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Biomedical subjects

K Appel

Publications and source records attributed to K Appel.

At least 19 recordsLinked to original sources
PubMed2003

Predictive variables for the biological behaviour of basal cell carcinoma of the face: relevance of morphometry of the nuclei.

We did a morphometric analysis of 130 histological sections of basal cell carcinoma (BCC) of the face to find out whether morphometric variables in the structure of the nuclei of BCC cells could serve as predictors of the biological behaviour. We considered the following variables: maximum and minimum diameters, perimeter, nuclear area and five form factors that characterise and quantify the shape of a structure (axis ratio, shape factor, nuclear contour index, nuclear roundness and circumference ratio). We did a statistical analysis of primary and recurring tumours and four histology-based groups (multifocal superficial BCCs, nodular BCCs, sclerosing BCCs and miscellaneous forms) using a two-sided t test for independent samples. Multifocal superficial BCCs showed significantly smaller values for the directly measured variables (maximum and minimum diameters, perimeter and nuclear area). Morphometry could not distinguish between primary and recurring tumours.

Adolescent↗
PubMed2000

In vitro identification of the cytochrome P450 isoform responsible for the metabolism of alpha-dihydroergocryptine.

1. The in vitro metabolism of alpha-dihydroergocryptine (DHEC, Almirid), an ergot-derived dopamine agonist for the treatment of Parkinson's disease, has been studied in cultured cell lines following incubation with DHEC. Human hepatocytes as well as two sets of metabolically competent cell lines expressing one single human cytochrome P450 (1A1, 1A2, 1B1, 2A6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4) were used. 2. Mono- and dihydroxy metabolites of DHEC could only be detected in the culture media of the cell line expressing human cytochrome CYP3A4. The same metabolites were found in the media of cultured human hepatocytes derived from three different donors. After 24-h incubation with 1 microM DHEC, approximately 60% mono- and approximately 20% dihydroxy metabolites were detected, i.e. approximately 80% of DHEC was metabolized. Further, DHEC demonstrated an inhibitory effect on CYP3A4-mediated testosterone metabolism and additionally could induce CYP3A4 and CYP2E1 mRNA when added at 10 microM to cultured human hepatocytes. 3. The data suggest that DHEC metabolism in humans is primarily mediated by the CYP3A4 isoform. The results are in accordance with findings derived from other ergot alkaloids.

Aged↗
PubMed2000

[Liver cell culture in bioreactors for in vitro drug studies as an alternative to animal testing].

An important consideration for the utilisation of in vitro culture models for studies on drug metabolism as an alternative to animal testing is the maintenance of a defined degree of cell differentiation. Thus, in vitro conditions reflecting as near as possible the in vivo situation of the cells within the whole organ are required. A bioreactor was developed for the cultivation of liver cells which allows the reorganisation of hepatocytes and non-parenchymal cells of the liver in coculture to form three-dimensional, tissue-like structures including extracellular matrix components produced by the cells. In this study, the vitality and metabolic activity of isolated rat hepatocytes was investigated over a two week culture period in bioreactors. The results show that after a reorganisation phase, the cells preserve specific functions, such as protein and urea synthesis capacity and specific cytochrome P450 activities during the culture period, with maximal values during the first week. Possible applications of the model in pharmaceutical industry are studies on metabolite patterns, enzyme induction, drug-drug-interactions, first pass effects and long-term toxicity of drugs.

Animal Testing Alternatives↗
PubMed1998

Effect of phenytoin on cytochrome P450 2B mRNA expression in primary rat astrocyte cultures.

Studies on cytochrome P450 2B (CYP2B) in the brain have essentially been focused on protein characterization and regional distribution. Due to the high sequence homology between the closely related CYP2B1 and 2B2 isoforms and the low amounts of the corresponding mRNAs few efforts have been made to analyze the expression, regulation, and inducibility of these P450 genes in a specific cell type. In the present study, we investigated CYP2B mRNA expression in primary rat astrocyte cultures under the influence of the anti-epileptic drug phenytoin, which is known to be a CYP2B inducing agent in liver. In situ hybridization with a digoxigenin (DIG)-labeled cRNA probe demonstrated that 30-40% of the astrocytes strongly expressed a CYP2B mRNA-specific signal within the first week of cultivation. With increasing age (> 14 days) a greater percentage of cells (>90%) expressed mRNA for P450 2B. However, the level of transcriptional activity was substantially lower than in younger cultures. To discriminate between the 2B1 and 2B2 isoforms the reverse transcription/polymerase chain reaction (RT/PCR) procedures were proved for rat hepatic mRNA as a control assay. Subsequently, the application of this method on cultured astrocytes confirmed that these brain cells may express CYP2B1 mRNA. CYP2B2 mRNA could not be detected in astrocyte cultures at any age examined. Phenytoin led to the down regulation of CYP2B1 mRNA, which contrasts with the drug inducing effect on hepatic CYP2B1 and 2B2 levels. After 4 hr of exposure of phenytoin to the astrocytes no amplification product could be detected at all. Phenytoin did not induce either CYP2B1 or 2B2 expression.

