Biomedical subjects

U Warskulat

Publications and source records attributed to U Warskulat.

At least 19 recordsLinked to original sources

PubMed2004

Light deprivation slows but does not prevent the loss of photoreceptors in taurine transporter knockout mice.

UNLABELLED: Taurine transporter knockout mice show severe retinal degeneration at an early age. The study was designed to determine whether degeneration also takes place in the absence of light. Mice were maintained up to 6 weeks of age in cyclic lighting or in total darkness. Degeneration took place in both groups, but was more rapid in animals exposed to standard cyclic illumination. At the ultrastructural level the retinas showed features characteristic of apoptosis but not of necrosis. CONCLUSIONS: Cell differentiation is not seriously affected by the lack of a functional taurine transporter but mature photoreceptor cells do not survive without an intact transporter, even in the dark.

Animals↗

PubMed2003

Taurine-induced long-lasting enhancement of synaptic transmission in mice: role of transporters.

Taurine, a major osmolyte in the brain evokes a long-lasting enhancement (LLETAU) of synaptic transmission in hippocampal and cortico-striatal slices. Hippocampal LLETAU was abolished by the GABA uptake blocker nipecotic acid (NPA) but not by the taurine-uptake inhibitor guanidinoethyl sulphonate (GES). Striatal LLETAU was sensitive to GES but not to NPA. Semiquantitative PCR analysis and immunohistochemistry revealed that taurine transporter expression is significantly higher in the striatum than in the hippocampus. Taurine transporter-deficient mice displayed very low taurine levels in both structures and a low ability to develop LLETAU in the striatum, but not in the hippocampus. The different mechanisms of taurine-induced synaptic plasticity may reflect the different vulnerabilities of these brain regions under pathological conditions that are accompanied by osmotic changes such as hepatic encephalopathy.

Algorithms↗

PubMed2001

Hypermethylation of the tumor necrosis factor receptor superfamily 6 (APT1, Fas, CD95/Apo-1) gene promoter at rel/nuclear factor kappaB sites in prostatic carcinoma.

DNA hypermethylation of CpG-rich promoter sequences is associated with tumor suppressor gene inactivation in many human cancers, notably in carcinoma of the prostate and the urinary bladder. Recently, the mouse homologue of the tumor necrosis factor receptor superfamily 6 (TNFRSF6) gene was reported to be inactivated by DNA methylation in various cell types. The Fas (CD95, Apo-1) protein encoded by the TNFRSF6 gene is an important mediator of apoptosis, which also is downregulated in different types of human carcinoma. We therefore investigated the methylation of the TNFRSF6 promoter in prostatic and bladder carcinomas and cell lines. In a restriction enzyme polymerase chain reaction assay, four of 32 prostatic carcinomas and three of 15 advanced bladder carcinomas showed evidence of hypermethylation at the rel/nuclear factor kappaB (NFkappaB) binding sites essential for promoter activity. The DU145 cell line derived from a metastasis of a prostate carcinoma also displayed hypermethylation in this assay, which was confirmed by bisulfite sequencing. Treatment of DU145 cells with the methylation inhibitor deoxyazacytidine slightly increased Fas protein expression, as detected by flow cytometry analysis. In vitro methylation of the TNFRSF6 promoter at the rel/NFkappaB sites completely abolished its activity. Thus, although the TNFRSF6 gene can be inactivated efficiently by DNA methylation, hypermethylation occurs neither frequently nor extensively in human carcinomas and appears to play a limited role in downregulation of Fas expression.

Aged↗

PubMed2001

Identification of osmosensitive and ammonia-regulated genes in rat astrocytes by Northern blotting and differential display reverse transcriptase-polymerase chain reaction.

