TRPA1 channel activation induces cholecystokinin release via extracellular calcium.

TRPA1 channels are non-selective cation channels activated by plant derived pungent products including allyl isothiocyanate (AITC) from mustard. Therefore, possible intestinal secretory functions of these channels were investigated. We detected TRPA1 mRNA in mouse and human duodenal mucosa and in intestinal mouse neuroendocrine STC-1 cells. Stimulation of STC-1 cells with AITC increased intracellular calcium ([Ca(2+)](i)) and significantly stimulated cholecystokinin secretion by 6.7-fold. AITC induced cholecystokinin release was completely blocked by TRPA1 antagonist ruthenium red and depletion of extracellular calcium and reduced by 36% by nimodipine and nifedipine. This suggests that spices in our daily food might stimulate digestive functions.

Anchored reference loci in loblolly pine (Pinus taeda L.) for integrating pine genomics.

Anchored reference loci provide a framework for comparative mapping. They are landmarks to denote conserved chromosomal segments, allowing the synthesis of genetic maps from multiple sources. We evaluated 90 expressed sequence tag polymorphisms (ESTPs) from loblolly pine (Pinus taeda L.) for this function. Primer sets were assayed for amplification and polymorphism in six pedigrees, representing two subgenera of Pinus and a distant member of the Pinaceae, Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). On average, 89% of primer sets amplified in four species of subgenus Pinus, 49% in one species of subgenus Strobus, and 22% in Douglas-fir. Polymorphisms were detected for 37-61% of the ESTPs within each pedigree. Comparative mapping in loblolly and slash pine (P. elliottii Englm.) revealed that ESTPs mapped to the same location. Disrupted synteny or significant disruptions in colinearity were not detected. Thirty-five ESTPs met criteria established for anchor loci. The majority of those that did not meet these criteria were excluded when map location was known in only a single species. Anchor loci provide a unifying tool for the community, facilitating the creation of a "generic" pine map and serving as a foundation for studies on genome organization and evolution.

c-met expression in pancreatic cancer and effects of hepatocyte growth factor on pancreatic cancer cell growth.

Hepatocyte growth factor (HGF) is a widely expressed growth factor secreted by cells of mesenchymal origin, which has been shown to be involved in growth processes of multiple cell types. The HGF receptor, the product of the c-met protooncogene, is expressed mainly by epithelial cells. Increased expression of the HGF receptor has been observed in various tumors. To investigate the expression of the HGF receptor in the pancreas, we analyzed rat and human normal tissue and pancreatic carcinoma by Western blot analysis. We observed weak expression of c-met reactivity in normal pancreas but markedly enhanced expression in both rat and human pancreatic cancer. To test the possibility that HGF could act as a growth factor on pancreatic carcinoma, the effects of HGF on DNA synthesis in a rat and two human pancreatic carcinoma cell lines were analyzed. HGF induced dose-dependent [3H]thymidine incorporation, reaching 320, 210, and 180% above unstimulated controls in AR4-2J, PancTu-1, and 818/4 cells, respectively. The activation of signal transduction pathways by HGF was further analyzed in AR4-2J cells. After stimulation, a rapid and intense increase in receptor tyrosine phosphorylation was detected. Furthermore, HGF induced a time- and dose-dependent induction of c-fos expression. The addition of tyrphostin, a specific tyrosine kinase inhibitor, prevented c-fos induction and inhibited HGF-induced [3H]thymidine incorporation. In summary, our results demonstrate strongly increased HGF receptor expression in pancreatic carcinoma and support the assumption that HGF could act as a growth promoting factor on this cancer via stimulation of tyrosine kinases.

Influence of heparin treatment on biochemical markers of an activation of the coagulation system.

