNA-ISH, regardless of the duration of formalin fixation. Based on our results we recommend MP as a reliable and inexpensive method to enhance, standardized and simplify RNA-ISH.

Formaldehyde↗
PubMed1996

Testis-specific protein, Y-encoded (TSPY) expression in testicular tissues.

TSPY, the 'testis-specific protein, Y-encoded', is the product of a tandem gene cluster on human proximal Yp. In order to gain insight into the function of this locus, we have analysed (I) the diversity of RNAs transcribed from the cluster, (II) the sequence homology of the deduced TSPY to other proteins, and (iii) its protein properties both in tissue extracts and in tissue sections, using a TSPY-specific antiserum. We have identified a set of distinct TSPY transcripts with diverse exon compositions. We show that TSPY has homology with other human and non-human proteins, including SET and NAP, factors that are suggested to play a role in DNA replication. Protein analysis revealed TSPY to occur mainly in a modified, putatively phosphorylated form. By immunostaining it was detected in distinct subsets of spermatogonia. TSPY was also strongly immunostained in early testicular carcinoma in situ (CIS), while seminomatous tumour cells stained less intensely. The spermatogonial cells of two XY-TFM-females gave a strong immune response. The data presented here point to a phosphorylation-dependent TSPY-function in early spermatogenesis, immediately prior to the spermatogonia-to-spermatocyte transition, and in early testicular tumorigenesis.

Amino Acid Sequence↗
PubMed1996

Fluorescence in situ hybridization (FISH) is a reliable diagnostic tool for detection of the 9;22 translocation.

The fluorescence in situ hybridization (FISH) technique for detection of the 9;22 translocation was compared with the "gold standard" of conventional cytogenetics. For this purpose, both methods were applied to 81 bone marrow aspirates and/or peripheral blood specimens comprising 50 CML cases and controls from 31 patients without CML. Independently, core biopsies of these 81 patients were investigated by three histopathologists. Conventional karyotype analysis from unstimulated bone marrow cells was successful in 71/81 cases and demonstrated the Ph-chromosome in 42/46 CML patients. With FISH, results were obtained in all 81 cases investigated, confirming fusion of the ber and abl genes in all cytogenetically Ph-positive patients. Among the five Ph-chromosome-negative specimens bcr/abl fusions were detected in only one patient. The percentage of cells found to be Ph-positive by both methods was correlated, but in individual cases considerable differences in the numbers of Ph-positive cells were observed. Different results may be due to selection of cells after in vitro cultivation predominantly. FISH proved to be a very reliable technique for specimens that do not contain dividing cells. With FISH, large numbers of cells can easily be scored which is an advantage compared to conventional cytogenetics. Therefore, this method is particularly suitable for those whose therapy is being monitored or a relapse is suspected. However, the FISH results should be evaluated critically with respect to the practical limit of sensitivity since non-specific fusion signals can also be observed in a small percentage of cells in non-CML cases. It is suggested that each laboratory define its own threshold of bcr/abl fusion signals for diagnosing Ph-positive CML by FISH.

Adolescent↗
PubMed1996

Preservation of functional and regulatory domains of expressed bcl-2 genes in non-Hodgkin's lymphoma.

The most common translocation in human lymphoma, t(14;18)(q32;q21), recombines the bcl-2 gene with the immunoglobulin (Ig) heavy-chain locus leading to the production of high levels of chimeric RNAs and the resulting 26 kDa bcl-2 protein. The oncogenic role of the bcl-2 gene has been shown by the suppression of a variety of programmed cell deaths (apoptosis). Bcl-2 is able to interact with other members of the bcl-2 family through at least one of its conserved dimerization domains. Although overproduction of the wild-type protein appears sufficient for conferring a selective growth or a survival advantage to hematopoietic cells, the mode of activation of the proto-oncogene remains to be elucidated. In a first step, we examined and quantitated the expression of the bcl-2 gene in primary biopsies of non-Hodgkin's lymphomas (NHL) as well as in cell lines derived from NHLs. The results show that bcl-2 expression is found in a variety of hematopoietic lineages, but is most strongly associated with the B cell lineage. Within the B cell lineage, the expression levels vary depending on the differentiation as well as on the t(14;18) rearranged status. The quantitative measurements show high steady-state mRNA levels in early and in t(14;18) arranged B cells, whereas bcl-2 expression decreases with further B cell maturation and differentiation. In a second step we analyzed the bcl-2 mRNA for secondary genetic alterations, which may alter regulatory regions rendering it more tumorigenic. For this purpose, we chose a combined RT-PCR/SSCP method in order to screen out mutations of alleles which are not expressed. Different migration patterns of SSCP products were found only in two cell lines and subsequent sequencing revealed that the functional domains are not affected. Our data suggest that the dimerization properties of this protein are preserved in tumor cells and that modifications of the bcl-2 gene by the somatic hypermutation mechanism are not involved and do not influence the pathobiology of NHL.

