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Biomedical subjects

W E Schmidt

Publications and source records attributed to W E Schmidt.

At least 55 records · Page 3Linked to original sources
PubMed1999

Stem cell factor influences neuro-immune interactions: the response of mast cells to pituitary adenylate cyclase activating polypeptide is altered by stem cell factor.

Mast cells degranulation can be elicited by a number of biologically important neuropeptides, but the mechanisms involved in mast cell-neuropeptide interactions have not been fully elucidated. Stem cell factor (SCF), also known as c-kit or kit ligand, induces multiple effects on mast cells, including proliferation, differentiation, maturation, and prevents apoptosis. We investigated the ability of SCF to affect mast cell responsiveness to the neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). PACAP 1-27, PACAP1-38, or VIP failed to induced preformed mediator release from mouse bone-marrow-cultured mast cells (BMCMC) derived in concanavalin A-stimulated spleen conditioned medium (CM). By contrast, BMCMC grown in SCF-containing medium or freshly isolated peritoneal mast cells exhibited significant 3H-hydroxytrypamine (5-HT) release in response to PACAP peptides or VIP. Deoxyglucose and the mitochondrial inhibitor antimycin significantly inhibited PACAP-induced 5-HT release indicating that the central event induced by PACAP peptides was exocytosis. The G(alpha)i inhibitor, pertussis toxin, significantly diminished PACAP-induced 5-HT release from BMCMCs in SCF suggesting the involvement of heterotrimeric G-proteins. Western blot analysis using antibodies directed against the human VIP type I/PACAP type II receptor demonstrated a 70-72 kD immunoreactive protein expressed in greater amounts in BMCMC grown in SCF compared with BMCMC in CM. We conclude that SCF induces a mast cell population that is responsive to PACAPs and VIP involving a heterotrimeric G-protein-dependent mechanism.

Animals↗
PubMed1999

The putative apoptosis inhibitor IEX-1L is a mutant nonspliced variant of p22(PRG1/IEX-1) and is not expressed in vivo.

IEX-1L has been claimed to act as an apoptosis inhibitor involved in NFkappaB-mediated survival in Jurkat cells [Wu et al. (1998) Science 281, 998-1001]. It represents a mutant nonspliced variant of the early response gene p22(PRG1/IEX-1) exhibiting one insertion and two deletions compared to the genomic sequence of p22(PRG1/IEX-1). Direct DNA sequencing of PCR products generated from human genomic DNA only detected the regular genomic sequence of p22(PRG1/IEX-1). No IEX-1L mRNA could be identified by RT-PCR analysis and subsequent DNA sequencing of total, nuclear, or cytoplasmic RNA fractions from PMA-stimulated Jurkat cells. The only functional transcript residing in the cytoplasm is regularly spliced p22(IEX-1/PRG1) mRNA. Substantial amounts of nonmutated nonspliced p22(IEX-1/PRG1) pre-mRNA were identified in the nucleus. Thus, IEX-1L seems to be a mutant variant of p22(IEX-1/PRG1) not existing in vivo. Antiapoptotic effects obviously represent transdominant negative inhibition of endogenous p22(PRG1/IEX-1) in Jurkat cells and several other tumor cell lines.

Alternative Splicing↗
PubMed1999

Mast cells contribute to PACAP-induced dermal oedema in mice.

In the present study the effect of intradermal PACAP-injection on dermal oedema in mice was investigated and the contribution of mast cells to this response was assessed. The injection of PACAP 1-38 into the ears of C57BL/6 mice evoked a dose-dependent response, which, after higher doses of PACAP 1-38, lasted at least 24 h. Histological examination showed significant mast cell degranulation induced by PACAP. Using mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) mice and the congenic mice, we demonstrated that the the early phase (30 min to 6 h) of PACAP-induced ear swelling response was significantly diminished in mast cell-deficient mice, suggesting that mast cell degranulation contributes to this phase of the response. When mast cell-deficient WBB6F1-Kit(W)/Kit(W-v) mice were locally and selectively reconstituted by adoptive mast cell transfer, the dermal oedema was almost equal to that of control animals in the early phase of PACAP injection. These results show that mast cell degranulation contributes to PACAP-induced dermal oedema in mice.