Monitoring of anticoagulant treatment is not yet satisfactory. Otherwise optimal treatment control in special situations as low dose heparin prophylaxis in hip surgery or high dose anticoagulant treatment in patients after coronary stent implantation may be desirable. Recently available thrombin markers were analyzed in 51 patients under low dose (group 1) and 30 patients under high dose therapy with unfractionated heparin (group 2a and b) as well as in controls (n = 26). Before therapy these parameters were significantly elevated in both patient groups. Elevated thrombin-antithrombin III-complexes (TAT) despite adequate prolongation of aPTT under high dose heparin in 38.2% of patients indicate that therapeutic concentrations of heparin in these cases are insufficient for depressing this parameter completely. During low dose therapy only prothrombin fragment (F1 + 2) significantly decreased. This may be explained by catalytic induction of TAT-complex formation by heparin. Decrease of D-Dimer under heparin therapy in both groups does not parallel with TAT and F1 + 2 but was more prolonged. This can be explained by dependence of the D-Dimer level on spontaneous fibrinolytic activity and by a longer plasma half-life as well as a chronic and continuous fibrinolytic process in an older thrombus. In conclusion, thrombin markers seem to be helpful in estimating anticoagulant treatment efficacy. As a consequence, anticoagulant treatment has to be intensified in high-risk patients for complete depression of these markers. Whether the benefit of higher heparin doses is worth the risk of drug-induced hemorrhage, however, remains to be clarified in clinical studies.

Unusual course of colonic tattooing with India ink.

We here report the successful but unusual course of colonic tattooing in a patient with a carcinoma in situ in a polyp. A 70-year-old woman was admitted for persistent diarrhea, occult fecal blood, and anemia. During colonoscopy, a pedunculated large polyp in the sigmoid colon occluding the lumen was removed successfully. Histopathological examination revealed a carcinoma in situ with a resection of the peduncle in sano. At the second sigmoidoscopy, the polypectomy site was marked with India ink to facilitate the recovery of the polypectomy site in follow-up endoscopies. Three months later, the India ink had spread in the submucosal layer and a segment measuring 15 cm was colored dark blue. At the original polypectomy site, an uncolored flat mucosal proliferation was found above the dark colonic wall. After mucosectomy, the tissue was classified as hyperplastic. Six weeks later, a second control sigmoidoscopy did not show any suspicious mucosal alterations.

Effect of medium- and long-chain fatty acid diets on PPAR and SREBP-1 expression and glucose homeostasis in ACBP-overexpressing transgenic rats.

AIM: Acyl-CoAs are important intermediates and regulators of lipid metabolism. Binding proteins like acyl-CoA binding protein (ACBP) can influence their regulatory functions. ACBP has also been shown to exert direct effects on gene regulation in vitro. As the physiological relevance of ACBP in the regulation of lipid metabolism under high fat diets is unclear, we investigated the influence of such diets on the metabolic responses in ACBP-overexpressing rats. METHODS: A transgenic rat line overexpressing the ACBP gene was used to study the effects of 4 weeks of feeding with medium- (MC) or long-chain (LC) fatty acid-containing diets. Glucose tolerance tests were performed. Expression of transcription factors was measured by quantitative RT-PCR and protein levels of AMP-activated protein kinase were determined by western blotting. RESULTS: Transgenic animals fed the MC diet had an improved glucose tolerance and lower serum insulin levels compared with controls. Their liver PPARgamma (by 43%) and SREBP-1 (by 35%) mRNA levels were found to be decreased, while adipose tissue PPARgamma expression was increased by 31%. Tg animals fed the LC diet did not exhibit changes in glucose or insulin levels but exhibited increased mRNA levels of liver PPARs and SREBP-1 (1.5-3.5 times) and decreased protein levels of AMPKalpha (by 48%). CONCLUSIONS: Our results demonstrate that ACBP overexpression affects metabolic responses to diets with distinct difference in their fatty acid chain lengths. The molecular regulatory mechanism behind these effects seems to be an ACBP-induced tissue-specific regulation of expression of PPARs and SREBP.

[Short version of the updated S3 (level 3) guidelines for diagnosis and treatment of gallstones of the German Society for Digestive and Metabolic Diseases and the German Society for the Surgery of the Alimentary Tract]. / Kurzfassung der aktualisierten S3-Leitlinie der DGVS und DGVC zur Diagnostik und Behandlung von Gallensteinen.

