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R M Schmid

Publications and source records attributed to R M Schmid.

At least 73 records · Page 4Linked to original sources
PubMed1998

Direct gene transfer into the rat pancreas using DNA-liposomes.

BACKGROUND: Pancreatic cancer represents a malignancy with very poor clinical prognosis and limited therapeutic potential. Recent developments of gene transfer technology offer new therapeutic avenues by delivering recombinant genes directly into normal or neoplastic tissue in vivo. METHODS: Here we show that the LacZ marker gene, complexed to cationic liposomes, can be introduced into the pancreas by either intraductal or intra-arterial injection. Expression of the beta-galactosidase gene product was monitored by polymerase chain reaction and histochemistry. RESULTS: Up to 28 days after in vivo gene transfer, beta-galactosidase activity could be demonstrated in the pancreas. Intraductal application induced gene expression in lining duct cells preferentially. Twenty-four hours after intraductal injection of liposomes, a dose-dependent, transient increase in serum amylase levels was detected. Nevertheless, no histological signs of pancreatitis were evident. Intra-arterial injection resulted in beta-galactosidase expression in endothelial cells of intrapancreatic arteries, as well as in the spleen, lymph nodes and liver, but not in ductal cells of the pancreas. Only occasionally were acinar cells positive for blue staining by either type of treatment. CONCLUSION: These experiments demonstrate that in vivo gene transfer into the pancreas is feasible using DNA-liposome complexes. Furthermore, the route of administration largely determines cell type specificity and side-effects. This technique might have an impact for the development of gene therapy strategies for pancreatic diseases.

Amylases↗
PubMed1998

NF-kappaB2 is a putative target gene of activated Notch-1 via RBP-Jkappa.

NF-kappaB2 (p100/p52), a member of the NF-kappaB/Rel family of transcription factors, is involved in the regulation of a variety of genes important for immune function. Previously, we have shown that the NF-kappaB2 gene is regulated in a positive and a negative manner. Two kappaB elements within the NF-kappaB2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, we have shown that there exists a transcriptional repression in the absence of NF-kappaB. To identify a DNA binding activity responsible for this transcriptional repression, we have partially purified a nuclear complex, named Rep-kappaB. Here we further analyze this putative repressive binding activity. Detailed examination of Rep-kappaB-DNA interaction revealed the sequence requirements for binding to be almost identical to those of recombination signal binding protein Jkappa (RBP-Jkappa), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su(H)]. In addition, in electromobility shift assays, Rep-kappaB binding activity is recognized by an antibody directed against RBP-Jkappa. By performing transient-transfection assays, we show that human RBP-Jkappa represses basal as well as RelA (p65)-stimulated NF-kappaB2 promoter activity. Studies in Drosophila melanogaster have shown that Su(H) is implicated in the Notch signaling pathway regulating cell fate decisions. In transient-transfection assays we show that truncated Notch-1 strongly induces NF-kappaB2 promoter activity. In summary, our data clearly demonstrate that Rep-kappaB is closely related or identical to RBP-Jkappa. RBP-Jkappa is a strong transcriptional repressor of NF-kappaB2. Moreover, this repression can be overcome by activated Notch-1, suggesting that NF-kappaB2 is a novel putative Notch target gene.

DNA↗
PubMed1998

bax, but not bcl-2, influences the prognosis of human pancreatic cancer.