Alternative Splicing↗
PubMed1998

Molecular biology of microglia cytokine and chemokine receptors and microglial activation.

Activation of brain microglial cells can be subdivided into a number of stages. Early stages likely are proliferation and migration to sites of cell damage. These two stages have been studied exemplarily on the IL-3 receptor beta-subunit and on the CC-chemokine receptor 5 using molecular biological methods. First, IL-3 receptor beta-subunit cDNA has been cloned in full length from rat microglia. Since cultured microglia are already activated to some extent, mRNA of this subunit has been detected in the isolated cells, but was absent in normal rat brain. Lipopolysaccharide (LPS) increased this mRNA in the cultured cells and LPS injected into the circulation of rats induced the mRNA specifically in brain microglia as revealed by in situ hybridizations. Next, we obtained partial cDNAs of receptor-coupled protein tyrosine kinases JAK 1 and JAK 2. These mRNAs were present both in cultured microglia and in rat brain, but were not influenced by LPS. Finally, a full-length cDNA of the rat chemokine receptor 5 has been obtained by PCR methodology. Its mRNA was increased by administration of LPS both in cultured microglia and in vivo. It is expected, that further investigations on these receptors could help to develop improved strategies to combat chronic inflammatory events in the brain.

Animals↗
PubMed1997

Immune responsiveness of splenocytes after chronic daily melatonin administration in male Syrian hamsters.

The interrelationships between the immune system and the pineal hormone, melatonin, have been explored recently. The present studies investigated the effects of daily melatonin injections on reproductive and spleen function in male Syrian hamsters. Testes weights and serum testosterone levels were depressed after 8-10 weeks of daily melatonin injections. Melatonin-treated hamsters exhibited increased splenic lymphoproliferative responses to a polyclonal T-cell mitogen (concanavalin A (Con-A)), but decreased proliferation following stimulation with a polyclonal B-cell mitogen (lipopolysaccharide). It appears that daily melatonin injections in male hamsters increase the T-cell-mediated immune capacity while reducing the antibody-mediated immune potential. These data suggest that chronic, daily melatonin alters immune system responsiveness in hamsters by shifting the balance of cellular and humoral reactivity.

Animals↗
PubMed1997

Lipopolysaccharide induces expression of tumour necrosis factor alpha in rat brain: inhibition by methylprednisolone and by rolipram.

1. We have investigated the effects of the phosphodiesterase (PDE) type IV inhibitor rolipram and of the glucocorticoid methylprednisolone on the induction of tumour necrosis factor alpha (TNF-alpha) mRNA and protein in brains of rats after peripheral administration of lipopolysaccharide (LPS). 2. After intravenous administration of LPS, a similar time-dependent induction of both TNF-alpha mRNA and protein was observed in rat brain. Peak mRNA and protein levels were found 7 h after administration of LPS. 3. In situ hybridization experiments with a specific antisense TNF-alpha riboprobe suggested that the cells responsible for TNF-alpha production in the brain were microglia. 4. Intraperitoneal administration of methylprednisolone inhibited the induction of TNF-alpha protein in a dose-dependent manner. A maximal inhibition of TNF-alpha protein production by 42.9+/-10.2% was observed at a dose regimen consisting of two injections of each 30 mg kg(-1) methylprednisolone. 5. Intraperitoneal administration of rolipram also inhibited the induction of TNF-alpha protein in a dose-dependent manner. The maximal inhibition of TNF-alpha protein production was 96.1+/-12.2% and was observed at a dose regimen of three separate injections of each 3 mg kg(-1) rolipram. 6. In situ hybridization experiments showed that the level of TNF-alpha mRNA induced in rat brain by LPS challenge was reduced by intraperitoneal administration of methylprednisolone (2 x 15 mg kg(-1)) and of rolipram (3 x 3 mg kg(-1)). 7. We suggest that peripheral administration of LPS induces a time-dependent expression of TNF-alpha in rat brain, presumably in microglial cells, and that methylprednisolone and rolipram inhibit LPS-induced expression of TNF-alpha in these cells via a decrease of TNF-alpha mRNA stability and/or TNF-alpha gene transcription.

