Biomedical subjects

Wolfgang Gartner

Publications and source records attributed to Wolfgang Gartner.

9 recordsLinked to original sources

PubMed2006

Immunoreactivity of calcium binding protein secretagogin in the human hippocampus is restricted to pyramidal neurons.

Disturbed calcium homeostasis plays a crucial role in the aetiology of Alzheimer's disease (AD) and the aging process. We evaluated immunoreactivity of secretagogin, a recently cloned calcium binding protein, in hippocampus and adjacent entorhinal cortex of 30 neuropathologically examined post mortem brains (m:f=12:18; mean age, 79.8+/-15.1 years). The study group consisted of 15 cases fulfilling the criteria for high probability of AD according to the NIA-Reagan Institute Criteria and 15 cases with no to medium probability. Sections were incubated with secretagogin-specific antibodies and the number of immunoreactive neurons as well as staining intensities in both neurons and neuropil were assessed. Both cellular and neuropil immunoreactivity were restricted to subiculum and Ammons horn. Cellular immunoreactivity was further restricted to pyramidal neurons and showed a hierarchical distribution: the mean percentage of immunoreactive neurons was highest in sector CA3 (64.41%), followed by CA2 (44.09%), CA4 (34.38%), CA1 (10.9%), and the subiculum (2.92%; P<0.001, except CA2-CA4, P>0.05), while it did not differ significantly between groups with different degrees of AD pathology. The pattern of secretagogin immunoreactivity resembles that of calcium sensor proteins as it is restricted to a subset of neurons and therefore secretagogin could serve highly specialized tasks in neuronal calcium signalling.

Adult↗

PubMed2006

Long-term in vitro growth of human insulin-secreting insulinoma cells.

OBJECTIVE: Long-term in vitro maintenance of human insulin-secreting insulinoma cells. METHODS: (1) Cell culture of ex vivo-derived insulinoma cell suspensions from 8 individual human donors, using various cell culture medium supplementations; (2) determination of insulin synthesis and secretion using immunocytochemistry and insulin and pro-insulin radioimmunoassays; (3) nestin-immunostaining of long-term in vitro grown insulinoma cell suspensions, and (4) phase-contrast light microscopy for analyzing the in vitro growth characteristics of the insulinoma cells. RESULTS: (1) Parallel persistence of in vitro insulinoma cell proliferation as well as insulin-synthesizing and -secreting capacity depended on both the co-culture of insulinoma cells with human fibroblasts and the supplementation of the cell culture medium with tissue culture supernatant derived from the rodent pituitary adenoma cell line GH-3; (2) immunostaining for insulin and secretagogin confirmed the neuroendocrine origin of the insulinoma cells grown in vitro; (3) insulin secretion capability persisted up to an observation period of 25 weeks; (4) insulin secretion rates after 6 weeks of in vitro growth ranged from 3.5 to 83.3 muU/ml/h/60,000 cells plated, and (5) after long-term in vitro growth of insulinoma-derived cell suspensions with persistent insulin-secreting capacity, nestin staining was observed predominantly in co-cultured fibroblasts. CONCLUSION: Our data describe for the first time the long-term in vitro culture of insulin-secreting human insulinomas and highlight the importance of beta-cell trophic factors for insulinoma cell growth.

Adult↗

PubMed2006

Secretagogin is a novel marker for neuroendocrine differentiation.

Our previous microarray-based studies identified secretagogin to be highly expressed in normal colon mucosa compared to basal expression in colon adenocarcinomas. The aim of this study was to analyze the differential expression of secretagogin in normal mucosa, adenocarcinomas, and neuroendocrine tumors. Western blotting, immunohistochemistry, immunofluorescence microscopy and ELISA were applied. Western blot analysis detected a 32-kDa secretagogin band in samples from normal mucosa. Immunohistochemical analyses on tissue specimens showed that secretagogin is exclusively expressed in neuroendocrine cells and nerve cells in normal mucosa of the digestive tract. Tissues adjacent to benign hyperplasic polyps and adenomas showed a decreased number of secretagogin-expressing neuroendocrine cells. Secretagogin co-localized with neuroendocrine markers (chromogranin A, neuron-specific enolase, synaptophysin) in neuroendocrine cells in crypts of normal mucosa, and in tumor cells of carcinoids. Secretagogin was strongly expressed in the cytosol and the nucleus of 19 well-differentiated neuroendocrine carcinoids and carcinoid metastases, as well as in neuroendocrine tumors from the lung, pancreas and adrenal gland. Secretagogin was detected in plasma from carcinoid patients with distant metastasis. Combined immunohistochemical analysis of secretagogin and FK506-binding protein 65, a protein de novo synthesized in adenocarcinomas, distinguished well-differentiated carcinoids, adenocarcinoids and undifferentiated carcinomas. We conclude that secretagogin is a novel marker for neuroendocrine differentiation.

