PubMed Health⌕ Search

Biomedical subjects

D Koczan

Publications and source records attributed to D Koczan.

At least 19 recordsLinked to original sources
PubMed2005

Protective effect of endotoxin preconditioning in pancreas: analysis with DNA chip arrays.

Recently we demonstrated a protective effect of endotoxin preconditioning 24 hours before pancreatic ischemia-reperfusion injury, which has also been described for other organs. The mechanisms underlying this phenomenon, such as differential gene expression, are poorly investigated. We chose to approach this question by investigating differential gene expression in the rat pancreas over the time course of endotoxin pretreatment. Male Wistar rats (5 groups, 5 animals per group) were pretreated with endotoxin intraperitoneally (1 mg/kg of body weight). After treatment at 30 minutes, and at 3 and 24 hours the pancreas was removed. Untreated animals and animals with injection of saline solution served as controls. After RNA isolation, RNA was pooled and hybridized to Affymetrix chips to measure the relative mRNA levels of 7000 genes and 1000 expression sequence tags. Three hours after administration of endotoxin there was an activation of proinflammatory transcription factors and other proinflammatory genes. After 24 hours there was a clear decrease of these proinflammatory genes, but a remaining and increasing upregulation of important antiapoptotic genes, antiproteases, and other probably protective genes. There was also a significant upregulation of complement factors. It was surprising that heat-shock proteins and other typical immediate early genes of the AP-1 complex were not upregulated. Our data show that 24 hours after endotoxin stress there is a regulation of a network of genes that represents a multifaceted preconditioning. As most important factors, inhibition of apoptosis and antiproteatic strategies are identified. Heat-shock proteins seem to play no important role in the mechanism of endotoxin preconditioning.

Animals↗
PubMed2005

Gene expression profiling of arthritis using a QTL chip reveals a complex gene regulation of the Cia5 region in mice.

One of the major quantitative trait loci (QTLs) associated with arthritis in crosses between B10.RIII and RIIIS/J mice is the Cia5 on chromosome 3. Early in the congenic mapping process it was clear that the locus was complex, consisting of several subloci with small effects. Therefore, we developed two novel strategies to dissect a QTL: the partial advanced inter-cross (PAI) strategy, with which we recently found the Cia5 region to consist of three loci, Cia5, Cia21 and Cia22, and now we introduce the QTL-chip strategy, where we have combined congenic mapping with a QTL-restricted expression profiling using a novel microarray design. The expression of QTL genes was compared between parental and congenic mice in lymph node, spleen and paw samples in five biological replicates and in dye-swapped experiments at three time points during the induction phase of arthritis. The QTL chip approach revealed 4 genes located in Cia21, differently expressed in lymph nodes, and 14 genes in Cia22, located within two clusters. One cluster contains six genes, differently expressed in spleen, and the second cluster contains eight genes, differently expressed in paws. We conclude the QTL-chip strategy to be valuable in the selection of candidate genes to be prioritized for further investigation.

Animals↗
PubMed2004

[The extracellular matrix structure in keratoconus].

PURPOSE: Keratoconus is a non-inflammatory disease characterized by progressive thinning of the central cornea. There are indications for an autosomal dominant heredity. Our purpose was to find correlations between gene expression and structural changes in extracellular matrix components. METHODS: Stromal RNA (keratoconus and comparison) was isolated from corneas, transformed to cRNA and analysed using biochips (Affymetrix). Structural investigations were performed by laser scanning and transmission electron microscopy. RESULTS: In keratoconus corneas there was an upregulation of different extracellular matrix components (collagen XV, metalloproteases) and a down-regulation of collagen IV (alpha1, alpha3) and versican. The morphological changes correlated to genetic obtained data. The orthogonal arrangement of the collagen fibrills (anteriorly and central) was altered in the collagen matrix of keratoconus corneas. CONCLUSIONS: The changes point on deregulation of the matrix arrangement. The interest in the cause of the disease is focused on the interfibrillar arrangement, the interaction between collagen and proteoglucanes.

Adult↗
PubMed2004

DNA microarray technology and its applications in dermatology.