Animals↗
PubMed1999

On the effect of xenin and xenin fragments on exocrine pancreas secretion in vivo.

A stimulatory effect on exocrine pancreas secretion could be demonstrated with high concentrations of the 25-amino-acid peptide xenin in non-anesthetized dogs. This peptide has been isolated from gastric mucosa and it is part of a structural coat protein. It has close structural similarities to neurotensin. The longer C-terminal fragments xenin-(13--25) and xenin-(18--25) are essential for the stimulation of exocrine pancreas secretion in vivo. The smaller peptide fragments xenin-(21--25) and xenin-(22--25) failed to stimulate the pancreas as well as the N-terminal peptide fragment xenin-(1--23). The stimulatory effects of xenin may be mediated via neural neurotensin pathways, because neurotensin receptor blockade abolished the stimulatory effect on pancreatic secretion. Cholinergic pathways are not involved, because atropine had no inhibiting effect.

Adjuvants, Anesthesia↗
PubMed1999

[ZAP genes: characterizing the protein structure of a new family of proliferation associated genes in the exocrine pancreas].

While interested in proliferation-dependent gene regulation in a pancreatic carcinoma cell line, we cloned a set of proteins (ZAP) characterized by a conserved region consisting of consecutive zinc finger, ankyrin repeat and PH domains. Functional aspects of these domains were obtained by comparison with proteins involved in several signal transduction pathways and cell cycle regulation. The members of the ZAP protein family are individually characterized by different types of supplementary protein domains, their chromosomal localization and their tissue specific gene transcription. All results indicate a wide spectrum of protein-protein interactions. Up to now specific binding partners have not been identified. In summary, the multiplicity of conserved regions and transcriptional data indicate a scaffold function for ZAP proteins in the complex network of proliferation associated intracellular signal transduction pathways.

Amino Acid Sequence↗
PubMed1999

Identification of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type 1 receptor in human skin: expression of PACAP-38 is increased in patients with psoriasis.

Morphological and biochemical evidence is presented for the presence of pituitary adenylate cyclase activating peptide (PACAP) and the high-affinity PACAP-1 receptor subtype in human skin. Immunohistochemical analysis revealed PACAP-immunoreactivity (IR) to be present predominantly in dermal nerve fibers close to the dermal-epidermal border, hair follicles, blood vessels and sweat glands. Radioimmunoassay, chromatographic analysis and Western blotting revealed this PACAP-IR to be PACAP-38 whereas the second molecular form, PACAP-27, is absent. In tissue of psoriasis patients significantly more PACAP-38 protein was detected as compared to normal skin. Using RT-PCR, the expression of a high-affinity PACAP-1 receptor in human skin was observed. These results indicate a possible role for PACAP-38 in inflammatory processes of psoriasis.

Antibodies, Monoclonal↗
PubMed1999

Structural motifs of pituitary adenylate cyclase-activating polypeptide (PACAP) defining PAC1-receptor selectivity.

Pituitary adenylate cyclase-activating polypeptide (PACAP) interacts with three types of PACAP/VIP-receptors. The PAC1-receptor accepts PACAP as a high affinity ligand but not vasoactive intestinal peptide (VIP) similarly binding to VPAC1- and VPAC2-receptors. To identify those amino acids not present in VIP defining PAC1-receptor selectivity of PACAP, radio receptor binding assays on AR4-2J cells were performed. It could be shown that PACAP(1-27) exhibited a distinct and much higher susceptibility to VIP-amino acid substitutions, compared to PACAP(1-38). Positions 4 and 5 seem to be most important for receptor binding of PACAP(1-27), whereas position 13 was identified to be crucial for maximal affinity of PACAP(1-38). PACAP(29-38) extension analogues of VIP revealed a stabilizing effect of the C-terminus of PACAP(1-38) on the optimal peptide conformation. The substitution analogues were also checked for their capacity to stimulate IP3 and cAMP formation in AR4-2J cells. Compared to PACAP(1-27) and PACAP(1-38), most analogues revealed potencies reduced congruously to their lower binding affinities. However, one of the analogues, PACAP(1-27) substituted in position 5, may represent a weak antagonist since this peptide was less potent in inducing second messengers than in label displacement. Our findings indicate that PACAP(1-27) and PACAP(1-38) differ in terms of their requirement of the amino acids in positions 4, 5, 9, 11 and 13 for maximal interaction with the PAC1-receptor.

