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A Braun

Publications and source records attributed to A Braun.

At least 73 records · Page 4Linked to original sources
PubMed2000

Cell cultures as tools in biopharmacy.

A survey is given on a few selected cell culture models that are used for transport studies. They are characterised for growth, transcellular electrical resistance and cytoarchitecture. The importance of standardisation in view of their use as transport models is documented. Their potential for studies on passive permeation and P-glycoprotein-mediated transport is explored and related to published data. Transport studies are presented that were performed in a two-chamber set-up, the Costar "vertical diffusion system". A series of non-homologous compounds showed similar permeability data (P(app)) in the different cell cultures. The origin of the cell type had no remarkable influence on passive transcellular permeation. MDCK cells, an epithelial cell line of canine kidney origin, are perfectly suited to screen for passive permeation. They have low expression of transporter proteins and low metabolic activity. In general, they probably represent the best-known epithelial cell line with respect to genetics as well as lipid and protein composition. MDCK cells are easy to handle. Transport experiments can be done between 7 and 14 days after seeding, when the stationary growth phase is reached. To screen for P-glycoprotein substrates, efflux and uptake studies were performed with mdr1-transfected MDCK cells (MDR1-MDCK) in a one-chamber system in the presence or absence of verapamil or cyclosporin A as inhibitor. Evidence is presented why the transfected cells, which express large amounts of P-glycoprotein, are not suitable for two-chamber transport studies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Replication and virulence of early protein 0 and long latency transcript deficient mutants of the Aujeszky's disease (pseudorabies) virus.

Early protein 0 (EP0)-deficient recombinant Aujeszky's disease viruses, Ka-ep0lac and Ba-ep0lac derived from strains Kaplan and Bartha, respectively, were constructed to explore the impact of the mutation on replication, virulence and latency of the virus. Inactivation of the EP0 gene resulted in a mutation of long latency transcript (Cheung et al., 1991) that is located on the complementary DNA strand of EP0 and immediate early protein (IE)175 genes. In infection of immortalized porcine kidney cells, the growth rate and yield of both EP0(-) mutant strains were significantly smaller than that of wild-type virus. Ka-ep0lac was found to be highly virulent, while Ba-ep0lac showed an attenuated phenotype in mice. PCR assay and immunohistochemistry showed that the Ba-ep0lac virus was able to establish latency in the mouse trigeminal ganglia. However, latent virus was not able to reactivate in explant reactivation assays. Accordingly, latent Ba-ep0lac has the potential to be exploited as vectors for the delivery of foreign genes to the nervous system.

Animals↗
PubMed2000

Formation of multilayers in the caco-2 cell culture model: a confocal laser scanning microscopy study.

PURPOSE: To introduce confocal laser scanning microscopy (CLSM) combined with digital image restoration to characterise Caco-2 cells under different culture conditions, and thus to define additional valid criteria for the optimisation of culture models. METHODS: Growth curves were established and transepithelial electrical resistance (TEER) measured for cells grown in EMEM or DMEM medium on Cyclopore membranes. Cytoskeleton, cell nuclei and tight junctions (TJ) were investigated by CLSM. RESULTS: Cultures reached a plateau of approximately 4.5 x 10(5) cells/cm2 after approximately 10 days. At the same time TEER reached 750 omega cm2. An irregular, fairly complete network of TJ was present at confluence (approximately 2 d). Between 15 and 30 days a regular TJ network was established. Cells formed mixed mono- and multilayers under most conditions with two exceptions: flat monolayers were observed on polycarbonate filters with EMEM and with the Biocoat intestinal epithelium differentiation environment system. In multilayers TJ were found in the upper as well as in the lower cell layers although the regular vertical polarity was disturbed. CONCLUSIONS: CLSM represents an important tool to investigate the cytoarchitecture of Caco-2 cells. 3D-analysis of confocal data gives important clues on the characteristics of cell layers and thus helps to validate optimisation strategies.

Adenocarcinoma↗
PubMed2000

Profilin II is alternatively spliced, resulting in profilin isoforms that are differentially expressed and have distinct biochemical properties.

