PubMed Health⌕ Search

Biomedical subjects

M M Lerch

Publications and source records attributed to M M Lerch.

At least 19 recordsLinked to original sources
PubMed1998

Intra-acinar cell activation of trypsinogen during caerulein-induced pancreatitis in rats.

Supramaximal stimulation of the pancreas with the CCK analog caerulein causes acute edematous pancreatitis. In this model, active trypsin can be detected in the pancreas shortly after the start of supramaximal stimulation. Incubation of pancreatic acini in vitro with a supramaximally stimulating caerulein concentration also results in rapid activation of trypsinogen. In the current study, we have used the techniques of subcellular fractionation and both light and electron microscopy immunolocalization to identify the site of trypsinogen activation and the subsequent fate of trypsin during caerulein-induced pancreatitis. We report that trypsin activity and trypsinogen-activation peptide (TAP), which is released on activation of trypsinogen, are first detectable in a heavy subcellular fraction. This fraction is enriched in digestive enzyme zymogens and lysosomal hydrolases. Subsequent to trypsinogen activation, both trypsin activity and TAP move to a soluble compartment. Immunolocalization studies indicate that trypsinogen activation occurs in cytoplasmic vacuoles that contain the lysosomal hydrolase cathepsin B. These observations suggest that, during the early stages of pancreatitis, trypsinogen is activated in subcellular organelles containing colocalized digestive enzyme zymogens and lysosomal hydrolases and that, subsequent to its activation, trypsin is released into the cytosol.

Acute Disease↗
PubMed1998

Zymogen proteolysis within the pancreatic acinar cell is associated with cellular injury.

The pathological activation of digestive zymogens within the pancreatic acinar cell probably plays a central role in initiating many forms of pancreatitis. To examine the relationship between zymogen activation and acinar cell injury, we investigated the effects of secretagogue treatment on isolated pancreatic acini. Immunofluorescence studies using antibodies to the trypsinogen-activation peptide demonstrated that both CCK (10(-7) M) hyperstimulation and bombesin (10(-5) M) stimulation of isolated acini resulted in trypsinogen processing to trypsin. These treatments also induced the proteolytic processing of procarboxypeptidase A1 to carboxypeptidase A1 (CA1). After CCK hyperstimulation, most CA1 remained in the acinar cell. In contrast, the CA1 generated by bombesin was released from the acinar cell. CCK hyperstimulation of acini was associated with cellular injury, whereas bombesin treatment did not induce injury. These studies suggest that 1) proteolytic zymogen processing occurs within the pancreatic acinar cell and 2) both zymogen activation and the retention of enzymes within the acinar cell may be required to induce injury.

Animals↗
PubMed1997

Cellular redistribution of protein tyrosine phosphatases LAR and PTPsigma by inducible proteolytic processing.

Most receptor-like protein tyrosine phosphatases (PTPases) display a high degree of homology with cell adhesion molecules in their extracellular domains. We studied the functional significance of processing for the receptor-like PTPases LAR and PTPsigma. PTPsigma biosynthesis and intracellular processing resembled that of the related PTPase LAR and was expressed on the cell surface as a two-subunit complex. Both LAR and PTPsigma underwent further proteolytical processing upon treatment of cells with either calcium ionophore A23187 or phorbol ester TPA. Induction of LAR processing by TPA in 293 cells did require overexpression of PKCalpha. Induced proteolysis resulted in shedding of the extracellular domains of both PTPases. This was in agreement with the identification of a specific PTPsigma cleavage site between amino acids Pro821 and Ile822. Confocal microscopy studies identified adherens junctions and desmosomes as the preferential subcellular localization for both PTPases matching that of plakoglobin. Consistent with this observation, we found direct association of plakoglobin and beta-catenin with the intracellular domain of LAR in vitro. Taken together, these data suggested an involvement of LAR and PTPsigma in the regulation of cell contacts in concert with cell adhesion molecules of the cadherin/catenin family. After processing and shedding of the extracellular domain, the catalytically active intracellular portions of both PTPases were internalized and redistributed away from the sites of cell-cell contact, suggesting a mechanism that regulates the activity and target specificity of these PTPases. Calcium withdrawal, which led to cell contact disruption, also resulted in internalization but was not associated with prior proteolytic cleavage and shedding of the extracellular domain. We conclude that the subcellular localization of LAR and PTPsigma is regulated by at least two independent mechanisms, one of which requires the presence of their extracellular domains and one of which involves the presence of intact cell-cell contacts.

