[Mechanical stimulation of venous blood flow in below-the-knee plaster cast]. / Mechanische Förderung des venösen Rückflusses im Unterschenkelgipsverband.

Physical methods became recently more important as an alternative to anticoagulation for prophylaxis of thromboembolism and were studied for their efficacy. The AV-impulse-system proved efficient in reducing thromboembolic complications in patients undergoing hip surgery by increasing the return of venous blood in the deep veins of the leg. In a preclinical trial we studied the influence of the AV-impulse-system and of active forefoot movement on venous blood return in 12 lower extremities of 6 healthy individuals immobilized in below the knee plaster casts. Our results show a significant increase in venous blood flow caused by the AV-impulse-system (p < 0.05) and by active forefoot movements (p < 0.05). Prevention of thromboembolic complications in trauma and orthopaedic patients immobilized in plaster cast seems possible by using the AV-impulse-system which significantly increases the venous blood flow independent from patient compliance.

Mutation analysis of the cationic trypsinogen gene in patients with pancreatic cancer.

Recently, an Arg to His mutation at residue 117 of the cationic trypsinogen gene (Arg117His) has been shown to be associated with hereditary pancreatitis (hp). A serious complication of hp is development of pancreatic cancer. Patients suffering from hp have been reported to have a 53-fold increased risk to die from pancreatic cancer. However, the quantitative contribution of mutations in the cationic trypsinogen gene to all pancreatic cancer cases is unknown. A relevant contribution of the Arg117His-mutation to pathogenesis of pancreatic cancer might be possible, since also asymptomatic individuals have been reported to carry this mutation and individuals with only mild symptoms may be undiagnosed as hp. In the present study we analyzed genomic DNA obtained from pancreatic cancer tissue from 34 patients and corresponding normal tissue from 28 of these individuals. The third exon of the cationic trypsinogen gene was amplified by nested PCR and digested with AflIII, since the Arg117His mutation creates an AflIII-restriction site. None of the examined samples carried the Arg117His mutation, whereas the amplification product obtained from a patient with known hp was clearly positive. Sequencing of the complete third exon of the cationic trypsinogen gene in 10 of the pancreatic cancer patients resulted exclusively in the wild-type sequence. In addition DNA obtained from venous blood of 116 further patients with pancreatic cancer did not carry the Arg117His mutation. Our results show that the Arg117His mutation does not contribute to pathogenesis of a substantial fraction of all pancreatic adenocarcinomas. In contrast to most oncogenes or tumor suppressor genes the cationic trypsinogen gene (3rd exon) does not contain mutational hot spots.

Gastrin is a target of the beta-catenin/TCF-4 growth-signaling pathway in a model of intestinal polyposis.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene occur in most colorectal cancers and lead to activation of beta-catenin. Whereas several downstream targets of beta-catenin have been identified (c-myc, cyclin D1, PPARdelta), the precise functional significance of many of these targets has not been examined directly using genetic approaches. Previous studies have shown that the gene encoding the hormone gastrin is activated during colon cancer progression and the less-processed forms of gastrin are important colonic trophic factors. We show here that the gastrin gene is a downstream target of the beta-catenin/TCF-4 signaling pathway and that cotransfection of a constitutively active beta-catenin expression construct causes a threefold increase in gastrin promoter activity. APC(min-/+) mice overexpressing one of the alternatively processed forms of gastrin, glycine-extended gastrin, show a significant increase in polyp number. Gastrin-deficient APC(min-/+) mice, conversely, showed a marked decrease in polyp number and a significantly decreased polyp proliferation rate. Activation of gastrin by beta-catenin may therefore represent an early event in colorectal tumorigenesis and may contribute significantly toward neoplastic progression. The identification of gastrin as a functionally relevant downstream target of the beta-catenin signaling pathway provides a new target for therapeutic modalities in the treatment of colorectal cancer.

Clinical consequences of molecular diagnosis in families with mismatch repair gene germline mutations.

