Biomedical subjects
Publications and source records attributed to K Ziegler.
The genes encoding subunits A and B of V-ATPase in Cyanidium caldarium were cloned and sequenced. While the gene encoding subunit A is not interrupted by introns, the gene encoding subunit B contains seven introns ranging from 36 to 60 nucleotides.
The hydrophobic cyclohexapeptide cyclo(Phe-Thr-Lys-Trp-Phe-DPro) (008), an analog of somatostatin with retro sequence, was previously shown to competitively inhibit the uptake of cholate and taurocholate into isolated rat liver cells. Conversely, the competitive uptake inhibition of 008 into isolated rat hepatocytes by bile acids confirmed the observation of common binding and transport sites by bile acids and cyclosomatostatin. Furthermore the transport characteristics of 008 uptake revealed a significant and rapid binding to cell membranes. In this context it was of special interest to investigate the specificity of the binding component since specific binding of the substrate to membrane proteins could be responsible for the low Km of 008-transport. Therefore, the cyclohexapeptide 008 could be used as the ligand in affinity chromatography in order to isolate such binding proteins. The gel matrix used did not interact non-specifically with octylglucoside-solubilized proteins from isolated rat liver plasma membranes. In affinity chromatography of octylglucoside-solubilized plasma membranes, two dominant proteins with apparent molecular masses of 60 and 58 kDa bound specifically to the 008 ligand. When used as ligands in affinity chromatography, these membrane-associated 60 and 58 kDa proteins bound exclusively to aromatic cyclopeptides, e.g. cyclosomatostatin 008, but not to linear peptides or taurocholate derivatives. The amino acid sequences of tryptic digests of the 008-affinity-purified 58 kDa protein were identical to the sequence of a microsomal pI6.1 carboxylesterase. Immunofluorescence of intact hepatocytes showed that this xenobiotic metabolizing enzyme is also located in sinusoidal rat liver plasma membranes and could therefore account for the extensive and specific binding of the cyclosomatostatin to sinusoidal plasma membranes of rat liver.
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A 22 year old male with infectious mononucleosis, who stated a history of trauma to his left thoracic wall, developed an acute abdomen with symptoms of shock. A ruptured spleen was suspected. This working diagnosis, which was supported by ultrasound led to a splenectomy the initial diagnosis. The case report of this rare complication--due to the increased vulnerability of the spleen capsula in infectious mononucleosis--is highlighting the possible danger of splenic rupture. As of today, emergency splenectomy without any compromise ist still the safest state of the art therapy.
Certain peptide drugs, such as the linear hydrophobic renin-inhibitor EMD 51921, are rapidly eliminated via the bile. At the sinosoidal membrane of liver cells EMD 51921 is taken up via a sodium-independent carrier-mediated mechanism, competing for the uptake of bile acids. Until now, the mechanisms of biliary excretion of EMD 51921 were unknown. In this study we describe an ATP-dependent transport system for the enzymatically and metabolically stable hydrophobic linear renin-inhibiting peptide EMD 51921. The ATP-dependent uptake into the osmotic reactive intravesicular space is saturable (Km 12 microM, Vmax 663 pmol/min per mg protein), temperature dependent and specifically requires ATP. Transport is inhibited by vanadate but not by ouabain, EGTA or NaN3, and does not function in basolateral plasma membrane vesicles. Transport is not altered in canalicular membrane vesicles isolated from Tr- rats lacking the canalicular ATP-dependent transport of cysteinyl leukotrienes and related anions. Transport is inhibited by taurocholate, a typical substrate of the canalicular ATP-dependent bile acid transporter, but also by vincristine and daunomycin, substrates of P-glycoproteins. EMD 51921, however, only inhibits the uptake of taurocholate, whereas the transport of daunomycin is not influenced. Taurocholate and EMD 51921 are mutually non- or un-competitive transport inhibitors. Incubation of rat liver canalicular membranes with micromolar concentrations of EMD 51921 resulted in a 1.8-2.5-fold increase in the rate of ATP-hydrolysis. In contrast, ATP-hydrolysis was not affected by fragments of the peptide that are not transported in an ATP-dependent manner. The apparent Km value (EMD) for ATP-hydrolysis is 68 microM. Vmax is 0.032 U/mg protein. ATPase activity is pH dependent. Stimulation of ATP-hydrolysis is inhibited by vanadate, NEM, hydroxymercuribenzoate and ascorbate, but is not affected by ouabain, EGTA or NaN3. EMD 51921 does not stimulate the ATPase activity of the Na+/K(+)-ATPase isolated from kidney medulla. The EMD-stimulatable ATPase seems to be distinct from the glutathione-S-conjugate stimulatable ATPase and the mdr 1a/b gene products and differs in its characteristics from that of the canalicular ecto-ATPase.
