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M Werner

Publications and source records attributed to M Werner.

At least 163 records · Page 9Linked to original sources
PubMed1998

Short oligonucleotides as external guide sequences for site-specific cleavage of RNA molecules with human RNase P.

Human RNase P recognizes a small model substrate consisting of only the 5' leader sequence, aminoacyl acceptor stem, and T stem and loop of a tRNA precursor. It was demonstrated here that a bimolecular construct in which the T loop is opened between G57 and A58 (tRNA numbering system) is still processed by RNase P. The strand that is cleaved can be considered the target RNA, whereas the other strand serves as an external guide sequence (EGS). The nucleotides corresponding to nt 58-60 in the T loop could be deleted without affecting cleavage of the substrate. Thus, the complete T loop can be replaced by the single-stranded sequence UUCG or UUCA (nt 55-57 in the T loop). The four nucleotides UUCR possibly form a structure that resembles the uridine turn in the T loop of tRNA. Because recognition by RNase P is independent of the helical sequence, this motif can be used for targeting RNA molecules for EGS-directed cleavage by human RNase P. Chemically modified EGSs with 2'-O-methyl groups also showed activity in inducing RNase P cleavage. Several 13-mer EGSs targeted to the 2.1-kb surface antigen mRNA of hepatitis B virus (HBV) were designed and tested using a co-transcriptional cleavage assay with a 2.1-kb HBV transcript. Some of the new EGSs were capable of inducing cleavage of the HBV RNA by RNase P.

Base Sequence↗
PubMed1998

Biotransformation, excretion, and nephrotoxicity of the hexachlorobutadiene metabolite (E)-N-acetyl-S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine sulfoxide.

Hexachlorobuta-1,3-diene (HCBD) is nephrotoxic in rodents. Its toxicity is based upon a multistep bioactivation pathway. Conjugation with glutathione by glutathione S-transferases to form (E)-S-(1,2,3,4,4-pentachlorobutadienyl)-L-glutathione (PCBG), further processing to the corresponding cysteine S-conjugate, and finally processing to a reactive thioketene are thought to be responsible for the observed nephrotoxic effects. A novel metabolite, identified as (E)-N-acetyl-S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine sulfoxide (N-AcPCBC-SO), was described after administration of [14C]HCBD to male Wistar rats. This metabolite is formed by sulfoxidation of N-acetyl-S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine (N-AcPCBC) mediated by cytochrome P450 3A and has been found to be cytotoxic to proximal tubular cells in vitro without activation by beta-lyase. In rats, given HCBD in vivo, only one diastereomer of the sulfoxide is excreted; however, in rat hepatic microsomes two diastereomers, (R)- and (S)-N-AcPCBC-SO, are formed. This study focuses on the mechanisms responsible for this discrepancy and on a possible contribution of N-AcPCBC-SO to the nephrotoxicity of HCBD in vivo. (R,S)-N-AcPCBC-SO (1:1 mixture of both diastereomers) and N-acetyl-alpha-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-d, L-cysteine sulfoxide (alpha-Me-N-AcPCBC-SO) were administered iv to male and female Wistar rats (20, 40, and 80 micromol/kg of body weight). alpha-Me-N-AcPCBC-SO cannot be cleaved by cysteine conjugate beta-lyase even if alpha-Me-N-AcPCBC-SO is deacetylated by acylases. Excretion of gamma-glutamyltranspeptidase, protein, and glucose in the urine, indicative for kidney damage, and histopathological examination of the kidneys showed marked differences in the renal damage in male and female rats after application of N-AcPCBC-SO and alpha-Me-N-AcPCBC-SO. Necroses of the kidney tubules were only found in male, but not female, rats. Major sex-specific differences were observed in the elimination of sulfoxides; the (R)-isomer was excreted in a 5-10-fold excess to the (S)-isomer after application of (R,S)-N-AcPCBC-SO. After purification, both isomers were administered to male rats resulting in the urinary excretion of (R)-N-AcPCBC-SO after giving the (R)-isomer; treatment with (S)-N-AcPCBC-SO, however, revealed the formation of (S)-N-acetyl-S-(2-glycinylcystein-S-yl-1,3,4, 4-tetrachlorobutadienyl)-L-cysteine. The results show major sex-specific differences in the nephrotoxic potency of N-AcPCBC-SO and alpha-Me-N-AcPCBC-SO. However, both N-AcPCBC-SO and alpha-Me-N-AcPCBC-SO are nephrotoxic in males, suggesting the formation of a vinyl sulfoxide as an additional, beta-lyase-independent mechanism in HCBD-caused nephrotoxicity.