BACKGROUND/AIMS: The hepatic encephalopathy (HE) is in part the result of astrocyte swelling with alterations of glial function. Detoxification of ammonia may be one mechanism by which astrocyte swelling is triggered in HE. METHODS: The differential display polymerase chain reaction (DDRT-PCR) and Northern blot analysis were used for study the functional consequence of cell volume changes and ammonia on gene expression in primary rat astrocytes. RESULTS: Differentially expressed cDNA products were identified with about 92% homology to genes coding for mouse proline rich protein expressed in brain (PRTB), rat clusterin, elongin, and human Kelch motif containing protein. As shown by Northern blot analysis, PRTB and clusterin mRNA levels were upregulated by 19-64% after 4-8 h by both ammonia and hypoosmolarity. Elongin mRNA expression increased by 97% in response to ammonia but slightly by hypoosmolarity. Further, hypoosmotic exposure for 1-24 hours but not ammonia led to an increase of 80% in KMCP mRNA levels. CONCLUSIONS: The identification of these genes offers the opportunity to identify unrecognized molecular mechanisms of HE. The finding that several genes are induced by both, hypoosmolarity and ammonia, supports the view that astrocyte swelling is a major, but not the only pathogenetic event in HE.

Ammonia↗

PubMed2000

Defining CD95 as a tumor suppressor gene.

The CD95 (Apo-1/Fas) receptor-ligand system is one of the key regulators of apoptosis and is particularly important for the maintenance of lymphocyte homeostasis. There is now broad evidence that susceptibility of tumor cells towards CD95-mediated apoptosis is largely reduced. In the human, germline and somatic mutations of the CD95 gene are associated with a high risk of both lymphoid and solid tumors. Based on these observations a new concept defining CD95 as a tumor suppressor gene is discussed. In addition to CD95, its natural ligand (CD95L) is also implicated in malignant progression. Compared to their nonmalignant counterparts, malignant cells frequently exhibit aberrant de novo expression of CD95L and are able to induce CD95L-mediated apoptosis in bystander cells. The role for neoplastic CD95L expression in local tissue destruction, invasion, and metastatic spread has been established for many tumor types. CD95L expression by malignant cells may counteract the host's antitumor immunity and favors immune escape of the tumor. On this basis, the significance of loss of CD95 and gain of CD95L expression for tumor progression is discussed.

Apoptosis↗

PubMed2000

CD95 ligand expression as a mechanism of immune escape in breast cancer.

Interaction of CD95 (Apo-1/Fas) and its ligand (CD95L) plays an important role in the regulation of the immune response, since CD95+ lymphocytes may be killed after engagement of the CD95 receptor. Studying the CD95/CD95L system in 40 cases of breast cancer, the malignant cells expressed CD95L, but lost CD95 expression, when compared with non-malignant mammary tissue. Jurkat T cells incubated on breast cancer sections underwent CD95L-specific apoptosis. The rate of apoptosis correlated with the CD95L mRNA levels of the tissue samples. In four breast cancer cell lines, CD95L expression was increased by interferon-gamma (IFN-gamma), which resulted in higher levels of CD95L-specific apoptosis in co-cultured Jurkat T cells. Since IFN-gamma is mainly secreted by activated T cells, up-regulation of CD95L in breast cancer cells in response to IFN-gamma may thus counterselect activated tumour-infiltrating T cells and favour the immune escape of breast cancer. As demonstrated by inhibition of matrix metalloproteinases, CD95L expressed on breast cancer cells can also be shed from the cell membrane into the culture supernatant. Supernatants derived from cultured breast cancer cells induced apoptosis in Jurkat T cells via CD95L. In breast cancer patients, depletion of CD4+ and CD8+ peripheral blood lymphocytes was significantly correlated with CD95L expression in the tumours. This might be suggestive for a relationship between CD95L expression by breast cancer and systemic immunosuppression.

Apoptosis↗

PubMed1999

Dexamethasone- and osmolarity-dependent expression of the multidrug-resistance protein 2 in cultured rat hepatocytes.