Alternative Splicing↗
PubMed1999

Biological activity of GLP-1-analogues with N-terminal modifications.

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and improves glycemic control in type 2 diabetes. In serum the peptide is degraded by dipeptidyl peptidase IV (DPP IV). The resulting short biological half-time limits the therapeutic use of GLP-1. Therefore, various GLP-1 analogues with alterations in cleavage positions were synthesized. GLP-1-receptor binding was investigated in RINm5F cells. Biological activity of the GLP-1 analogues was investigated in vitro by measuring cAMP production in RINm5F cells. GLP-1 analogues with modifications in position 2 were not cleaved by DPP IV and showed receptor affinity and in vitro biological activity comparable to native GLP-1. Analogues with alterations in positions 2 and 8, 2 and 9 or 8 and 9 showed a significant decrease in receptor affinity and biological activity. In vivo biological activity was tested in pigs. GLP-1 analogues were administered subcutaneously followed by an intravenous bolus injection of glucose. Plasma glucose and insulin were monitored over 4 h. Compared to native GLP-1, analogues with an altered position 2 showed similar or increased potency and biological half-time. Other GLP-1 analogues were less active. Despite the lack of degradation of these GLP-1 analogues by DPP IV in vitro, their biological action is as short as that of GLP-1, except for desamino-GLP-1, indicating that other degradation enzymes are important in vivo. Alterations of GLP-1 in positions 8 or 9 result in a loss of biological activity without extending biological half-time.

Cyclic AMP↗
PubMed1999

Comparison of the effect of native glucagon-like peptide 1 and dipeptidyl peptidase IV-resistant analogues on insulin release from rat pancreatic islets.

BACKGROUND: Glucagon-like peptide 1 (GLP-1) stimulates insulin secretion and may improve glycaemic control in type 2 diabetes. Therapeutic use is limited by its rapid degradation, primarily by dipeptidyl peptidase IV. MATERIALS AND METHODS: Five GLP-1 analogues with alterations at cleavage positions were synthesized according to the Fmoc strategy and tested for metabolic stability by incubation with rat kidney membranes containing dipeptidyl peptidase IV activity. Their insulinotropic effect was compared in isolated rat pancreatic islets after 24 h maintenance in tissue culture. Ten islets per vial were incubated for 30 min; insulin was measured radioimmunologically. Each analogue was compared with GLP-1 in the same experiment. RESULTS: All analogues were biologically active in isolated islets in the potency order da2d8 = da2 > d2d9 > da2ds8 > desamino. At 16.7 mmol L-1 glucose, GLP-1 and GLP-1 analogues altered as position 2, or 2 and 8 significantly (P < 0.05) increased insulin release at 10(-9) mol L-1. N-terminal modification of GLP-1 confers resistance to dipeptidyl peptidase IV degradation in rat kidney membranes in vitro. CONCLUSIONS: The analogues tested are biologically active and resistant to degradation by dipeptidyl peptidase IV. Their greater metabolic stability may help to realize the potential of GLP-1 analogues in diabetes therapy.

Amino Acid Sequence↗
PubMed1999

Interaction of the tyrosine phosphatase inhibitor ortho-vanadate on stimulus--secretion coupling in pancreatic acinar cells.