Animals↗
PubMed1998

Long-term results of the co-operative German-Austrian-Swiss osteosarcoma study group's protocol COSS-86 of intensive multidrug chemotherapy and surgery for osteosarcoma of the limbs.

BACKGROUND: In an effort to intensify osteosarcoma therapy, systemic ifosfamide was added pre- and postoperatively to an already aggressive three-drug regimen. In a subgroup of patients, loco-regional treatment intensification was attempted by using the intraarterial route to give cisplatin. PATIENTS AND METHODS: Patients < or = 40 years at diagnosis of a localised, de novo high-grade central extremity osteosarcoma were eligible for inclusion into study COSS-86 if registered within three weeks from biopsy. Doxorubicin, high-dose methotrexate, and cisplatin were given to all patients. Patients who fulfilled one or more of three defined high-risk criteria received early systemic treatment intensification by adding ifosfamide as the fourth agent. Preoperatively, these high-risk patients received cisplatin either intraarterially or intravenously. RESULTS: 171 eligible patients were entered, of which 128 were stratified into the high-risk group. When all 171 were analysed by intention-to-treat, actuarial overall and event-free survival rates at ten years were 72% and 66%, respectively. No benefit of intraarterial cisplatin application was detected. Cumulative treatment toxicity was considerable. CONCLUSIONS: In a multicenter setting, intensive treatment of osteosarcoma according to protocol COSS-86 led to long-term disease-free survival for two thirds of patients. We saw no benefit of using the intraarterial route to administer cisplatin.

Adolescent↗
PubMed1998

Direct and rapid induction of migration in human CD4+ T lymphocytes within three-dimensional collagen matrices mediated by signalling via CD3 and/or CD2.

Specific activation of T cells requires stable cell-cell interaction; however, little is known how the transition from a previously motile state into a sessile state following activation is achieved. We investigated the direct effect of T-cell receptor (TCR)/CD3 complex engagement and/or stimulation of the accessory molecule CD2 on the locomotion of peripheral human T cells within three-dimensional (3-D) collagen lattices. Simultaneous engagement of CD3 and CD2 very potently stimulated T-cell migration, resulting in the recruitment of previously sessile cells (about 24% of the total population was additionally recruited) as well as an increase in the mean duration of active locomotion. This induction of migration was accompanied by an increased tyrosine phosphorylation of a 125 000 MW substrate corresponding to the focal adhesion kinase. Using confocal laser scanning microscopy we detected antibody-induced receptor capping into the uropod of migrating T cells whereas untreated control cells displayed an even distribution of CD3 and CD2 on the cell surface. Less pronounced induction of locomotion was achieved following triggering of CD3 or CD2 alone. Thus, in 3-D collagen lattices specific T-cell activation did not lead to cessation of cellular migration but rather induced cytoskeletal activity that ultimately resulted in vigorous locomotory activity.

Antibodies, Monoclonal↗
PubMed1998

A birth in non-mosaic Klinefelter's syndrome after testicular fine needle aspiration, intracytoplasmic sperm injection and preimplantation genetic diagnosis.