The use of DNA microarray technology in biomedical research has dramatically increased during the past years. In the present report, we provide an overview on the basic DNA microarray technology and biostatistical methods for gene expression analysis. A focus is then put on its applications in dermatological research. In recent years, a series of gene expression studies have been performed for various dermatological diseases, such as malignant melanoma, psoriasis and lupus erythematosus. These analyses have identified interesting target genes as well as putative disease susceptibility loci. However, further functional studies will be needed for a more complete understanding of the pathogenesis of these diseases. This may be performed by means of the recently developed RNA interference technology. Besides its role in large-scale gene expression studies, DNA microarray technology has proved to be a valuable tool for genomic screens of genetic alterations, e.g. single nucleotide polymorphisms. These play a role in tumour development and progression, and also function as genetic markers for disease susceptibility. Taken together, DNA microarray technology opens enormous perspectives for dermatologists. It may help us understand the complex pathogenesis of a wide variety of dermatologic diseases and identify their genetic background.

Cell Differentiation↗
PubMed2003

[Gene expression in keratoconus. Initial results using DNA microarrays].

INTRODUCTION: Keratoconus is a non-inflammatory ocular disease characterised by conical deformation, progressive thinning and scarring of the central cornea. Despite intensive investigations, the exact cause of the disease still remains unclear. Clinical studies provide strong indications of a major genetic role in the aetiology. We set out to examine the involvement in the manifestation of keratoconus of any of the 5,600 gene specificities available on the Affymetrix GeneChip HuGeneFL. METHODS: After examination of two corneas they were stored in RNAlater, RNA was extracted and hybridised on the chips. Using a combination of dyes it was possible to read the chips with laser detection and to visualise the gene expression pattern. RESULTS: We found an upregulation of collagens, versican, metalloproteinases and cell adhesion proteins. A downregulation was observed for TIGR protein, cytokeratins, eyes absent homologue (Eab1) and the proteins for radical treatment selenoprotein P and monooxygenase. CONCLUSIONS: Our results indicate that keratoconus is a process in which repair and scar-formation mechanisms operate at the same time. As candidate genes for this mechanism, collagen IV and related proteoglycans were favoured.

Cicatrix↗
PubMed2002

Differential expression of thrombospondin 2 in primary and metastatic malignant melanoma.

In the present report we used oligonucleotide microarray analysis for the identification of genes characterizing the late-stage metastatic phenotype of malignant melanoma. A panel of 5,600 genes was analysed in a low aggressive and a highly aggressive (metastatic) human malignant melanoma cell line, respectively. More than 300 differentially regulated genes were identified. High metastatic potential correlated with upregulated mRNA expression in the groups of cytoskeletal proteins, apoptosis and cell cycle proteins, GTP binding proteins and oncogenes, extracellular ligands and receptors, transcription and translation factors. In contrast, most angiogenesis factors, extracellular matrix molecules, and melanoma-specific antigens were downregulated. Particular target genes were further analysed by in situ hybridization and immunohistochemical staining of primary malignant melanomas and melanoma metastases. Here, we show that thrombospondin 2, an extracellular matrix molecule which was differentially regulated in the microarray analysis, was strongly expressed in melanoma metastases, but not in primary tumours. The identification of thrombospondin 2 as a target molecule emphasizes the importance of cell-matrix interactions for the pathogenesis of malignant melanoma metastasis and may open future perspectives for treatment of this tumour.

Blotting, Northern↗
PubMed2002

Microarray analysis of differentially expressed genes in placental tissue of pre-eclampsia: up-regulation of obesity-related genes.

Susceptibility genes present in both mother and fetus most likely contribute to the risk of pre-eclampsia. Placental biopsies were therefore investigated by high-density DNA microarray analysis to determine genes differentially regulated within chorionic villous tissue in pre-eclampsia and normal pregnancy. The pooled RNAs of pre-eclamptic and normotensive subjects were hybridized to the HuGeneFL array representing sequences from approximately 5600 full-length human cDNAs. The differentially expressed genes that were detected could be categorized into nine groups: adhesion molecules, obesity-related genes, transcription factors/signalling molecules, immunological factors, neuromediators, oncogenic factors, protease inhibitors, hormones and growth factor-binding proteins. Among those, the obesity-related genes included putative candidate genes associated with the pathogenesis of pre-eclampsia. One of the most up-regulated transcripts was the obese gene (43.6-fold change), and this was reflected by elevated leptin protein levels. In the case of feto-maternal contribution of polymorphic genes to pre-eclampsia, expression analysis of placental tissue has lead to numerous target genes waiting for large scale genetic linkage analyses.

