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Biomedical subjects

E Schulze

Publications and source records attributed to E Schulze.

At least 37 records · Page 2Linked to original sources
PubMed2000

Characterization of caveolins from human knee joint catilage: expression of caveolin-1, -2, and -3 in chondrocytes and association with integrin beta1.

Interactions between the extracellular matrix (ECM) and chondrocytes are of great importance for structure and function of cartilage. The present study was undertaken to answer the question whether caveolins take part in integrin-mediated cell-ECM interactions in the human cartilage. In samples of human knee joint cartilage, we detected the caveolin subtypes -1, -2, and -3 by immunohistochemical methods. Double-label experiments revealed a colocalization of caveolin with beta1-integrin. Results of immunoprecipitation and immunoblotting assays show that beta1-integrins associate with all three caveolin subtypes in human chondrocytes and indicate that they are part of the same complexes. Furthermore, immunoelectron microscopy shows the localization of beta1-integrin in caveolae-like structures of the cell membrane. The data stimulate further investigations on the role of the caveolin-integrin complex for integrin-mediated signaling pathways in chondrocytes.

Cartilage↗
PubMed2000

Metabolism of glucocorticoids and mineralocorticoids in patients with adrenal incidentalomas.

BACKGROUND: Adrenal incidentalomas are mostly nonfunctioning adrenocortical adenomas (NFI). However, in 5%-12% of the patients a preclinical Cushing's syndrome (PCS) with autonomous cortisol production by the tumour is present. Since urinary free cortisol excretion is not sensitive enough to determine subclinical hypercortisolism, in the present study more sensitive indicators of daily cortisol production were measured. DESIGN: (1) Tetrahydrocortisol, tetrahydrocortisone, urinary free cortisone together with urinary free cortisol were measured in 35 patients with adrenal incidentalomas (29 NFI and six PCS) and in 35 healthy controls. (2) Since little is known about daily aldosterone production, aldosterone metabolite excretions were measured. (3) As recently reported, ACTH stimulation revealed an increased response of precursors of the glucocorticoid and mineralocorticoid pathway. To find out which steroidogenic enzymes have altered activities, a 1-24ACTH stimulation test (250 microg i.v.) was carried out in 25 patients and 18 healthy controls with determination of multiple steroids. (4) Finally, since it was assumed that 21-hydroxylase deficiency or even 11b-hydroxylase deficiency may be involved in adrenal tumourigenesis, the prevalence of germline CYP21B and CYP11B1 mutations were studied in the same patients, who had underwent the ACTH stimulation test. RESULTS: (1) Glucocorticoid metabolites were within the normal range in all but three patients with NFI. As a group, the patients had subtle alterations in cortisol metabolism. Tetrahydrocortisol excretion was elevated in NFI and PCS compared with normal subjects (2.1 +/- 0.2 and 2.5 +/- 0.5 vs. 1.5 +/- 0.1 mg 24 h(-1); P < 0.05). Accordingly, the twofold elevation of the tetrahydrocortisol/free cortisol ratio indicates an increased 5beta-reduction of cortisol in the liver. (2) Tetrahydroaldosterone and aldosterone-18-glucuronide excretions were not different to controls. (3) In patients with incidentalomas an increased response to ACTH was seen for 17-hydroxyprogesterone (595 +/- 133 vs. 160 +/- 25 ng dL(-1)), 21-desoxycortisol (105 +/- 25 vs. 29 +/- 9 ng dL(-1)) and 11-desoxycortisol (401 +/- 40 vs. 293 +/- 17 ng dL(-1)). (4) In only one of 25 patients, a heterozygous deletion in exon 3 of the CYP21B gene was detected. CONCLUSIONS: (1) In conclusion, even the excretion of the main glucocorticoid metabolites is not a marker sensitive enough to distinguish between NFI and PCS. However, it is also possible that alterations in cortisol secretion are qualitative rather than quantitative. (2) Zona glomerulosa function is not influenced. (3) The elevation of 21-desoxycortisol argues against an impairment of 11beta-hydroxylase and favours a decreased activity of 21-hydroxylase. All others had wild-type sequences of both genes. (4) In conclusion, neither 21-hydroxylase deficiency nor 11beta-hydroxylase deficiency are predisposing factors for adrenal tumourigenesis.