Non-mosaic Klinefelter patients are generally azoospermic due to primary testicular failure. Nevertheless, in some cases, testicular spermatozoa may be recovered and utilized to fertilize oocytes via intracytoplasmic sperm injection (ICSI). As the risk for an increased number of gonosomes in these spermatozoa is unclear, preimplantation genetic diagnosis (PGD) may be attempted in the resulting embryos. In the present study, we report our experience with the combined approach of sperm retrieval by testicular fine needle aspiration (FNA), ICSI and PGD in seven consecutive non-mosaic Klinefelter individuals. In four patients, between one and five spermatozoa were retrieved in five out of nine consecutive attempts. In a fifth patient, only 10 round spermatids could be isolated. Mature spermatozoa were injected into a total of 16 metaphase-II oocytes, of which 11 (69%) remained intact. Two distinct pronuclei (2PN) were observed in four oocytes (36%) while a single pronucleus (1PN) was documented in two oocytes. Five cleavage stage embryos developed from the oocytes of two couples. Upon the request of one couple, their three embryos (two derived from 1PN oocytes) were transferred without PGD but pregnancy was not achieved. PGD by fluorescence in-situ hybridization (FISH) was performed in the two embryos of the other couple which were derived from normal fertilization. PGD results of one embryo were 18,18,X,X,Y, the embryo was not transferred and FISH analysis of the remaining blastomeres identified variable chromosome numbers in the nuclei. The second embryo was diagnosed as normal and was transferred, resulting in a successful pregnancy and birth. In conclusion, the results of this report indicate that a pregnancy and birth may be attained in azoospermic non-mosaic Klinefelter individuals by testicular FNA combined with ICSI. Due to the unknown risk of gonosomes aneuploidy in embryos from Klinefelter patients, PGD or prenatal diagnosis should be recommended.

Adult↗
PubMed1998

Intracytoplasmic sperm injection allows fertilization and development of a chromosomally balanced embryo from a binovular zona pellucida.

A binovular zona pellucida was found in two in-vitro fertilization (IVF) treatment cycles. In both cases, two oocytes of slightly unequal size were enclosed within a single zona pellucida, the larger oocyte appearing as a metaphase II oocyte while the smaller one as an immature oocyte with a germinal vesicle. Intracytoplasmic sperm injection performed in the mature oocyte of each pair led to normal fertilization and embryonic development in both cases. Results of genetic analysis performed by fluorescence in-situ hybridization in one of the two treatment cycles were consistent with a diploid chromosomal status of both the non-injected immature oocyte as well as the embryo which developed following the microinjection. These results indicate that, in this case, the binovular zona pellucida was most probably created when granulosa cells failed to separate two distinct oocytes during follicular formation. It may also imply that selective fertilization of a single mature oocyte in a binovular zona pellucida by intracytoplasmic sperm injection can lead to the development of a chromosomally balanced embryo and can prevent the undesired consequences that may result if the two oocytes are fertilized in the course of standard IVF.

Adult↗
PubMed1998

Expression of proliferation associated antigens and detection of numerical chromosome aberrations in primary human liver tumours: relevance to tumour characteristics and prognosis.

AIMS: To assess cell proliferation and the presence of numerical chromosome aberrations involving chromosomes 1 and 8 in benign and malignant liver tumours. METHODS: Cell proliferation was studied immunohistochemically in paraffin wax embedded material from 62 primary liver tumours (20 hepatocellular carcinomas, 16 cholangiocellular carcinomas, 15 liver cell adenomas, 11 focal nodular hyperplasias), and the results were compared with histological characteristics and clinical data. Copy numbers of chromosomes 1 and 8 were assessed by interphase fluorescence in situ hybridisation (FISH) with satellite probes in fresh tumour material. RESULTS: The expression of proliferation associated antigen Ki67, using the monoclonal antibody MIB-1, and proliferating cell nuclear antigen (PCNA), using the antibody PC10, was found to be significantly higher in malignant versus benign liver tumours. Neither Ki67 nor PCNA expression were independent prognostic parameters. However, there was a tendency for a worse outcome (survival < 12 months) for patients with a high MIB-1 labelling index (> 20%) compared with patients having the same tumour stage and a low MIB-1 index. Aneusomy for chromosomes 1 and 8 was demonstrated by FISH in malignant tumours (six of seven hepatocellular carcinomas, four of five cholangiocellular carcinomas) but not in benign tumours (none of nine) or non-neoplastic liver (none of nine). CONCLUSION: Both the determination of the proliferating cell fraction and FISH analysis are useful for distinguishing hepatocellular carcinoma from liver cell adenoma or focal nodular hyperplasia; high fractions of proliferating cells are predictive of an early relapse.