BACKGROUND: The effect of the tyrosine phosphatase inhibitor ortho-vanadate on stimulus-secretion coupling was investigated in isolated rat pancreatic acini. METHODS AND RESULTS: Ortho-vanadate (10(3)M) reduced cholecystokinin (CCK)-8 (10(10) M)-stimulated amylase release by 40% (IC50 = 5 x 10(4) M). In contrast, preincubation with 10(3) M ortho-vanadate increased secretin (5 x 10(9) M) and vasoactive intestinal peptide (VIP) (10(7) M)-induced amylase release by 65% and 80% (IC50= 3 x 10(-4) M), respectively. 8-Bromo-cyclic adenosine-5-monophosphate (cAMP) (10(-4) M) and phorbol ester (10(-5) M)-induced secretion was increased by 60% and 50%, respectively, whereas thapsigargin-induced amylase release was not affected. Ortho-vanadate did not affect CCK-8 binding or VIP-induced cAMP synthesis in isolated acini. In contrast, preincubation with 10(-4) M ortho-vanadate resulted in a significant reduction of CCK-8-induced intracellular calcium release. In streptolysin-O-permeabilized acini, ortho-vanadate reduced calcium-induced amlyase secretion by 50%. CONCLUSIONS: The present data provide indirect evidence of a differential involvement of protein tyrosine dephosphorylation in both cAMP- and IP3/Ca(2+)-mediated pancreatic secretion. The differential effects of ortho-vanadate on cAMP- versus calcium-mediated secretion correspond to the results obtained with receptor-independent intracellularly acting secretagogues. Further experiments must define the tyrosine phosphatases involved in both signal-transduction pathways.

Amylases↗
PubMed1999

Human PACAP response gene 1 (p22/PRG1): proliferation-associated expression in pancreatic carcinoma cells.

p22/PACAP response gene-1 (PRG1) is a novel rat early response gene expressed during induction of proliferation and stress response. In humans, a homolog of p22/PRG1, designated IEX-1/DIF-2, exists, yet the exact function of this gene remains elusive. To characterize the expression of p22/PRG1 in human cancers, we analyzed the expression of p22/PRG1 in the human pancreatic carcinoma cell lines 818-4, PT45, and PancTu1. Serum or growth factors, like epidermal growth factor (EGF) and hepatocyte growth factor (HGF), rapidly and transiently induced transcription of p22/PRG1 in these cells, correlating with the mitogenic response. Treatment with TNF-alpha was followed by a rapid increase of p22/PRG1 messenger RNA (mRNA) levels in PT45 and Panc-Tul cells, which proliferate in the presence of TNF-alpha, but not in 818-4 cells, which are growth-inhibited when treated with TNF-alpha. Our findings suggest that human p22/PRG1 is expressed in various pancreatic carcinoma cells as a growth-associated early response gene.

Cell Division↗
PubMed1999

Effects of ramipril on cardiac gene transcription levels of angiotensin II receptors after myocardial infarction.

AIM: To study the early changes of cardiac angiotensin (Ang) II receptor gene transcription after myocardial infarction (MI) in rats chronically treated with the angiotensin-converting enzyme (ACE) inhibitor ramipril. METHODS: MI was induced by left anterior descending coronary artery ligation in rats and sham-operated rats were used as control. Rats were treated daily with ramipril (1 mg.kg-1) or water, initiated 1 wk before surgery. Quantitative RT-PCR was applied to determine the Ang II receptors AT1, AT2 receptor gene mRNA levels in the non-infarcted myocardium. RESULTS: AT1 and AT2 mRNA levels increased time point-dependently in the cardiac septum after MI reaching a peak on d 1. There was no significant difference of the myocardial AT1 and AT2 receptor mRNA levels between the ramipril-treated and water-treated rats after MI. CONCLUSION: The AT1 and AT2 receptor gene transcription in the non-infarcted myocardium was associated with the process of cardiac remodeling after MI but not affected by ACE inhibition.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1998

p22/PACAP response gene 1 (PRG1): a putative target gene for the tumor suppressor p53.

In this study we describe a novel putative p53-responsive gene, designated p22/PACAP response gene 1 (PRG1), recently identified as a proliferation-associated early-response gene in rats. By means of electrophoretic mobility shift assay and CAT-reporter gene assay, we could demonstrate that the p53 binding site residing in the promoter of p22/PRG1 is functional in vitro. Furthermore, in clone 6 cells expression of p22/PRG1 is induced in parallel to p21/Waf1 under conditions permitting mutant p53 to adopt wild-type configuration. An increase of p22/PRG1 transcription was also observed in gamma-irradiated rat splenocytes, which undergo p53-dependent apoptosis. Our findings demonstrate that p22/PRG1 fulfills all essential criteria as a p53 target gene and might be implicated in p53-dependent apoptosis.

Animals↗