Cloning and molecular characterization of Dashurin encoded by C20orf116, a PCI-domain containing protein.

BACKGROUND: Characterization of gene products originating from undefined open reading frames and delineation of biological functions has become the task after the human genome has been decoded. METHODS: We cloned the human C20orf 116 and defined its transcript in liver, kidney and various brain regions by Northern analysis. Antibodies against recombinant protein used for immunofluorescence and immunoblots confirmed its expression in these tissues. With the focus on kidney, its tubular expression and presence in glomerula were shown. RESULTS: A 28 aa long signal peptide predicted by in silico analysis is reflected by visualization of size variants of approximately 3kDa difference suggesting a signal peptidase cleavage of the proform. Cell compartment separation confirmed the presence of Dashurin in peroxisomes/mitochondria, microsomes, cytosol and nucleus. This is in line with green fluorescent protein (GFP)-Dashurin fusion protein shuttling between cytosol and nucleus. Luciferase reporter studies revealed a 2-3 fold increase of promoter activities upon over-expression. Bioinformatic analysis identified a PCI-domain at the C-terminus providing protein-protein interaction capabilities. CONCLUSION: Our present findings suggest the involvement of Dashurin in gene transcription or mRNA translation. GENERAL SIGNIFICANCE: Dashurin shares the PCI-domain with three multisubunit protein complexes (26S proteasome, COP9 signalosome and eIF3 translation initiation factor).

Reduced TGF-beta1 expression and its target genes in human insulinomas.

Aiming to identify signalling pathways relevant for ss-cell growth we performed an explorative micro-array analysis comparing the gene expression profiles of three human insulinomas and one normal pancreatic islet preparation. This revealed an insulinoma-associated down-regulation of the transforming growth factor beta 1 (TGF-beta1) and its target genes. Comparative quantitative real-time PCR (qRT-PCR) including an expanded sample number of both insulinomas (n=9) and pancreatic islet preparations (n=4) confirmed the decreased TGF-beta1 expression and its target molecules (TGFBI, NNMT, RPN2) in insulinomas. Similarly, TGF-beta1 immunofluorescence analysis revealed reduced expression in insulinomas when compared to pancreatic islets. In contrast, TGFBR2 (transforming growth factor beta receptor II) was found up-regulated. However, the consistent down-regulation of the TGF-beta1 targets TGFBI (transforming growth factor, beta-induced), NNMT (nicotinamide N-methyltransferase), RPN2 (ribophorin II) indicates that the parallel up-regulation of TGFBR2 does not compensate for the only marginal TGF-beta1 expression levels in insulinomas. TGFBR2 expression was confirmed at the protein level in insulinomas. SMAD2/3 protein expression was found at higher levels in human pancreatic islets when compared with insulinomas by dual colour confocal microscopy. TGF-beta1 signalling is known to be involved in cell replication and is abrogated in ductal pancreatic tumours. The down-regulation of TGF-beta1 expression and its target molecules in insulinomas is a new aspect of this cytokine. Our data underline parallels in endocrine and exocrine pancreatic tumour development, which may implicate common progenitor cells.

Catabolic properties of aglycofibrinogen synthesized by tunicamycin-treated human hepatoma (HepG2) cells and rabbit hepatocytes.

Human hepatoma cell (HepG2) or rabbit hepatocyte monolayers were incubated with [35S]methionine in presence or absence of tunicamycin, a potent inhibitor of asparagine-linked glycosylation. The 35S-labeled nonglycosylated and control fibrinogens purified from the media were used to evaluate the influence of the oligosaccharide on the catabolic properties of this glycoprotein. Plasmin, pronase, cathepsin D or cathepsin B each degraded the nonglycosylated and control fibrinogens similarly, as evidenced by the release of trichloroacetic acid-soluble radioactivity and by SDS-polyacrylamide gel electrophoresis and autoradiography of plasmic digests. Nonglycosylated and control fibrin clots also showed no differences in susceptibility to plasmic digestion. The two forms of fibrinogen demonstrated the same plasma half-life in rabbits. These data indicate that the oligosaccharide does not influence the proteolytic stability or the in vivo plasma survival of fibrinogen, and suggest that other biochemical determinants may influence the catabolic properties of this molecule.

