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Biomedical subjects

B Wittig

Publications and source records attributed to B Wittig.

At least 73 records · Page 4Linked to original sources
PubMed1996

P53 and induction of apoptosis as a target for anticancer therapy.

p53 is the most frequently mutated gone in human cancer cells. Its wild-type gone encodes for a protein with pivotal functions: (i) interaction with key players in the cell cycle leading to cell cycle arrest; (ii) induction of programmed call death, or apoptosis. P53 may be seen as another member of the family of proteins involved in resistance to anticancer therapy, since mutations/deletions involving the p53 gene lead frequently to resistance of radiation/cytotoxic drug treatment. Consequently, patients with p53-mutated tumors may harbor a worse prognosis. On the other hand, reintroducing wild-type P53 may lead to an adequate function of the cellular cell cycle and/or apoptosis program, thus enabling efficient anti-cancer therapy even in the presence of mutated P53. Two options are being discussed: (i) gene therapy approaches; (ii) modulating mutated P53 with yet unknown molecules.

Animals↗
PubMed1995

Identification of transcriptionally induced Z-DNA segments in the human c-myc gene.

Using monoclonal antibodies against Z-DNA three AluI restriction fragments of the human c-myc gene were previously found to form Z-DNA in agarose-embedded, metabolically active permeabilized nuclei. The formation of Z-DNA in these fragments was dependent on negative supercoiling generated by transcription of the gene. Here we show which sequence elements of these three AluI restriction fragments adopt the Z conformation upon negative supercoiling. The three fragments (Z1, Z2 and Z3) were inserted in a suitable plasmid vector. Z-DNA forming elements were detected by comparing DEPC reactivity in relaxed circular and supercoiled plasmid DNA. Z1 and Z3 each contained one major Z-DNA forming region 20-25 nucleotides long, whereas Z2 contained two discrete regions 90 nucleotides apart one about 35 nucleotides the other about 20 nucleotides long.

Base Sequence↗
PubMed1995

Subunit composition of G(o) proteins functionally coupling galanin receptors to voltage-gated calcium channels.

The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels.

Amino Acid Sequence↗
PubMed1995

DNASTAT: a Pascal unit for the statistical analysis of DNA and protein sequences.

DNASTAT is a collection of Pascal routines for researchers who develop their own application programs for statistical analysis of DNA and protein sequences. Dynamic and file-based data structures allow users to process sets of sequences by simple loop control without limitations on the number of sequences and their individual sizes. This frees the programmer from potentially error-prone tasks like dynamic memory allocation and controlling array sizes. Sequences can be stored in databases along with biological and statistical attributes. Individual sequences can be accessed by column name and row number as with spread-sheets. DNASTAT allows large sets of sequences to be processed using a PC with standard configuration. Its small size, simplicity and free availability make it attractive to students of mathematical biology. Use of DNASTAT is illustrated by two sample programs that generate a database of coding regions from the GenBank entry of the tobacco chloroplast genome. A version of DNASTAT written in ANSI-C for PCs and Unix workstations is also available.

Base Sequence↗
PubMed1994

Effect of netropsin, distamycin A and chromomycin A3 on the binding and cleavage reaction of DNA gyrase.

The influence of netropsin (Nt), distamycin A (Dst-3) and chromomycin A3 (CHR) on the binding of gyrase from Streptomyces noursei to an 162 bp-fragment of pBR 322 containing a strong gyrase cleavage site and on the gyrase mediated cleavage of this fragment was analyzed. Binding of the enzyme to the fragment is effectively inhibited by the GC-specific drug CHR, but poorly influenced by Dst-3, while Nt is ineffective. Cleavage of the fragment catalysed by the enzyme is inhibited by all three ligands but to different extent. Dst-3 and Nt inhibit the enzyme cleavage reaction at 20- or 250-fold higher concentration than that required for CHR. The inhibitory mechanism of CHR on gyrase-DNA binding and cleavage may be related to a competitive interaction of the ligand to GC sequences located at and around the gyrase cleavage site. The fact that AT-specific minor groove binders Dst-3 and Nt poorly inhibit the binding of gyrase to the fragment due to the low amount of the AT basepair sequences contained in the fragment and their inhibitory influence on the cleavage step underlines the role of the DNA minor groove during enzyme action.