Animals↗
PubMed1996

Expression of tumor necrosis factor alpha after focal cerebral ischaemia in the rat.

Induction of tumor necrosis factor alpha was studied in the brain of rats after focal cerebral ischaemia by occlusion of the left middle cerebral artery. Using a specific antisense riboprobe for in situ hybridization histochemistry, cells positive for tumor necrosis factor alpha messenger RNA were detected within 30 min in the brain regions known to be necrotic within one to two days after onset of ischaemia. Their number increased over a time period of 1-8 h and then declined. Only a few tumor necrosis factor alpha messenger RNA positive cells could be detected four days after the onset of ischaemia. Reverse-transcription polymerase chain reaction experiments showed that maximal increase of tumor necrosis factor alpha messenger RNA level in the ischaemic brain hemisphere occurred 3 h after occlusion of the middle cerebral artery. Immunocytochemical experiments using an anti-tumor necrosis factor alpha antibody showed the presence of tumor necrosis factor alpha immunopositive cells as early as 30 min after occlusion of the middle cerebral artery in the same brain regions where tumor necrosis factor alpha messenger RNA positive cells were detected. Tumor necrosis factor alpha positive cells were highly abundant in the infarcted brain 8-24 h, but only few of them were detectable four days after the onset of ischaemia. Specificity of the anti-tumor necrosis factor alpha antibody and of the induction of tumor necrosis factor alpha protein was confirmed by western blot analysis. Tumor necrosis factor alpha messenger RNA- and protein-positive cells were also detected in the watershed zone and in some structures of the contralateral brain hemisphere. According to their morphology, tumor necrosis factor alpha-positive cells could be identified as microglial cells and macrophages at different states of activation. This assumption was further confirmed by double-labeling studies using the isolectin B4 from Griffonia simplicifolia, a specific microglial/macrophage cell marker. These results demonstrate that expression of tumor necrosis factor alpha is part of an intrinsic inflammatory reaction of the brain after ischaemia.

Animals↗
PubMed1996

Acute and chronic effects of melatonin as an anticonvulsant in male gerbils.

Melatonin, a hormone produced in the pineal gland and released into the general circulation on a diurnal basis, has been implicated in many behavioral processes, where it has been shown to have anxiolytic, sedative, and anticonvulsant effects. Male gerbils (Meriones unguiculatus) injected daily with melatonin (25 micrograms, s.c.) exhibited a reduced seizure response to pentylenetetrazol (PTZ, 60 mg/kg, s.c.). The present studies determined 1) whether melatonin's effect was related to the time of day that it was administered and 2) whether a single acute injection of melatonin at various doses could produce anticonvulsant activity. Gerbils provided with 13 weeks of daily melatonin injections (25 micrograms, s.c.) exhibited fewer convulsions after PTZ treatment irrespective of the time of day melatonin was injected. In addition, the melatonin-treated gerbils had lower mortality rates (1/12) than the untreated or vehicle-injected gerbils (5/12). On the other hand, single acute injections of melatonin (0.1-10 mg/kg, i.p.) produced no anticonvulsant activity. It appears that the anticonvulsant effects of melatonin occur only after the animals are chronically exposed to the indole. In addition, melatonin's anticonvulsant ability may utilize a different mechanism than those involved in its endocrine effects, since no diurnal difference in melatonin's anticonvulsant activity was observed.

Animals↗
PubMed1995

Mapping of the interaction sites of the growth suppressor protein p53 with the regulatory beta-subunit of protein kinase CK2.

p53 plays an essential role in cellular growth control. Some of its distinct biological functions are regulated by interaction with cellular proteins. We have previously (Wagner et al., 1994) shown that p53 binds to the regulatory subunit of protein kinase CK2. Using C-terminal protein fragments of p53 we now demonstrate that the region between amino acids 287 and 340 on the polypeptide chain of p53 is critical for binding of p53 to the beta-subunit of CK2. Neither phosphorylation at the p34cdc2 site (aa315) nor at the CK2 site (aa392) is necessary for binding of p53 to the beta-subunit of CK2. Using deletion mutants of the beta-subunit of CK2 we also show that an internal region between amino acids 72 and 149 of the beta-subunit of CK2 is necessary for binding to p53. Thus, this study defines new functional regions on the polypeptide chains of p53 and of protein kinase CK2.