Base Sequence↗
PubMed1996

[Therapy-induced changes in osteosarcoma after neoadjuvant chemotherapy (COSS 86 Therapy Study). Correlation between morphologic findings and clinical follow-up].

207 osteosarcomas were examined morphologically after neoadjuvant chemotherapy according to the COSS-86 protocol using representative slides of one whole tumor plane. The rate of responders 63%. In relapse-free patients both the whole tumors and the vital areas there of were smaller than in patients with relapse during a follow-up period of 5 years. Within the subgroup of osteoblastic osteosarcomas, metastases were observed following smaller tumors than in chondroblastic osteosarcomas. Therefore, in addition to degree of regression, histological subtype and tumor size should be considered in the prognostic evaluation of osteosarcomas.

Adolescent↗
PubMed1996

[DNA cytometry of solitary and aneurysmal bone cysts and low malignancy and high malignancy central osteosarcomas. Current significance within the scope of morphologic diagnosis of intraosseous cystic and osteoblastic lesions].

DNA cytometric investigations of intraosseous cystic lesions and osteoblastic tumors may be helpful in morphological diagnosis. The detection of a DNA-aneuploid stemline supports the diagnosis of a high-grade malignant bone neoplasm, even in small biopsies. Solitary and aneurysmal bone cysts have DNA-diploid cell populations. Low-grade central osteosarcomas may show single-cell aneuploidies of varying extent as well as a DNA-diploid stemline. Their presence may be a sign of genetic instability within the population of neoplastic cells before a DNA-aneuploid stemline is established and should prompt careful postoperative observation.

Aneuploidy↗
PubMed1996

Light-chain mRNA in lymphocyte-predominant and mixed-cellularity Hodgkin's disease.

There is growing evidence for a B-cell lineage of lymphocyte-predominant Hodgkin's disease (1). To support this assumption, in situ hybridization techniques were used to detect immunoglobulin light-chain mRNA in 44 formalin-fixed specimens of Hodgkin's lymphoma (22 of lymphocyte-predominant Hodgkin's disease; 22 of mixed-cellularity Hodgkin's disease). In addition, immunoglobulin light chains were evaluated by polyclonal antisera. All specimens had been unequivocally diagnosed histologically by the referees of the German Hodgkin Trial and been immunophenotyped by monoclonal antibodies against CD15, CD20, CD30, and CD45. Light-chain mRNA coding either for kappa or for lambda could be detected by an enhanced in situ hybridization protocol using microwave heating in the lymphocytic and histiocytic cells of 14 (64%) of 22 specimens of lymphocyte-predominant Hodgkin's disease tested. None of the specimens, however, belonging to one of the classic subtypes of Hodgkin's disease (mixed-cellularity Hodgkin's disease) showed positivity for mRNA in the giant tumor cells. Our results support the idea that lymphocyte-predominant Hodgkin's disease represents a B-cell malignancy that is a entity separate from classic Hodgkin's disease. Diverging results of former studies in assessing light-chain mRNA in lymphocytic and histiocytic cells probably reflect problems with the detection threshold, i.e., the sensitivity of the techniques applied.

Hodgkin Disease↗
PubMed1996

[Cystic degeneration of the adventitia of the popliteal artery after PTA of the superficial femoral artery].

Cystic adventitial degeneration is rare. Most frequently it occurs in the popliteal artery, occasionally it may be found in other arteries and even in veins. It is always localized in joint-adjacent vessel areas and appears to originate in ectopic synovial tissue occasionally with a stemlike connection with the adjacent joint. The diagnosis of a cystic adventitial degeneration should be considered in cases of isolated stenosis or occlusion of the popliteal artery. Realtime ultrasound or computerized tomography may help to establish the diagnosis. The treatment of choice is surgival removal of the cysts or reconstruction with an autologous vein interponate. A rare case of simultaneous occurrence of cystic adventitial degeneration and arteriosclerotic occlusive disease is described.

Angioplasty, Balloon↗
PubMed1995

Detection of p53 gene alteration in renal-cell cancer by micropreparation techniques of tumor specimens.