Base Sequence↗
PubMed1994

Enormous hemangiosarcoma of the heart.

This report describes a 26-year-old patient with hemangiosarcoma of the heart and summarizes the clinicopathological features in previous reports of patients with cardiac angiosarcoma. The patient was admitted to our hospital because of a syncope and one episode of nocturnal dyspnea and hemoptysis. In his history he complained of progressive weakness and loss of weight over the past 2 months. Echocardiography and computed tomography of the chest showed inhomogeneous masses in the pericardial cavity completely surrounding the heart and involving the ascending aorta and the superior vena cava. Histological examination of the tissue obtained from the mass by fine needle technique revealed a poorly differentiated malignant tumor of mesenchymal origin. Exploratory thoracotomy followed by tumor biopsies showed an inoperable cardiac hemangiosarcoma of enormous size with multiple metastases in both lungs. Palliative tumor resection was not performed. During the postoperative course the patient still required controlled ventilation. After 3 days of cytostatic chemotherapy no regression of tumor mass was seen by chest radiography. Cardiorespiratory insufficiency was progressive, and the patient died within 3 weeks after admission.

Adult↗
PubMed1993

Gi2 and protein kinase C are required for thyrotropin-releasing hormone-induced stimulation of voltage-dependent Ca2+ channels in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) and other secretion-stimulating hormones trigger an increase in the cytosolic Ca2+ concentration by two mechanisms. Ca2+ is released from intracellular stores in response to inositol 1,4,5-trisphosphate and can enter the cell through voltage-dependent L-type Ca2+ channels. Stimulation of these channels is sensitive to pertussis toxin, indicating that a pertussis toxin-sensitive heterotrimeric guanine nucleotide-binding regulatory protein (G protein) is involved in functional coupling of the receptor to the Ca2+ channel. We identified the G protein involved in the stimulatory effect of TRH on the Ca2+ channel by type-selective suppression of G-protein synthesis. Antisense oligonucleotides were microinjected into GH3 cell nuclei, and 48 h after injection the TRH effect was tested. Whereas antisense oligonucleotides hybridizing to the mRNA of G(o) or Gi1 alpha-subunit sequences did not affect stimulation by TRH, oligonucleotides suppressing the expression of the Gi2 alpha subunit abolished this effect, and oligonucleotides directed against the mRNA of the Gi3 alpha subunit had less effect. The requirement of a concurrent inositol phospholipid degradation and subsequent protein kinase C (PKC) activation for the TRH effect on Ca(2+)-channel activity was demonstrated by inhibitory effects of antisense oligonucleotides directed against Gq/G11/Gz alpha-subunit sequences and treatment of GH3 cells with PKC inhibitors, respectively. Our results suggest that TRH elevates the cytosolic Ca2+ concentration in GH3 cells transiently via Ca2+ release from internal stores, followed by a phase of sustained Ca2+ influx through voltage-dependent Ca2+ channels stimulated by the concerted action of Gi2 (and Gi3) plus PKC.

Animals↗
PubMed1993

Selectivity in signal transduction determined by gamma subunits of heterotrimeric G proteins.

Various heterotrimeric guanine nucleotide-binding proteins have been identified on the basis of the individual subtypes of their alpha subunits. The beta gamma complexes, composed of beta and gamma subunits, remain tightly associated under physiological conditions and have been assumed to constitute a common pool shared among various guanosine triphosphate (GTP)-binding (G) protein heterotrimers. Particular alpha and beta subunit subtypes participate in the signal transduction processes between somatostatin or muscarinic receptors and the voltage-sensitive L-type calcium channel in rat pituitary GH3 cells. Among gamma subunits the gamma 3 subtype was found to be required for coupling of the somatostatin receptor to voltage-sensitive calcium channels, whereas the gamma 4 subtype was found to be required for coupling of the muscarinic receptor to those channels.

Animals↗
PubMed1993

Expression of intercellular adhesion molecule 1 (ICAM-1, CD54) in colonic epithelial cells.