Heat shock protein 70 (Hsp70) subtype expression in neuroendocrine tissue and identification of a neuroendocrine tumour-specific Hsp70 truncation.

In order to identify neuroendocrine tumour-specific protein expression, we generated monoclonal antibodies (mAbs) with a tumour-related reaction pattern using a human insulinoma as immunogen. One of the generated mAbs (mAb 1D4) exhibited striking immunoreactivity against various neuroendocrine tumours without staining pancreatic islets of Langerhans. Furthermore, mAb 1D4 immunostained a characteristic subtype of hypothalamic neurones. Using two-dimensional (2-D) gel electrophoresis, mAb 1D4 immunoblotting and mass spectrometry, heat shock protein 70 (Hsp70) isoforms were identified as the mAb 1D4-specific antigen. In hypothalamic tissue, the presence of two different Hsp70 isoforms (Hsp70-8 and Hsp70-1) was revealed by 2-D gel immunoblots and consecutive mass spectrometric peptide analysis. In contrast, insulinoma and other neuroendocrine tumours displayed solely Hsp70-8 expression. Moreover, the tumour-specific presence of an additional mAb 1D4 immunoreactive protein of 40 kDa was observed in eight out of eight tested neuroendocrine tumours. For this variant, exclusively, peptides derived from the C terminus excluding the 299 amino-terminal residues were detected. In cultured tumour-derived fibroblasts, expression of the truncated Hsp70-8 subtype was not present. In conclusion, we have demonstrated a neuroendocrine tumour-specific expression pattern of Hsp70 isoforms and identified an as yet unknown N-terminally truncated Hsp70-8 variant.

Detection of BLT substrate-specific proteases in individual human peripheral blood leucocytes and bone marrow cells. Application of the method to the classification of leukaemia.

A trypsin-like serine esterase (SE) is known to be present in cultured cells with cytolytic potential. The distribution pattern of this enzyme in haematological cells and body tissues has been assessed using a method which permits rapid identification of individual cells. Cells and tissue sections were fixed and immersed in the substrate N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT)/Fast Blue BB chromogen solution. To identify the phenotype of SE+ cells the cytochemical stain was followed by the application of monoclonal antibody and alkaline phosphatase-anti-alkaline phosphatase (APAAP) complex immunocytochemical procedures. CD8+ and CD57+ lymphocytes showed SE+ granules. Neutrophil granulocytes and progenitors other than undifferentiated myeloblasts developed a dense stain while eosinophils were negative. 35% of monocytes showed positivity mainly in the area of nuclear indentation. Tumour-infiltrating SE+ lymphocytes could also be demonstrated with this method.

In vivo and in vitro primed lymphocytes. Correlation of cytochemically detected BLT-specific lymphoid serine protease with cytotoxic activity.

We describe the validation of a cytochemical method to detect a cytolytic cell-specific lymphoid serine protease which can be upregulated during viral infection and allogeneic stimulation. The cytolytic cell specificity was ascertained by demonstrating a high correlation between BLT substrate-specific serine protease (SP) activity and cytotoxicity of in vivo and in vitro stimulated lymphocytes. The presence of SP in peripheral blood lymphocytes was compared with their capacity to kill K562 targets in a lectin-dependent cytotoxicity assay. The correlation coefficient was 0.92 and 0.93 at E:T ratios 10:1 and 20:1 respectively. In allogeneic mixed lymphocyte cultures an increase of SP activity in effector lymphocytes was paralleled by an augmentation of cytotoxic capacity towards stimulator target cells. SP+ granules showed intracellular polarization to the effector/target cell interface during conjugate formation. These results together with previous studies suggest that this method provides a sensitive assay which predicts the cytolytic potential present in a lymphocyte population.

