Biomedical subjects

Erno Duda

Publications and source records attributed to Erno Duda.

10 recordsLinked to original sources

PubMed2006

Gene expression dynamics in deer antler: mesenchymal differentiation toward chondrogenesis.

Annual re-growth of deer antler represents a unique example of complete organ regeneration. Because antler mesenchymal cells retain their embryonic capacity to develop into cartilage or bone, studying antler development provides a natural system to follow gene expression changes during mesenchymal differentiation toward chondrogenic/osteogenic lineage. To identify novel genes involved either in early events of mesenchymal cell specialization or in robust bone development, we have introduced a 3 K heterologous microarray set-up (deer cDNA versus mouse template). Fifteen genes were differentially expressed; genes for housekeeping, regulatory functions (components of different signaling pathways, including FGF, TGFbeta, Wnt), and genes encoding members of the Polycomb group were represented. Expression dynamics for genes are visualized by an expression logo. The expression profile of the gene C21orf70 of unknown function is described along with the effects when over-expressed; furthermore the nuclear localization of the cognate protein is shown. In this report, we demonstrate the particular advantage of the velvet antler model in bone research for: (1) identification of mesenchymal and precartilaginous genes and (2) targeting genes upregulated in robust cartilage development.

Amino Acid Sequence↗

PubMed2006

Pentoxifylline and its major oxidative metabolites exhibit different pharmacological properties.

Previous investigations indicate that some of the metabolites of the hemorheological agent pentoxifylline (PTX), namely 1-(5-hydroxyhexyl)-3,7-dimethylxanthine (M1), 1-(4-carboxybutyl)-3,7-dimethylxanthine (M4) and 1-(3-carboxypropyl)-3,7-dimethylxanthine (M5), concur to some of the biological effects of the drug. However, information on the bioactivity of the major circulating oxidative metabolites of PTX (M4 and M5) is scanty. Here, we compared the effects of M4 and M5 with that of PTX and its major reductive metabolite, M1, on TNF-alpha production and cytotoxicity, endothelial cell proliferation and on the ATPase activity related to some ATP-binding cassette (ABC) transporters. Unlike PTX and M1, M4 and M5 poorly inhibited lipopolysaccaride-stimulated tumor necrosis factor-alpha (TNF-alpha) release by RAW 264.7 murine macrophages, and did not affect at all cell proliferation and upregulation of TNF-alpha-induced vascular cell adhesion molecule-1 (VCAM-1) in H5V endothelioma cells. By contrast, M4 and M5 were more effective than PTX and M1 in protecting WC/1 murine fibrosarcoma cells from TNF-alpha cytotoxicity. Moreover, results from ATP hydrolase assays indicated that neither PTX nor its tested metabolites interacted significantly with the human multidrug resistance transporters p-glycoprotein/multidrug resistance 1 (MDR1), multidrug resistance-related protein 1 (MRP1), and breast cancer resistance protein (BCRP). Based on these results and literature data, M5, retaining some of the PTX effects but lacking in significant inhibition of TNF-alpha production, may be a promising candidate drug for certain pathologic conditions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

In vitro and in vivo nuclear factor-kappaB inhibitory effects of the cell-penetrating penetratin peptide.

Penetratin is a cationic cell-penetrating peptide that has been frequently used for the intracellular delivery of polar bioactive compounds. Recent studies have just revealed the major role of polyanionic membrane proteoglycans and cholesterol-enriched lipid rafts in the uptake of the peptide. Both proteoglycans and lipid-rafts influence inflammatory processes by binding a wide array of proinflammatory mediators; thus, we decided to analyze the effect of penetratin on in vitro and in vivo inflammatory responses. Our in vitro luciferase gene assays demonstrated that penetratin decreased transcriptional activity of nuclear factor-kappaB (NF-kappaB) in tumor necrosis factor (TNF)-stimulated L929 fibroblasts and lipopolysaccharide-activated RAW 264.7 macrophages. Penetratin also inhibited TNF-induced intercellular adhesion molecule-1 expression in human endothelial HMEC-1 cells. Exogenous heparan sulfate abolished the in vitro NF-kappaB inhibitory effects of the peptide. Uptake experiments showed that penetratin was internalized by all of the above-mentioned cell lines in vitro and rapidly entered the cells of the lung and pancreas in vivo. In an in vivo rat model of acute pancreatitis, a disease induced by elevated activities of stress-responsive transcription factors like NF-kappaB, pretreatment with only 2 mg/kg penetratin attenuated the severity of pancreatic inflammation by interfering with IkappaB degradation and subsequent nuclear import of NF-kappaB, inhibiting the expression of proinflammatory genes and improving the monitored laboratory and histological parameters of pancreatitis and associated oxidative stress.

Amino Acid Sequence↗

PubMed2005

The proteasome inhibitor MG132 protects against acute pancreatitis.