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Neuroendocrine tumors (NETs) of the foregut type are frequently smaller than 2 cm in diameter and mainly located in the pancreas or the gastric and duodenal wall. Conventional cross-sectional imaging techniques, such as transabdominal ultrasonography (US), computed tomography (CT), and magnetic resonance imaging (MRI) are limited by their inability to detect small tumors and especially those located within the gastrointestinal wall. Endoscopic ultrasonography (EUS) allows detailed visualization of the whole pancreas and almost all parts of the gastric and duodenal walls. Therefore, EUS is an important diagnostic tool for the preoperative localization of NETs of the foregut type. Several studies performed in a retrospective manner, as well as two studies performed in a prospective manner, indicate a clear superiority of EUS as compared to CT, US, MRI, and also angiography in detecting NETs of the foregut type. Somatostatin-receptor scintigraphy (SRS) also detects NETs of the foregut type in a very high percentage of cases, and the combination of EUS and SRS appears to increase the sensitivity even more. Thus EUS and also SRS should be employed early if NETs of the foregut type are suspected. Conventional imaging procedures such as US, CT, and MRI should be mainly used to exclude local and distant metastases.
The hydrophilic HMG-CoA reductase inhibitor pravastatin is not taken up via a carrier-mediated system into Hep G2 cells. Therefore, Hep G2 cells are not a good model for human hepatocytes with respect to elucidation of the effect of hydrophilic HMG-CoA reductase inhibitors. Sulfobromophthalein (BSP), on the other hand, is taken up into Hep G2 cells by carrier systems with Km and Vmax values almost identical to freshly isolated hepatocytes. These results indicate that the hepatocellular BSP transporting proteins expressed in Hep G2 cells (bilitranslocase and BSP/bilirubin binding protein) are not involved in the hepatocellular uptake of pravastatin. In contrast to the hepatocellular sodium-taurocholate cotransporter, which is not functioning in Hep G2 cells, we found a saturable transport of cholate with Km and Vmax values identical to those in cultured rat hepatocytes in the presence of sodium.
The bile acids cholate and taurocholate on the one hand and the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, on the other hand, display mutually competitive transport inhibition into isolated rat hepatocytes. This indicates a common transport system for bile acids and cyclosomatostatins in sinusoidal rat liver plasma membranes. In order to identify and isolate common binding and/or transport proteins for bile acids and the cyclopeptides by affinity chromatography, the bile acid derivative 4'-amino-7-benzamidotaurocholate (ABATC) and the cyclosomatostatin-analog 008 were attached to a gel matrix. Two methods were used to prepare integral membrane proteins: (1) alkaline EDTA extraction and (2) Triton X-114 phase separation. Octyl glycoside solubilized, alkaline EDTA-extracted integral basolateral membrane proteins with apparent molecular masses of 52 and 48 kDa bound specifically to the ABATC affinity matrix. Two-phase Triton X-114 separated integral membrane proteins of the same molecular masses bound specifically to the cyclosomatostatin ligand. The 48 kDa ABATC and 008 binding protein was shown to be present in the basolateral plasma membrane fraction and in the microsomal fraction. The isolated 52 kDa ABATC binding protein was localized only in basolateral plasma membranes and could not be found in isolated microsomes.