Female↗
PubMed2002

The KOX zinc finger genes: genome wide mapping of 368 ZNF PAC clones with zinc finger gene clusters predominantly in 23 chromosomal loci are confirmed by human sequences annotated in EnsEMBL.

The chromosome locations of 368 human Kruppel-type zinc finger (ZNF) PAC clones were physically mapped by FISH to human chromosomes in support of recent efforts of assigning KOX cDNAs (KOX1-KOX32) to zinc finger gene clusters. Recent mapping results were validated and confirmed by sequence comparisons to zinc finger gene sequences automatically annotated in EnsEMBL. In toto, 799 Kruppel-type zinc finger genes have been annotated in EnsEMBL of which 290 genes are found to encode KRAB domains. Sequence homologies of the zinc finger domains were used to establish phylogenic trees of KOX zinc finger genes as well as of all KRAB containing human zinc finger and KOX genes documenting the evolution of KRAB zinc finger genes late in primate evolution. A list of 368 assigned ZNF PAC clones is available under http://www.pzr.uni-rostock.de/supplements.

Base Sequence↗
PubMed2001

Gene expression profiling of the nervous system in murine experimental autoimmune encephalomyelitis.

Multiple sclerosis is thought to be a polygenic disease driven by dysregulation of the immune system leading to an autoimmune response against one or several antigens of cerebral white matter tissue. Experimental autoimmune encephalomyelitis (EAE) is a mouse model that is used to study the aetiology and pathogenesis of multiple sclerosis and new therapeutic approaches. We used oligonucleotide microarrays to determine gene expression profiles of the inflamed spinal cords of EAE mice at the onset and at the peak of the disease. Of the approximately 11 000 genes studied, 213 were regulated differentially and 100 showed consistent differential regulation throughout the disease. Inflammation resulted in a profile of increased gene expression of immune-related molecules, extracellular matrix and cell adhesion molecules and molecules involved in cell division and transcription, and differential regulation of molecules involved in signal transduction, protein synthesis and metabolism. Of the 104 genes with defined chromosomal locations, 51 mapped to known EAE-linked quantitative trait loci and as such are putative candidate genes for susceptibility to EAE.

Animals↗
PubMed2001

Pancreatic adenocarcinoma cell lines show variable susceptibility to TRAIL-mediated cell death.

BACKGROUND AND AIMS: Programmed cell death via the Fas receptor/Fas Ligand and DR4, DR5/TRAIL plays a major role in tumor escape and elimination mechanisms. It also promises to be an effective therapy alternative for aggressive tumors, as has been recently shown for colon, breast, and lung cancer cells. We attempted to clarify the role of these molecules in aggressivity of pancreatic carcinomas and to identify possible pathways as targets for therapy. METHODS: Five pancreatic cell lines were investigated for the expression of FasL/Fas, DcR3, DR4, DR5/TRAIL, DcR1, DcR2, and other death pathways related molecules such as Bax, bcl-xL, bcl-2, FADD, and caspase-3 by flow cytometry, immunoblotting, and RT/PCR, both semiquantitative and real time (TaqMan). The susceptibility of these cell lines to apoptosis mediated by recombinant TRAIL was investigated. The effect of therapeutic agents (gemcitabine) on their susceptibility to TRAIL induced apoptosis was studied as well. RESULTS: Pancreatic adenocarcinomas expressed high levels of apoptosis-inducing receptors and ligands. They showed differential susceptibility to cell death induced by TRAIL, despite expressing intact receptors and signaling machineries. Treatment with commonly used therapeutic agents did not augment their susceptibility to apoptosis. This could be explained by the fact that they expressed differentially high levels of decoy receptors, as well as molecules known as inhibitors of apoptosis. CONCLUSIONS: The data suggest that pancreatic carcinoma cells have developed different mechanisms to evade the immune system. One is the expression of nonfunctional receptors, decoy receptors, and molecules that block cell death, such as bcl2 and bcl-xL. The second is the expression of apoptosis-inducing ligands, such as TRAIL, that could induce cell death of immune cells. The success in treating malignant tumors by recombinant TRAIL might apply to some but not all pancreatic tumors because of their differential resistance to TRAIL-induced cell death.