The expression of intercellular adhesion molecule-1 (ICAM-1, CD54) was examined in 16 surgically removed colonic tumours and two colonic carcinoma cell lines. Immunohistochemistry showed a varying percentage of ICAM-1 positive colonic carcinoma cells in 9/16 tissue specimens, while normal colonic tissue (apart from a slight reactivity of endothelial cells) was not stained. The presence of the ICAM-1 molecule on the cell surface and the expression of ICAM-1 mRNA were investigated for two colonic carcinoma cell lines. It was possible to enhance the expression of ICAM-1 considerably by incubating the cells in the presence of inflammatory cytokines in HT-29 and CaCo-2 cells. The responsiveness to either interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha), or interleukin 1 beta (IL-1 beta) treatment was different in each cell line. Interestingly, ICAM-1 is shed by colonic carcinoma cells because soluble sICAM-1 was detected in the cell culture supernatants. In comparison with normal serum samples, the mean value of sICAM-1 in 63 samples of patients with colonic carcinoma and in 20 cases of active inflammatory bowel disease is raised about twofold. It remains to be clarified what part both forms of ICAM-1 play in the course of colonic cancer, ulcerative colitis, and Crohn's disease.

Base Sequence↗
PubMed1992

Different beta-subunits determine G-protein interaction with transmembrane receptors.

Regulatory GTP-binding proteins (G proteins) are membrane-attached heterotrimers (alpha, beta, gamma) that mediate cellular responses to a wide variety of extracellular stimuli. They undergo a cycle of guanine-nucleotide exchange and GTP hydrolysis, during which they dissociate into alpha-subunit and beta gamma complex. The roles of G-protein alpha-subunits in these processes and for the specificity of signal transduction are largely established; the beta- and gamma-subunits are essential for receptor-induced G-protein activation and seem to be less diverse and less specific. Although the complementary DNAs for several beta-subunits have been cloned, isolated subunits have only been studied as beta gamma complexes. Functional differences have been ascribed to the gamma-subunit on the basis of extensive sequence similarity among beta-subunits and apparent heterogeneity in gamma-subunit sequences. Beta gamma complexes can interact directly or indirectly with different effectors. They seem to be interchangeable in their interaction with pertussis toxin-sensitive alpha-subunits, so we tested this by microinjecting antisense oligonucleotides into nuclei of a rat pituitary cell line to suppress the synthesis of individual beta-subunits selectively. Here we show that two out of four subtypes of beta-subunits tested (beta 1 and beta 3) are selectively involved in the signal transduction cascades from muscarinic M4 (ref. 4) and somatostatin receptors, respectively, to voltage-dependent Ca2+ channels.

Animals↗
PubMed1992

Transcription of human c-myc in permeabilized nuclei is associated with formation of Z-DNA in three discrete regions of the gene.

When human U937 cells are placed in agarose microbeads and treated with a detergent, the cytoplasmic membrane is lysed and the nuclear membrane is permeabilized. However, the nuclei remain intact and maintain both replication and transcription. Biotin labeled monoclonal antibodies against Z-DNA have been diffused into this system and used to measure the amount of Z-DNA present in the nuclei. It has previously been shown that the amount of Z-DNA present decreases due to relaxation by topoisomerase I and increases as the level of transcription increases. Here we measure the formation of Z-DNA in the c-myc gene by crosslinking the antibodies to DNA using laser radiation at 266 nm for 10 ns. The crosslinked DNA is isolated by restriction digestion, separation of antibody labeled fractions through the biotin residue, and subsequent proteolysis to remove the crosslinked antibody. Three AluI restriction fragments of the c-myc gene are shown to form Z-DNA when the cell is transcribing c-myc. The Z-DNA forming segments are near the promoter regions of the gene. However, when U937 cells start to differentiate and transcription of the c-myc gene is down-regulated, the Z-DNA content goes to undetectable levels within 30-60 min.

Base Sequence↗
PubMed1992

[The clinical relevance of anti-CEA immunoscintigraphy with the 99mTc-labelled monoclonal antibody BW 431/26. A critical assessment after 119 studies].