This short version of the guidelines summarizes the evidence-based key recommendations for the diagnosis and treatment of gallstones. The guidelines were developed by an interdisciplinary team of gastroenterologists, surgeons, radiologists, geneticists, and patient support groups, under the auspice of the German Society for Gastroenterology and Metabolic Diseases and the German Society for General Surgery and Surgery of the Alimentary Tract. It used structural level 3 consensus-based methodology and includes statements on clinical practice, prevention, quality assurance, outcome analysis, and integration of outpatient and inpatient care for patients with gallstone disease.

[S3-guidelines for diagnosis and treatment of gallstones. German Society for Digestive and Metabolic Diseases and German Society for Surgery of the Alimentary Tract]. / S3-Leitlinie der Deutschen Gesellschaft für Verdauungs- und Stoffwechselkrankheiten und der Deutschen Gesellschaft für Viszeralchirurgie zur Diagnostik und Behandlung von Gallensteinen.

This guideline provides evidence-based key recommendations for diagnosis and therapy of gallstones and upgrades version 2000. It was developed by an interdisciplinary team of gastroenterologists, surgeons, radiologists, geneticists, external comparative quality assurance and patient support groups under the auspices of the German Society for Digestive and Metabolic Diseases and the German Society for Surgery of the Alimentary Tract. The guideline used structural S3 consensus-based methodology and includes statements on clinical practice, prevention, outcome analysis, and integration of outpatient and inpatient care for patients with gallstone diseases.

The influence of alpha- and beta-galactose residues and sialic acid O-acetyl groups of rat erythrocytes on the interaction with peritoneal macrophages.

The significance of glycoconjugates on the surface of rat erythrocytes was studied in the interaction of these cells with homologous peritoneal macrophages. The erythrocytes exposing terminal alpha-galactose and thus of B blood group specificity, as well as sialic acid are not bound by the macrophages. beta-Galactose residues exposed by sialidase induced strong binding and additional alpha-galactosidase treatment enhanced the binding. beta-Galactose exposed on glycolipids after pronase and alpha-galactosidase treatment induced no binding. An intact protein core of the glycoproteins on the erythrocyte surface was necessary for interaction with macrophages. Partial de-O-acetylation of sialic acids prior to sialidase treatment stimulated subsequent binding of the erythrocytes.

Synergistic stimulation of DNA synthesis by bradykinin and vasopressin in Swiss 3T3 cells.

Vasopressin and bradykinin bind to receptors coupled to GTP-binding proteins and rapidly induce polyphosphoinositide breakdown leading to Ca2+ mobilization and activation of protein kinase C. Both peptides are known to induce mitogenesis in the presence of growth factors that act through receptors with intrinsic tyrosine kinase activity. Surprisingly, addition of a combination of vasopressin and bradykinin to Swiss 3T3 cells synergistically stimulates DNA synthesis in the absence of any other growth factors. This effect is induced at nanomolar concentrations of the peptides and could be inhibited by addition of specific receptor antagonists or broad spectrum neuropeptide antagonists. Bradykinin, which stimulates transient activation of protein kinase C, induces DNA synthesis in synergy with substances that cause long-term activation of protein kinase C, like vasopressin or phorbol 12,13-dibutyrate. Down-regulation of protein kinase C inhibited the induction of mitogenesis by the combination of vasopressin and bradykinin, thus demonstrating the importance of long-term activation of this enzyme for DNA synthesis. Analysis of tyrosine phosphorylated proteins of M(r) = 110,000-130,000 and M(r) = 70,000-80,000 revealed a biphasic response after stimulation with bradykinin, whereas the response induced by vasopressin declined after the initial maximum. The combination of bradykinin with vasopressin caused an enhanced and prolonged increase in tyrosine phosphorylation of these proteins as compared with the individual peptides. Inhibition of tyrosine phosphorylation by tyrphostin was paralleled by inhibition of DNA synthesis. Together, these results demonstrate synergistic stimulation of DNA synthesis by bradykinin and vasopressin via prolonged stimulation of multiple signaling pathways and imply that the interactive effects of Ca(2+)-mobilizing peptides on mitogenesis may be more general than previously thought.

Down-regulation of bradykinin receptors and bradykinin-induced Ca2+ mobilization, tyrosine phosphorylation, and DNA synthesis by autocrine factors, tumor necrosis factor alpha, and interferon beta in Swiss 3T3 cells.