Adenocarcinoma↗

PubMed2006

Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin.

To identify proteins with colorectal cancer-specific regulation, comparative 2-DE of individual-matched normal and neoplastic colorectal tissue specimens was performed. We found 15 protein spots with concordantly increased and 20 protein spots with concordantly decreased intensity in tumor tissue (expression regulation more than fivefold). Nine of these proteins were identified by MS/MS. Interestingly, one of the proteins, which exhibited a marked down-regulation in colorectal cancer tissues, was the recently identified endocrine cell-expressed protein secretagogin. The reduction of the secretagogin content in colorectal cancer tissues was confirmed by comparative immunoblotting (n = 17) and RT-PCR (n = 22) as well as by immunohistochemistry (n = 45) of individual-matched neoplastic and normal colorectal tissue specimens. Immunohistochemistry revealed absence of secretagogin-expressing cells in most of the colorectal cancer tissue specimens. However, some colorectal cancers were characterized by secretagogin-expressing cells. In normal mucosa, positively stained cells exhibited a neuroendocrine cell-characteristic morphology and mucosal location. In colorectal cancer tissues, secretagogin-expressing cells were characterized by a malignant morphology. Our findings might represent the basis for the clinical application of secretagogin as a biomarker for a distinct subgroup of colorectal cancers.

Biomarkers, Tumor↗

PubMed2005

Setagin and secretagogin-R22: Posttranscriptional modification products of the secretagogin gene.

We describe two new variants of the recently identified hexa-EF-hand calcium binding protein secretagogin. The first variant (secretagogin-R22) is characterized by one single amino acid exchange (Q/R) at codon 22, most likely due to RNA editing. The second variant of secretagogin (setagin) consists of 49 amino acids. Due to a frame shift, only the first 27 amino acids are identical to secretagogin. We demonstrate that this protein truncation results in complete loss of the calcium binding capacity. Setagin expression was found in considerable amounts in the pancreas whereas secretagogin and secretagogin-R22 were also found in the central nervous system and organs containing neurendocrine cells.

Alternative Splicing↗

PubMed2005

A newly identified RET proto-oncogene polymorphism is found in a high number of endocrine tumor patients.

Multiple RET proto-oncogene transcripts, due to genomic variations and alternate splicing, have been described. To investigate endocrine tumor tissue characteristic RET proto-oncogene expression, we performed quantitative RT-PCR, Northern blot and Southern blot analyses of benign and malignant endocrine-derived tissues. We newly describe RET proto-oncogene expression in carcinoid-, gastrinoma- and insulinoma-derived tissue samples. In addition, the presence of a 3'-terminally truncated RET proto-oncogene mRNA variant in benign and malignant thyroid neoplasias, as well as in a pheochromocytoma, an ovarian carcinoma and a medullary thyroid carcinoma, is demonstrated. Southern blot analysis revealed no evidence of gross RET proto-oncogene rearrangements or deletions. As the underlying cause for a bi-allelic TaqI restriction fragment length polymorphism (RFLP), a C (allele 1)/T (allele 2) transition within intron 19, was characterized. This polymorphism is close to a recently described polyadenylation site and lies within a binding site for the nucleic acid binding protein Pbx-1. Screening of healthy subjects and of patients suffering from various endocrine malignancies revealed exclusively allele 1 homozygous and allele 1/allele 2 heterozygous genotypes. Heterozygous genotypes were found in a significantly higher percentage in samples derived from endocrine tumor patients when compared with those from healthy control subjects. Homozygosity for allele 2 was found exclusively in somatic DNA derived from endocrine tumors with high malignant potential. Analysis of DNA derived from varying regions within individual anaplastic thyroid carcinomas revealed an allele 1/allele 2 switch of the RFLP banding pattern, indicating loss of heterozygosity at the RET proto-oncogene locus. In conclusion, our data demonstrate presence of a 5'-terminal RET proto-oncogene transcript in endocrine tissues and reveal a bi-allelic RET proto-oncogene polymorphism. A heterozygous genotype for this polymorphism is found in a considerable number of endocrine tumor patients.