The cell-permeant MG132 tripeptide (Z-Leu-Leu-Leu-aldehyde) is a peptide aldehyde proteasome inhibitor that also inhibits other proteases, including calpains and cathepsins. By blocking the proteasome, this tripeptide has been shown to induce the expression of cell-protective heat shock proteins (HSPs) in vitro. Effects of MG132 were studied in an in vivo model of acute pancreatitis. Pancreatitis was induced in male Wistar rats by injecting 2 x 100 microug/kg cholecystokinin octapeptide intraperitoneally (ip) at an interval of 1 h. Pretreating the animals with 10 mg/kg MG132 ip before the induction of pancreatitis significantly inhibited IkappaB degradation and subsequent activation of nuclear factor-kappaB (NF-kappaB). MG132 also increased HSP72 expression. Induction of HSP72 and inhibition of NF-kappaB improved parameters of acute pancreatitis. Thus MG132 significantly decreased serum amylase, pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, proinflammatory cytokine concentrations, and the expression of pancreatitis-associated protein. Parameters of oxidative stress (GSH, MDA, SOD, etc.) were improved in both the serum and the pancreas. Histopathological examinations revealed that pancreatic specimens of animals pretreated with the peptide demonstrated milder edema, cellular damage, and inflammatory activity. Our findings show that simultaneous inhibition of calpains, cathepsins, and the proteasome with MG132 prevents the onset of acute pancreatitis.

Acute Disease↗

PubMed2005

Adenovirus infection dramatically augments lipopolysaccharide-induced TNF production and sensitizes to lethal shock.

We observed a remarkable synergism of adenoviruses and LPS in triggering the production of TNF in intact animals. We found that in mice pre-exposed to adenoviruses, LPS injections generated extremely high levels of TNF with altered kinetics. The elevated TNF synthesis stemmed mostly from posttranscriptional up-regulation of TNF production, although transcription of the TNF gene was also induced. Adenoviruses and LPS exhibited a significant but less dramatic synergism in the induction of IL-6, IFN-gamma, and NO. Only marginal changes were detected in the synthesis of a panel of other cytokines. Different serotypes of the virus showed practically identical effects. As deletion mutants lacking indispensable viral genes or UV inactivated virions exhibited similar activities as the infectious, wild-type virus, it seems unlikely that the viral genome plays any significant role in the phenomenon. Published data indicate that other viruses also show some kind of synergism with LPS, although by different cellular mechanisms. T cells and their IFN-gamma production--crucial in the synergism of influenza viruses and LPS--were dispensable in our experiments. We suggest that the phenomenon is probably a general one: an overlap between different molecular mechanisms detecting bacterial and viral pathogens and inducing mediators of nonspecific cell-mediated host defense. The synergism of viruses and LPS (bacteria) could be a concern in medical practice as well as in gene therapy experiments with high doses of recombinant adenoviruses.

Adenoviridae Infections↗

PubMed2005

A nuclear import inhibitory peptide ameliorates the severity of cholecystokinin-induced acute pancreatitis.

AIM: To assess the effect of our novel cell-permeable nuclear factor-kappaB (NF-kappaB) inhibitor peptide PN50 in an experimental model of acute pancreatitis. PN50 was produced by conjugating the cell-penetrating penetratin peptide with the nuclear localization signal of the NF-kappaB p50 subunit. METHODS: Pancreatitis was induced in male Wistar rats by administering 2X100 mug/kg body weight of cholecystokinin-octapeptide (CCK) intraperitoneally (IP) at an interval of 1 h. PN50-treated animals received 1 mg/kg of PN50 IP 30 min before or after the CCK injections. The animals were sacrificed 4 h after the first injection of CCK. RESULTS: All the examined laboratory (the pancreatic weight/body weight ratio, serum amylase activity, pancreatic levels of TNF-alpha and IL-6, degree of lipid peroxidation, reduced glutathione levels, NF-kappaB binding activity, pancreatic and lung myeloperoxidase activity) and morphological parameters of the disease were improved before and after treatment with the PN50 peptide. According to the histological findings, PN50 protected the animals against acute pancreatitis by favoring the induction of apoptotic, as opposed to necrotic acinar cell death associated with severe acute pancreatitis. CONCLUSION: Our study implies that reversible inhibitors of stress-responsive transcription factors like NF-kappaB might be clinically useful for the suppression of the severity of acute pancreatitis.

Active Transport, Cell Nucleus↗

PubMed2005

Synergistic effect of Avemar on proinflammatory cytokine production and Ras-mediated cell activation.

Macrophages activated by lipopolysaccharide and/or phorbol esters exhibited high sensitivity to Avemar, a fermented wheat germ extract. Avemar synergized with lipopolysaccharide and PMA in the induction of the transcription of cytokine genes and release of inflammatory cytokines. At higher concentrations the preparation had a significant negative effect on the proliferation and survival of activated myeloid cell types. Avemar treatment induced the synthesis of ICAM-1 and synergized with the ICAM-inducing effect of TNF, but had no effect on VCAM-1 expression on microvascular endothelial cells. The effect of Avemar on signaling pathways, which are involved in cell activation was studied on HeLa cells as a model system. Avemar treatment increased the activity of stress kinases in a concentration-dependent way, resulting in the activation of AP-1 transcription factor. NF-kappa B-sensitive reporters were also activated by Avemar; in contrast, no effect of the preparation was observed on PKA-sensitive signaling pathways.