Antigens, Neoplasm↗
PubMed1998

[Cytogenetic changes in low grade central osteosarcomas].

Low grade central osteosarcomas are a group of rare bone neoplasms morphologically simulating benign bone tumors or tumor-like lesions (fibrous dysplasia, nonossifying fibroma, osteoblastoma, chondromyxoidfibroma or aneurysmal bone cyst). Because of the apparent bland histology they may be misinterpreted as benign lesions despite their primary low malignant behavior and late metastatic potential including the capability to transform into a highly malignant sarcoma. In contrast to high grade osteosarcomas cytogenetic studies of these entities are very rare. Karyotyping including FISH analysis with painting- and centromere-specific probes was done in 2 cases of low grade central osteosarcomas (fibrous dysplasia like type). One case was a primary lesion in the upper femur, the other was a recurrent lesion in the upper humerus. Additionally a static DNA cytometry using Feulgen stained imprints was carried out in both cases. In the first case clonal aberrations with a Robertsonian translocation between chromosomes #14 und #15, balanced translocations including chromosomes #5 and #21, #12 and #20, and a supernumary ring chromosome derived from chromosome #1 were detected in a near diploid karyotype. The second case displayed the development from a DNA-diploid to a DNA-peritetraploid lesion during a follow up of six years. Cytogenetic different marker chromosomes and again ring chromosomes were detected in a near tetraploid karyotype. Low grade central osteosarcomas of fibrous dysplasia like type may be characterized by chromosomal aberrations including ring chromosomes. Regarding the last feature these cytogenetic findings resemble those found in low grade parosteal osteosarcomas and other mesenchymal lesions of borderline behavior or low malignancy grade (for example dermatofibrosarcoma protuberans, low grade liposarcoma).

Adolescent↗
PubMed1998

Laparoscopic cholecystectomy alleviates pain in patients with acalculous biliary disease.

Our goal was to determine whether laparoscopic cholecystectomy is a safe and effective means of treatment for patients with acalculous cholecystitis. We reviewed the charts of 243 patients diagnosed with acalculous cholecystitis or biliary dyskinesia. Follow-up telephone interviews to measure degree of patient satisfaction and relief of preoperative symptoms were conducted. Hepatobiliary scanning results and postoperative symptom resolution were compared. One hundred seventy-one patients (94.5%) reported complete or partial resolution of symptoms postoperatively. Although 99 patients had symptoms reproduced with cholecystokinin injection during scanning, there was no significant correlation between these findings and alleviation of pain with cholecystectomy. There was no significant correlation between pain resolution after cholecystectomy and abnormal pathologic findings. Patients who suffered symptoms for a longer period of time preoperatively were more likely to be satisfied with the result of laparoscopic cholecystectomy. Laparoscopic cholecystectomy alleviates symptoms in many patients with acute or chronic acalculous cholecystitis or biliary dyskinesia with minimal morbidity.

Adolescent↗
PubMed1997

Dual role of the C34 subunit of RNA polymerase III in transcription initiation.

The C34 subunit of yeast RNA polymerase (pol) III is part of a subcomplex of three subunits which have no counterpart in the other two nuclear RNA polymerases. This subunit interacts with TFIIIB70 and is therefore thought to participate in pol III recruitment. To study the role of C34 in transcription, we have mutagenized RPC34, the gene encoding C34, and found that mutations affecting growth also altered C34 interaction with TFIIIB70. The two mutant pol III that were purified had catalytic properties indistinguishable from those of the wild-type pol III on a poly[d(A-T)] template, while specific transcription of pol III genes in the presence of general transcription factors was impaired. The defect of the C34-1124 mutant enzyme could be compensated by increasing the amount of pol III present in the reaction, suggesting that the enzyme had a lower affinity for pre-initiation complexes. In contrast, the C34-1109 mutant enzyme was defective in transcription initiation due to impaired open complex formation. These observations demonstrate that the C34 subunit is a major determinant in pol III recruitment by the pre-initiation complex and further acts at a subsequent stage that involves the configuration of an initiation-competent form of RNA polymerase.

