Ahn HK, Chung JY, Park SK
… +3 more, Joo SM, Park SK, Koh YW
Biochem Mol Biol Int
· 1999 May · PMID 10365242
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Human thrombopoietin (hTPO) variant cDNAs truncated in the C-terminal regions of wild-type hTPO (332 amino acids) were constructed by PCR and expressed in Trichoplusia ni (Tn5) insect cells using a baculovirus expression...Human thrombopoietin (hTPO) variant cDNAs truncated in the C-terminal regions of wild-type hTPO (332 amino acids) were constructed by PCR and expressed in Trichoplusia ni (Tn5) insect cells using a baculovirus expression system. Each variant, hTPO163 (amino acids 1-163), hTPO198 (1-198) and hTPO245 (1-245), was produced in insect cells with very low efficiency in comparison with wild-type hTPO. Immunoblot analysis showed that the predicted 20, 25 and 34 kDa molecular sizes corresponding to hTPO163, hTPO198 and hTPO245, respectively, were barely detected in culture medium and most of the proteins remained within the cell. These results suggest that C-terminal regions containing potential N-glycosylation sites of hTPO are required for the secretion of hTPO into culture medium as well as expression in insect cells.
The sodium hydrogen exchanger isoform, NHE-1 plays an important role in electrolyte and water homeostasis. These functions are compromised in pregnancies complicated with preeclampsia. At present it is not known whether...The sodium hydrogen exchanger isoform, NHE-1 plays an important role in electrolyte and water homeostasis. These functions are compromised in pregnancies complicated with preeclampsia. At present it is not known whether NHE-1 expression is altered during preeclampsia. In the present study the placental level of NHE-1 protein was measured using immunoblotting. Since prostaglandins regulate the secretory and absorptive functions, the levels of prostaglandin E-2 as well as the expression of cyclooxygenase-1 and -2 were also estimated. The amount of NHE-1 protein and cyclooxygenase-2 was reduced in preeclamptic placentas, whereas the level of cyclooxygenase-1 remained unaltered. In contrast, prostaglandin E-2 concentration was higher in preeclampsia. Suppression of NHE-1 might render the placenta with impaired uptake of water and electrolytes and therefore may be involved in the pathogenesis of preeclampsia. While prostaglandin E-2 may play a role in preeclampsia, these findings discount the induction of cyclooxygenase-genes for this increase.
Anti-tumor active polysaccharide against Sarcoma 180 was isolated by DEAE-Sepharose CL-6B and Sepharose 4B column chromatography from the hot-water soluble fraction of the mycelium of liquid-cultured Agaricus blazei mill...Anti-tumor active polysaccharide against Sarcoma 180 was isolated by DEAE-Sepharose CL-6B and Sepharose 4B column chromatography from the hot-water soluble fraction of the mycelium of liquid-cultured Agaricus blazei mill. This polysaccharide did not react with antibodies of anti-tumor polysaccharides such as lentinan, gliforan, and FIII-2-b which is one of anti-tumor polysaccharides from Agaricus blazei. Moreover, the analyses of 13C-NMR and GC-MS suggested that this polysaccharide was preliminarily glucomannan with a main chain of beta-1,2-linked D-mannopyranosyl residues and beta-D-glucopyranosyl-3-O-beta-D-glucopyranosyl residues as a side chain. This polysaccharide was completely different from the anti-tumor polysaccharide from fruiting body of Agaricus blazei, beta-1,6-glucan.
The isolation and partial characterization of a serine protease with arginine ester hydrolase activity from Bothrops jararacussu snake venom are described. The purification procedure consisted of a gel filtration of the...The isolation and partial characterization of a serine protease with arginine ester hydrolase activity from Bothrops jararacussu snake venom are described. The purification procedure consisted of a gel filtration of the crude venom on Sephadex G-75 followed by an ion-exchange chromatography of the active fraction on DEAE-cellulose and a rechromatography on Bio-Rex 70 resin. The esterase fraction (DI-III), M(r) = 25,000 by SDS-PAGE, showed proteolytic activity on fibrinogen and casein. After 2 hr incubation, the A alpha and B beta chains of fibrinogen were intensely hydrolysed, while the gamma chain kept apparently intact, even after 20 hr of incubation. In spite of that, DI-III did not clot fibrinogen. DI-III induced edema in the rat paw. Although unable to release bradykinin, it induced contractions of the isolated rat uterus. DI-III did not catalyse the hydrolysis of bradykinin. Its arginine ester hydrolase activity was completely inhibited by diisopropyl fluorophosphate after 1 hr incubation, but not by phenylmethylsulfonyl fluoride under the same conditions.
The polymerization of actin induced by dilution has previously been reported, where a 1000-fold molar excess of ATP over actin resulted when actin was diluted to 4.0 micrograms/ml in low salt buffer A (0.1 mM ATP, 0.1 mM...The polymerization of actin induced by dilution has previously been reported, where a 1000-fold molar excess of ATP over actin resulted when actin was diluted to 4.0 micrograms/ml in low salt buffer A (0.1 mM ATP, 0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 5 mM 2-mercaptoethanol, 1 mM NaN3). Filaments formed by the addition of ATP to a 1000-fold molar excess over actin in buffer B (0.1 mM CaCl2, 2 mM Tris-HCl, pH 8.0, 1 mM NaN3) were then separated by gel-filtration. When ATP was removed from these filaments using Dowex-1, depolymerization occurred. Thus, the reversible polymerization induced by the dilution of actin or by addition of ATP can be ascribed to the binding of ATP at the low affinity site of actin.
