Cloning of a new Kunitz-type protease inhibitor with a putative transmembrane domain overexpressed in pancreatic cancer.
Muller-Pillasch F. Wallrapp C. Bartels K. Varga G. Friess H. Buchler M. Adler G. Gress TM.
Department of Internal Medicine I, University of Ulm, Germany. firstname.lastname@example.org
In a previous large scale screen for differentially expressed genes in pancreatic cancer, we identified a gene highly overexpressed in cancer encoding a novel putative transmembrane protein with two Kunitz-type serine protease inhibitor domains. The identified gene named kop (Kunitz domain containing protein overexpressed in pancreatic cancer) was assigned to chromosome 19 in the region 19q13.1. Kop was detected at high levels in pancreatic cancer cell lines and was overexpressed in pancreatic cancer tissues as compared to both, normal pancreas and chronic pancreatitis tissues. Being a member of the Kunitz-type serine protease inhibitor family, this new gene may participate in tumour cell invasion and metastasis and in the development of the marked desmoplastic reaction typical for human pancreatic cancer tissues. In this context, the fact that kop has a putative transmembrane domain may have functional implications of particular interest.
Correlation of hepatocyte growth factor-induced proliferation and calcium-activated potassium current in human gastric cancer cells.
Liu SI. Chi CW. Lui WY. Mok KT. Wu CW. Wu SN.
Department of Surgery, Veterans General Hospital-Kaohsiung, Taiwan.
Hepatocyte growth factor (HGF) has been found to stimulate proliferation and migration of human gastric carcinoma cells. Whether the HGF-induced responses are correlated with the expressed level of HGF receptors or the changes of ionic currents is not clear. The present study investigated the effects of HGF on the proliferation and ionic currents of two human gastric adenocarcinoma cell lines, which were found to express different amounts of HGF receptor. Results showed that HGF induced a dose-dependent growth stimulation and accelerated cell cycle progression in SC-M1 cells. In patch clamp study, HGF treatment induced an outward K+ current and increased the slope conductance at -80 mV from 110+/-15 pS/pF to 207+/-15 pS/pF. The HGF-induced K+ current was abolished when tetraethylammonium chloride was added in bathing solution or a low Ca2+ solution was included in the recording pipette. Furthermore, HGF (10 ng/ml) induced an oscillatory Ca2+-activated K+ current with a lag period of 5+/-3 min in SC-M1 cells. In contrast, HGF did not induce mitogenesis, cell cycle progression and changes in ionic currents in KATO-III cells, although this cell line expressed a higher level of HGF receptors than SC-M1 cells did. These findings provide evidence that the activity of Ca2+-activated K+ channel may be involved in the HGF-induced cell proliferation in human gastric cancer cells, but it did not correlate with the density of HGF receptors.
Hexokinase isoenzymes in normal and cirrhotic human liver: suppression of glucokinase in cirrhosis.
Lowes W. Walker M. Alberti KG. Agius L.
Department of Medicine, Human Diabetes and Metabolism Research Centre, The University of Newcastle upon Tyne, UK.
The activities of hexokinase isoenzymes I-IV (EC 22.214.171.124) and of N-acetylglucosamine kinase (EC 126.96.36.199) were determined in normal human liver and in alcoholic liver disease and primary biliary cirrhosis after FPLC fractionation of high-speed supernatants on Mono-Q with a linear NaCl gradient. In control human liver the hexokinase activities were: I, 3.6; II, 0.7; III, 3.5, IV, 4.8 (mUnits/mg supernatant protein). The activity of N-acetylglucosamine kinase was 8 mU/mg of protein. In alcoholic liver disease and primary biliary cirrhosis, the activity of hexokinase IV (glucokinase) was suppressed to less than 10% of control activity and the activity of hexokinase I was increased 3-fold. The activity of hexokinase II was increased approximately 7-fold in alcoholic liver disease. The activities of hexokinase III and N-acetylglucosamine kinase were unchanged in cirrhosis. Hexokinase III showed 50% substrate inhibition at 100 mM glucose as compared with 0.5mM glucose. The high activity of hexokinase III in human liver (approximately 50% of the low-Km activity and 70% of glucokinase activity) results in a significant underestimation of glucokinase activity as determined by the conventional spectrometric assay while the activity of N-acetylglucosamine kinase may contribute to an overestimation of glucokinase activity in the radiochemical assay. Furthermore glucokinase is dramatically suppressed in liver disease, which although partly compensated for by the increase in hexokinase I (and II), accounts in part for the well-known glucose intolerance of liver cirrhosis.