Amino Acid Sequence↗
PubMed1997

A circus flea.

Explore the source record for details and available documents.

Animals↗
PubMed1997

Dissociation and reassembly of adherens junctions during experimental acute pancreatitis.

BACKGROUND & AIMS: The initial pathophysiological events that characterize acute pancreatitis include the formation of pancreatic edema. An interstitial accumulation of fluid, however, is incompatible with the presence of intact intercellular junctions between acinar cells. This study examined the major components of adherens junctions, E-cadherin, alpha-catenin, beta-catenin, and actin, during the initial phase of experimental pancreatitis. METHODS: Pancreatitis was induced in rats by 10 micrograms.kg-1.h-1 intravenous cerulein for up to 12 hours. Adherens junction proteins were localized by immunocytochemistry for fluorescence microscopy or electron microscopy, their expression was studied by slot blot analysis, and their association was investigated by immunoprecipitation and Western blot. RESULTS: During a rapid increase of E-cadherin-encoding RNA, E-cadherin protein declined only moderately and, unlike its cytoskeletal binding partner actin, was not proteolytically cleaved during pancreatitis. Morphologically, E-cadherin and beta-catenin were localized at the basolateral cell membrane from where they rapidly dissociated early in pancreatitis and to where they slowly relocalized during the subsequent course. E-cadherin/beta-catenin complexes disintegrated and reassembled completely in parallel on immunoprecipitation experiments. CONCLUSIONS: The dissociation of adherens junctions and the internalization, relocalization, and reassembly of their major components seem to represent the critical biochemical event at cell-cell contacts during edema formation and resolution in acute pancreatitis.

Actins↗
PubMed1997

Common biliary-pancreatic conduit stenosis induces pancreobiliary reflux.

A striking pancreatic juice reflux toward the biliary tree (PR) has frequently been found in subjects with common bile-pancreatic conduit (CBPC) and gallstone-induced acute pancreatitis (SAP). Our aim was to determine the role of the CBPC functional diameter as a potential trigger of PR. The CBPC was calibrated in three groups of patients: (A) patients with gallbladder stones using intraoperative direct cholangiometry (n = 24), (B) patients after surgical removal of common bile duct stones using postoperative cholangiometry via T-drain (n = 6), and (C) patients with SAP using the same procedures as in group B (n = 6). In all patients in groups B and C, aliquots of bile were collected and assayed for pancreatic amylase content. The mean functional diameter of the CBPC varied from 0.80 mm in patients in groups A and B to 0.47 mm in group C (p < 0.001). The bile samples of group B contained low concentrations of amylase, whereas high values could be found in all samples of group C. We conclude that PR is a frequent event occurring after bile duct revision concerning small amounts of pancreatic juice. However, CBPC functional stenosis as seen in patients with SAP obviously induces PR with high amounts of pancreatic juice.

Acute Disease↗
PubMed1997

Expression of the protooncogene jun is induced in the rat pancreas by cerulein infusion.

Cholecystokinin (CCK) can stimulate secretion and DNA synthesis in pancreatic acinar cells. Hyperstimulation with cerulein (a CCK analogue) induces acute edematous pancreatitis. To study the effects of in vivo pancreatic stimulation with cerulein, we analyzed the expression of the protooncogenes jun, myc, and fos on the mRNA and protein levels. RNA and protein were extracted from the pancreas of rats administered an infusion of cerulein, 10 micrograms/kg/h (Group A) or 0.25 microgram/kg/h (Group B), or saline (Group C) and sacrificed 2, 4, and 6 h after beginning the infusion and 0, 12, and 24 h and 2, 4, and 6 days after completing the infusion period. Transcript levels were studied using slot-blot analysis. Protein expression was studied using Western blot and immunohistochemistry. No changes were found for the expression of protooncogenes myc and fos on either the transcript or the protein levels. Higher jun mRNA levels were found in Group A than in Group B or C, particularly after 2 h of infusion and 12, 24, and 48 h after the end of a 12-h cerulein infusion. No significant difference was observed in Groups B and C. The jun protein behavior was similar in Groups A and B, revealing two peaks: one early during infusion and a second one after the end of a 12-h cerulein infusion. Jun protein was found mainly in the acinar cells. In conclusion, (1) acinar cells in the rat pancreas respond to cerulein stimulation by increasing the expression of jun; (2) in vivo high doses of cerulein increase the jun mRNA and jun protein levels, whereas low doses raise only the protein levels; and (3) myc and fos are apparently uninfluenced by cerulein administration.