Hereditary nonpolyposis colorectal cancer (HNPCC), clinically defined by the Amsterdam criteria, is associated with mismatch repair gene germline mutations. This study was performed to evaluate the efficiency of combined clinical and molecular diagnostics in identifying carriers of a mutated gene in families meeting criteria of the Bethesda guidelines and to examine the influence of molecular diagnosis on clinical decision-making in carriers and noncarriers. Seventy-two patients meeting criteria of the Bethesda guidelines were tested for microsatellite instabilities (MSI). MSI-H tumors were found in 38 (52.8%) index patients. Complete sequencing of hMLH1 and hMSH2 in 38 MSI-H patients and of hMSH6 in one of these patients revealed 15 pathogenic germline mutations, including three novel mutations, and three novel unclassified germline variants. Twelve of the 15 pathogenic mutations were found in patients fulfilling the Amsterdam I/II criteria. Surgical and genetic counseling was offered to the affected families; as a result of molecular diagnosis in the 15 families, 26 index patients and affected carriers and 8 asymptomatic carriers of a mutated mismatch repair gene were included in the surveillance program, and 26 noncarriers were excluded from this program. Although germline mutations are detected in only 20.8% of patients fulfilling criteria of the Bethesda guidelines, family history and MSI-H tumor classification are both strong indicators for germline mutations in hMSH2, hMLH1, and hMSH6 genes, resulting in a 51.9% mutation detection rate. Identification of individual mutation status allows clear-cut decisions on whether or not inclusion in surveillance programs is indicated.

Quantitative differences between aberrant transcripts which occur as common isoforms and due to mutation-based exon skipping of the mismatch repair gene hMLH1.

About one-third of hereditary non-polyposis colorectal cancer-related mutations in the mismatch repair gene hMLH1 result in the loss of entire exons from the wild type transcripts. Here we describe quantitative differences of hMLH1 transcripts without exon 15, exon 16 or exon 17 in several members of a family with hereditary non-polyposis colorectal cancer. The transcript lacking exon 15 is caused by a G to A transition affecting the last nucleotide of the respective exon and results in a truncated protein. The transcripts lacking exon 16 or exon 17, which are in-frame deletions, were also found in all tested samples of a normal population and represent common isoforms. Reverse transcription-polymerase chain reaction-based relative quantification revealed about 50 % signal intensity for the mutation-based transcript, but less than 10% for the common isoforms, if compared to the wild type. All aberrant transcripts were detected from blood-derived cDNAs but not from samples of normal colon epithelium. Although the biological significance of the common isoforms is unknown, they might lead to false risk assessment in hereditary non-polyposis colorectal cancer cases.

Drug metabolizing capacity of cryopreserved human, rat, and mouse liver parenchymal cells in suspension.

The phase I and phase II drug-metabolizing capacity of freshly isolated and cryopreserved parenchymal cells (PC) from human, rat, and mouse liver held in suspension at 37 degrees C for up to 120 min after thawing was compared. Although 7-ethoxycoumarin-O-deethylase activity was strongly reduced in freshly isolated as well as in cryopreserved PC from human, rat, and mouse liver after 120 min, 7-ethoxyresorufin-O-deethylase activity as well as the profile and formation rates of hydroxylated testosterone metabolites in general remained constant throughout the 2-h incubation period in cryopreserved PC from all three species and was similar to that measured in freshly isolated PC. The activity of glutathione S-transferase (GST) and that of UDP-glucuronosyltransferase (UDP-GT) toward 4-methylumbelliferone significantly decreased, whereas the activities of UDP-GT activity toward 4-hydroxybiphenyl and sulfotransferase in cryopreserved human PC were similar to those measured in freshly isolated PC. The activities of GST, UDP-GT toward 4-methylumbelliferone, and sulfotransferase in cryopreserved rat PC showed a significant decrease when compared with the activities in freshly isolated PC. The phase II enzyme activities in cryopreserved mouse PC proved to be far more stable, being similar to the activities of freshly isolated mouse PC at all four time points measured with the exception of GST, which showed a decay from t = 60 min onward. In conclusion, phase I drug-metabolizing enzyme activities in cryopreserved human, rat, and mouse PC are very similar to those of freshly isolated PC, whereas phase II enzyme activities are affected by cryopreservation.