Cell Line↗

PubMed2003

The anti-inflammatory effect of methylprednisolone occurs down-stream of nuclear factor-kappaB DNA binding in acute pancreatitis.

Glucocorticoids are potent anti-inflammatory drugs. The molecular mechanisms underlying these effects have not yet been fully revealed. The aim of the present study was to establish whether methylprednisolone pretreatment is beneficial and if it can block the pancreatic DNA binding of the transcription factor nuclear factor-kappaB (NF-kappaB) and proinflammatory cytokine synthesis during cholecystokinin-octapeptide (CCK)-induced acute pancreatitis in rats. Additionally, we set out to investigate the potential effects of methylprednisolone and CCK on pancreatic heat shock protein (HSP) synthesis. The dose-response (5-40 mg/kg) and time-course (6-72 h) curves of methylprednisolone on pancreatic HSP60 and HSP72 synthesis were evaluated following methylprednisolone treatment. We demonstrated that methylprednisolone specifically and dose-dependently induced HSP72 in the pancreas of rats, while it did not have a significant effect on HSP60 expression. The pancreatitis was induced near the peak level of HSP72 synthesis (2 x 30 mg/kg body weight [b.w.] methylprednisolone i.m. at an interval of 12 h, followed by a 12-h recovery period after the second injection of methylprednisolone) by administering 2 x 100 microg/kg CCK subcutaneously at an interval of 1 h. The injections of CCK in the vehicle-pretreated group significantly elevated the levels of pancreatic HSP60 and HSP72 2-4 h after the second CCK injection. Methylprednisolone pretreatment ameliorated many of the examined laboratory (the pancreatic weight/body weight [p.w./b.w.] ratio, the serum amylase activity, the plasma trypsinogen activation peptide concentration, the pancreatic levels of tumor necrosis factor-alpha and interleukin-6, the degree of lipid peroxidation, protein oxidation, nonprotein sulfhydryl group content and the pancreatic myeloperoxidase activity) and morphological parameters of the disease. Methylprednisolone pretreatment did not influence pancreatic NF-kappaB DNA binding, but decreased proinflammatory cytokine synthesis in this acute pancreatitis model. The findings suggest that the anti-inflammatory effect of large doses of methylprednisolone in secretagogue-induced pancreatitis occurs downstream of NF-kappaB DNA binding, and that increased pancreatic HSP72 synthesis may play a role in the protective effect of the drug.

Acute Disease↗

PubMed2003

Truncated chicken interleukin-1beta with increased biologic activity.

Chicken interleukin-1beta (ChIL-1beta) is synthesized as a precursor molecule that unlike its mammalian counterpart, lacks a typical caspase-1 cleavage site. Therefore, it was unclear if proteolytic cleavage of ChIL-1beta can occur and if cleavage might modulate the biologic activity of this cytokine. Using an avian indicator cell line that carries an NF-kappaB-regulated luciferase reporter gene, we established a sensitive and highly specific bioassay for ChIL-1beta. Experiments with a rabbit antiserum indicated that the NF-kappaB-stimulating activity in supernatants of lipopolysaccharide (LPS)-treated chicken HD-11 macrophages is largely due to IL-1beta and that proteolytic processing of natural and recombinant ChIL-1beta is not very efficient. Functional analyses further revealed that cDNAs for either full-length or N-terminally truncated chicken ChIL-1beta yielded active cytokine. A truncated molecule that closely resembled putative mature ChIL-1beta exhibited more than 100-fold enhanced biologic activity after expression in mammalian cells, indicating that precursor cleavage is indeed of critical importance for maximal activity.

Animals↗

PubMed2002

Flow cytometric cytotoxicity assay for measuring mammalian and avian NK cell activity.

BACKGROUND: Flow-cytometric assays are convenient alternatives to classic radioactive natural killer (NK) tests. MitoTracker Green FM, a green fluorescent intracellular probe serving originally for staining mitochondria, seemed especially suitable for labeling NK target cells. Materials and Methods NK target cells were labeled with MitoTracker Green FM. After incubation with effector spleen cells, cell suspensions were stained with propidium iodide (PI), and flow-cytometric analysis was performed. RESULTS: MitoTracker Green FM stained efficiently each cell type we assayed, including resting cells, and it was not released from dead cells. NK assays were set up using mouse spleen effector cells and K562 NK target cells. MitoTracker Green FM and PI double staining allowed a discrimination of live and dead target cells, and the cytotoxicity values were in the expected range. Then the method was applied to a less well-known chicken model. We found that chicken-skin fibroblasts had a definite sensitivity to autologous splenic NK cells, sometimes as high as the sensitivity of classic NK targets. CONCLUSIONS: Convenient flow-cytometric NK tests can be performed by MitoTracker Green FM and PI staining. Using this method, we demonstrated that chicken fibroblasts are sensitive to the cytotoxic effect of autologous NK cells.

Aldehydes↗