Expression of the conjugate export pump multidrug-resistance protein 2 (MRP2) in liver is regulated by endotoxin and anti-tumour agents. This paper reports on the effects of dexamethasone and osmolarity on MRP2 expression. MRP2 expression was studied at the protein, mRNA, immunocytochemical and functional levels in cultured rat hepatocytes. Protein and mRNA expression of MRP2 in rat hepatocytes 24 and 48 h after isolation were largely dependent on the presence of dexamethasone (100 nmol/l) in the culture medium. MRP2 was localized at the pseudocanalicular membrane and increased expression of MRP2 was accompanied by a widening of the pseudocanaliculi. In presence of dexamethasone, hypo-osmolarity (205 mosmol/l) led to a strong induction of MRP2 mRNA and protein, whereas expression was decreased by hyperosmolarity (405 mosmol/l). Also, a decay of MRP2 protein and mRNA following dexamethasone withdrawal was osmosensitive. Expression of dipeptidylpeptidase IV, another canalicular protein, was unaffected by dexamethasone and osmolarity. It is concluded that glucocorticoids are strong inducers of MRP2 in liver. Besides short-term carrier insertion/retrieval, osmoregulation of MRP2 also involves a long-term effect on MRP2 expression.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1999

Anisoosmotic regulation of the Nopp140 mRNA in H4IIE rat hepatoma cells and primary hepatocytes.

Using the differential display polymerase chain reaction osmosensitive regulation of mRNA levels of the nucleolar phosphoprotein of 140 kDa (Nopp140) was found in H4IIE rat hepatoma cells. These levels were downregulated after hypoosmotic exposure in H4IIE cells and primary rat hepatocytes. Hyperosmotic incubation increased Nopp140 mRNA levels in H4IIE cells but not in hepatocytes. Inhibition of p38MAPK or MAP kinase kinase upstream of Erk-1 and Erk-2 decreased Nopp140 mRNA levels but did not prevent their osmosensitivity. Because Nopp140 is involved in the regulation of transcriptional activity it could play a role in the osmosignalling pathway towards gene expression in H4IIE cells and hepatocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Regulation of CD95 (Apo-1/Fas) ligand and receptor expression in squamous-cell carcinoma by interferon-gamma and cisplatin.

CD95 (Apo-1/Fas) ligand (CD95L) expression has been observed in various malignancies. In human primary cell lines from a squamous cell carcinoma (SCC) of the vulva, the effect of cisplatin (CDDP) and IFNgamma on the expression of CD95L and its 2 receptor isoforms, CD95 transmembrane (CD95tm) and CD95 soluble receptor, was studied at the mRNA and protein levels. Addition of CDDP and IFNgamma increased CD95L mRNA levels in the primary cell line 6-fold and 1.7-fold, respectively. In comparison, CD95tm mRNA levels were diminished by CDDP but increased 8-fold upon IFNgamma challenge. CD95L expressed by SCC cells was functionally relevant since these cells were able to induce CD95-specific apoptosis in autologous lymphocytes from the SCC-bearing patient. Thus, CD95L expression in SCC may contribute to tumor-associated immunosuppression, which may be modulated by CDDP and IFNgamma. In tumor samples of the primary SCC, CD95L expression was enhanced in the area of the border between invasive tumor tissue and surrounding stroma cells. The locally restricted over-expression of CD95L was congruent with the arrangement of apoptotic stroma cells in the direct vicinity of invading tumor tongues, suggesting a role as invasion factor for CD95L.

Antineoplastic Agents↗

PubMed1999

CD95 ligand expression in dedifferentiated breast cancer.