Protein lipid interaction in bile: effects of biliary proteins on the stability of cholesterol-lecithin vesicles.
Luk AS. Kaler EW. Lee SP.
Department of Chemical Engineering, Center for Molecular and Engineering Thermodynamics, University of Delaware, Newark, DE, USA.
The nucleation of cholesterol crystals is an obligatory precursor to cholesterol gallstone formation. Nucleation, in turn, is believed to be preceded by aggregation and fusion of cholesterol-rich vesicles. We have investigated the effects of two putative pro-nucleating proteins, a concanavalin A-binding protein fraction and a calcium-binding protein, on the stability of sonicated small unilamellar cholesterol-lecithin vesicles. Vesicle aggregation is followed by monitoring absorbance, and upon addition of the concanavalin A-binding protein fraction the absorbance of a vesicle dispersion increases continuously with time. Vesicle fusion is probed by a fluorescence contents-mixing assay. Vesicles apparently fuse slowly after the addition of the concanavalin A-binding protein, although inner filter effects confound the quantitative measurement of fusion rates. The rates of change of absorbance and fluorescence increase with the concentration of the protein, and the second-order dimerization rate constant increases with both the protein concentration and the cholesterol content of the vesicles. On the other hand, the calcium-binding protein has no effect on the stability of the vesicle dispersion. This protein may therefore affect cholesterol crystal formation not by promoting the nucleation process, but by enhancing crystal growth and packaging. Our results demonstrate that biliary proteins can destabilize lipid vesicles and that different proteins play different roles in the mechanism of cholesterol gallstone formation.
Hepatic responses to inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase: a comparison of atorvastatin and simvastatin.
Bergstrom JD. Bostedor RG. Rew DJ. Geissler WM. Wright SD. Chao YS.
Department of Biochemistry, Merck Research Laboratories, Rahway, NJ 07065-0900, USA. email@example.com
We have compared the cellular responses to simvastatin (Simva) and atorvastatin (Atorva), two potent HMG-CoA reductase inhibitors. The two drugs exhibited similar IC50's for inhibition of either rat or human reductase, and single oral dosing in rats showed the compounds to be nearly equipotent at inhibiting hepatic cholesterol synthesis. Treatment of rats with Simva or Atorva in the feed for four days yielded comparable inductions of hepatic reductase activity and reductase protein. For example, 0.05% Simva induced reductase activity 27.3 +/- 9.1 fold and 0.05% Atorva induced activity 26.9 +/- 4.7 fold. This adaptive response was also studied in HepG2 cells, a human hepatoblastoma line, cultured for 24 h in delipidated serum and then for an additional 24 h with Simva or Atorva. Over a broad range (10 nM-10 microM), both drugs caused similar inductions of reductase activity, reductase protein, and reductase mRNA. Under all conditions, the drugs induced similar changes in the ratio of mRNA/protein suggesting that Simva and Atorva have similar effects on both transcriptional and post-transcriptional regulatory machinery. Moreover, reductase in cells treated with Simva or Atorva for 22 h responded similarly to subsequent challenge with 25-hydroxycholesterol. Finally, we measured the ability of the two reductase inhibitors to reduce ApoB secretion by HepG2 cells. Simva and Atorva at 0.5 microM inhibited ApoB secretion nearly identically, 38% and 42% respectively. We conclude that these two drugs induce similar adaptive responses in cells and that their actions are qualitatively and mechanistically identical. Human studies have shown that plasma is cleared of Atorva much more slowly than it is of Simva. The large pharmacokinetic difference in man, rather than some difference in mechanism, is the most likely explanation for the finding that the equipotent dose ratio for cholesterol lowering in humans of Simva to Atorva is about 2/1.