Tissue expression of the vesicle protein pantophysin.

The cell-type restricted expression of cytoplasmic microvesicle membrane protein isoforms may be a consequence of the functional adaptation of these vesicles to the execution of specialized processes in cells of different specialization. To characterize the expression of the vesicle protein pantophysin, an isoform of the synaptic vesicle proteins synaptophysin and synaptoporin, we have prepared and characterized antibodies useful for the immunological detection of pantophysin in vitro and in situ. Using these reagents, we show by immunoblot analyses that pantophysin expression is not homogeneous but differs significantly between various bovine tissues. Furthermore, these differences are not exactly paralleled by the expression of other vesicle proteins of the SCAMP (secretory carrier-associated membrane protein) and VAMP (vesicle-associated membrane protein) types that have previously been localized to pantophysin vesicles in cultured cells. By immunofluorescence microscopy, pantophysin expression is seen predominantly in non-neuroendocrine cells with pronounced membrane traffic. Pantophysin staining codistributes with SCAMP and VAMP immunoreactivities in most instances but differs in some. Remarkably, pantophysin staining in liver is restricted to cells surrounding sinusoids and is not detectable in hepatocytes, similar to that of the SCAMP and VAMP isoforms as detected by our reagents in tissue sections.

[Promoting venous return in plaster cast by AV impulse system. A preclinical study]. / Förderung des venösen Rückstroms im Liegegipsverband durch das AV-Impulssystem. Eine präklinische Untersuchung.

A new pneumatic compression pump--the AV-impulse system--causes increased return of venous blood from the lower limbs to the heart and increases total blood flow in the lower limbs by emptying the plantar venous plexus. Up to date there exist no experiences with using this system in plaster cast. We studied the maximum venous blood flow, the venous blood flow per minute and the venous diameters above the popliteal and femoral vein by duplexsonography in 12 lower limbs of 6 healthy persons before and after applying below-the-knee plaster casts. After applying the plaster cast we observed a slight increase in venous diameter (p = 0.02). By using the AV-impulse-system we observed a significant increase in maximum venous blood flow and venous blood flow per minute (p < 0.05). We demonstrated a significant increase of venous blood return in the deep veins of the lower limbs after applying a lower limb plaster cast by using the AV-impulse-system. These results indicate the possible benefit of using the AV-impulse-system as a physical method of thromboprophylaxis in orthopaedic and trauma patients with plaster cast immobilisation of the leg.

Cytoplasmic and peroxisomal catalases of the guinea pig liver: evidence for two distinct proteins.

Catalase, a peroxisomal marker enzyme in the liver of most mammals, is found by immuno-electron microscopy in guinea pig (GP) hepatocytes not only in peroxisomes, but also in the cytoplasm (Beier et al. (1988) Eur. J. Cell Biol. 46, 129-135). We have been able to distinguish in GP liver homogenates between the cytosolic catalase and that part of the enzyme activity which is due to leakage of the enzyme from peroxisomes by adding 4% polyethylene glycol to the homogenization medium. This approach revealed that approximately 40% of the total catalase activity and almost all of alpha-hydroxy-acid oxidases are peroxisomal, while 60% of catalase is of genuine cytosolic origin. The cytosolic and peroxisomal catalases of guinea pig were purified to homogeneity and were analyzed by SDS-PAGE and isoelectric focussing. The cytosolic catalase exhibited a slightly higher Mr (approximately 1000) and a less acidic pI than the peroxisomal enzyme. Limited proteolysis and amino-acid analysis revealed also slight differences between the two molecular forms of catalase. Total RNA was isolated from guinea pig liver and translated in vitro by using a rabbit reticulocyte lysate system. Immunoprecipitation with an antibody against guinea pig catalase followed by high-resolution polyacrylamide gel electrophoresis revealed two polypeptide bands differing slightly in Mr. These observations suggest strongly, that cytoplasmic and peroxisomal catalases in guinea pig liver are two closely related but distinct proteins.