In order to localise neuroendocrine tumours of the foregut type (that is, of the stomach, duodenum, and pancreas), 18 patients were studied prospectively by endoscopic ultrasonography, computed tomography, transabdominal ultrasonography, magnetic resonance imaging, and somatostatin receptor scintigraphy. These 18 patients had a total of 25 primary tumour lesions which were verified histologically in tissue obtained by surgery or by ultrasound or endoscopy guided biopsy. Tumours were found in the stomach (n = 1), duodenum (n = 6), pancreas (n = 17), and liver (n = 1). Endoscopic ultrasonography had the highest sensitivity for tumour detection, followed by somatostatin receptor scintigraphy, computed tomography, transabdominal ultrasonography, and magnetic resonance imaging (88%, 52%, 36%, 32%, and 24% respectively). Endoscopic ultrasonography was especially sensitive in tumours smaller than 2 cm in diameter (88% v somatostatin receptor scintigraphy 35%; computed tomography 12%; transabdominal ultrasonography 6%; and magnetic resonance imaging 0%). Of 17 tumours located in the pancreas, endoscopic ultrasonography showed a sensitivity of 94% (somatostatin receptor scintigraphy 47%; computed tomography 47%; transabdominal ultrasonography 41%; and magnetic resonance imaging 29%). Of eight extrapancreatic tumours, six were identified by endoscopic ultrasonography, five by somatostatin receptor scintigraphy, and only one by computed tomography, transabdominal ultrasonography, and magnetic resonance imaging. One neuroendocrine tumour that was not detected by endoscopic ultrasonography was correctly identified by somatostatin receptor scintigraphy. Endoscopic ultrasound allowed correct determination of the tumour size and tumour spread into parapancreatic structures, especially the large vessels (T stage), in all 14 patients operated upon. The lymph node stage (N stage) was correctly determined in 10 of these 14 patients. In summary, endoscopic ultrasonography and somatostatin receptor scintigraphy were the most sensitive imaging methods for the localisation of these tumours and should be used as early diagnostic procedures to accurately stage neuroendocrine tumours of the foregut type.
The uptake of the bile acids cholate (CHA) and taurocholate (TCHA) was studied in isolated hepatocytes from rainbow trout (Oncorhynchus mykiss). Both CHA and TCHA were taken up in a concentration- and temperature-dependent manner with optimum temperature at 15 degrees C and a strikingly efficient uptake even at low temperatures (0-5 degrees C). The total uptake was a combination of a saturable [Michaelis-Menten constant (Km) for CHA, 20 microM; Km for TCHA, 19 microM] and a nonsaturable component. The maximal uptake rate of the saturable component was 416 and 805 pmol.mg protein-1.min-1 for CHA and TCHA, respectively. The uptake of both bile acids was shown to be energy dependent, since it was inhibited by the metabolic inhibitors antimycin A, oligomycin and carbonyl cyanide m-chlorophenylhydrazone. The uptake was clearly Na+ independent, since isosmotic replacement of extracellular Na+ by Li+, choline, or K+ did not inhibit the uptake. Furthermore, it seemed to be independent of the presence of extracellular Cl-, since it was not inhibited by replacement of Cl- with sodium gluconate. On the whole, our results show that the hepatocellular uptake of bile acids in rainbow trout is mediated by a Na(+)-independent carrier system, with characteristics resembling the corresponding transport component in mammalian hepatocytes, but with high efficiency even at low temperatures.