Preincubation of quiescent Swiss 3T3 cells in fresh synthetic medium caused a reduction of the lag period prior to bradykinin-stimulated DNA synthesis as well as a leftward shift in the dose-response curve (half-maximum effect at 2 nM and 8 nM for preincubated cells and control cells, respectively). These enhancing effects were selective for bradykinin since vasopressin-stimulated DNA synthesis was not affected by preincubation in synthetic medium. Preincubation in synthetic medium also caused a marked enhancement (five- to sixfold increase) of bradykinin-induced Ca2+ mobilization from intracellular stores. This enhancement was time-dependent, peaked after 12 h of preincubation, and was prevented by inhibition of RNA or protein synthesis. Furthermore, preincubation in synthetic medium did not enhance the Ca2+ mobilization by bombesin, vasopressin, or PDGF. Additionally, bradykinin-induced tyrosine phosphorylation was also enhanced by prior incubation in fresh medium. Scatchard analysis of [3H]bradykinin binding revealed a doubling of the number of bradykinin receptors without any significant change of affinity after preincubation, thus providing an explanation for the increased cellular responsiveness to bradykinin. This enhancement of responsiveness to bradykinin was caused by the removal of an inhibitory factor present in conditioned medium which is produced by the cells and accumulates gradually in the medium. Addition of tumor necrosis factor alpha or interferon beta to synthetic medium substituted for conditioned medium in preventing the increase in responsiveness to bradykinin. These findings demonstrate a novel mechanism that regulates cellular sensitivity to bradykinin via an autocrine factor(s).

[Chronic granulomatous chorioretinitis in tuberculosis]. / Chronische granulomatöse Chorioretinitis bei Tuberkulose.

BACKGROUND: Though the incidence of tuberculosis decreased substantially during the second half of the century, a steady increase of new cases has been observed within the last decade. Among other reasons, the growing number of immunodeficient patients (e.g. HIV, therapy with immunosuppressive agents) and migration from underdeveloped to industrial countries contribute to this finding. PATIENT: A 57-year-old male patient presented with a history of chronic bilateral chorioretinitis of unknown origin. During the last months a marked decrease in visual acuity of the left eye was noted. Prior diagnostic attempts had not led to a specific diagnosis. Notable was the history of long-term systemic corticosteroid therapy for chronic obstructive lung disease. Funduscopy of the right eye revealed a choroidal granuloma with adjacent serous retinal detachment. Both eyes showed multiple dot-like lesions in the retinal pigment epithelium of the posterior pole. Finally the diagnosis could be made by isolating Mycobacterium tuberculosis from sputum samples and gastric aspirate. Within a few weeks after starting tuberculostatic therapy the ocular symptoms regressed and the visual acuity improved significantly. DISCUSSION: Securing the diagnosis of tuberculous uveitis is often difficult. The differential diagnosis includes other granulomatous ocular inflammations. The detection of Mycobacterium tuberculosis and the clinical course make the diagnosis most likely. CONCLUSION: Tuberculosis should always be included in the differential diagnosis as a possible etiology for uveitis, particularly in those cases taking a chronic course. Despite the recent emergence of drug-resistant strains in most cases tuberculosis is a well curable disease.

High complication rate of bile duct stents in patients with chronic alcoholic pancreatitis due to noncompliance.

BACKGROUND AND STUDY AIMS: Biliary obstruction in chronic pancreatitis is frequently treated by endoscopic insertion of a plastic stent into the common bile duct, a therapy regarded as having a low complication rate. The aim of this study is to analyze the frequency and severity of complications caused by biliary stents in patients with chronic alcoholic pancreatitis. PATIENTS AND METHODS: We retrospectively analyzed all our patients with chronic pancreatitis (n = 14) who were provided with a plastic stent for biliary stenosis between June 1993 and December 1997. Stent exchanges were followed until December 1998. RESULTS: Stent insertion was performed without early complications and was successful in each patient. Only two patients were admitted after 3-4 months at the scheduled dates for stent exchange, both without complications. In one of these patients, the bile duct stenosis was reopened after two stent exchanges over a total period of 8 months. Most of our patients (n=12) did not come at the arranged dates for stent exchange. They were repeatedly admitted (mean 2.9 times/patient, range 1-5) as emergency cases with severe complications of biliary obstruction, such as cholangitis or biliary sepsis. Reopening of the bile duct stenosis was not achieved in these patients. CONCLUSIONS: We associate the high rate of complications with the noncompliance of our patients, who were all alcoholics. The high incidence of late complications in noncompliant patients is a limitation of biliary stenting, and appears to be potentially harmful.