Adenocarcinoma↗
PubMed2001

Transcriptional repression mediated by the KRAB domain of the human C2H2 zinc finger protein Kox1/ZNF10 does not require histone deacetylation.

The KRAB domain of human Kox1, a member of the KRAB C2H2 zinc finger family, confers strong transcriptional repressor activities even to remote promoter positions. Here, HDAC inhibitors were used to demonstrate that histone deacetylation is not required for mediating transcriptional repression of KRAB zinc finger proteins. Two reporter systems with either stably integrated or transiently transfected templates, both under control of strong viral promoters, were analyzed. Under all circumstances, HDAC inhibition did not alter the repression potential of the KRAB domain. In case of the stably integrated luciferase reporter gene system, neither expression levels of the KRAB fusion protein nor complex formation with its putative co-repressor TIF1beta were significantly changed. Furthermore, the TIF1beta/KRAB complex was devoid of mSin3A and HDAC1. In the transient transfection system, the transcriptional repression induced by TIF1beta and HP1alpha was not diminished by HDAC inhibitors, whereas the repressory activity of TIF1alpha was significantly affected. Thus, KRAB, TIF1beta and HP1alpha are likely to be functionally linked. In conclusion, HDAC activity is not essential for the strong transcriptional repressor activity mediated by the KRAB domain of Kox1 in particular and, presumably, by KRAB domains in general. This feature might be helpful in identifying and characterizing target genes under the control of

Chromobox Protein Homolog 5↗
PubMed2001

Activation of c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma.

Mitogen-activated protein kinase (MAPK) signaling was examined in malignant melanoma cells exposed to hypoxia. Here we demonstrate that hypoxia induced a strong activation of the c-Jun NH2-terminal kinase (JNK), also termed stress-activated protein kinase (SAPK), in the melanoma cell line 530 in vitro. Other members of the MAPK family, e.g., extracellular signal-regulated kinase and p38, remained unaffected by the hypoxic stimulus. Activated JNK/SAPK could also be observed in the vicinity of hypoxic tumor areas in melanoma metastases as detected by immunohistochemistry. Functional analysis of JNK/SAPK activation in the melanoma cell line 530 revealed that activation of JNK/SAPK is involved in hypoxia-mediated tumor cell apoptosis. Both a dominant negative mutant of JNK/SAPK (SAPKbeta K-->R) and a dominant negative mutant of the immediate upstream activator of JNK/SAPK, SEK1 (SEK1 K-->R), inhibited hypoxia-induced apoptosis in transient transfection studies. In contrast, overexpression of the wild-type kinases had a slight proapoptotic effect. Inhibition of extracellular signal-regulated kinase and p38 pathways by the chemical inhibitors PD98058 and SB203580, respectively, had no effect on hypoxiainduced apoptosis. Under normoxic conditions, no influence on apoptosis regulation was observed after inhibition of all three MAPK pathways. In contrast to recent findings, JNK/SAPK activation did not correlate with Fas or Fas ligand (FasL) expression, suggesting that the Fas/FasL system is not involved in hypoxia-induced apoptosis in melanoma cells. Taken together, our data demonstrate that hypoxia-induced JNK/SAPK activation appears to play a critical role in apoptosis regulation of melanoma cells in vitro and in vivo, independent of the Fas/FasL system.

Apoptosis↗
PubMed2000

FasL:Fas ratio--a prognostic factor in breast carcinomas.

Programmed cell death (apoptosis) is primarily mediated by Fas ligand (FasL; CD95L) and the Fas receptor (Fas; CD95). In this study, FasL was detected by immunohistochemical analysis in 85% of breast carcinomas and 14% of fibroadenomas randomly chosen, indicating that high expression of FasL might play a role in tumor pathology. FasL and Fas levels as well as FasL:Fas ratios were further ascertained in 215 human breast tumors, including 199 invasive ductal carcinomas, by real-time quantitative reverse transcription-PCR and compared with expression levels and ratios found in 25 normal human tissues, in 37 fibroadenomas, and in 5 normal breast tissues. Among breast carcinomas, high FasL mRNA expression seems to be positively correlated with histological grading (n = 212; P<0.0001). A ratio of FasL:Fas mRNA transcripts >1 is found to be significantly associated with decreased patient's disease-free survival (n = 211; P<0.03) and increased mortality (n = 211; P = 0.19). A FasL:Fas ratio >1 is related to tumor progression scored by histological grading (n = 212; P<0.02). The selection process leading to highly aggressive breast tumor variants might be enhanced by FasL-mediated tumor fratricide, eventually a possible target for novel therapeutic strategies.