Adenoma↗
PubMed2000

Functionalized and [a]-anellated carbazoles as potential B-DNA ligands: experimental studies of DNA binding and molecular modeling of intercalation complexes.

Three synthetically available carbazole derivatives 3-5 were investigated for DNA binding (ethidium bromide displacement assay, DNA unwinding assay), for inhibition of topoisomerase I and for cell cytotoxicity (antitumour cell lines). In addition molecular modeling studies of DNA complexes were performed by semiempirical quantum chemistry, force field calculations and molecular dynamics calculations. In summary, combining the results from experiments and molecular modeling, the naphthoquinone anellated carbazole 4 emerges as a promising antitumour active candidate for further drug design studies in carbazole chemistry.

Animals↗
PubMed1999

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8.

Until now, many extracellular matrix proteins, e.g. osteopontin and osteonectin, have been used to determine a cell's osteogenic maturation. The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage. In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies. For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium. This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells. In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level. E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts. Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems.

Animals↗
PubMed1999

Immunohistochemical localization of the differentiation marker E11 in dental development of rats.

E11 antigen, originally characterized in a rat osteosarcoma cell line, is known to be expressed during late stages of the osteogenic cell lineage both in vitro and in vivo. The aim of the present study was to monitor the occurrence and distribution patterns of the E11 antigen using monoclonal antibodies (mAb E11 and MEP-1) during different stages of tooth germ development of new-born rats by means of immunohistochemistry. Both antibodies strongly bound to plasma membranes of ameloblasts in presecretory and secretory stages. In addition, odontoblasts and cells of the periodontium were immunoreactive for E11 and MEP-1. During maturation, the immunoreactivity of ameloblast plasma membranes decreased significantly. Our data suggest that E11 and MEP-1 might be important as markers for cell differentiation and mineralization processes during tooth germ development.

Ameloblasts↗
PubMed1999

Chronic liver injury alters basal and stimulated nitric oxide production and 3H-thymidine incorporation in cultured sinusoidal endothelial cells from rats.

BACKGROUND/AIM: Under pathological conditions the nitric oxide synthase (NOS)-mediated nitric oxide production of sinusoidal endothelial cells might be altered. Therefore, studies were performed to evaluate the nitrite formation by cultured sinusoidal endothelial cells from rat livers chronically injured by thioacetamide and the effect of endogenously or exogenously generated nitric oxide on their proliferative activity. METHODS: Basal and stimulated nitrite formation, expression of NOS and DNA synthesis were examined in sinusoidal endothelial cells isolated and cultivated from livers with incipient or advanced chemically-induced cirrhosis. RESULTS: Cultured sinusoidal endothelial cells from injured livers exhibited a reduced basal and an increased lipopolysaccharide-stimulated nitrite production when compared with controls. Western blot analysis revealed a markedly reduced protein expression of endothelial NOS (eNOS) and inducible NOS (iNOS) in sinusoidal endothelial cells from both experimental groups when compared with controls. Lipopolysaccharide stimulated iNOS expression in sinusoidal endothelial cells from control livers only marginally, and from those with cirrhosis more strongly. There was no clear correlation between the amount of enzyme and nitrite formation. Cultured sinusoidal endothelial cells from livers with incipient cirrhosis showed a higher proliferative activity than controls. Endogenously-produced nitric oxide inhibited DNA synthesis in all groups in a cGMP-independent way. Exogenously-generated nitric oxide affected DNA synthesis differently in sinusoidal endothelial cells from controls and injured livers. CONCLUSION: The results provide evidence that cultured sinusoidal endothelial cells from controls and livers with incipient or advanced cirrhosis differ with respect to basal and lipopolysaccharide-stimulated nitrite production. The data can be taken as evidence that in sinusoidal endothelial cells from livers chronically injured by thioacetamide, eNOS and iNOS are aberrantly expressed and differently regulated.