Animals↗
PubMed1997

Combined treatment with C1 esterase inhibitor and antithrombin III improves survival in severe acute experimental pancreatitis.

BACKGROUND: Patients with severe acute pancreatitis die of complications closely related to the systemic activation of protease cascades. AIM: To examine the effects of human C1 esterase inhibitor (C1 INH) and antithrombin III (AT III) on two experimental models of acute pancreatitis. METHODS: Oedematous pancreatitis was induced by continuous intravenous infusion of caerulein and haemorrhagic pancreatitis by retrograde injection of sodium taurocholate into the biliopancreatic duct. C1 INH and AT III were given intravenously, either before or after the induction of pancreatitis. Treatment with C1 INH and AT III had no beneficial effect on oedematous pancreatitis. On the other hand, combined C1 INH and AT III therapy improved the survival in haemorrhagic pancreatitis compared with treatment with human serum albumin. This reduction in mortality was found regardless of whether the treatment was given prophylactically or therapeutically. CONCLUSIONS: Treatment with C1 INH and AT III represents a promising therapeutic concept for patients with severe haemorrhagic pancreatitis.

Acute Disease↗
PubMed1997

Failure of a prolyl 4-hydroxylase inhibitor to alter extracellular matrix deposition during experimental pancreatitis.

In chronic pancreatitis the exocrine pancreatic tissue is replaced by extracellular matrix deposits from fibroblasts. We have stimulated fibrogenesis in the pancreas by inducing a single episode of cerulein pancreatitis (10 microg/kg/h for 12 h). We have used a spectrophotometrical assay to measure the tissue hydroxyproline content and immunohistochemistry to study the transient deposition of extracellular matrix in the pancreas. We have investigated whether a potent prolyl 4-hydroxylase inhibitor (HOE-077) can influence the deposition of extracellular matrix in the pancreas. Three days after the induction of the pancreatitis we found the maximum increase in pancreatic hydroxyproline content (by 63%), the maximum decrease in total protein content and amylase activity (by -39 and -86%, respectively), as well as a significant increase in DNA content and the deposition of interstitial collagen fibers on electron microscopy. By immunohistochemistry the largest expansion of extracellular matrix components was found for fibronectin. HOE 077, regardless of the concentration administered, failed to affect any of these parameters. We conclude that the induction of a single episode of cerulein pancreatitis and the serial determination of pancreatic hydroxyproline content represents a simple method to induce and monitor experimental fibrogenesis in the pancreas. Prolyl 4-hydroxylase inhibition did not affect the course of extracellular matrix deposition in the pancreas.

Acute Disease↗
PubMed1996

Association of human protein-tyrosine phosphatase kappa with members of the armadillo family.

We have identified a human receptor-like protein-tyrosine phosphatase (PTP) in the mammary carcinoma cell line SK-BR-3, which represents the human homolog of murine PTPkappa (Jiang, Y.-P., Wang, H., D'Eustachio, P., Musacchio, J. M., Schlessinger, J., and Sap, J. (1993) Mol. Cell. Biol. 13, 2942-2951) and was therefore termed hPTPkappa. We show here that hPTPkappa expression is dependent on cell density and find it colocalized with two members of the arm family of proteins, beta-catenin and gamma-catenin/plakoglobin, at adherens junctions. Using both in vitro and in vivo binding assays, we demonstrate specific complex formation between endogenous hPTPkappa and beta- and gamma-catenin/plakoglobin. In addition, we present evidence that suggests that beta-catenin may represent a substrate for the catalytic activity of hPTPkappa. The identification of specific binding partners for this receptor-like PTP provides insight into the mechanisms of its biological action and suggests a role for hPTPkappa in the regulation of processes involving cell contact and adhesion such as growth control, tumor invasion, and metastasis.