3' Untranslated Regions↗

PubMed2005

Differential expression of alphaB-crystallin and Hsp27-1 in anaplastic thyroid carcinomas because of tumor-specific alphaB-crystallin gene (CRYAB) silencing.

Expression of the small heat shock protein alphaB-crystallin in differentiated thyroid tumors has been described recently. In this study, we investigated the molecular mechanisms that affect the expression of alphaB-crystallin in benign goiters (n = 7) and highly malignant anaplastic thyroid carcinomas (ATCs) (n = 3). AlphaB-crystallin expression was compared with that of Hsp27-1. Immunoblot and quantitative real-time (RT) polymerase chain reaction revealed marked downregulation of alphaB-crystallin in all the tested ATCs and the ATC-derived cell line C-643 . In contrast, considerable expression of Hsp27-1 in benign and malignant thyroid tissue was demonstrated. Immunofluorescence analysis revealed no relevant topological differences between benign and malignant thyrocytes in the cytoplasmic staining of both proteins. Consistent and marked downregulation of TFCP2L1 was identified as one of the main mechanisms contributing to CRYAB gene silencing in ATCs. In addition, CRYAB gene promoter methylation seems to occur in distinct ATCs. In silico analysis revealed that the differential expression of alphaB-crystallin and Hsp27-1 results from differences between the alphaB-crystallin and Hsp27-1 promoter fragments (712 bp upstream from the transcriptional start site). Biological activity of the analyzed promoter element is confirmed by its heat shock inducibility. In conclusion, we demonstrate downregulation of alphaB-crystallin expression in highly dedifferentiated ATCs because of a tumor-specific transcription factor pattern. The differential expression of alphaB-crystallin and Hsp27-1 indicates functional differences between both proteins.

Animals↗

PubMed2004

Do iodine-containing contrast media induce clinically relevant changes in thyroid function parameters of euthyroid patients within the first week?

Little is known about the reaction of normal thyroid glands to the iodine load given by x-ray dyes. We have therefore investigated the short-term effects of high doses of iodine on thyroid parameters in euthyroid patients. We measured free triiodothyronine (FT3), free thyroxine (FT4), and thyrotropin (TSH) serum concentrations before and daily for 1 week after parenteral application of x-ray dyes (coronary angiography: n = 16; computed tomography [CT] of either thorax or abdomen: n = 6; iodine dose range from 300-1221 mg of iodine per kilogram). Inclusion criteria were as follows: normal FT4, normal TSH, negative thyroid antibodies, urinary iodine excretion below 30 microg/dL, no palpable goiter and no euthyroid sick syndrome. All but one patient reacted with a TSH increase. Mean TSH values increased significantly 3-5 days after the iodine load within the normal range. Four patients (18%) had a TSH increase above normal, the maximal observed value being 6.4 microU/mL. Basal TSH values of these four patients were above 2 microU/mL. The day peak TSH concentrations were reached varied from day 1 to day 7, the majority (32%) having the peak on day 3. Peak TSH was significantly correlated with basal TSH values (r = 0.794, p < 0.0001). FT4 and FT3 remained unchanged and there was no significant correlation between the dose of iodine and the TSH reaction. In conclusion, iodine-containing contrast media can induce transiently subclinical hypothyroidism even in euthyroid patients. The TSH reaction seems to depend on the preexisting state of thyroid function.

Adult↗

PubMed2003

The ATP-dependent helicase RUVBL1/TIP49a associates with tubulin during mitosis.

RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.

ATPases Associated with Diverse Cellular Activitie↗