Binding Sites↗
PubMed1995

Expression of interleukin-3 and tumor necrosis factor-beta mRNAs in cultured microglia.

The function of interleukin-3 (or multi-CSF) in the hemopoietic system has been studied in great detail. Although its growth promoting activity on brain microglial cells has been confirmed both in vitro and in vivo, its presence in the brain and even in cultured brain cells has repeatedly been questioned. We have shown recently that isolated rat microglia express mRNA(IL-3) and synthesize IL-3 polypeptide. It is shown here by use of the PCR method, that mRNA(IL-3) is found also in C6 glioblastoma, in rat aggregate cultures, and in newborn and adult rat brain. Quantitation of amplified cDNA(IL-3) was achieved by non-competitive RT-PCR using an elongated internal standard. IL-3 messenger RNA was almost undetectable in vivo and low in (serum-free) aggregate cultures. In isolated microglia, mRNA(IL-3) was increased upon treatment with LPS, PHA, with the cytokines IL-1 or TNF-alpha, with retinoic acid, dbcAMP or the phorbol ester TPA. Effects of LPS were inhibited by dexamethasone, while the glucocorticoid by itself had no effect on basal IL-3 expression. LPS increased mRNA(IL-3) in a concentration-dependent manner beginning with 10 pg/ml and reaching plateau levels at 10 ng/ml. LPS also increased mRNAs of TNF-alpha and TNF-beta. TNF-alpha mRNA was already detectable in untreated microglia and LPS-increased levels were sustained for a few days. In contrast, TNF-beta mRNA was observed only between 4 and 16 h of LPS incubation. It was absent in LPS-free microglia, and after 24 h of LPS-treatment or later.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Cloning of rat interleukin-3 receptor beta-subunit from cultured microglia and its mRNA expression in vivo.

The high-affinity receptors for interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of a ligand binding (alpha-) and a transducing (beta-) subunit. Two distinct transducing subunits (clones AIC2A and AIC2B) have been cloned from mouse, whereas in humans, only one (common) beta-subunit (beta c) has been found. A PCR-based cloning strategy was used to obtain a full-length cDNA sequence from rat microglia including 5'-untranslated regions. Sequence analysis revealed a number of features indicative of the presence of only one beta-subunit in the rat. Most likely, the new rIL-3R beta cDNA is the rat equivalent of human respective murine (AIC2B) beta c subunits. Regulation of rIL-3R beta mRNA expression was investigated in cultured microglia and in vivo. Purified microglia expressed significant amounts of rIL-3R beta mRNA. Addition of lipopolysaccharide (LPS) resulted in a marked upregulation of rIL-3R beta mRNA within approximately 4 hr. No downregulation was observed within 1 week's treatment. No rIL-3R beta mRNA was detectable in normal rat brain. However, 3 hr after a single injection of LPS into the tail vein of a rat, a marked induction of receptor mRNA occurred in a variety of brain regions. Transcriptional rates subsided significantly after 24 hr. rIL-3R beta mRNA was visualized by in situ hybridizations with cRNA antisense probes in ramified cells formerly characterized as microglial cells. rIL-3R beta mRNA was also induced in rat brain after occlusion of middle cerebral artery (MCAO).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Changes of beta-amyloid precursor protein splice patterns in brain cell aggregate cultures.

The splice pattern of beta-amyloid precursor protein (beta-APP) has been studied in a variety of neuronal and glial cells and in brain cell aggregate cultures by the polymerase chain reaction (PCR). The brain-typical pattern, in which beta-APP695 is the dominant form, has been found only in aggregate cultures but not in any of the other cell types including neuronal cell lines. Selective elimination of glial cells from aggregates resulted in increased quantities of beta-APP695, whereas removal of neurons led to a reduction of beta-APP695 and to an elevation of beta-APP751 and beta-APP770. This shift of splice pattern was not observed in cocultures of the neuronal cell line PC 12 with primary astrocytes combined in a variety of cellular ratios. Blood serum, which is an essential component of these cultures, tested on aggregates, did not reduce the amount of beta-APP695 or have any marked effects on splice patterns generally. From these results it is concluded that investigations on brain-typical splicing of beta-APP require primary neurons. Neuronal cell lines may be no suitable model systems. Splicing events favoring production of beta-APP695 may mark an important, very early step of amyloid formation in the brain.

Amyloid beta-Protein Precursor↗