Lysophosphatidylcholine increases apolipoprotein B secretion by enhancing lipid synthesis and decreasing its intracellular degradation in HepG2 cells.
Zhou Z. Luchoomun J. Bakillah A. Hussain MM.
Department of Pathology, The Allegheny University of the Health Sciences, MCP Hahnemann School of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA.
Free fatty acids and lysophosphatidylcholine (lysoPC) are the major lipids bound to human plasma albumin. The effects of fatty acids on the hepatic production of Apolipoprotein B (apo B) have been studied but those of lysoPC have not. In HepG2 cells, lysoPC increased apo B secretion in different experiments by 50-120%, but did not affect the flotation properties of secreted lipoproteins. LysoPC affected neither the cellular protein levels nor apo A-I secretion suggesting that its effect was specific to apo B. Apo B secretion was maximum after incubating cells for 6 h with 0.2 mM lysoPC as equimolar fatty acid free bovine serum albumin (BSA) complexes. LysoPC was metabolized by cells and its fatty acids were used for the synthesis of phosphatidylcholine and triglycerides (TG). Experiments were performed to understand the mechanism of lysoPC action. LysoPC increased the incorporation of 3H-glycerol into newly synthesized cellular (3-fold) and secreted (4-fold) triglycerides, and increased the synthesis (40%) and secretion (4-fold) of phospholipids. LysoPC did not affect apo B synthesis, but inhibited the intracellular degradation of apo B and increased its secretion. Triacsin C (5 microM), an inhibitor of long chain acyl-CoA synthase, completely inhibited the induction of lipid synthesis and abolished the effect of lysoPC on apo B secretion. These studies indicated that lysoPC increased apo B secretion by inducing lipid synthesis; newly synthesized lipids probably protected apo B from intracellular degradation and enhanced secretion. These studies are consistent with the hypothesis that physiologic concentrations of lysoPC can be an important modulator for hepatic apo B secretion.
Appearance of cross linked proteins in human atheroma and rat pre-fibrotic liver detected by a new monoclonal antibody.
Itabe H. Jimi S. Kamimura S. Suzuki K. Uesugi N. Imanaka T. Shijo H. Takano T.
Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan. firstname.lastname@example.org
A new monoclonal antibody against malondialdehyde (MDA)-treated low density lipoprotein (LDL) was raised using homogenate of human atheroma as immunogen. This antibody, DLH2, was obtained by selecting the clones which did not react to native LDL but did react to copper-induced oxidized LDL (OxLDL). DLH2 showed a greater reactivity to MDA-LDL than to OxLDL. When LDL was treated with various aldehyde containing reagents, treatment of LDL with glutaraldehyde or MDA greatly increased the reactivity to the antibody, while LDL treated with 2,4-hexadienal or 4-hydroxynonenal was not reactive. Among many proteins tested, high density lipoprotein, bovine serum albumin and hemoglobin showed significant reactivity to DLH2 after they were treated with MDA or glutaraldehyde. When low density and high density lipoproteins treated with MDA were subjected to immunoblot analysis, newly formed products larger than the original apolipoproteins were detected with the antibody, suggesting that this antibody recognizes aggregated proteins with divalent short chain cross linkers. The antigenic materials were shown by immunohistochemical analysis to be present in foamy macrophages in human atheromatous lesions. DLH2 antigen did not colocalize either with apolipoprotein B. Furthermore, we found a massive accumulation of the antigenic material in Kupffer cells in the liver of rats treated with alcohol and carbonyl iron, a model of hepatic fibrosis due to oxidative stress. These results suggest the presence of cross linked proteins in damaged tissues.