Pravastatin and pravastatin-lactone are not taken up into extrahepatic cells such as fibroblasts, or hepatoma cells such as AS-30D ascites hepatoma cells or FAO cells. In contrast, pravastatin is taken up into isolated rat hepatocytes by a carrier mediated, saturable, temperature-dependent and energy-dependent mechanism. The kinetic parameters for the saturable uptake are Km 27 microM, Vmax 537 pmol/mg per min. The permeability coefficients were determined to be 9.829 x 10(-7) cm/s at 4 degrees C, 1.76 x 10(-6) cm/s at 7 degrees C, 3.85 x 10(-6) cm/s at 17 degrees C and 5.82 x 10(-6) cm/s at 37 degrees C. The activation energy is 60 kJ/mol for 100 microM pravastatin at 37 degrees C. The Q10 values are between 1.7 and 2.8. In the presence of metabolic inhibitors and in the absence of oxygen, transport is inhibited. Uptake of pravastatin is not dependent on an extracellular to intracellular sodium-gradient. Replacement of chloride by sulfate, nitrate, gluconate or thiocyanate significantly inhibits the uptake of pravastatin. Uptake is competitively inhibited by cholate and taurocholate in the presence and absence of sodium. Pravastatin, however, competitively inhibits the uptake of cholate and taurocholate only in the absence of sodium. In addition, pravastatin-lactone enters liver cells via an energy-dependent, carrier-mediated uptake system. For the saturable energy-dependent part of the hepatocellular uptake a Km value of 9 microM and a Vmax value of 621 pmol/mg per min was determined. The permeability coefficient of pravastatin-lactone uptake is calculated to be 5.41 x 10(-6) cm/s at 37 degrees C. The uptake of pravastatin-lactone is competitively-noncompetitively inhibited by pravastatin and by lovastatin and vice versa. These results indicate that the hepatoselectivity of pravastatin is due to its carrier-mediated uptake into rat hepatocytes via a sodium-independent bile acid carrier. Pravastatin-lactone resembles pravastatin-sodium in its hepatoselectivity.
The hepatic uptake of a hydrophilic, cationic linear peptide with renin inhibitory activity [5(4-amino-piperidyl-1-carbonyl)-L-2,6[3H]phenyl-alanyl-beta-alanyl-(4S- amino-3S-hydroxy-5-cyclohexyl)-pentan-carbonyl-L-isoleucyl-amin ome thyl-4-amino-2-methyl-pyrimidine-citrat] (code number EMD 56133; EMD, E. Merck, Darmstadt) was investigated in isolated rat hepatocytes. EMD 56133 was taken up by isolated rat liver cells in a time-, concentration-, energy- and temperature-dependent manner. The uptake was a combination of diffusion and a carrier-mediated process. EMD 56133 was accumulated 4.5-fold in liver cells. Eighty-three per cent of the accumulated peptide was found in the cytosol, not bound to membrane proteins. Seventeen per cent was associated with membrane proteins after cell fractionation and centrifugation at 100,000 g. The permeability coefficient of the non-saturable uptake of EMD 56133 was P = 1.973 x 10(-6) cm/sec. The kinetic constants for the carrier-mediated transport are Km = 92 microM and Vmax = 128 pmol/mg x min. Various substrate analogs inhibited the uptake of EMD 56133. AS-30D ascites hepatoma cells and Reuber hepatoma cells did not accumulate EMD 56133. The absence of oxygen or a decreased cellular ATP content blocked the hepatocellular uptake of the renin inhibitor. Temperatures above 20 degrees increased the transport; the activation energy was determined to be Aapp = 41 kJ/mol. The apparently active uptake of EMD 56133 was not sodium dependent. In contrast, the membrane potential might be a driving force for the transport of the positively charged EMD 56133.