Regulation of the actin cytoskeleton by p125 focal adhesion kinase in rat pancreatic acinar cells.

BACKGROUND/AIMS: Activation of p125 focal adhesion kinase by cholecystokinin (CCK)-8 has recently been demonstrated in pancreatic acinar cells. The purpose of this study is to examine downstream events of this kinase. METHODS: Activation of p125 focal adhesion kinase in freshly isolated rat pancreatic acinar cells was determined by Western blot analysis. Actin cytoskeletal changes were visualized on TRITC-phalloidin-stained cryosections. Amylase release was measured by colorimetric assay. RESULTS: CCK-8 caused dose-dependent activation of p125 focal adhesion kinase. Time-course analysis showed rapid activation with maximum between 5 and 10 min and stimulation still detectable after 60 min. Preincubation with 2 microM cytochalasin D specifically inhibited p125 focal adhesion kinase, but not p42 mitogen-activated protein kinase or increases in intracellular calcium concentrations. The actin cytoskeleton showed rapid reorganization after stimulation, with an initial increase in fluorescence followed by a decline after 30 min. Preincubation with cytochalasin D prevented cytoskeletal changes. Amylase release at concentrations up to 0.1 nM CCK-8 was not influenced by cytochalasin D. In contrast, supramaximal inhibition of amylase release was less pronounced after cytochalasin D incubation. CONCLUSION: p125 focal adhesion kinase in acinar cells appears to be part of a signalling pathway leading to changes in cellular morphology via the actin cytoskeleton. Maximal activation of this signalling pathway might participate in supramaximal inhibition of enzyme secretion.

C-met protooncogene expression and its regulation by cytokines in the regenerating pancreas and in pancreatic cancer cells.

BACKGROUND: Activation of the receptor c-met stimulates motility, mitosis, morphogenesis, processes involved in organ regeneration, or progression of malignancies. In the present study we investigated the expression of c-met protein in the regenerating pancreas and characterized the influence of cytokines on c-met expression. METHODS: Acute pancreatitis was induced in rats by cerulein injection. Rat acini and rat and human pancreatic cancer cells were stimulated with interleukin-1alpha (IL-1alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1). C-met expression was analyzed by means of Western blotting and localization in pancreatic tissue by immunohistochemistry. RESULTS: C-met protein expression was significantly upregulated in the regenerating pancreas and localized in areas of regenerating tissue. Stimulation with cytokines resulted in a two- to threefold increase of c-met expression in vitro. CONCLUSION: Enhanced c-met expression after acute pancreatitis suggests that HGF/met has an important role in pancreatic regeneration, which is probably mediated by cytokines. This regulatory mechanism is also of importance in pancreatic cancer.

Isolation of tobacco SSU genes: characterization of a transcriptionally active pseudogene.

Genomic clones containing three genes for the small subunit (SSU) of ribulose bisphosphate carboxylase were isolated from tobacco. Detailed analysis was performed on two of these clones to give a clearer picture of this multigene family in tobacco. This analysis demonstrated that one of the clones contained a pseudogene that was unusual in that it was transcriptionally active. This is the first transcriptionally active pseudogene that has been reported in plants. In addition, another clone was found to contain coding sequences which are 100% homologous to a previously-cloned tobacco SSU gene (Mazur, B.J. and Chiu, C-F. [1985] Nuc. Acids Res. 13, 2372-2386), indicating that gene duplication and/or gene conversion may have played a role in the evolution of the tobacco SSU family.

Cell-specific expression of plant histone H2A genes.