We deduced the structure of the mouse profilin II gene. It contains five exons that can generate four different transcripts by alternative splicing. Two transcripts encode different profilin II isoforms (designated IIa and IIb) that have similar affinities for actin but different affinities for polyphosphoinositides and proline-rich sequences. Profilins IIa and IIb are also present in humans, suggesting that all mammals have three profilin isoforms. Profilin I is the major form in all tissues, except in the brain, where profilin IIa is most abundant. Profilin IIb appears to be a minor form, and its expression is restricted to a limited number of tissues, indicating that the alternative splicing is tightly regulated. Western blotting and whole-mount in situ hybridization show that, in contrast to the expression of profilin I, the expression level of profilin IIa is developmentally regulated. In situ hybridization of adult brain sections reveals overlapping expression patterns of profilins I and IIa.

Alternative Splicing↗
PubMed2000

Sequential development of airway hyperresponsiveness and acute airway obstruction in a mouse model of allergic inflammation.

BACKGROUND: Mouse models have been established mirroring key features of human bronchial asthma including airway hyperresponsiveness (AHR). Acute airway obstruction in response to an allergen challenge, however, remains to be demonstrated in these models. OBJECTIVE: A mouse model of allergic lung inflammation was employed to analyze the development of specific (allergen-induced) and nonspecific (methacholine-induced) airway obstruction. METHODS: Mice were sensitized to ovalbumin (OVA) and challenged with OVA aerosol twice each week during four weeks. Changes in lung functions were determined by noninvasive head-out body plethysmography. The development of acute airway obstruction after OVA challenge and AHR after methacholine aerosol application were assessed by a decrease in the mid-expiratory flow rate (EF(50)). RESULTS: Two airway challenges were sufficient to induce AHR (5.7 vs. 15 mg/ml methacholine). Further OVA challenges reduced the baseline EF(50) from 1.85 to 1.20 ml/s (4th week) and induced acute airway obstruction. The OVA-induced obstruction was maximal in the 4th week (EF(50) = 0.91 ml/s). CONCLUSION: The development of acute airway obstruction in allergen-sensitized mice was demonstrated by means of head-out body plethysmography. In our model, AHR was observed before the development of airway obstruction.

Airway Obstruction↗
PubMed2000

Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders. CAH is most often caused by deficiency of steroid 21-hydroxylase. The frequency of CYP21-inactivating mutations and the genotype-phenotype relationship were characterized in 155 well defined unrelated CAH patients. We were able to elucidate 306 of 310 disease-causing alleles (diagnostic sensitivity, 98.7%). The most frequent mutation was the intron 2 splice site mutation (30.3%), followed by gene deletions (20.3%), the I172N mutation (19.7%) and large gene conversions (7.1%). Five point mutations were detected that have not been described in other CAH cohorts. Genotypes were categorized in 4 mutation groups (null, A, B, and C) according to their predicted functional consequences and compared to the clinical phenotype. The positive predictive value for null mutations (ppv(null)) was 100%, as all patients with these mutations had a salt-wasting phenotype. In mutation group A (intron 2 splice site mutation in homozygous or heterozygous form with a null mutation), the ppv(A) to manifest with salt-wasting CAH was 90%. In group B predicted to result in simple virilizing CAH (I172N in homozygous or compound heterozygous form with a more severe mutation), ppv(B) was 74%. In group C (P30L, V281L, P453S in homozygous or compound heterozygous form with a more severe mutation), ppv(C) was 64.7% to exhibit the nonclassical form of CAH, but 90% when excluding the P30L mutation. Thus, in general, a good genotype-phenotype relationship is shown in patients with either the severest or the mildest mutations. A considerable degree of divergence is observed within mutation groups of intermediate severity. As yet undefined factors modifying 21-hydroxylase gene expression and steroid hormone action are likely to account for these differences in phenotypic expression.

Adrenal Hyperplasia, Congenital↗
PubMed2000

Experiences with external quality assessment (EQA) in molecular diagnostics in clinical laboratories in Germany. Working Group of the German Societies for Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM).

The German Societies for Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM) have established an official working group on molecular diagnostics in the field of laboratory medicine. The group's objectives are to support the establishment of molecular biology methods for the use in diagnostics in German clinical laboratories. Towards this end, we have defined specific aims, which are 1) the implementation and extension of external quality assessment (EQA) schemes and methodological exercises offered to clinical diagnostic laboratories, 2) the establishment of a proficiency network and data base within the societies, 3) the implementation of an educational program in molecular diagnostic procedures for clinical chemists and laboratory physicians through the organisation of symposia and workshops and 4) the cooperation with other DGKC/DGLM working groups on shared aspects of laboratory analysis, e.g. preanalytics. The focus of this presentation is to introduce some of these goals in more detail with particular emphasis on the first two program aspects and to discuss experiences with these activities.