Alterations in the p53 tumor-suppressor gene have been identified in a variety of human malignancies, including renal-cell cancer. A technique for the isolation of tumor areas from tissue specimens to analyze formalin-fixed and paraffin-embedded tumors and try to avoid a disturbance of the results due to genetic background signal by the presence of tumor-infiltrating lymphocytes, was established. The presence of lymphocytes within the tumor areas investigated was determined by immunohistochemical staining for CD3, a lymphatic surface antigen. Following the isolation of about 100-200 tumor cells, PCR-directed molecular genetic analysis was performed. A highly informative allelotyping approach for the detection of loss of heterozygosity (LOH), determining BstU1- and VNTR-polymorphisms, a 100-bp marker directly localized in intron 1 of the p53 gene, as well as screening for mutations by single-strand conformation polymorphism analysis (SSCP) in exons 5-8, were used. Out of 44 renal-cancer specimens, 33 (75%) were informative for PCR-directed RFLP-analysis. Allelic loss at the p53 gene locus was observed in 10 of 33 cases (33%). No correlation between p53 gene alteration and T-stage, histological grade or histological differentiation could be observed. Alterations in the p53 gene, as detected by a molecular genetic as well as an immunohistochemical approach, were correlated to overall survival. During univariate analysis histological grade, lymphnode status and the presence of distant metastases could be identified as prognostic parameters for overall survival. During multivariate analysis none of the factors investigated remained an independent prognosticator for survival. Summarizing these results, it seems unlikely that p53 gene alterations will serve as an important new factor for the clinical prognosis of patients with renal-cell cancer.

Adult↗
PubMed1995

[Ectopic thyroid tissue in the heart--two case reports].

A 61-year-old previously healthy woman had been suffering from a dry cough for several weeks. On auscultation she had a 3/6 holosystolic murmur. The electrocardiogram (ECG) showed incomplete right bundle branch block (IRBBB) and abnormal repolarisation. Conventional and oesophageal echocardiography revealed a right ventricular tumour and significant coronary heart disease. At operation a tumour, arising from the interventricular septum and histologically consisting of thyroid tissue, was resected. In addition, the left mammary artery was anastomosed to the anterior interventricular branch of the left coronary artery. A 34-year-old woman developed increasing exertion-independent dyspnoea. For years she had had occasional attacks of palpitations and restlessness, once even a syncope. The ECG showed IRBBB, echocardiography demonstrated a tumour attached to the right side of the interventricular septum. The tumour was removed and found histologically to consist of colloid-containing thyroid follicles. Both tumours were benign. 45 and 36 months, respectively, later both patients are free of symptoms.

Adult↗
PubMed1995

A universally conserved region of the largest subunit participates in the active site of RNA polymerase III.

The largest subunits of the three eukaryotic nuclear RNA polymerase present extensive sequence homology with the beta' subunit of the bacterial enzymes over five major co-linear regions. Region d is the most highly conserved and contains a motif, (Y/F)NADFDGD(E/Q)M(N/A), which is invariant in all multimeric RNA polymerases. An extensive mutagenesis of that region in yeast RNA polymerase III led to a vast majority (16/22) of lethal single-site substitutions. A few conditional mutations were also obtained. One of them, rpc160-112, corresponds to a double substitution (T506I, N509Y) and has a slow growth phenotype at 25 degrees C. RNA polymerase III from the mutant rpc160-112 was severely impaired in its ability to transcribe a tRNA gene in vitro. The transcription defect did not originate from a deficiency in transcription complex formation and RNA chain initiation, but was mainly due to a reduced elongation rate. Under conditions of substrate limitation, the mutant enzyme showed increased pausing at the intrinsic pause sites of the SUP4 tRNA gene and an increased rate of slippage of nascent RNA, as compared with the wild-type enzyme. The enzyme defect was also detectable with poly[d(A-T)] as template, in the presence of saturating DNA, ATP and UTP concentrations. The mutant enzyme behavior is best explained by a distortion of the active site near the growing point of the RNA product.

Adenosine Triphosphate↗
PubMed1995

The effect of base mismatches in the substrate recognition helices of hammerhead ribozymes on binding and catalysis.

The ability of the hammerhead ribozyme to distinguish between matched and mismatched substrates was evaluated using two kinetically defined ribozymes that differed in the length and sequence of the substrate recognition helices. A mismatch in the innermost base pair of helix I affected k2, the chemical cleavage step, while more distal mismatches had no such effect. In contrast, mismatches in any of the four innermost base pairs of helix III affected k2. Chase experiments indicated that mismatches also increased the rate of substrate dissociation by at least 20-100-fold, as expected from the stabilities of RNA helices.

Base Composition↗
PubMed1995

Nephrotoxicity of xenobiotics.