BACKGROUND: bcl-2 and bax belong to the bcl-2-related gene family, which marks a new class of genes that influence apoptosis. The bcl-2 oncogene acts as a broad antiapoptotic factor and extends both normal and tumour cell survival. In contrast, the bax gene is a promoter of apoptosis. AIMS: To analyse the expression of bcl-2 and bax in pancreatic cancer and correlate the results with clinical parameters. PATIENTS: Pancreatic cancer tissue samples were obtained from 28 female and 32 male patients (median age 63, range 43-79 years) having surgery for pancreatic cancer. Normal pancreatic tissues obtained from 18 previously healthy organ donors served as controls. METHODS: The levels of bcl-2 and bax mRNA expression were analysed by northern blot and the exact site of mRNA transcription was determined by in situ hybridisation. The presence of the corresponding proteins was determined by immunohistochemistry. RESULTS: Northern blot analysis indicated that, in comparison with the normal pancreas, bcl-2 mRNA was overexpressed in 30% and bax mRNA in 61% of the pancreatic cancer samples. Concomitant overexpression of bcl-2 and bax was present in 26% of the cancer samples. Pancreatic adenocarcinomas exhibited 3.7-fold and 5.4-fold increases (p < 0.001) in bcl-2 and bax mRNA levels respectively. In situ hybridisation showed that both bcl-2 and bax mRNA were expressed in the cancer cells. Immunohistochemical analysis showed positive Bcl-2 and Bax immunostaining in 28 and 83% of the cancer samples respectively. In multivariate analysis (Cox regression model), bax expression was found to be a strong indicator of survival (p < 0.001). Patients whose tumours exhibited Bax immunostaining lived significantly longer (12 months) than those whose tumours were Bax negative (five months) (p < 0.039). In contrast, no relation was found between Bcl-2 and survival time. CONCLUSIONS: The data indicate that genes that are involved in the regulation of apoptosis are upregulated in human pancreatic cancer cells. Prolonged survival times in patients in whom apoptosis promoting factors are upregulated indicate that apoptotic pathways are of biological significance in pancreatic cancer.

Adolescent↗
PubMed1998

K-ras mutations at codon 12 are rare events in chronic pancreatitis.

BACKGROUND/AIMS: The activation of the K-ras gene at codon 12 is thought to be an early genetic event in the multistep pathogenesis of pancreatic cancer. Since the risk of pancreatic cancer is significantly elevated in subjects with chronic pancreatitis, the aim of the present study was to determine the frequency of K-ras mutations in chronic pancreatitis. METHODS: Pancreatic DNA from intraoperatively resected tissues of 60 patients with chronic pancreatitis and 11 patients with histologically confirmed pancreatic carcinoma was evaluated by PCR amplification and restriction fragment length polymorphism analysis. RESULTS: In none of the 60 samples of chronic pancreatitis could K-ras mutations be identified using two independent PCR assays. In 5 of 11 patients with pancreatic carcinoma, K-ras mutations in codon 12 were detected. CONCLUSION: These data indicate that K-ras mutations are rare events in chronic pancreatitis. Alternatively, it is possible that the time span between the occurrence of K-ras mutations and malignant transformation is rather short.

Carcinoma↗
PubMed1998

Colon epithelium can be transiently transfected with liposomes, calcium phosphate precipitation and DEAE dextran in vivo.

BACKGROUND: The introduction of recombinant DNA into cells is the initial step toward the development of gene therapy. It has been shown that cationic liposomes are useful vehicles to introduce DNA into colon epithelial cells in vivo. METHODS: In the present study we compared the efficacy of different nonviral transfection methods into the colon wall. In anesthetized rats, a double balloon catheter was advanced into the colon and a chloramphenicol acetyltransferase (CAT) reporter plasmid complexed to liposomes, mixed with DEAE dextran, or precipitated with calcium phosphate was instilled. Following 2 days CAT activity was determined in the transfected colon segments. RESULTS: DEAE dextran and liposomes were more effective than calcium phosphate, whereas naked DNA was not taken up by the colon epithelial cells. Reporter gene expression was dose-dependent. Expressing cell types did not differ utilizing the various transfection methods as judged by X-gal staining of colon sections after transfection with a LacZ reporter plasmid. CONCLUSION: These data indicate that in addition to liposomes, plasmid DNA mixed with DEAE dextran can be taken up by colon epithelial cells. This transfection techniques may prove useful in the development of gene therapy approaches for colon disease.

Animals↗
PubMed1997

Germline configuration of nfkb2, c-rel and bcl3 in childhood acute lymphoblastic leukemia (ALL).

The well-known family of NF-kappaB/Rel transcription factors is a central regulator of growth, differentiation and apoptosis in hematopoietic cell lineages. There is increasing evidence for their role in malignant transformation, especially in lymphomas. To study the possible involvement of NF-kappaB/Rel genes in the development of pediatric acute lymphoblastic leukemia (ALL), DNA samples from 140 patients were examined by Southern blot analysis. All samples revealed germline configuration of nfkb2, c-rel, and bcl3, indicating that structural alterations of these members of the NF-kappaB/Rel family are extremely rare, if existing at all in childhood ALL.