Clinical Laboratory Techniques↗
PubMed2000

Serum ras (p21) as a marker for occupationally derived lung cancer?

Certain subsets of the population are especially sensitive to carcinogens, and this can be determined using molecular biological methods. In the literature there has been evidence presented for the use of p21ras (ras) as a tumor marker for human carcinogenic substances such as asbestos, polycyclic aromatic hydrocarbons, and vinyl chloride in the workplace. In this study we have examined whether serum ras could serve as a biomarker for the early detection of occupationally derived lung cancer, with an emphasis on Schneeberger (radon-induced) lung cancer. Sera were taken from 65 male tumor patients. Fifty-nine patients suffered from primary lung cancer (including 18 patients with Schneeberger lung cancer and 12 patients with asbestos-related lung cancer). Additionally, 29 patients with non-malignant lung disease, and a healthy control group (44) including 32 former uranium miners of SDAG Wismut exposed to ionizing radiation (radon and its decay products) were examined. Ras protein was determined via three different methods: 1) immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting; 2) SDS-PAGE using 5-17% gradient gels followed by Western blotting; 3) pre-incubation with Blue Sepharose, SDS-PAGE on 5-17% gradient gels, and Western blotting. The results show that 1 ng ras protein was measurable in serum standards. This protein could not be detected in patient sera or in sera from any of the study groups. Thus, ras cannot be considered useful as a marker for the early detection of asbestos-induced or Schneeberger lung cancer.

Aged↗
PubMed2000

Sequence and expression analyses of the UL37 and UL38 genes of Aujeszky's disease virus.

Previously, we sequenced the HSV-1 Ul39-Ul40 homologue genes of Aujeszky's disease virus (ADV), also designated as pseudorabies virus (Kaliman et al., 1994a, b). Now we report the nucleotide sequence of the adjacent DNA that encodes Ul38, the 5'-region (750 bp) of Ul37, and the promoter regions between these divergently arranged two genes. The ADV Ul38 gene encodes a protein of 368 amino acids. Amino acid sequence comparison of ADV Ul38 with that of other herpesviruses revealed significant structural homology. In a transcription study using RNase protection assay and Northern blot hybridization, we found that the Ul38 gene had one initiation site, but the Ul37 gene was initiated at two transcription sites with two potential initiator AUGs, one of which was dominant. Comparison of ADV Ul37, Ul38 and ribonucleotide reductase gene expression showed that these genes belong to the same temporal class with early kinetics. Data of structural and transcriptional studies suggest that regulation of the expression of these two ADV genes could differ from that of the HSV-1 virus.

Animals↗
PubMed1999

Chip-based genotyping by mass spectrometry.

Silicon chips with immobilized target DNAs were used for accurate genotyping by mass spectrometry. Genomic DNAs were amplified with PCR, and the amplified products were covalently attached to chip wells via N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) chemistry. Primer annealing, extension, and termination were performed on a 1-microl scale directly in the chip wells in parallel. Diagnostic products thus generated were detected in situ by using matrix-assisted laser desorption ionization mass spectrometry. This miniaturized method has the potential for accurate, high-throughput, low-cost identification of genetic variations.

Base Sequence↗
PubMed1999

Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology.

The high density lipoprotein (HDL) receptor SR-BI (scavenger receptor class B type I) mediates the selective uptake of plasma HDL cholesterol by the liver and steroidogenic tissues. As a consequence, SR-BI can influence plasma HDL cholesterol levels, HDL structure, biliary cholesterol concentrations, and the uptake, storage, and utilization of cholesterol by steroid hormone-producing cells. Here we used homozygous null SR-BI knockout mice to show that SR-BI is required for maintaining normal biliary cholesterol levels, oocyte development, and female fertility. We also used SR-BI/apolipoprotein E double homozygous knockout mice to show that SR-BI can protect against early-onset atherosclerosis. Although the mechanisms underlying the effects of SR-BI loss on reproduction and atherosclerosis have not been established, potential causes include changes in (i) plasma lipoprotein levels and/or structure, (ii) cholesterol flux into or out of peripheral tissues (ovary, aortic wall), and (iii) reverse cholesterol transport, as indicated by the significant reduction of gallbladder bile cholesterol levels in SR-BI and SR-BI/apolipoprotein E double knockout mice relative to controls. If SR-BI has similar activities in humans, it may become an attractive target for therapeutic intervention in a variety of diseases.