Nephrotoxicity can be grouped by the xenobiotics place of action, by the clinical presentation or by the generic toxic effect. The latter can be dose related, indirect, idiosyncratic or allergic. Nephrotoxicity of lithium, demeclocycline, aminoglycosides, cyclosporine, mercuric ion, nonsteroidal anti-inflammatory drugs, methoxyflurane, ethylene glycol, D-penicillamine and methicillin is reviewed in light of all these three viewpoints, but emphasis is on toxic mechanisms.

Aminoglycosides↗
PubMed1995

A mutation in the C31 subunit of Saccharomyces cerevisiae RNA polymerase III affects transcription initiation.

The C31 subunit belongs to a complex of three subunits (C31, C34 and C82) specific to RNA polymerase (pol) III that have no counterparts in other RNA polymerases. This complex is thought to play a role in transcription initiation since it interacts with the general initiation factor TFIIIB via subunit C34. We have obtained a conditional mutation of pol III by partially deleting the acidic C-terminus of the C31 subunit. A Saccharomyces cerevisiae strain carrying this truncated C31 subunit is impaired in in vivo transcription of tRNAs and failed to grow at 37 degrees C. This conditional growth phenotype was suppressed by overexpression of the gene coding for the largest subunit of pol III (C160), suggesting an interaction between C160 and C31. The mutant pol III enzyme transcribed non-specific templates at wild-type rates in vitro, but was impaired in its capacity to transcribe tRNA genes in the presence of general initiation factors. Transcription initiation, but not termination or recycling of the enzyme, was affected in the mutant, suggesting that it could be altered on interaction with initiation factors or on the formation of the open complex. Interestingly, the C-terminal deletion was also suppressed by a high gene dosage of the DED1 gene encoding a putative helicase.

Alleles↗
PubMed1995

Appropriate utilization and cost control of the hospital laboratory: panel testing and repeat orders.

Analysis of hospital laboratory utilization shows a bi-exponential relationship between the many different test combinations ordered and their frequency of use. This suggests two opposite strategies to reduce test volume. Policy could discourage use of the most popular request combinations to reduce volume, or policy could discourage use of the least popular request combinations to reduce order variety. Analysis of biochemical test orders further suggests that the largest part of test requests is associated with a small number of pathophysiological issues, namely those involving electrolytes, fluid balance, blood lipids, heart, liver, kidney or bone disease. Therefore, efficiency results when a limited number of standard test panels addressing these issues reduce order variety by discouraging infrequently used test combinations. Standard panels can be developed from an analysis of the most popular existing orders, accepting some degree of discrepancy among otherwise overlapping requests. This empiric approach avoids sterile debate about the composition of ideal panels. Since the resources of the laboratory, the mix of patients served and local medical tradition affect test orders, each institution providing health care must develop the standard panels best suited to its needs. Analysis of standing repeat orders suggests they are often used to anticipate potential pathology in stable patients. Therefore, economy results when test volume is reduced by discouraging series of futile repeat tests with unchanging normal results. Unfortunately, an empiric approach to regulate repeat orders is not available and only a more cumbersome process to build a locally agreed upon consensus between providers and users of laboratory services offers itself as a promising solution.

Alabama↗
PubMed1995

Interphase cytogenetic analysis of distinct X-chromosomal translocation breakpoints in synovial sarcoma.

Synovial sarcomas show a specific translocation involving chromosomes X and 18, t(X;18)(p11.2;q11.2). Two distinct X-chromosomal breakpoints occur in different synovial sarcoma tumour samples. These breakpoints are located within two related genomic regions containing ornithine aminotransferase-like sequences, termed OATL1 and OATL2. Preliminary observations indicated the potential correlation of OATL1-associated breakpoints with biphasic tumours and OATL2-associated breakpoints with monophasic fibrous tumours. The present study uses interphase cytogenetics to investigate the nature of chromosomal aberrations in frozen synovial sarcoma tissue samples. Two-colour fluorescence in situ hybridization (FISH) was performed using probes specific for the centromeres of chromosome X or 18, along with yeast artificial chromosome probes corresponding to the distinct breakpoint regions on Xp. One monophasic epithelial and two monophasic fibrous synovial sarcomas showed an OATL2-associated breakpoint, while a biphasic tumour revealed a hybridization pattern indicating a breakpoint within the OATL1 region. These results confirm our previous suggestion of a relationship between alternative breakpoints in Xp11.2 and different histological phenotypes observed in synovial sarcomas. They also demonstrate the utility of the two-colour hybridization approach for the identification of chromosomal changes in interphase nuclei isolated from frozen tissues.

Chromosomes, Human, Pair 18↗