Constitutive and interferon-gamma-induced expression of the human proteasome subunit multicatalytic endopeptidase complex-like 1.
Foss GS. Larsen F. Solheim J. Prydz H.
Biotechnology Centre of Oslo, University of Oslo, Norway.
Proteasomes generate peptides from intracellular endogenous and viral proteins for presentation by MHC class I molecules. During viral infection, interferon-gamma (IFN-gamma) acts as a cytokine altering the catalytic specificity of proteasomes by inducing the synthesis of the three proteasome subunits, low molecular weight protein (LMP) 2, LMP7 and multicatalytic endopeptidase complex-like 1 (MECL1). LMP2 and LMP7 have been shown to favour the presentation of certain antigenic peptides. These subunits are constitutively expressed in cell lines related to the immune system and IFN-gamma-inducible in other cell lines. Less is known about MECL1. To reveal the extent of constitutive and IFN-gamma-induced expression of MECL1, we studied MECL1 in different cell lines by Northern and Western blotting. The two B cell lines IM9 and Reh showed high constitutive expression of MECL1, only slightly induced by IFN-gamma stimulation. The B cell line Daudi and the monocyte cell line THP-1 expressed MECL1 constitutively at an intermediate level. The MECL1 protein level in the THP-1 cells increased markedly in response to IFN-gamma. In cells unrelated to the immune system, a very low constitutive expression of MECL1 was detected, highly inducible by IFN-gamma. These results indicate that, similar to LMP2 and LMP7, MECL1 is constitutively expressed at high levels only in certain cell lines and can be induced by IFN-gamma in other cell lines. The differential expression of MECL1 may be of importance for which antigenic peptides are presented by different cells as well as by the same cells at different IFN-gamma levels.
Evidence that photodynamic stress kills Zellweger fibroblasts by a nonapoptotic mechanism.
Spisni E. Cavazzoni M. Griffoni C. Calzolari E. Tomasi V.
Department of Experimental Biology, University of Bologna, Italy.
Zellweger fibroblasts, which are devoid of peroxisomes and fail to synthesize plasmalogens, are very sensitive to the killing effect triggered by UV-activated 12-(1-pyrene) dodecanoic acid (P12). Although in some studied performed, it is assumed that reactive oxygen species (ROS) may damage plasma membrane causing necrosis, other studies suggest that ROS are involved in apoptotic cell death induced by a wide variety of stimuli. Analysing the P12 dose-response in Zellweger fibroblasts, we observed that at high doses (1-2 microM), more than 75% of the cells died after 24 h. This behaviour suggested that, at high doses, P12 kills the cells by unspecific lytic mechanisms or by necrosis, while at low doses (0.1-0.5 microM), an apoptotic mechanism could be involved. Cytofluorimetric analysis of Zellweger fibroblasts-treated with activated P12 (0.5 microM) did not show morphological modifications typical of apoptotic cell death. This was supported by comparative staining of fibroblast nuclei, DNA gel electrophoresis and identification of poly(ADP-ribose) polymerase (PARP) cleavage and Bcl-2 expression, assayed by Western blots. Thus, our results, while confirming the importance of plasmalogens in the protection against ROS, establish that apoptosis is not involved in photodynamic death induced by activated P12. Therefore, we can expect that in gene transfer experiments, the rescue of Zellweger cells will be dependent only on the correction of peroxisomal biogenesis.
Studies on the regulation of CTP:phosphocholine cytidylyltransferase using permeabilized HEP G2 cells: evidence that both active and inactive enzyme are membrane-bound.
Weinhold PA. Barrett D.