Animals↗
PubMed1999

Differential modulation of human immunoglobulin isotype production by the neuropeptides substance P, NKA and NKB.

The modifying effects of tachykinins substance P, neurokinin A and neurokinin B on immunoglobulin production were analyzed in an in vitro culture system. Purified human T- and B-cells were stimulated with TGFbeta2 and IL-5 to induce preferential IgA production. Neuropeptides had the following effects. (1) The levels of IgA and IgG4 production were enhanced by IL-5 and TGFbeta2; IgA levels remained constant or were slightly augmented by neuropeptides, whereas IgG4 was further augmented. (2) IL-5 and TGFbeta2 did not alter IgG3 production, but neuropeptides stimulated secretion of this subclass. (3) IgG1 and IgM production were inhibited by IL-5 and TGFbeta2. This effect was prevented by neuropeptides. (4) Other isotypes including IgG2 and IgE remained unaffected. Except for IgM, these effects were blocked by specific receptor antagonists indicating specificity. The tachykinin receptor NK-1 mRNA was detected in B- and T-cells, whereas NK-3 mRNA was only present in T- and B-cell coculture following activation. Furthermore, neuropeptide effects depended on cytokine co-stimulation and the presence of T-cells. These results suggest that neuropeptides are potent modifiers of preferential IgA synthesis.

B-Lymphocytes↗
PubMed1999

Serum levels of pantropic p53 protein and EGF-receptor, and detection of anti-p53 antibodies in former uranium miners (SDAG Wismut).

BACKGROUND: The oncogene product EGF-receptor (EGF-R), the tumor suppressor gene product p53, and anti-p53 antibodies are detectable in serum of certain cancer patients. Increased levels of some of these products were reported in lung cancer patients after occupational asbestos exposure, after exposure to polycyclic aromatic hydrocarbons or vinyl chloride. This molecular epidemiological study investigated the use of serum EGF-R, p53-protein, and anti-p53 antibodies as biomarkers for detection of effects of radon and its decay products. METHODS: Serum EGF-R, p53-protein, and anti-53 antibodies were measured using ELISA in former uranium miners of SDAG Wismut without lung disease (n=106) and miners with Schneeberg lung cancer (n=22). They were compared with healthy subjects (n=23), patients with lung cancer not due to ionizing radiation (n=88), and patients with non-malignant lung or pleural diseases (n=50). RESULTS: No significantly elevated or decreased serum values for p53 protein, EGF-R, or anti-p53 antibodies could be found. There was no correlation of these with Working Level Months (WLM). CONCLUSIONS: p53 protein, EGF-R, or anti-p53 antibodies in serum are not useful as biomarkers for detection of lung cancer related to ionizing radiation (i.e., Schneeberg lung cancer).

Aged↗
PubMed1999

A novel frameshift mutation (141delT) in exon 1 of the 21-hydroxylase gene (CYP21) in a patient with the salt wasting form of congenital adrenal hyperplasia. Mutation in brief no. 255. Online.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disease with a wide range of clinical manifestation. In 90-95% of the cases it is caused by 21-hydroxylase deficiency (OMIM #201910) due to mutations of the CYP21 gene (GDB Accession #M12792). In most cases the CYP21-inactivating point mutations are transferred by apparent gene conversions from CYP21P to CYP21. In only a few cases point mutations have been described, which are not present in the pseudogene. Using Southern blot analysis and DNA sequencing we have identified a novel mutation (141delT) of the CYP21 gene in a patient suffering from the salt wasting form of CAH. This results in a premature termination of a truncated protein at amino acid position 51 (L51X), which is likely to result in an enzyme with no activity. This novel mutation has not been reported to occur in the CYP21P alleles and it was not found in the CYP21P alleles in this CAH family.

Adrenal Hyperplasia, Congenital↗
PubMed1999

Abundant production of brain-derived neurotrophic factor by adult visceral epithelia. Implications for paracrine and target-derived Neurotrophic functions.