The results of 119 radioimmunoscintigraphies (RIS) in 113 patients with the 99mTc-labeled monoclonal anti-CEA-antibody BW 431/26 (Behring) have been analysed. The aim of our study was the estimation of the method's sensitivity and specificity under different aspects to find out for which indications and questions the 99mTc-RIS is useful. Colorectal primary tumours in 19 patients were scintigraphically detected with a sensitivity of 83% and a specificity of 100%; 3 out of 7 other tumour sites were localised correctly. 55 patients were examined during the follow-up of colorectal cancer. There were 17 out of 22 true positive findings of local recurrences (sensitivity 77%, specificity 88%). Liver metastases were imaged as hot lesions with only 41% sensitivity and 86% specificity. The detection of extrahepatic tumour sites is difficult because of the persistently high blood-pool activity of the monoclonal antibody and, in the pelvic area, the unspecific bowel activity. Skeletal metastases were recognised in 7 out of 9 cases. In 14 patients with other non-colorectal carcinomas, RIS was successful in single cases. It is not helpful, however, when searching for tumours of unknown origin or for the screening of patients with elevated CEA levels without tumour history. The high technical, methodological and time effort required by RIS is justified in the follow-up of cancer patients when conventional diagnostic procedures are inconclusive or the status of morphological findings remains unclear. The use of RIS as an unspecific screening tool in tumour diagnosis must be rejected because of the not completely explored risks of the examination. Repeated applications of monoclonal antibodies require controls of the patients' HAMA titers before performing RIS.

Adult↗
PubMed1991

Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents.

The inhibition of voltage-dependent Ca2+ channels in secretory cells by plasma membrane receptors is mediated by pertussis toxin-sensitive G proteins. Multiple forms of G proteins have been described, differing principally in their alpha subunits, but it has not been possible to establish which G-protein subtype mediates inhibition by a specific receptor. By intranuclear injection of antisense oligonucleotides into rat pituitary GH3 cells, the essential role of the Go-type G proteins in Ca(2+)-channel inhibition is established: the subtypes Go1 and Go2 mediate inhibition through the muscarinic and somatostatin receptors, respectively.

Animals↗
PubMed1991

Transcription is associated with Z-DNA formation in metabolically active permeabilized mammalian cell nuclei.

Mammalian cells have been encapsulated in agarose microbeads, and from these cells metabolically active permeabilized nuclei were prepared. Previously, we showed that biotin-labeled monoclonal antibodies against Z-DNA can be diffused into the nuclei and, over a specific concentration range, they will bind to Z-DNA within the nucleus in a concentration-independent manner. By using radiolabeled streptavidin, we showed that the amount of Z-DNA antibody bound is related to the torsional strain of the DNA in the nucleus. Relaxation of the DNA results in a decrease of Z-DNA formation, whereas increasing torsional strain through inhibiting topoisomerase I results in increased Z-DNA formation. Here we measure the influence of RNA transcription and DNA replication. Transcription is associated with a substantial increase in the binding of anti-Z-DNA antibodies, paralleling the increased level of RNA synthesized as the level of ribonucleoside triphosphate in the medium is increased. DNA replication yields smaller increases in the binding of Z-DNA antibodies. Stopping RNA transcription with inhibitors results in a large loss of Z-DNA antibody binding, whereas only a small decrease is associated with inhibition of DNA replication.

Antibodies, Monoclonal↗
PubMed1991

Lack of correlation between DNA methylation and transcriptional inactivation: the chicken lysozyme gene.

We have analyzed the methylation state of all nine CpG sites in the transcription start region (-420 to +250 base pairs) of the chicken lysozyme gene by genomic sequencing. One of these sites, at -81, lies within the promoter, seven are clustered within the first exon, and the last is in the first intron. Five cell types and tissues have been investigated to study the relationship between methylation level and gene expression. For each cell type used, the majority of CpG sites showed a similar level of methylation. Of two gene-nonexpressing tissues, erythrocytes are hypomethylated, whereas liver is methylated at most of its CpG sites. For gene-expressing tissues, oviduct is completely unmethylated, whereas HD-11 culture cells are methylated. Thus no correlation is observed between degree of CpG methylation and level of expression of the lysozyme gene. The observed methylation patterns are discussed in terms of possible features of the local chromatin structure.

Animals↗