Histone H2A is a component of eukaryotic chromatin whose expression has not been studied in plants. We isolated and characterized a tomato and a pea cDNA encoding histone H2A. We found that in tomato H2A is encoded by a small gene family and that both the pea and the tomato mRNAs are polyadenylated. Tomato H2A has 82% amino acid residue identity to pea H2A, 83% to wheat, and 65% to human and yeast H2A. Plant H2As differ from fungal and animal H2As in their amino-terminal and carboxy-terminal regions. Carboxy-terminal plant H2A regions contain the motif SPKK, a peptide implicated in binding of A/T-rich DNA regions. By using RNA gel blot analysis, we determined that the steady-state mRNA level of these genes was abundant in apices and early developing fruit and very low in mature tissues. In situ RNA hybridization showed strong spatial regulation because the mRNA was abundant in some cells and not detectable in others. In tomato shoot tips, H2A-expressing cells were distributed irregularly in or near meristems. In tomato or pea root tips, expressing cells were concentrated near the apex, and their distribution was consistent with that expected of cycling cells. Other H2A transcripts were found in nondividing cortical cells that are known to undergo endoduplication during the late maturation phase of primary development.

Thyroid autoantibodies and thyroid dysfunction during treatment with interferon-alpha for chronic hepatitis C.

Interferon-alpha (2a or 2b) is increasingly used for treatment of chronic hepatitis C virus (HCV) infection. Recent reports suggested a correlation between increases in thyroid autoantibodies and the development of thyroid dysfunction during interferon-alpha therapy. In this study, we analyzed thyroid hormones and antithyroid antibodies at monthly intervals in 53 patients who received interferon alpha for chronic active hepatitis C infection. Of five patients with initially elevated levels of antithyroid peroxydase antibodies (anti-TPO), the antibodies increased further in two of them. Ten patients, who started interferon therapy with normal antibody levels, developed elevated anti-TPO antibodies for limited times during treatment. Levels of anti-TPO antibodies showed a marked fluctuation, and only three patients had increased anti-TPO antibodies persisting for longer than 3 mo. Antithyroglobulin antibodies appeared in four patients, all of whom were also positive for anti-TPO antibodies. No changes in TRAB levels were observed. All of these patients with elevated antithyroid antibodies remained in an euthyroid state. One patient with normal antithyroid antibodies developed thyroiditis with severe thyrotoxicosis after 9 wk of interferon therapy. These findings suggest that the induction of antithyroid antibodies during treatment with interferon-alpha does not indicate clinical relevant thyroid dysfunction. Routine measurement of antithyroid antibodies during interferon-alpha therapy does not seem to be mandatory.

Enhanced production of insecticidal proteins in Bacillus thuringiensis strains carrying an additional crystal protein gene in their chromosomes.

A two-step procedure was used to place a cryIC crystal protein gene from Bacillus thuringiensis subsp. aizawai into the chromosomes of two B. thuringiensis subsp. kurstaki strains containing multiple crystal protein genes. The B. thuringiensis aizawai cryIC gene, which encodes an insecticidal protein highly specific to Spodoptera exigua (beet armyworm), has not been found in any B. thuringiensis subsp. kurstaki strains. The cryIC gene was cloned into an integration vector which contained a B. thuringiensis chromosomal fragment encoding a phosphatidylinositol-specific phospholipase C, allowing the B. thuringiensis subsp. aizawai cryIC to be targeted to the homologous region of the B. thuringiensis subsp. kurstaki chromosome. First, to minimize the possibility of homologous recombination between cryIC and the resident crystal protein genes, B. thuringiensis subsp. kurstaki HD73, which contained only one crystal gene, was chosen as a recipient and transformed by electroporation. Second, a generalized transducing bacteriophage, CP-51, was used to transfer the integrated cryIC gene from HD73 to two other B. thuringiensis subsp. kurstaki stains. The integrated cryIC gene was expressed at a significant level in all three host strains, and the expression of cryIC did not appear to reduce the expression of the endogenous crystal protein genes. Because of the newly acquired ability to produce the CryIC protein, the recombinant strains showed a higher level of activity against S. exigua than did the parent strains. This two-step procedure should therefore be generally useful for the introduction of an additional crystal protein gene into B. thuringiensis strains which have multiple crystal protein genes and which show a low level of transformation efficiency.