CD95 ligand expression has been observed in various malignancies. Studying the CD95 ligand (CD95L) and receptor (CD95) system in eight non-malignant mammary tissues and 40 breast cancer tissues, mRNA and protein expression was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence. mRNA levels of CD95L correlated positively (r=0.90; p< 0.01) and transmembrane CD95 inversely (r=-0.88; p< 0.01) with histopathological grading of the breast tumours: CD95L mRNA levels were low in adenomas, but increased by 20-fold in grade I, 120-fold in grade II, and 310-fold in grade III breast cancer. In contrast, CD95 mRNA levels were low in high-grade carcinomas, but high in benign mammary tissues. Since CD95L acts as an efficient inducer of apoptosis in CD95(+) cells, apoptotic cells were identified on the tissue sections. Tumour-infiltrating lymphocytes and stromal cells in close proximity to CD95L-expressing breast cancer underwent apoptosis. As a functional test, CD95(+) target cells were cultured on breast cancer tissue sections. The target cells underwent apoptosis when cultured on breast cancer sections, but could be rescued when CD95L was specifically blocked by a CD95-Fc fusion molecule. The data suggest an inverse regulation of CD95 ligand and receptor expression during dedifferentiation of breast cancer. Killing of bystander cells by the CD95L-expressing breast tumour could be involved in tissue invasion.

Adult↗

PubMed1999

Involvement of soluble CD95 in Churg-Strauss syndrome.

Deficiency of CD95 (Apo-1/Fas)-mediated apoptosis has recently been found in some autoimmune lymphoproliferative disorders due to inherited mutations of the CD95 gene. In this study, impairment of CD95 ligand-mediated killing of lymphocytes and eosinophils in Churg-Strauss Syndrome (CSS), which was a result of variation of CD95 receptor isoform expression, is demonstrated. Compared to those from healthy individuals, peripheral blood lymphocytes from eight CSS patients exhibit a switch from the membrane-bound CD95 receptor expression to its soluble splice variant, which protects from CD95L-mediated apoptosis. In five out of seven CSS patients recurrent oligoclonal T cell expansions were found, all using a Vbeta-gene from the Vbeta21 family associated with similar CDR3 motifs, indicating the predominance of T cell clones of a similar specificity in the CSS patients. In two of them, the effect of immunosuppressive therapy was studied. In both cases aberrant overexpression of the soluble CD95 receptor isoform and deviations from normal TCR Vbeta-gene usage normalized in parallel with the clinical improvement. Furthermore, soluble CD95 was identified as a survival factor for eosinophils rescuing eosinophils from apoptosis in the absence of growth factors in vitro. Given the role of eosinophils as effector cells in CSS, these findings suggest that soluble CD95 may be mechanistically involved in the disease.

Adult↗

PubMed1999

Regulation of the multidrug resistance protein 2 in the rat liver by lipopolysaccharide and dexamethasone.

BACKGROUND & AIMS: Endotoxin lipopolysaccharide (LPS) induces cholestasis and down-regulates the multidrug resistance protein 2 (MRP2). This study intends to characterize the short-term effects of LPS on MRP2. METHODS: The effects of LPS and dexamethasone on excretion of bromosulphalein (BSP), MRP2 messenger RNA (mRNA) levels, and subcellular MRP2 localization were studied by means of liver perfusion, Northern blots, and confocal microscopy. RESULTS: LPS treatment for 3-12 hours decreased biliary BSP excretion (10 micromol/L) by 40%. Hyposmolarity stimulated BSP excretion to control levels 3 hours after LPS injection, but was ineffective after 12 hours or in saline-treated controls. LPS led to a strong decrease of MRP2 mRNA after 12 hours, but not during the first 6 hours. LPS induced the appearance of MRP2 in intracellular vesicles in the immediate vicinity of the canaliculi within 3 hours, and these vesicles were remote from the canaliculi after 6 and 12 hours. The MRP2-containing vesicles did not stain for dipeptidylpeptidase IV (DPPIV). Dexamethasone counteracted the LPS effects on MRP2 mRNA levels, subcellular distribution, and BSP excretion. CONCLUSIONS: LPS induces cholestasis due to an early retrieval of MRP2 from the canalicular membrane, whereas down-regulation of MRP2 mRNA is a later event. LPS-induced MRP2 retrieval from the canalicular membrane is not associated with the retrieval of DPPIV, suggestive for selectivity of the process.