Animals↗
PubMed1998

Spectrofluorimetric quantification of malondialdehyde for evaluation of cyclooxygenase-1/thromboxane synthase inhibition.

The in vitro assay developed by Hartmann and Ledergerber (1995) utilizing the spectrofluorimetric quantification of malondialdehyde after reaction with thiobarbituric acid was modified and used for further investigations. The human whole blood was replaced by a platelet suspension of pig blood, and calcium ionophore A23187 was used instead of collagen for inducing the arachidonic acid cascade. The modified assay represents a simple, time and cost saving method for the evaluation of cyclooxygenase-1/thromboxane synthase inhibition. The reproducibility and comparability of results is given. Additional experiments allow classification of selective phospholipase A2, cyclooxygenase-1, and thromboxane synthase inhibitors. Further studies of malondialdehyde formation show that the cyclooxygenase and/or the thromboxane synthase are competitively inhibited by reaction products of the cyclooxygenase pathway by a negative feedback mechanism.

Animals↗
PubMed1998

Pre-treatment of peripheral blood progenitor cells with macrophage inflammatory protein-1alpha induces prolonged survival of early progenitor cells over 6 weeks of long-term culture.

It has previously been shown that a combination of macrophage inflammatory protein-1alpha (MIP-1alpha) and interleukin (IL)-3 maintained human bone marrow (BM)-derived long-term culture-initiating cells (LTC-IC) for at least 8 weeks in vitro. We investigated colony- and cobblestone area-formation potential of peripheral blood progenitor cells (PBPC) at week 6 of long-term culture (LTC) in the absence of exogenous MIP-1alpha. but using cells which had been pre-incubated in the presence of MIP-1alpha for 40 h in liquid culture. The recovery of colony-forming cells (CFC) and cobblestone-area-forming cells (CAFC) after pre-incubation of PBPC with MIP-1alpha was up to threefold higher (P<0.05, n=5) than that of untreated controls. These results demonstrate that short-term pre-treatment of PBPC with MIP-1alpha induces long-lasting survival effects on early PBPC-derived progenitors in vitro.

Cell Survival↗
PubMed1998

Steroid 21-hydroxylase mutations and 21-hydroxylase messenger ribonucleic acid expression in human adrenocortical tumors.

Twenty-one hydroxylase (P450c21) is a key enzyme essential for normal zona glomerulosa and fasciculata function. Recently, 21-hydroxylase deficiency has been implicated in the pathogenesis of adrenocortical tumors. Therefore, we investigated the mutational spectrum of the CYP21B gene and the messenger RNA expression of P450c21 in six aldosterone-producing adenomas, seven cortisol-producing adenomas, two nonfunctional incidentally detected adenomas, and four adrenal carcinomas. DNA from leukocytes and tumors was amplified by PCR using primers specific for the CYP21B gene. The 10 exons, intron 2, intron 7, all other exon/intron junctions, and 380 bp of the promoter region of CYP21B were automatically sequenced. Poly(A) RNA was extracted from tumor tissue, dot blotted on a nylon membrane, and hybridized with 32P-labeled P450 side-chain cleavage, P450 17-alpha-hydroxylase, and P450c21 complementary DNA probes. We detected heterozygous germline mutations (exon 7, Val 281Leu) in two patients, one with a cortisol-producing adenoma and the other with an androgen-secreting adrenocortical carcinoma. A somatic, heterozygous microdeletion was found in exon 3 of one aldosterone-producing adenoma. The P450c21 gene expression correlated with the clinical phenotype of the tumor, with low P450c21 messenger RNA expression in nonfunctional adenomas (18.8%, 1.5%) compared with high P450c21 expression in aldosterone- and cortisol-producing adenomas (84 +/- 8% and 101 +/- 4%, respectively, vs. normal adrenals, 100 +/- 10%). In conclusion, the prevalence of heterozygous germline mutations in the CYP21B gene was higher in patients with adrenocortical tumors (11%; 95% confidence interval, 1-34%) than in the general European population (2%; 95% confidence interval, 1.93-2.06%), but this difference is questionable because of the low number of subjects in our series. The pathophysiological significance of this finding in the presence of one normal CYP21B gene seems to be low, suggesting that 21-hydroxylase deficiency is not a major predisposing factor for adrenal tumor formation.