Adolescent↗
PubMed1997

Direct gene transfer into the colon using a double-balloon catheter.

BACKGROUND AND STUDY AIMS: In spite of the many advances that have been made in understanding the molecular basis for diseases, a major obstacle to the treatment of human disorders remains the inability to express genes at specific sites in vivo. Recent progress in gene transfer technology has provided access to a variety of recombinant gene products that can be applied in clinical medicine for therapeutic purposes. MATERIALS AND METHODS: In an animal model, we describe here the way in which a marker gene can be introduced into the colon using a double-balloon catheter. Cationic liposomes were used as vehicles to introduce DNA into the living organism. RSV-LacZ plasmid coding for the enzyme beta-galactosidase was used as a marker gene. Cells expressing beta-galactosidase can be stained using the chromogen X-gal. Positive cells show a blue coloration in the cytoplasm. RESULTS: Both absorptive cells and goblet cells were successfully transduced with the marker gene. No evidence of similar staining was observed in control animals receiving a control plasmid or liposomes alone. CONCLUSIONS: The method used is a simple, safe, and nontoxic way of delivering genes of interest to specific sites in the colon. Gene transfer may offer fresh potential for endoscopic interventions in colonic disease.

Animals↗
PubMed1997

Liposome mediated gene transfer into the rat oesophagus.

BACKGROUND: Cancer of the oesophagus has so far eluded every attempt at pharmacological treatment. The recent advent of somatic gene therapy offers a new therapeutic approach to malignant tumours. AIM: To investigate whether and how gene transfer into the oesophagus can be achieved. METHODS: A LacZ reporter gene was used as marker and transferred into the oesophagus of rats using cationic liposomes. Gene transfer was achieved by either luminal instillation into a closed segment using a double balloon catheter, or by intramural injection through a needle. Expression of beta-galactosidase was monitored in the oesophagus and various control tissues by histochemistry, polymerase chain reaction (PCR), reverse transcriptase PCR, and Southern blotting. RESULTS: Up to 100 days after in vivo gene transfer beta-galactosidase activity could be demonstrated in the oesophagus. Following luminal application, the transgene was expressed in epithelial cells whereas intramural injection induced preferential expression in fibroblasts. CONCLUSION: In vivo gene transfer into the esophagus is feasible and safe, and the route of administration largely determines cell type specificity. This novel approach will enable in vivo studies of growth, differentiation, and malignant transformation in the oesophagus, and may open new avenues to the confinement of oesophageal malignancies.

Animals↗
PubMed1994

Structural and functional analysis of NF-kappa B. Determinants of DNA binding specificity and protein interaction.

The NF-kappa B transcription factors display a high degree of sequence conservation in a domain initially described in the rel oncogene. Two family members, NF-kappa B1 and NF-kappa B2, have distinct DNA binding properties and functionally distinct effects on different enhancers. NF-kappa B1, for example, binds to the kappa B site from the human immunodeficiency virus (HIV) with approximately 15-fold higher affinity than NF-kappa B2. In this study, we have defined regions within the Rel domain which determine DNA binding specificity and interaction with other proteins. We find that the COOH-terminal putative Rel dimerization domain of NF-kappa B1 is required for preferential binding to the HIV kappa B site. In contrast, preferential stimulation of the HIV enhancer by NF-kappa B2 with RelA(p65) is determined by both the NH2- and COOH-terminal Rel domains of NF-kappa B2. These two regions of NF-kappa B2 also mediate preferential synergy with Bcl3. These data suggest that a specific subdomain of the Rel conserved region has evolved to control the fine specificity of DNA binding, and two distinct subregions within the Rel domain determine the specificity of interaction with other transcription factors. These specific Rel-conserved domains therefore determine the specificity of NF-kappa B interactions and contribute to selective gene activation.

Base Sequence↗
PubMed1994

Transcriptional regulation of NF-kappa B2: evidence for kappa B-mediated positive and negative autoregulation.