Adolescent↗
PubMed2000

A novel secreted ribonuclease from Bacillus intermedius: gene structure and regulatory control.

A second secreted ribonuclease, designated binase II, has been detected in Bacillus intermedius 7P, and its structural gene was cloned and sequenced. Unlike the well-known binase I, a 109-amino acid guanyl-specific enzyme, the 292-residue binase II is closely related to the B. subtilis nuclease Bsn, in structure and in its enzymatic properties. Binase II is also insensitive to inactivation by barstar, an inhibitor protein that is specific for guanyl-specific ribonucleases. While both B. intermedius enzymes are induced upon phosphate starvation, only the gene for binase I belongs to the pho regulon system and carries pho-box elements adjacent to its promoter sequence. The gene for binase II is similar to that for Bsn in lacking such elements. The birB gene coding for binase II appears to be located next to the 3'-end of a ferric ion transport operon, with which it convergently overlaps. This would allow attenuator control over binase II expression under conditions of starvation for ferric ions.

Amino Acid Sequence↗
PubMed2000

Absolute quantification of human chorionic gonadotropin-beta mRNA with TaqMan detection. 4.

We describe a reverse transcriptase-polymerase chain reaction (RT-PCR) for determination of human chorionic gonadotropin-beta (HCG beta) mRNA copies using the TaqMan system. To evaluate our quantitative assay, we analyzed HCG beta transcripts of all protein coding genes (HCG beta 5, 3, 8, and 7) in human RNA panels of different normal tissues and in glycodelin-A-stimulated trophoblast cell cultures. Absolute quantification using HCG beta TaqMan probe was found to be highly reproducible. Our study of RNA panels confirms recently published results that expression of HCG beta transcripts is a common feature of a great variety of different normal tissues. High levels of HCG beta mRNAs (> 1,000 molecules per 200 ng RNA) were detected in placenta, uterus, and testis. An increase of HCG beta mRNA expression (1.7-fold) was detected at 150 micrograms/mL glycodelin-A treatment in trophoblast cell culture. Time-dependence study showed that the increase in HCG beta mRNA level was evident at 60 min after glycodelin-A treatment. In summary, we have developed a highly sensitive one-tube, one-enzyme quantitative RT-PCR system that is time-saving and avoids postamplification procedures.

Adolescent↗
PubMed2000

Human chorionic gonadotrophin-beta transcripts correlate with progesterone receptor values in breast carcinomas.

The pathophysiological role for the expression of human chorionic gonadotrophin (hCG) in malignant neoplasms is currently speculative. We investigated the overall expression of genes hCG-beta 5, 3, 8 and 7 in breast carcinoma (n=214), fibroadenoma (n=37) and macromastia (n=10) by quantitative reverse transcriptase-PCR. Eighty (37.4%) of the breast cancer samples revealed positive hCG-beta mRNA expression and the mean value was 67. 9 copies per 200 ng total RNA (range: 0-1743; 95% confidence interval (CI) for mean: 44-92). Fibroadenomas had more frequently detected (56.8%) and greater hCG-beta copy numbers (mean 86.9; range: 0-845; 95% CI for mean: 35-138). Macromastia probes yielded no positive hCG-beta mRNA. The hCG-beta mRNA expression was significantly different in the three histological subgroups (P=0. 006). Among breast carcinomas, a positive correlation was detected between hCG-beta mRNA copy numbers and progesterone receptor (PgR) values (P<0.001). No significant differences were seen regarding disease-free (P=0.87) and overall survival (P=0.20) depending on hCG-beta mRNA status. Finally, our findings do not support a role for hCG-beta in malignant transformation of human breast cells and indicate a possible involvement of hCG-beta in benign breast disease. The relationship with PgR expression may suggest that progestins regulate the expression of hCG in breast epithelial cells.

Adult↗