Adrenal Cortex Neoplasms↗
PubMed1998

[Biological monitoring of persons in areas with increased soil mercury, arsenic and antimony content].

Part of the northern Palatinate region in Germany is characterised by elevated levels of mercury, arsenic and antimony in the soil due to the presence of ore sources and former mercury mining activities. In a biomonitoring study, roughly 200 residents of this region were investigated for a putative increased absorption of these elements. Urine and scalp hair samples were examined. The results did not show a correlation between the mercury and antimony contents in the soil of the housing area and those in urine and hair. On the other hand, slightly but presumably non-hazardous, elevated arsenic contents in urine and scalp hair could be correlated to an increased arsenic content in the soil. According to multiple regression analysis this increase also correlated with the consumption of poultry, eggs and vegetables kept and cultivated respectively in grounds contaminated with arsenic. In urine, sex differences could be seen for antimony: male probands showing significantly higher mean urinary antimony levels than female probands. Urinary mercury contents were correlated with the amalgam area. Similarly, scalp hair of men contained significantly higher levels of arsenic and antimony in comparison to women. Mercury contents in scalp hair were correlated with the consumption of seafood. Children aged 2 to 6 years had significantly lower levels of mercury, arsenic and antimony in urine than the adult subjects. Thus, an elevated health hazard to these children by e.g. intake of contaminated soil is not expected.

Adolescent↗
PubMed1998

Determination of 13C/12C-ratios in rumen produced methane and CO2 of cows, sheep and camels.

Naturally produced methane shows different delta 13C-values with respect to its origin, e.g., geological or biological. Methane-production of ruminants is considered to be the dominant source from the animal kingdom. Isotopic values of rumen methane--given in literature--range between -80/1000 and -50/1000 and are related to feed composition and also sampling techniques. Keeping cows, camels and sheep under identical feed conditions and sampling rumen gases via implanted fistuale we compared delta PDB 13C-values of methane and CO2 between the species. Referring to mean values obtained from 4 or 5 samples at different times of 11 animals (n = 47) we calculated delta PDB 13C-medians resulting in small but not significant differences within and significant differences between the species for CO2 and methane. The delta PDB 13C-differences between methane and CO2 were statistically equal within and also between the species. Therefore a linear regression of methane values on CO2 is appropriate and leads to: delta PDB 13C(methane)/1000 = 1.57 * delta PDB 13C(CO2)/1000 - 47/1000 with a correlation coefficient of r = 0.87.

Animals↗
PubMed1997

The histone H1 genes of the dipteran insect, Chironomus thummi, fall under two divergent classes and encode proteins with distinct intranuclear distribution and potentially different functions.