Amino Acid Sequence↗
PubMed1997

[Effect of early diagnosis and therapy on prognosis of Parkinson syndrome].

Parkinson's disease is a chronic-progressive neuro-degenerative syndrome, which should be diagnosed as early as possible because of the therapeutic success in the beginning. Knowing the initial symptoms and using modern technical devices like SPECT and PET we shall be able to influence the course and the prognosis of the disease positively. The application of a current combined drug-therapy and in addition physiotherapy, speech-therapy and psychotherapy enable us to provide quality of life or to restore it. Repeated treatments in Parkinson medical centers intensify the efficacy of therapeutic efforts. Nevertheless even in early stages patients and physicians can be confronted with social medical problems like defining the degree of disability or the time for receiving a pension. The knowledge of specific disease-related findings will help to judge all these problems in accordance with the needs of the patients.

Combined Modality Therapy↗
PubMed1997

Immunohistochemical detection of E-cadherin in differentiated thyroid carcinomas correlates with clinical outcome.

A retrospective immunohistochemical analysis of the adhesion molecule E-cadherin (E-CD) was performed in 112 differentiated thyroid carcinomas and 38 synchronous and 20 relapse metastases primarily from operations performed at the Medical School Hanover between 1982 and 1992. E-CD-specific antibody 5H9 was applied to paraffin-embedded tissues. All patients were clinically followed for a maximal period of 12 years. Lack of E-CD expression i.e., <5% of tumor cells positive) occurred in 18 of 112 (16.1%) cases, whereas the majority showed either low (24.1%), medium (35.7%), or high (24.1%) positivity. No difference was found between papillary (n = 88) and follicular (n = 24) carcinomas. Univariate statistical analysis for survival (Kaplan-Meier) showed that lack of E-CD expression (P < 0.024) is an adverse prognostic factor for differentiated thyroid carcinomas. The highest significance was seen among patients without lymph node involvement at first presentation (pN(0); P = 0.0068) and among females (P = 0.0033). Multivariate analysis (Cox model) indicated that E-CD staining is an independent prognostic factor (corrected risk factor, 3.7; P < 0.03) in addition to distant metastasis (pM1) and tumor size. A comparison of E-CD stainings between primary tumors and their metastatic lesions showed similar results in both synchronous and relapse metastases after therapy. In conclusion, E-CD immunostaining is an independent prognostic indicator for differentiated thyroid carcinomas. It may help to uncover the small group of patients with differentiated thyroid carcinomas carrying a high risk of suffering an unfavorable clinical outcome.

Biopsy↗
PubMed1997

Linking analytic performance goals to medical outcome.

As laboratorians relate analytic performance to medical goals, they face complex choices among competing subjective and objective criteria for the assessment of acceptable analytic error. Defining desirable performance as some fraction of physiologic variability provides potentially excessive benchmarks for quality. More important than the recognition of health are the clinical decisions which deal with diseases, particularly the latters' degrees of severity and the different medical actions these prompt. It is equally essential to take into account the reasoning by which physicians arrive at these decisions, since their mental processes condition desirable performance goals. Considering these modalities, a universal model of analytic performance requirements uniformly applicable to all measured parameters clearly cannot be devised. Rather, tolerance limits for analytic error must be tailored to specific medical problems. To facilitate this seemingly Herculean task, this paper develops concepts and principles derived from operation research, and illustrates their application by three examples. The generic conclusion evidenced by the latter is that the linkage between analytic performance goals and medical strategies is reciprocal, namely that outcome can just as well be optimized by tailoring medical strategy to existing analytic performance as by adapting analytic performance to medical strategy.