Prevention of upper gastrointestinal bleeding in long-term ventilated patients. Sucralfate versus ranitidine.

Thirty-two long-term ventilated patients were randomly selected for a study of the efficacy of sucralfate (1 g six times per day via gastric tube) versus ranitidine (six 50-mg to six 100-mg doses per day intravenously) for the prevention of upper gastrointestinal bleeding. The patients of the two treatment groups (each 16) were comparable with respect to diseases precipitating acute respiratory failure and risk factors of bleeding, e.g., renal failure, thrombopenia, coagulopathy, and anticoagulant treatment. Mean duration of mechanical ventilation was 7.4 in sucralfate- and 7.7 days in ranitidine-treated patients. During mechanical ventilation, macroscopically visible bleeding developed in three of the sucralfate-treated (18.7 percent) and seven of the ranitidine-treated (43.7 percent) patients. Until the end of the study, only three of the sucralfate-treated but nine of the ranitidine-treated (56.2 percent) patients bled; the difference between the two treatment groups was at all times significant (p less than 0.05). Packed red blood cells had to be administered to the three bleeding patients in the sucralfate group and to seven bleeding in the ranitidine group. Therefore it seems that sucralfate prevented mostly minor bleeding. The high bleeding rate during ranitidine treatment was presumably due to the high number of pH-nonresponders, as almost 30 percent of the gastric aspirates of this group had a pH less than 5. During treatment no difference was found in positive blood culture specimens and bronchial secretions between the two groups. However, nosocomial pneumonia developed in two ranitidine-treated patients, whereas that complication developed in none of the sucralfate-treated patients. In long-term ventilated patients, sucralfate prevented minor upper gastrointestinal bleeding significantly better than ranitidine. However, this does not imply that major upper gastrointestinal bleeding can be prevented by either sucralfate or ranitidine in these patients.

Culture of human insulinoma cells: development of a neuroendocrine tumor cell- and human pancreatic islet cell-specific monoclonal antibody.

We report on the culture of human insulinoma cells derived from a 32-year-old male patient with hyperinsulinism due to an insulinoma of the pancreas. A single-cell suspension was made by passing insulinoma fragments through a fine-gauge stainless-steel mesh. Cluster-forming insulinoma cells resembling pancreatic islets grew in the presence of fibroblasts. The insulinoma cell clusters could be differentiated from fibroblasts by using in situ pan optic staining and specific immunocytochemical staining (anti-human insulin and anti-human insulinoma monoclonal antibody (mAb) D24). mAb D24 was generated using insulinoma cells as antigen for immunization of a Balb/C mouse and cell fusion by the hybridoma cell technique. The anti-insulinoma cell mAb recognized a 32 kDa protein on immunoblot analysis of neuroendocrine tumor cells. D24 mAb also reacted immunohistochemically with normal pancreatic beta-cells and tumors such as vipoma, gastrinoma and carcinoid. Insulinoma cell clusters separated from fibroblasts by micromanipulation and plated into multiwell culture dishes exhibited an insulin-secretion rate of approximately 30 U/100 cells per 24 h with no insulin-secretory response to elevated glucose concentration. Purified insulinoma cells incubated with 1 ng/ml human nerve growth factor expressed neurofilament and neurite extension. These findings together with earlier observations in animal models suggest that human pancreatic beta-cells share some properties with neurons and are related to other neuroendocrine cells in the gastrointestinal tract.

Gastrointestinal perforation early after peripheral blood stem cell transplantation for AL amyloidosis.

The morbidity and mortality of AL amyloidosis is caused by the deposition of Ig light chains as amyloid protein in vital organs. With conventional therapy median survival of patients with AL amyloidosis is 10-14 months. With high-dose chemotherapy clinical remissions of organ-specific disease have been reported. Here, we present a patient with high-risk AL amyloidosis who was given high-dose therapy and a peripheral blood stem cell transplant. Four days later she died of gastrointestinal perforation due to amyloid infiltrations.