Amino Acid Sequence↗
PubMed1996

Characterization of PCP-2, a novel receptor protein tyrosine phosphatase of the MAM domain family.

DNA sequences encoding a novel member of the receptor protein tyrosine phosphatase (R-PTP) family, termed PCP-2, were identified in a human pancreatic adenocarcinoma cDNA library. Human PCP-2 cDNA predicts a protein of 1430 amino acids with a calculated Mr of 160 kDa. The predicted PCP-2 enzyme consists of a 740 amino acid extracellular region, a single transmembrane domain, and a 666 amino acid intracellular portion. The extracellular sequence contains a MAM (meprin/A5/PTPmu) domain, an immunoglobulin-like domain and four fibronectin type III-like repeats, suggesting that it is a member of the PTPkappa and PTPmu subfamily. The intracellular region contains two tandemly-repeated protein tyrosine phosphatase domains. Northern blot analyses revealed a single transcript of 5.5 kilobases, which is expressed at different levels in many human tissues except spleen and placenta. Upon transfection of PCP-2 cDNA into human embryonic kidney fibroblast 293 cells, a protein with an apparent Mr of 180 000 was detected by immunoblot analysis. This size was reduced to the predicted Mr upon treatment with endoglycosidase F, indicating that PCP-2 is glycosylated and, hence, expressed at the cell surface. A potential role of PCP-2 in cell-cell recognition and adhesion is supported by its co-localization with cell adhesion molecules, such as catenin and E-cadherin, at sites of cell-cell contact.

Adenocarcinoma↗
PubMed1996

Characterization of a non-invasive, vascular model of acute necrotizing pancreatitis.

One of the vasoactive peptides that has been implicated in the progression from edematous to necrotizing pancreatitis is bradykinin. We have investigated the effect of bradykinin administration and bradykinin inhibition on an edematous model of acute pancreatitis in rats (10 micrograms/kg/h of caerulein i.v.). Within six hours i.v. bradykinin reduced circulating serum amylase levels significantly but neither affected tissue edema nor morphology. A bradykinin antagonist (HOE-140), on the other hand, reduced pancreatic edema by 70% and converted edematous pancreatitis into a hemorrhagic and necrotizing variety of the disease. In further experiments we determined the time course and the minimal dosage required for the induction of this severe and non-invasive disease variety. A single dose of caerulein (40 micrograms/kg i.p.) together with a single administration of the bradykinin antagonist HOE-140 (100 micrograms/kg s.c.) consistently resulted in hemorrhagic necrosis of the pancreas within six hours. We conclude that this simple protocol allows for the non-invasive induction of a vascular model of necrotizing pancreatitis and appears ideally suited to study the development of this severe form of the disease.

Acute Disease↗
PubMed1995

Cloning, characterization, and differential expression of MDK2 and MDK5, two novel receptor tyrosine kinases of the eck/eph family.

Using a polymerase chain reaction-based strategy for the cloning of developmentally regulated receptor tyrosine kinases, we identified two novel members of the eck/eph-related subfamily which, in analogy with the recently identified mouse developmental kinase 1 (MDK1), were designated MDK2 and MDK5. MDK2 is highly homologous to the mouse kinase Myk-1 and the human kinase Htk, whereas MDK5 represents the mouse homologue of human Hek2. Northern blot analyses of adult mouse tissues revealed a 4.7 kb transcript of MDK2 and a 4.8 kb transcript of MDK5 in various organ systems, including lung, liver, kidney, intestine, muscle, heart, and, in the case of MDK5, also the brain. In addition to the full-length transcripts, smaller fragments were identified that probably represent truncated receptors. Northern blot analysis and in situ hybridization of mouse embryos indicated abundant expression during embryonic development, with preferential involvement of tissues of epithelial and endothelial origin for both kinases and of the spinal cord gray matter for MDK5. Unlike most other members of the eck/eph-related subfamily, the expression of MDK2 and MDK5 is not primarily restricted to neuronal structures, and their abundant presence in various organ systems during embryonic development suggests an important role in gestational growth and differentiation.