Animals↗

PubMed1999

Involvement of CD95 (Apo-1/Fas) ligand expressed by rat Kupffer cells in hepatic immunoregulation.

BACKGROUND & AIMS: CD95 (Apo-1/Fas) ligand suppresses inflammatory responses in immune-privileged organs. In this study, modulation of the hepatic CD95 receptor/ligand system by interferon gamma and cyclosporin A was investigated. METHODS: CD95 receptor and ligand expression were measured at the messenger RNA level by using quantitative reverse-transcription polymerase chain reaction and immunocytochemistry in primary cultures of rat Kupffer cells, hepatocytes, and T lymphocytes. Soluble CD95 in culture supernatants was detected by enzyme-linked immunosorbent assay and apoptosis by the TUNEL method. RESULTS: Interferon gamma treatment led to an increase in CD95 ligand messenger RNA levels in Kupffer cells followed by an overexpression of the soluble CD95 receptor. Supernatants derived from 24-hour but not from 48-hour interferon gamma-treated Kupffer cells killed lymphocytes by a CD95-dependent mechanism. Cyclosporin A inhibited CD95 ligand expression in Kupffer cells and lymphocyte killing. In liver parenchymal cells, interferon gamma increased messenger RNA levels of the transmembrane CD95 isoform and sensitivity of these cells toward CD95-mediated apoptosis. CONCLUSIONS: The expression pattern of CD95 receptor and ligand in response to interferon gamma points to a coordinated interplay between Kupffer cells, hepatocytes, and T lymphocytes in which Kupffer cells may regulate programmed cell death of T lymphocytes and hepatocytes.

Animals↗

PubMed1999

Lack of osmosensitive CD9 expression in rat liver cells.

CD9 mRNA was found to be strongly expressed in H4IIE rat hepatoma cells and rat liver macrophages (Kupffer cells), whereas the expression was weak in primary rat liver parenchymal cells. An osmosensitive regulation of CD9 mRNA levels was not detectable in all three cell types, whereas this has recently been described for Madin Darby canine kidney cells and rabbit renal papillary cells (see text). The findings suggest that osmoregulation of specific genes may exhibit cell type specificity.

Animals↗

PubMed1999

Regulation of bile salt export pump mRNA levels by dexamethasone and osmolarity in cultured rat hepatocytes.

The major canalicular bile salt export pump (Bsep) of mammalian liver is downregulated by endotoxin. This study reports on the effects of dexamethasone and osmolarity on Bsep mRNA expression in cultured rat hepatocytes and its functional relevance in rat liver. Expression of Bsep mRNA in rat hepatocytes 24 and 48 h after isolation was dependent on the presence of dexamethasone (100 nM) in the culture medium. Bsep was functionally active at the pseudocanalicular membrane in cells cultured for 4 days in medium containing dexamethasone. Hypoosmolarity (205 mosmol/l) led to an induction of Bsep mRNA levels, whereas expression was decreased by hyperosmolarity (405 mosmol/l). Also the decay of Bsep mRNA following dexamethasone withdrawal was osmosensitive. In rat liver, dexamethasone counteracted the lipopolysaccharide (LPS)-induced down-regulation of Bsep mRNA levels after 12 hours and abolished the LPS-induced inhibition of taurocholate excretion. These results indicate that glucocorticoids are strong inducers of Bsep in liver. Furthermore, Bsep mRNA levels are osmosensitively regulated. The data suggest a longterm control of Bsep mRNA by osmolarity in addition to the short-term effects on canalicular bile acid excretion, which were reported recently.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Anisoosmotic regulation of the Mi-2 autoantigen mRNA in H4IIE rat hepatoma cells and primary hepatocytes.