Lung perforation by nasogastric feeding tubes.

Endotracheal misdirection of narrow bore nasogastric feeding tubes resulted in perforation of the lung, pneumothorax and hydrothorax in two intensive care patients. Both were intubated with cuffed endotracheal low pressure tubes, one patient was on respirator therapy with neuromuscular relaxation. Feeding tubes were inserted by experienced personnel with the assistance of a steel stylet without difficulties. Aspiration of fluid was misinterpreted as proof of correct positioning, the liquid being however pleural effusion and not gastric juice. Similarly auscultation of gurgling sounds in the upper epigastrium was not a reliable sign of intragastric position. Insertion of nasoenteric feeding tubes may be complicated by perforation of the upper gastrointestinal tract and lung in poorly responsive patients with cuffed endotracheal devices during neuromuscular blockage. In these patients a laryngoscope and forceps should be used to ensure free passage of the tube into the oesophagus. Röntgenographic confirmation of correct positioning of the tube immediately after insertion is mandatory.

[Intestinal lavage solution for orthograde intestinal irrigation]. / Darmspüllösung zur orthograden Darmspülung.

Gut lavage by ingestion of large volumes of electrolyte solutions has been shown to be an effective method of cleansing the colon before colonoscopy, barium enema or surgery. Absorption of water and electrolytes, which might be hazardous to patients who are unable to readily excrete an additional sodium and/or water load, is prevented by addition of non-absorbable substances to the solutions, but systematic studies are lacking. We have evaluated the influence of three solutions for gut lavage with different electrolyte composition (sodium concentration 67 mmol/l and 125 mmol/l) and addition of different non-absorbable substances (mannitol and polyethylene glycol [PEG]) on water and electrolyte homeostasis and subjective tolerance, both in healthy volunteers and in patients before endoscopy of the colon. In a randomized, blind study 6 liters of the three solutions were administered via a nasogastric tube to 6 healthy volunteers during 4 hours (i.e. 1.5 l/h). Body weight, serum concentrations of sodium, potassium and of phosphate were measured before infusion of the solution and after the last rhythmic rectal effluent. No significant changes were observed in any of the studied parameters and the incidence of side effects (nausea, abdominal cramps) was comparable. In an additional clinical double blind study, 26 patients before diagnostic colonoscopy were asked to drink 4 liters of the gut lavage solutions as quickly as possible in order to clean out the colon. The time for drinking was significantly shorter in patients using the mannitol and low sodium solution (204 +/- 70 minutes) than in patients drinking the solution with polyethylene glycol and a high sodium concentration (387 +/- 137 minutes). There was a tendency to a longer drinking period in patients ingesting the solution with polyethylene glycol and low sodium (306 +/- 106 minutes). Thus, the acceptance for solutions containing polyethylenglycol and high sodium concentration is reduced because of low palatibility. Again no influence on serum electrolyte concentrations or body weight could be observed in any patient, the spectrum of side effects was similar and the cleansing effect of all three solutions was adequate. In conclusion solutions for gut lavage containing a balanced electrolyte concentration and nonresorbable substances such as mannitol or polythylenglycol are equivalent. However, solutions containing mannitol and a low sodium concentration are better tolerated by the patients but the use of mannitol is limited because of the risk of releasing explosive gases during interventional endoscopy. To enhance the acceptance and palatibility of solutions for gut lavage containing polethylenglycol the addition of flavoured substances is recommended.

[Circulatory behavior in critically ill patients during hemodialysis]. / Kreislaufverhalten kritisch kranker Patienten während der Hämodialyse.

Haemodynamic data were obtained during haemodialysis on 21 occasions in 7 patients with septicaemia and pancreatitis, and in 5 patients with primary renal failure without septicaemia or pancreatitis. In the former group of patients there was a lowering of the blood pressure 30, 60, 90 and 120 minutes after haemodialysis had been initiated, which was significantly greater than in the later group. The drop in the blood pressure was caused by a decreased cardiac output. The pulmonary wedge pressure dropped in all patients. Peripheral resistance and heart rate did not change during the whole procedure.