To define the endogenous transport system responsible for the hepatocellular uptake of hydrophilic linear peptides, interactions between the cationic renin-inhibitor, [5(4-amino-piperidyl-1-carbonyl-L-2,6[3H]phenyl-alanyl-beta-alanyl(4S- amino-3S-hydroxy-5-cyclo-hexyl)-pentan-carbonyl-L-isoleucyl-ami nom ethyl-4-amino-2-methyl-pyrimidine-citrat] (code number EMD 56133; EMD, E. Merck, Darmstadt) and substrates of endogenous transport systems of liver cells were studied in isolated rat hepatocytes. EMD 56133 competitively inhibited the uptake of ouabain (Ki = 75 microM) and vice versa (Ki = 200 microM). In contrast, the sodium-dependent as well as the sodium-independent uptake of cholate and the total uptake of taurocholate were non-competitively blocked, whereas EMD 56133 decreased the uptake of the cyclosomatostatin 008 in an uncompetitive manner. EMD 56133 did not interfere with transport systems for monovalent organic cations, amino acids and long chain fatty acids. The uptake of rifampicin, however, was increased in the presence of EMD 56133. The transport of EMD 56133 was non-competitively inhibited by cholate (Ki = 126 microM) and taurocholate (Ki = 44 microM), and uncompetitively inhibited by the linear peptide EMD 51921. In contrast, the uncharged compound ouabain (Ki = 200 microM) and the bivalent organic cation d-tubocurarine (Ki = 370 microM) competitively inhibited the uptake of the renin inhibitor. Several substrates of other endogenous transport systems (e.g. bilirubin, cyclopeptides, monovalent cations, dipeptides, amino acids, fatty acids, hexoses) did not interfere with the transport of EMD 56133. Our results suggest that transport systems for bivalent organic cations or uncharged compounds (ouabain) are able to eliminate the linear hydrophilic peptide tested.
Two proteins, in previous work labeled by affinity markers derived from taurocholic acid, were purified and partially sequenced. Antibodies were raised against purified proteins, and cross-reactions were carefully checked. The influence of these antibodies upon taurocholic acid import into vesicles from rat liver plasma membranes was measured, and showed a distinct inhibition of transport in the case of the 54 kD protein.
From 1986 to 1990 a prospective comparative study was undertaken to compare the relative accuracy of computed tomography, endogastric ultrasonography, and intraoperative surgical assessment in evaluating the depth of invasion (T category) and involvement of lymph nodes (N category) of patients with gastric carcinoma. One hundred and eight consecutive patients, who were treated by total gastrectomy and previously evaluated with computed tomography, endogastric ultrasonography, and intraoperative surgical assessment, entered the study. Results (T and N category) were compared with those of histopathological staging (pT and pN category). T categories were correctly staged in 43% of cases with computed tomography, 86% with endogastric ultrasonography, and 56% with intraoperative surgical assessment. Computed tomography scanning correctly staged 51% of all N1 and N2 lymph nodes compared with 74% for endogastric ultrasonography and 54% for intraoperative surgical assessment. In general, computed tomography was more accurate for advanced stages of cancer and showed a tendency to overstage the T category and understage N category of gastric tumours. By contrast, endogastric ultrasonography was equally accurate for all T categories and showed an understaging for N categories. Intraoperative surgical assessment overstaged early T stages, understaged T4 tumours, and was equally accurate for all grades of N categories. Computed tomography scanning and intraoperative surgical assessment of T and N categories were of little value in staging of gastric carcinoma. Endogastric ultrasonography is more accurate than computed tomography scanning and intraoperative surgical assessment. Therefore endogastric ultrasonography should be introduced in the preoperative assessment of patients with gastric carcinoma.
A 35 year old women patient with Crohn's disease and previous multiple abdominal operations presented with a calcified stone of 12 mm diameter in the cystic duct giving rise to cholecystitis. The surgeons declined to operate because of extensive intra-abdominal adhesions caused by multiple intestinal resections and chronic enterocutaneous fistulas. It was possible to fragment the stone in three lithotripsy sessions. The fragments were excreted spontaneously through the ductus choledochus and the cholecystitis was cured by antibiotic treatment. The patient remained symptom free after 12 months.
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