Clinical Medicine↗
PubMed1997

What constitutes long-term survival after surgery for hilar cholangiocarcinoma?

BACKGROUND: Hilar cholangiocarcinomas are rarely considered to be resectable and specific data regarding long-term survival are not available. METHODS: A retrospective single center experience from 1971 and 1995 details long-term survival among 339 patients who underwent surgery for adenocarcinoma of the hepatic duct bifurcation. Tumor removal was accomplished by resection of the bile duct bifurcation either alone (33 patients), in combination with hepatic resection (77 patients), or combined with hepatic and vascular resection (41 patients). Thirty-two patients underwent total hepatectomy and liver transplantation. In 188 patients the tumor was unresectable and palliative internal or external biliary drainage was established. The resectability rate was 49.2% and rose to 54% after inclusion of liver transplantation. The clinicopathologic features of 32 5-year survivors were compared with patients whose survival was less than 5 years. RESULTS: Thirty-two patients survived longer than 5 years after surgical treatment of hilar cholangiocarcinoma; 22 of these patients (69%) were still alive at last follow-up. Twenty-six long-term survivors had primarily undergone resection; 4 received liver transplants. There was 1 long-term survivor beyond 5 years after both exploratory laparotomy and subsequent percutaneous transhepatic drainage, and palliative hepatojejunostomy. Long-term survival after resection was significantly associated with less advanced tumor stages. Lymph node involvement and residual tumor were the primary prognostic factors both in a multivariate analysis after resection and in the cohort of long-term survivors. Nevertheless, long-term survivors were still at a significant risk to die from tumor recurrence. CONCLUSIONS: Radical resection offers the best possibility of prolonged survival with a good quality of life for patients with hilar cholangiocarcinoma.

Bile Duct Neoplasms↗
PubMed1997

Rapid detection of karyotype changes in interphase bone marrow cells by oligonucleotide primed in situ hybridization (PRINS).

Fluorescence in situ hybridization (FISH) using DNA probes of several hundred or thousand base pairs in length enables the visualization of chromosomal aberrations in interphase nuclei. A new method for in situ labelling of chromosomes is the oligonucleotide primed in situ labelling (PRINS) technique. So far, this has mainly been used to demonstrate subtle changes in metaphase spreads. The aim of the present study was to investigate the suitability of PRINS for detecting chromosome gains or losses in interphase nuclei. This technique was compared with FISH analysis by examining the bone marrow cells of ten patients in whom the karyotypes were known from conventional chromosome banding. Corresponding results by both PRINS and FISH were obtained for chromosomes 1, 3, 7, 8, and Y in five patients with normal chromosome patterns, as well as in five patients with clonal karyotype changes, e.g., monosomy 7, trisomy 8, or loss of the Y chromosome. Being faster and approximately ten times less expensive, PRINS can replace FISH for detecting numerical karyotype changes.

Adult↗
PubMed1997

Influence of postmortem changes on immunohistochemical reactions in skin.

The influence of postmortem damage of tissues on the immunohistochemical diagnosis of wound age has not as yet been clarified. We utilized antibodies against the proteinase inhibitors alpha-1-antichymotrypsin and alpha-2-macroglobulin, fibronectin and lysozyme to study samples of skin which had been intact intravitally, but were damaged postmortem either by autolysis or compression with a surgical clamp at the time of dissection. Even in the absence of autolysis, antibodies against the proteinase inhibitors and fibronectin exhibited staining of tissue margins. Autolysis caused an increase in false positive results. In contrast, antibodies against lysozyme did not give false positive staining. There were no antigens sensitive to postmortem clamping and false positive results were not observed. Antibodies against proteinase inhibitors are not useful for the diagnosis of wound age because of a high number of false positive reactions in marginal areas. Fibronectin also showed false positive band-shaped staining patterns at the tissue margin. In addition, autolytic processes increase the number of false positives. The antibody against lysozyme is much less sensitive to autolysis and no false positive reactions were observed in our series of tests.

Fibronectins↗