[Circulatory behavior of patients with liver insufficiency]. / Kreislaufverhalten von Patienten mit Leberinsuffizienz.

Haemodynamic data were obtained in 26 patients with hepatic failure admitted to the intensive care unit of the First Department of Medicine, Vienna University. There was a significant increase in heart rate (101 vs 78 beats/min) and decreased diastolic pressure (56 vs 71 mm Hg) as compared with healthy persons. The cardiac index was elevated (5.1 vs 3.5 l/m2) and the total peripheral resistance was lowered (621 vs 1130 dyn/sec/cm-5). The systolic blood pressure was within the normal range except in 8 patients whose illness was complicated by sepsis. In those 8 patients the systolic blood pressure (86 vs 128 mm Hg), the diastolic blood pressure (42 vs 61 mm Hg) and the total peripheral resistance (434 vs 764 dyn. sec. cm-5) were all decreased as compared with patients with hepatocellular disease without sepsis. The decreased total peripheral resistance, however, was not associated with a further increase in the heart rate or stroke volume. On the contrary, in these 8 patients the left ventricular performance was lowered. The increase in cardiac output was not associated with an increase in oxygen consumption in patients without sepsis. Oxygen consumption was increased in patients with hepatocellular insufficiency and sepsis (157 ml/m2 vs 123 ml/m2) and this was accompanied by a diminished oxygen extraction rate (16% vs 26% in these 8 patients.

[Effect of high-dose prednisolone on lung fluid in patients with non-cardiogenic lung edema]. / Einfluss von hochdosiertem Prednisolon auf das Lungenwasser bei Patienten mit nichtkardiogenem Lungenödem.

The influence of high-dose prednisolone on extravascular lung water (EVLW) was studied in a randomized trial in patients with noncardiac pulmonary edema. The patients were treated every 6 hours for 48 hours with 2 g of prednisolone-hemisuccinate or placebo. In the prednisolone-group (n = 7) EVLW decreased from 16.4 +/- 6.2 before to 11.8 +/- 5.1 ml/kg after treatment (p less than 0.05). Additionally alveolar-arterial oxygen gradient (AaDO2/FiO2), pulmonary vascular resistance and heart rate decreased, while arterial oxygen tension (PaO2/FiO2) and mean arterial pressure increased (p less than 0.05). In the placebo-group (n = 7) EVLW increased slightly from 17.5 +/- 3.1 before to 19.3 +/- 10.3 ml/kg after treatment. Additionally all other parameters did not change significantly in this group. Although no statistical significant difference was found between the two groups of treatment, a decrease in EVLW was observed in all prednisolone-treated patients, whereas a pronounced increase in EVLW was found in 3 placebo-treated patients. Probably, those patients would have benefited from high-dose prednisolone treatment. High-dose prednisolone reduced EVLW and improved hemodynamics and gas exchange in patients with noncardiac pulmonary edema, whereas placebo did not achieve comparable effects. Therefore, high-dose prednisolone appears beneficial in noncardiac pulmonary edema in respect of EVLW, hemodynamics, and gas exchange.

[Monotherapy of endemic Kaposi sarcoma with long-term vincristine infusion]. / Monotherapie des endemischen Kaposi-Sarkoms mit Vincristin-Dauerinfusion.

Twelve patients with endemic Kaposi's sarcoma (KS) were entered into a clinical trial of vincristine (VCR) infusion. Patients received 5-day courses of VCR, 0.25 mg/m2/day by continuous infusion, after an 0.5 mg intravenous bolus injection. Courses were repeated every four weeks. Stabilization of disease occurred in nine patients and could be maintained for a mean of 3 months (range: 2-7 months). Complete or partial remissions were not achieved with this protocol. Complications of therapy consisted of development of moderate neurotoxicity and paralytic ileus in one patient. Two patients developed opportunistic infections while on therapy. Hematologic toxicity, nausea or emesis did not occur. Single agent VCR by infusion is well tolerated by patients with the acquired immunodeficiency syndrome (AIDS) but appears to have only limited activity in the treatment of AIDS-related KS.