Amino Acid Sequence↗
PubMed1995

The role of nitric oxide in hemodynamic and metabolic alterations induced by prostaglandin F2 alpha in the perfused rat liver.

In the liver prostaglandins have been shown to be potent regulators of portal blood flow, carbohydrate metabolism and bile secretion. It is not known whether these effects represent a direct action of prostaglandins, and it has been suggested that nitric oxide (NO) might be a critical mediator for prostaglandin induced hepatic events. We have studied whether nitric oxide formation or inhibition alters the action of prostaglandin F2 alpha (PG F2 alpha) in a single-pass liver perfusion model. The liver of untreated rats (constitutive NO-synthase) or after pretreatment with endotoxin (inducible form of NO-synthase) was perfused at a constant pressure via the portal vein. Effluate were collected in 1-min intervals and bile in 5-min intervals. In both groups the addition of PG F2 alpha (10 microM) to the perfusate for 5 min resulted in a significant increase of glucose and lactate production, and in a significant decrease in portal blood flow (-0.56 +/- 0.04 ml/g per min), in bile flow (-60.7%) and in bile acid release (-60.6%). Inhibition of NO synthase by adding NG-monomethyl-L-arginine (L-NMMA, 100 microM) to the perfusate did not affect any of the alterations induced by PG F2 alpha. Substitution of the endogenous substrate for the NO synthase L-arginine (500 microM) in the perfusate completely prevented the hemodynamic alterations induced by PG F2 alpha in endotoxin pretreated livers and limited the flow reduction (0.15 +/- 0.04 ml/g per min) in the untreated group. The substitution of L-arginine in the perfusate of endotoxin pretreated livers raised nitrite (from 1.5 +/- 0.3 to 3.6 +/- 0.7 nmol/g per min) and urea release (from 65 +/- 25 to 294 +/- 68 nmol/g per min), but had no effect on any of the other metabolic parameters and bile secretion. We conclude that PG F2 alpha increases glucose and lactate production in the perfused rat liver and decreases portal flow bile secretion. The metabolic effects induced by PG F2 alpha appear to be independent of NO mediation and hemodynamic alterations. Portal flow alone can be influenced by endogenous NO formation.

Animals↗
PubMed1995

Expression and in-situ localization of genes coding for extracellular matrix proteins and extracellular matrix degrading proteases in pancreatic cancer.

Pancreatic cancer shows a strong desmoplastic reaction characterized by a remarkable proliferation of interstitial connective tissue (collagens type I and III, fibronectin). In this study we have analyzed the balance of expression of mRNAs encoding extracellular matrix components (collagens I, III and IV, laminin, fibronectin), extracellular matrix-degrading metalloproteinases (MMP-1, -2, -3 and -9) and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in pancreatic cancer and control pancreatic tissue by Northern-blot analysis and mRNA in situ hybridization. Transcripts for MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) were not detectable in pancreatic cancer and control tissues. Steady-state levels of transcripts encoding extracellular matrix proteins, MMP-2 (72-kDa collagenase IV), MMP-9 (92-kDa collagenase type IV), TIMP-1 and TIMP-2 were elevated in the majority of pancreatic-cancer tissue samples as compared to control pancreatic tissue. A good correlation was seen between overexpression of these MMPs and TIMPs and the steady-state levels of transcripts coding for extracellular matrix proteins, the amount of collagen protein and the severity of the desmoplastic reaction. In situ hybridization studies localized transcripts coding for collagens type I and III to spindle-shaped stromal cells, whereas transcripts for MMP-2, MMP-9, TIMP-1 and TIMP-2 were found in both stromal and tumor cells. However, MMP-2 transcripts appeared to be more abundant in stromal cells, TIMP-1 and TIMP-2 transcripts were evenly distributed over tumor and stromal cells and relatively more MMP-9 transcripts were found in tumor cells. We conclude that, in human pancreatic cancer, MMP-2, MMP-9, TIMP-1 and TIMP-2 may be involved in processes leading to the strong desmoplastic reaction observed in these tumors. Both stromal and tumor cells appear to be the source of MMPs and TIMPs in human pancreatic cancer.