Brain-derived neurotrophic factor (BDNF) plays a crucial role for the survival of visceral sensory neurons during development. However, the physiological sources and the function of BDNF in the adult viscera are poorly described. We have investigated the cellular sources and the potential role of BDNF in adult murine viscera. We found markedly different amounts of BDNF protein in different organs. Surprisingly, BDNF levels in the urinary bladder, lung, and colon were higher than those found in the brain or skin. In situ hybridization experiments revealed that BDNF mRNA was made by visceral epithelial cells, several types of smooth muscle, and neurons of the myenteric plexus. Epithelia that expressed BDNF lacked both the high- and low-affinity receptors for BDNF, trkB and p75(NTR). In contrast, both receptors were present on neurons of the peripheral nervous system. Studies with BDNF-/-mice demonstrated that epithelial and smooth muscle cells developed normally in the absence of BDNF. These data provide evidence that visceral epithelia are a major source, but not a target, of BDNF in the adult viscera. The abundance of BDNF protein in certain internal organs suggests that this neurotrophin may regulate the function of adult visceral sensory and motor neurons.

Animals↗
PubMed1999

p53 protein, EGF receptor, and anti-p53 antibodies in serum from patients with occupationally derived lung cancer.

The oncogene product epidermal growth factor receptor (EGF-R), the tumour suppressor gene product p53 and anti-p53 antibodies are detectable in the serum of certain cancer patients. Increased levels of some of these products were reported in lung cancer patients after occupational asbestos exposure and after exposure to polycyclic aromatic hydrocarbons or vinylchloride. In the first step, this study investigated the possible diagnostic value of serum EGF-R, p53-protein and anti-p53 antibodies, measured by an enzyme-linked immunosorbent assay, in lung tumour patients. In addition to being investigated on a molecular epidemiological basis, these parameters were examined as biomarkers of carcinogenesis, especially with regard to asbestos incorporation effects or of radon-induced lung cancers. Also, a possible effect of cigarette smoking and age dependence were studied. A total of 116 male patients with lung or pleural tumours were examined. The histological classification was four small-cell cancers, six large-cell cancers, 32 adenocarcinomas, 47 squamous carcinomas, 12 mixed lung carcinomas, five diffuse malignant mesotheliomas and ten lung metastasis of extrapulmonary tumours. Twenty-two lung cancers and all mesotheliomas were related to asbestos, 22 lung cancers were related to ionizing radiation and 61 patients had cigarette smoke-related lung cancer. Besides these patients 50 male patients with non-malignant lung or pleural diseases were included; of the latter eight subjects suffered from asbestosis. Controls were 129 male subjects without any lung disease. No significantly elevated or decreased serum values for p53 protein, EGF-R, or anti-p53 antibodies as a function of histological tumour type, age, or degree and type of exposure (asbestos, smoking, ionizing radiation) could be found. The utility of p53-protein, EGF-R and anti-p53 antibodies as routine biomarkers for screening occupationally derived lung cancers is limited.

Adenocarcinoma↗
PubMed1999

A new role for neurotrophins: involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control.

Neurotrophins exert many biological effects not directly targeted at neurons, including modulation of keratinocyte proliferation and apoptosis in vitro. Here we exploit the cyclic growth and regression activity of the murine hair follicle to explore potential nonneuronal functions of neurotrophins in the skin, and analyze the follicular expression and hair growth-modulatory function of BDNF, NT-4, and their high-affinity receptor, TrkB. The cutaneous expression of BDNF and NT-4 mRNA was strikingly hair cycle dependent and peaked during the spontaneous, apoptosis-driven hair follicle regression (catagen). During catagen, BDNF mRNA and immunoreactivity, as well as NT-4-immunoreactivity, were expressed in the regressing hair follicle compartments, whereas TrkB mRNA and immunoreactivity were seen in dermal papilla fibroblasts, epithelial strand, and hair germ. BDNF or NT-4 knockout mice showed significant catagen retardation, whereas BDNF-overexpressing mice displayed acceleration of catagen and significant shortening of hair length. Finally, BDNF and NT-4 accelerated catagen development in murine skin organ culture. Together, our data suggest that BDNF and NT-4 play a previously unrecognized role in skin physiology as agents of hair growth control. Thus, TrkB agonists and antagonists deserve exploration as novel hair growth-modulatory drugs for the management of common hair growth disorders.

Animals↗