Veterans Affairs Medical Center and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA. email@example.com
To obtain more insight into the mechanisms regulating CTP:phosphocholine cytidylyltransferase (CT), we determined the effect of oleate treatment on the rate of CT release from permeabilized Hep G2 cells and the distribution of the CT remaining in the permeabilized cells. When we permeabilized untreated cells in pH 7.5 buffer containing 0.15 M KCl, the rate of CT release was much slower than the release of lactate dehydrogenase. Oleate treatment caused a further decrease in CT release from cells. In untreated cells, 70-80% of the CT remaining in cells 10 min after permeabilization was recovered as soluble CT. Oleate treatment increased the amount of bound CT but over 50% of the CT in cells 10 min after permeabilization was recovered as soluble CT. In both control and oleate-treated cells, the increase in CT release with time correlated with a decrease in the amount of CT recovered from permeabilized cells as soluble CT. These results suggested that CT existed in a form that was not immediately available for release from permeabilized cells, but was recovered in the soluble fraction after cell disruption. When cells were permeabilized in 10 mM imidazole-20% glycerol-5 mM Mg2+ pH 6.5, over 80% of CT in control and over 90% of CT in oleate-treated cells was recovered bound to the particulate fraction. Essentially no CT was released from the cells. The recovery of CT in the particulate fraction required Mg2+ to be present when permeabilization was initiated. The addition of Mg2+, after cells were disrupted, did not increase CT in the particulate fraction. In untreated cells, 50% of bound CT was active. Oleate treatment increased the amount of active CT in the particulate fraction to over 70% of total. About 50% of particulate CT in untreated cells but only 15% in oleate-treated cells was extracted with 0.15 M KCl. Inactive CT was preferentially extracted by KCl. The bound CT was recovered in isolated nuclei. Overall, the results suggested that both inactive and active CT are bound to nuclear membranes, and that the activation of CT involves conversion of CT loosely bound to membrane to a form more tightly bound to membranes perhaps by hydrophobic interaction with phospholipids. This model does not involve translocation from a soluble pool.
Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers.
Toncheva V. Wolfert MA. Dash PR. Oupicky D. Ulbrich K. Seymour LW. Schacht EH.
Biomaterials and Polymers Research Group, University of Gent, Gent B9000, Belgium.
Complexes formed between DNA and cationic polymers are attracting increasing attention as novel synthetic vectors for delivery of genes. We are trying to improve biological properties of such complexes by oriented self-assembly of DNA with cationic-hydrophilic block copolymers, designed to enshroud the complex within a protective hydrophilic polymer corona. Poly(L-lysine) (pLL) grafted with range of hydrophilic polymer blocks, including poly(ethylene glycol) (pEG), dextran and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA), shows efficient binding to DNA and mediates particle self-assembly and inhibition of ethidium bromide/DNA fluorescence. The complexes formed are discrete and typically about 100 nm diameter, viewed by atomic force microscopy. Surface charges are slightly shielded by the presence of the hydrophilic polymer, and complexes generally show decreased cytotoxicity compared with simple pLL/DNA complexes. pEG-containing complexes show increased transfection activity against cells in vitro. Complexes formed with all polymer conjugates showed greater aqueous solubility than simple pLL/DNA complexes, particularly at charge neutrality. These materials appear to have the ability to regulate the physicochemical and biological properties of polycation/DNA complexes, and should find important applications in packaging of nucleic acids for specific biological applications.
Lack of 2,5-oligoadenylate-dependent RNase expression in the human hepatoma cell line HepG2.
Tnani M. Bayard BA.
UMR 5539 Centre National de la Recherche Scientifique, Universite de Montpellier II, France.