OMGE--Study on prevalence of hepatitis B surface antigen in different liver diseases.

The prevalence of hepatitis B viral infection has been evaluated by means of a questionnaire. Contributions were made by 160 institutions from 39 countries and involved more than 400 collaborators. HBsAg was identified by a variety of test kits which were available at the time of the questionnaire. Data are presented for the prevalence of HBsAg in acute viral hepatitis, chronic hepatitis, cirrhosis of the liver and primary liver cancer. Wide variations in antigenaemia were identified in different countries and between the various forms of liver disease. HBsAg is positive more often in chronic hepatitis than cirrhosis. More data using sensitive tests are needed but it appears as if at least one-fifth of the world population has had a previous hepatitis B virus infection.

Hypofibrinogenemia due to fibrin formation in subserosal type eosinophilic gastroenteropathy.

A 33-year-old woman presented with abdominal pain and distention, diarrhoea and marked eosinophilia in blood and ascites. As other causes could be excluded, the subserosal type of eosinophilic gastroenteropathy was diagnosed. The low plasma fibrinogen level (less than 100 mg/100 ml) found in this patient is an as yet undescribed feature. During prednisolone therapy it increased concurrently with the fall of blood eosinophils and the relief of clinical symptoms. Interest was further directed to the ascitic fluid where not only the presence of eosinophils (74%) enveloped in fibrin yarn and of basophils (2%) but also of 24% T lymphocytes (among them 75% CD4+, 24% CD8+, 4% CD25+, less than 1% CD19+, less than 1% natural killer cells) could be demonstrated. These lymphocytes are likely to be the source for lymphokine production chemoattracting eosinophils into the intestine. In addition they seem to be involved in IgE hyperproduction, which after adequate therapy and complete resolution of the clinical symptoms, tended to decrease slowly.

Binding and processing of fibrinogen by rabbit hepatocytes.

We describe a specific fibrinogen-hepatocyte interaction. Rabbit 125I-labeled fibrinogen (125I-FGN) was incubated at 4 degrees C with suspensions of rabbit hepatocytes (approximately 1 X 10(6) cells/ml). Bound ligand was separated from free by centrifugation of cells through oil and quantitated by gamma-scintillation counting. Specific binding, determined by subtraction of nonspecific binding in the presence of 8 mM EDTA from total binding in the presence of 2 mM CaCl2, required 3 h to plateau and represented approximately 70% of total binding. Specific binding was calcium-dependent and was negligible in buffer containing 2 mM MgCl2. Half-maximal saturation occurred at approximately 30 nM 125I-FGN with approximately 480,000 molecules/cell at saturation. Dilution experiments revealed comparable affinities for labeled and unlabeled fibrinogen. Total binding was irreversible as determined by addition of excess unlabeled fibrinogen or EDTA. Specific binding of 25 nM 125I-FGN was inhibited, in a concentration-dependent fashion, by unlabeled fibrinogen or fibrinogen fragment D95 (Mr = 95,000), but not by fibrinogen fragment E or Arg-Gly-Asp-containing peptides. Unlabeled fibrinogen (3.1 microM) completely abolished specific binding, whereas greater than 80% inhibition was achieved with 10 microM fragment D95. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography of 125I-FGN bound in the presence of calcium demonstrated disappearance of A alpha chains with formation of products of Mr greater than 200,000; EDTA or unlabeled fibrinogen prevented fibrinogen processing. These data describe a unique fibrinogen-hepatocyte interaction which differs considerably from the platelet-fibrinogen interaction, especially with regard to the processing of the fibrinogen molecule.