NF-kappa B is an inducible transcription factor complex which regulates the expression of a variety of genes which are involved in the immune, inflammatory, and acute-phase responses. The maintenance of NF-kappa B activity in stimulated cells requires ongoing protein synthesis, suggesting several modes of regulation. In this report, we have characterized the transcriptional regulation of one family member, NF-kappa B2. The genomic structure and sequence of NF-kappa B2 revealed the presence of two promoters and at least four kappa B regulatory elements, which mediate responsiveness to phorbol myristate acetate and tumor necrosis factor alpha. Similar to other NF-kappa B family members, NF-kappa B2 is positively autoregulated. In contrast to other family members, we find that kappa B elements in the NFKB2 promoter can also mediate transcriptional repression in the absence of NF-kappa B. We identified a nuclear complex which binds specifically to a subset of kappa B-related sites but not to the canonical kappa B element. Because of its putative inhibitory or repressive effect, this binding activity has been termed Rep-kappa B. This mechanism of repressing basal NF-kappa B2 transcription in an inactivated state enables the cell to tightly control NF-kappa B2 activity. These data demonstrate that a novel mode of kappa B-dependent regulation is mediated by specific kappa B sites in the NFKB2 promoter.

Base Sequence↗
PubMed1994

Liposome mediated in vivo gene transfer into different tissues of the gastrointestinal tract.

The possibility to transfer and express genetic material in mammalian cells represents a new approach to the treatment of genetic and acquired disorders. So far, most studies use in vitro techniques to introduce foreign DNA into cultured cells, followed by reintroduction of these genetically altered cells into living organisms. In the present study we demonstrate that the LacZ marker gene can be selectively delivered, by in vivo techniques, to various locations of the gastrointestinal tract. Genetic material was targeted to the stomach, the colon, the liver and the pancreas using cationic liposomes. For transfer into the stomach and colon an intraluminal application, in the liver a portal access and in the pancreas an intraductal infusion was chosen. 48 hours after administration, the LacZ gene product beta-galactosidase could be localized in these tissues by cytochemistry. These experiments suggest a new approach to study gastrointestinal physiology and may offer novel aspects for the treatment of gastrointestinal diseases.

Animals↗
PubMed1993

Purification of the major histocompatibility complex class I transcription factor H2TF1. The full-length product of the nfkb2 gene.

H2TF1 is a ubiquitous major histocompatibility complex (MHC) class I-specific transcription factor, which binds to the palindromic kappa B enhancer site upstream of MHC class I genes. Here we report that H2TF1 consists of a polypeptide with relative molecular mass 110,000, that corresponds to the predicted 100-kDa product (NF-kappa B2 p100) encoded by the candidate proto-oncogene nfkb2 (lyt-10). H2TF1 was purified by a novel affinity chromatography method and identified as the NF-kappa B2 p100 polypeptide by peptide sequencing as well as by reactivity with a specific antiserum. Purified H2TF1 binds the MHC kappa B site with high affinity (KD = 3 x 10(-11) M), in contrast with previous reports that NF-kappa B2 p100 did not bind DNA.

Amino Acid Sequence↗
PubMed1993

A cooperative interaction between NF-kappa B and Sp1 is required for HIV-1 enhancer activation.

The human immunodeficiency virus (HIV-1) long terminal repeat (LTR) contains two binding sites for NF-kappa B in close proximity to three binding sites for the constitutive transcription factor, Sp1. Previously, stimulation of the HIV enhancer in response to mitogens has been attributed to the binding of NF-kappa B to the viral enhancer. In this report, we show that the binding of NF-kappa B is not by itself sufficient to induce HIV gene expression. Instead, a protein-protein interaction must occur between NF-kappa B and Sp1 bound to an adjacent site. Cooperativity both in DNA binding and in transcriptional activation of NF-kappa B and Sp1 was confirmed by electrophoretic mobility shift gel analysis, DNase footprinting, chemical cross-linking and transfection studies in vivo. With a heterologous promoter, we find that the interaction of NF-kappa B with Sp1 is dependent on orientation and position, and is not observed with other elements, including GATA, CCAAT or octamer. An increase in the spacing between the kappa B and Sp1 elements virtually abolishes this functional interaction, which is not restored when these sites are brought back into the same helical position. Several other promoters regulated by NF-kappa B also contain kappa B in proximity to Sp1 binding sites. These findings suggest that an interaction between NF-kappa B and Sp1 is required for inducible HIV-1 gene expression and may serve as a regulatory mechanism to activate specific viral and cellular genes.

Avian Sarcoma Viruses↗