Blotting, Northern↗
PubMed1995

Prevention of postoperative adhesions by single intraperitoneal medication.

Postoperative adhesions account for a significant morbidity after abdominal, gynecological, or cardiac surgery. A large number of compounds have been suggested to prevent such adhesions, but none is generally accepted. We have compared eight different substances that could be beneficial for the prevention of postoperative adhesions in a new standardized rabbit model with measurement of the areas of adhesion. In 10 groups of 20 rabbits an area of abrasion of the serosa of the ileum, the appendix, and the abdominal wall measuring 10,000 mm2 was created by an emery piston during celiotomy. The controls received no medication. The treatment groups received a single intraperitoneal administration of 1 ml per 100 g body wt of normal saline (NaCl), 5 mg taurolidine (T), 0.5 U plasmin/300 U DNase (PD), 2000 IU streptokinase/500 IU streptodornase (SS), 7 mg phosphatidylcholine (PC), 4 mg hyaluronic acid (HA), 7 mg sphingolipid (SL), 7 mg galactolipid (GL), or 0.5 ml tetrachlorodecaoxide (TCDO), respectively. Ten days later the extent of adhesions was quantified by morphometry. The total area of adhesions (+/- SEM) was found to be 1998 +/- 124 mm2 in controls. The application of NaCl reduced the adhesions to 1368 +/- 58 mm2, of T to 1012 +/- 48 mm2, of PD to 673 +/- 33 mm2, of SS to 360 +/- 44 mm2, of PC to 335 +/- 84 mm2, of HA to 328 +/- 76 mm2, of SL to 278 +/- 80 mm2, of GL to 261 +/- 67 mm2, and of TCDO to 240 +/- 45 mm2. The effects of PD, SS, PC, HA, SL, GL, and TCDO were significant in comparison to controls and NaCl. Our experimental data suggest that the two new lipid substances, SL and GL, are the most likely candidates for routine clinical use in the prevention of postsurgical adhesions.

Abdominal Muscles↗
PubMed1995

Supramaximal secretagogue stimulation enhances heat shock protein expression in the rat pancreas.

Heat shock or stress proteins (HSPs) are synthesized by various cell types in response to different metabolic insults (e.g., hyperthermia). Although the function of HSPs is not fully understood, they are believed to be an evolutionary conserved intracellular defense mechanism. In an attempt to characterize the autoprotective potential of pancreatic acinar cells, we investigated the regulation of HSPs of the 70-kD family and the small HSP ubiquitin in vitro and in vivo during supramaximal cerulein stimulation. Infusion of the secretagogue cerulein induces a mild edematous form of pancreatitis in vivo and is characterized by a marked disturbance of the intracellular transport and segregation of enzymes. Synthesis of HSP70 mRNA is upregulated in isolated pancreatic lobules by either cerulein (100 nM) or hyperthermia (42 degrees C for 60 min). In contrast, expression of ubiquitin mRNA was not altered by either secretagogue treatment or hyperthermia. This heat shock-like response of pancreatic acinar cells could be reproduced in vivo: Pancreatitis was induced in male Wistar rats by intravenous infusion of supramaximal doses of cerulein (10 micrograms/kg/h). Analysis of mRNA expression revealed a significant upregulation of HSP70 RNA during supramaximal secretagogue stimulation. mRNA levels encoding for ubiquitin remained unchanged. Western blot analysis demonstrated that the transcriptional upregulation of HSP70 in vivo was reflected on the protein level. This study demonstrates that the marked intracellular disturbance observed in secretagogue-induced pancreatitis is associated with enhanced expression and synthesis of a major stress protein. Given the autoprotective potential of HSPs, this upregulation may indicate a self-defense mechanism of pancreatic acinar cells in experimental pancreatitis.

Animals↗