Four histone H1 genes of the midge, Chironomus thummi piger, and three H1 genes of the subspecies C. thummi thummi have been cloned and assigned to the four different H1 proteins from C. thummi larvae. Together with an earlier cloned H1 gene from C. thummi thummi [Hankeln, T. & Schmidt, E. R. (1991) Chromosoma 101, 25-31], these genes probably constitute the complete complement of H1 genes in both subspecies. They were found to fall under two classes that differ remarkably in their gene copy numbers, genomic organization, structure of flanking sequences, codon usage, and expression during embryonic development, and that encode H1 proteins of divergent structure. Histone H1 I-1 contains an inserted sequence, KAPKAPKAPKSPKAE in C. thummi piger, and KAPKAPKSPKAE in C. thummi thummi, that is lacking in the other H1 variants, H1 II-1, H1 II-2, and H1 III-1. In the immediate neighbourhood to the inserted sequence, a substitution in the H1 I-1 protein sequence dramatically enhances the potential to form a reversed turn. In early development, H1 I-1 is expressed at a higher rate than the other H1 genes. The transcripts have a size of about 1 kb; in addition, the H1 I-1 gene exhibited two minor transcripts of about 2.5 and > 3 kb size in middle blastoderm that are possibly polyadenylated. Together with our earlier finding that histone H1 I-1 is found in a limited number of polytene chromosome bands whereas the other H1 histones are uniformly distributed in chromatin, these results intimate functional differences between the two classes of H1 genes and their products.

Amino Acid Sequence↗
PubMed1997

The expression of the imprinted genes H19 and IGF-2 in choriocarcinoma cell lines. Is H19 a tumor suppressor gene?

H19 is a paternally imprinted gene with unknown function. It is located in close proximity to the maternally imprinted IGF-2 gene on chromosome 11p15.5. In this study no consistent relationship between the expression of these two genes in clones derived from JEG-3 and JAr cell lines could be detected. Nor could a consistent relationship be detected between the expression levels of these two genes and between certain characteristic tumorigenic properties of these clones. We included in this study clones, expressing low H19 levels, which after transfection with an H19 expression construct highly expressed the H19 gene. In tumors, formed by the injection of cells of JAr or JEG-3 clones into nude mice, the H19 expression was high and irrelevant to the expression level in the cells before the injection. The same phenomenon was found for IGF-2 expression during tumorigenesis caused by cells of different JEG-3 clones and in some but not all JAr derived clones. Both H19 and IGF-2 are biallelicly expressed in all the JAr and JEG-3 clones. In summary, our observations point to the conclusion that H19 is not a tumor suppressor gene. However, its high expression in all the tumors formed after injection of cells of the JAr and JEG-3 clones, leaves its role, if any, in choriocarcinogenesis an open question.

Animals↗
PubMed1997

Suppression of insulin-like growth factor type I receptor by a triple-helix strategy inhibits IGF-I transcription and tumorigenic potential of rat C6 glioblastoma cells.

Homopurine (AG) and homopyrimidine (CT) oligodeoxyribonucleotides predicted to form triple-helical (triplex) structures have been shown to specifically suppress gene expression when supplied to cultured cells. Here we present evidence that homopurine RNA (effector) sequences designed to form a triplex with a homopurine. homopyrimidine sequence 3' to the termination codon of the insulin-like growth factor type I receptor (IGF-IR) structural gene can efficiently suppress IGF-IR gene transcription. Transfection vectors were constructed to drive transcription of either AG or CT variant triplex-forming strands. To increase the probability of obtaining stable transfectants with adequate expression of effector sequences, these were designed to be transcribed together with cDNA sequences conferring neomycin resistance as a fusion transcript. Rat C6 glioblastoma cells transfected with the AG variant showed dramatic reduction of IGF-IR transcripts compared with untransfected cells. The AG transfectants also exhibited marked down-regulation of the IGF-I, and an enhanced accumulation of serine protease inhibitor nexin-I mRNA. Similar changes in gene expression were observed following transfection of C6 cells with constructs transcribing antisense RNA to IGF-IR transcripts, but were not observed in C6 cells transfected with either the CT triplex variant or with vector lacking triplex-forming sequences. Moreover, C6 cells transfected with AG triplex variant displayed a dramatic inhibition of tumor growth when injected into nude mice. The results suggest that a triple-helix strategy can be used to inhibit transcription elongation of the IGF-IR gene, and emphasize the efficacy of triplex-mediated gene inhibition in an animal model.