2',5'-adenylate oligonucleotide (2-5A)-dependent RNase and 2-5A-synthetase are two enzymes of the 2-5A system strongly implicated in the basal control of RNA decay of both interferon-treated and untreated cells. RNase is activated by a 2-5A produced by 2-5A-synthetase, both enzymes being overexpressed by type I-interferon (alpha/beta). We described here for the first time a cell line completely deficient in RNase and its mRNA, while p69 2-5A-synthetase was normally interferon alpha/beta-induced. The complete absence of this RNase in human hepatoma cells (HepG2) was shown using three different methods based on the binding of a [32P]-labeled 2-5A probe of high specific activity to its binding site. Negative Western blotting assay with a specific monoclonal antibody correlated the previous findings. RNase-specific mRNA was not detectable even after treatment of cells with 1000 units/ml of interferon alpha/beta. This is not due to a mutation of the gene because an intronless genomic DNA sequence encoding 2-5A-binding site was cloned and expressed. It is likely that the expression of 2-5A-dependent RNase was impaired at the transcriptional level while having the known IFN alpha/beta-transcriptional regulatory factors as revealed by induction of p69 2-5A-synthetase gene. This may account for a differential activation of 2-5A-dependent RNase and 2-5A-synthetase genes by type I-interferon, and suggests that other members of regulatory transcription factors, different from IRF-1 and STAT proteins, may participate in two different interferon alpha/beta signaling pathways.
Rise of intracellular free calcium levels with activation of inositol triphosphate in a human colonic carcinoma cell line (COLO 205) by heat-stable enterotoxin of Escherichia coli.
Bhattacharya J. Chakrabarti MK.
Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Calcutta 700 010, India.
The heat-stable enterotoxin (STa) produced by Escherichia coli has been found to increase rapidly two potential intracellular signals, inositol triphosphate and cytosolic free calcium in a human colonic cell line, COLO 205. Addition of STa to COLO 205 cells prelabelled with myo-[2-3H]inositol resulted in a rapid rise of [3H]inositol triphosphate. Using fluorescent indicator, Fura-2AM, intracellular free Ca2+ has been found to increase 5.12-fold compared to control. Suspension of cells in calcium-free buffer demonstrated STa-induced rapid rise of cytosolic Ca2+. The same result was found when extracellular calcium was chelated with EGTA. This effect was not observed with cells that were pretreated with dantrolene which suggest that the intracellular calcium rise might be due to mobilization from intracellular stores. This study demonstrated for the first time a change in cytosolic calcium in cultured human colonic cells by STa, which is accompanied by inositol triphosphate activation.
Secondary structure of P-glycoprotein investigated by circular dichroism and amino acid sequence analysis.
Dong M. Ladaviere L. Penin F. Deleage G. Baggetto LG.
Institut de Biologie et Chimie des Proteines, UPR 412 CNRS, 7 Passage du Vercors F-69367, Lyon Cedex 07, France.
P-glycoprotein (Pgp) is a plasma membrane protein known as an ATP-dependent drug-efflux pump that confers multidrug resistance to tumor cells. Structural analysis of Pgp was investigated by circular dichroism (CD) for the first time and in combination with amino acid sequence analysis. CD of highly purified Pgp from human, rat and murine Pgp-overexpressing drug resistant cells revealed slight variations in the spectral shape when recorded in the presence of dodecyl maltoside (DM). These species-dependent variations in CD shapes resulted from the interaction of the oligosaccharidic part with the protein core since they were abolished either in the presence of sodium dodecyl sulfate (SDS) or after deglycosylation, the latter not altering the Pgp ATP-dependent drug transport activity. Whatever the level of Pgp glycosylation and the detergent used (SDS or DM), the content in secondary structure deduced from deconvolution of CD spectra is almost the same for the three sources of Pgp and estimated to 43% alpha-helix, 16% beta-sheet, 15% beta-turn and 26% of other structures. These data, which constitute the first report of Pgp structure analysis by circular dichroism, are consistent with the 48% alpha-helix and 16% beta-sheets global contents predicted by using recently reported efficient secondary structure prediction methods. This consistency reinforces the reliability of the probable nature and localization of predicted Pgp secondary structure elements. This provides a good framework for precise 3D structure modeling of Pgp by homology with proteins of known 3D structure, as it is illustrated here for the A motifs of the ATP-binding domains of Pgp.