Using the differential display polymerase chain reaction (DDRT-PCR) a 169 bp cDNA product, which is 88.8% homologous to the human Mi-2beta autoantigen, was identified in H4IIE rat hepatoma cells. At protein level 100% homology was found. The Mi-2 mRNA was downregulated after hypoosmotic exposure and upregulated after hyperosmotic exposure in H4IIE cells and rat hepatocytes. The human Mi-2 is an autoantigen in dermatomyositis and is a member of the SNF/RAD 54 helicase family. Accordingly, Mi-2 may not only be a target of osmosignalling but could also be involved in the osmosignalling pathway towards gene expression in H4IIE and liver parenchymal cells.

Adenosine Triphosphatases↗

PubMed1998

Osmolyte strategy in human monocytes and macrophages: involvement of p38MAPK in hyperosmotic induction of betaine and myoinositol transporters.

Betaine and myoinositol are compatible organic osmolytes which are specifically accumulated by cells exposed to hyperosmotic medium. A role for compatible organic osmolytes in the regulation of immune function for rat liver macrophages has been described recently. This report describes an osmolyte strategy in human peripheral blood monocytes and human peripheral blood-derived macrophages. Hyperosmotic (405 mOsm) exposure of monocytes and macrophages led to an upregulation of betaine/gamma-amino-n-butyric acid (GABA) transporter BGT-1 and sodium-dependent myoinositol transporter SMIT in mRNA levels within 6 to 12 h. Induction of BGT-1 and SMIT mRNA occurred regardless of whether hyperosmolarity was induced by addition of NaCl (50 mM) or raffinose (100 mM). Betaine (5 mM) inhibited upregulation of BGT-1 as well as SMIT mRNA. After hyperosmotic (405 mOsm) exposure uptake of betaine and myoinositol was increased up to 10-fold compared to normoosmotic conditions. Hypoosmotic exposure led to a rapid efflux of betaine and myoinositol. Treatment of cells with the pyridinyl imidazole SB 203580 (10 microM), a specific inhibitor of p38 MAP kinase, inhibited the hyperosmolarity-induced increase in BGT-1 and SMIT mRNA as well as betaine and myoinositol uptake by 45-70%. The data show that human peripheral blood monocytes and human peripheral blood-derived macrophages use betaine and myoinositol are compatible organic osmolytes when exposed to osmotic stress and that p38MAPK is involved in hyperosmolarity-induced upregulation of osmolyte transporters BGT-1 and SMIT.

Betaine↗

PubMed1998

Regulation of CD95 (APO-1/Fas) receptor and ligand expression by lipopolysaccharide and dexamethasone in parenchymal and nonparenchymal rat liver cells.

The effect of lipopolysaccharide (LPS) on the expression of CD95 (APO-1/Fas) receptor and ligand (CD95L) was studied in primary cultures of rat liver Kupffer cells (KCs), sinusoidal endothelial cells (SECs), and parenchymal cells (PCs) at the messenger RNA (mRNA) level and by means of immunocytochemistry. LPS treatment of KCs and SECs led to a three- to five-fold increase in CD95L mRNA levels within 6 hours, which declined thereafter. Within 24 hours, the number of KCs and SECs staining positive for CD95L strongly increased. After a lag phase of 12 hours after LPS addition, in both cell types the mRNA levels for the soluble CD95 isoform increased approximately 10-fold; however, the number of KCs and SECs staining positive for transmembrane CD95 remained low and did not significantly increase. Compared with nonparenchymal cells, CD95L mRNA levels in primary hepatocyte cultures were low in the absence and presence of LPS. On the other hand, functionally active CD95 expression markedly increased in response to LPS in these cells. Dexamethasone diminished the LPS-induced stimulation of CD95L expression in nonparenchymal cells but markedly stimulated CD95L expression in PCs. Apoptosis of PCs and thymic lymphocytes was stimulated by the addition of supernatants derived from LPS-treated KC or SEC cultures and was apparently mediated by CD95L as assessed by its sensitivity to inhibitors of the CD95-dependent apoptotic pathway in PCs. The data suggest a complex and timely coordinated interplay between the various liver cell populations with respect to LPS-induced activation of the apoptotic machinery with potential relevance for immunoregulation.

Animals↗