Amyloid beta-Protein Precursor↗
PubMed1997

Immunohistochemical detection of human skin nerve fibers.

The autonomic nervous system is involved in different functions such as transduction of afferent sensory inputs, trophic actions, modulation of immunologic events and thermoregulation. In the present investigation, we studied the pattern of human autonomic skin innervation with special reference to its relation to blood vessels, hair follicles, sweat glands and sensory receptors. For the first time, two clinically important areas have been compared: the skin of the forearm and of the face. Using indirect immunohistochemistry, we analyzed the distribution of calretinin (CR), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP), neurokinin A (NKA), vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS), tyrosine hydroxylase (TH), histamine, serotonin, enkephalin, and, enzyme histochemically, NADPH-diaphorase (NADPH-d). In the epidermis, we found nerve fibers containing SP, NKA and CGRP. In the dermis, SP-, CR-, VIP-, CGRP- and NKA-positive nerve fibers were detected. Particularly the large nerve fibers contained CR. VIP-positive fibers occurred especially around hair follicles and sweat glands. CGRP-positive nerve fibers were located close to the epidermal basal membrane, in the wall of blood vessels, and to a lesser extent around hair follicles. Immunoreactivity for SP and NKA in the dermis was observed predominantly in the papillary layer near the epidermal basal membrane. All neuropeptides tested in this study were also detected in the nerve fibers of the subcutis. Most of them were CGRP- and VIP-positive. They occurred in association with sweat glands and large arteries. NPY-positive nerve fibers are predominant in the wall of arteries, arterioles and veins. Nerve fibers containing NKA and SP were less common and identified only in the walls of large arteries in deeper dermal layers. In double-staining experiments, the NADPH-d reaction and reactivity to tubulin revealed a partial co-localization in nerve fibers, blood vessel walls, around glands and ganglionic cells. VIP-positive fibers were more common in the face skin than in the forearm. However, in forearm we detected more NPY-, CGRP-, NKA- and SP-positive nerve fibers than in face skin. These findings are important for future studies on skin disorders, such as sensory neuropathies, inflammatory reactions or allergic responses of human skin.

Blood Vessels↗
PubMed1996

PCR-mediated DNA fingerprinting of atypical campylobacter strains isolated from surface and drinking water.

This study shows that typing through polymerase chain reaction (PCR) can be used to differentiate between strains of Campylobacter and Arcobacter, whereas by means of conventional biotyping Arcobacter might be identified as atypical Campylobacter. Microaerophilic "campylobacter-like" organisms were isolated from several drinking water treatment plants as well as environmental sources. The strains were characterized by biotyping and PCR amplification of Campylobacter flaA/flaB and 16s-rRNA Arcobacter butzleri-specific genes.

Animals↗
PubMed1996

Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees.

Steroid 21-hydroxylase deficiency is among the most common inborn errors of metabolism in man. Characterization of mutations in the 21-hydroxylase gene (CYP21) has permitted genetic diagnosis, facilitated by the polymerase chain reaction (PCR). The most common mutation is conversion of an A or C at nt656 to a G in the second intron causing aberrant splicing of mRNA. Homozygosity for nt656G is associated with profoundly deficient adrenal cortisol and aldosterone synthesis, secondary hypersecretion of adrenal androgens, and a severe form of congenital adrenal hyperplasia (CAH) characterized by ambiguous genitalia and/or sodium wasting in newborns. During the course of genetic analysis of CYP21 mutations in CAH families, we and others have noticed a number of relatives genotyped as nt656G homozygotes, yet showing no clinical signs of disease. A number of lines of evidence have led us to propose that the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one haplotype during PCR amplification of CYP21. For prenatal diagnosis, we recommend that microsatellite typing be used as a supplement to CYP21 genotyping in order to resolve ambiguities at nt656.

Adrenal Hyperplasia, Congenital↗