Plasma lipid profile and abdominal obesity have been associated with breast cancer risk, however published results have been inconsistent. To clarify these associations we studied lipid and lipoprotein alterations, obesi...Plasma lipid profile and abdominal obesity have been associated with breast cancer risk, however published results have been inconsistent. To clarify these associations we studied lipid and lipoprotein alterations, obesity degree and body fat distribution, in 30 newly diagnosed breast cancer patients without treatment and 30 controls matched by age and menopausal status. Both pre and postmenopausal breast cancer patients presented higher body mass index, waist/hip ratio and insulin levels than their matched controls. An increase in triglycerides and a decrease in HDL-cholesterol, especially in the HDL2 subfraction, were observed in patients with breast cancer. Besides, HDL particle from these patients showed increased apo A1/HDL-cholesterol ratio. These alterations were correlated with waist/hip ratio. The association between lipoprotein alterations and abdominal obesity independent of menopausal status, in untreated newly diagnosed breast cancer patients is reported for the first time in this study.
Because the expression of the isoforms of protein kinase C (PKC) in human basal keratinocytes is not understood, the expression of PKC isoforms were screened in specimens of epidermal tissue from postburn skin and the no...Because the expression of the isoforms of protein kinase C (PKC) in human basal keratinocytes is not understood, the expression of PKC isoforms were screened in specimens of epidermal tissue from postburn skin and the normal locations for skin grafts in patients with second or higher degrees of flame injury. The expression of individual isoform was determined by Western blot technique. Only PKC alpha and zeta were detected in the epidermal tissues of normal and postburn skin and translocation occurred in PKC alpha. Patients without antibiotic treatment after flame injury had higher expressions of PKC alpha and zeta. These findings indicate that the mechanisms of cellular differentiation and growth in postburn epidermal tissue may be related to the expression and translocation of PKC alpha induced by intra- and extracellular stimulation. These changes in PKC alpha further activate the DAG/PKC signal transduction pathways.
The interaction between acetylsalicylic acid (aspirin) and membrane was studied by NMR spectra. (1) NMR spectra showed acetylsalicylic acid did not insert into membrane; (2) 1H NMR spectrum recorded by spin-echo pulse se...The interaction between acetylsalicylic acid (aspirin) and membrane was studied by NMR spectra. (1) NMR spectra showed acetylsalicylic acid did not insert into membrane; (2) 1H NMR spectrum recorded by spin-echo pulse sequence showed protons of the aromatic ring interacted with membrane; (3) the change of spin-lattice relaxation (T1) of 31P was ascribed to the association of acetylsalicylic acid to the polar head of lecithin; (4) the self-diffusion coefficient measured by pulsed field gradients NMR showed the mobility of acetylsalicylic acid was restricted by membrane and that acetylsalicylic acid changed membrane viscosity. Based on the results, the relationship between the interaction and the mechanism of the wide pharmacological functions of acetylsalicylic acid is discussed.
The human CR1 is a single chain membrane glycoprotein that is a member of the group of regulators of the complement activation system. In order to clarify the regulatory mechanisms of human CR1 gene expression, the 5'-fl...The human CR1 is a single chain membrane glycoprotein that is a member of the group of regulators of the complement activation system. In order to clarify the regulatory mechanisms of human CR1 gene expression, the 5'-flanking region of the human CR1 gene was isolated and its promoter was characterized. The CR1 expression was found to be transcriptionally up-regulated in HL60 cells by stimulation with DMSO. The cloned CR1 gene promoter was sequenced and computer analyzed. The potential promoter region lacks a distinct TATA box sequence. The transcription initiation site was determined by primer extension and several possible regulatory elements for transcription were found in the promoter region.
Cu,Zn-superoxide dismutase (SOD) can catalyze hydroxyl radical generation using H2O2 as a substrate. Lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system was investigated. When linoleic acids micelles or phosphati...Cu,Zn-superoxide dismutase (SOD) can catalyze hydroxyl radical generation using H2O2 as a substrate. Lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system was investigated. When linoleic acids micelles or phosphatidylcholine liposomes were incubated with Cu,Zn-SOD and H2O2, lipid peroxidation was gradually increased in a time-dependent manner. The extent of lipid peroxidation was proportional to Cu,Zn-SOD and H2O2 concentrations. Hydroxyl radical scavengers and copper chelator inhibited lipid peroxidation induced by the Cu,Zn-SOD and H2O2 system. These results suggest that lipid peroxidation is mediated by the Cu,Zn-SOD and H2O2 system via the generation of hydroxyl radicals by a combination of the peroxidative reaction of Cu,Zn-SOD and the Fenton-like reaction of free copper released from oxidatively damaged SOD.
The Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disease characterized by chronic conjugated hyperbilirubinemia. The phenotype of this syndrome is thought to be caused by the impaired expression of th...The Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disease characterized by chronic conjugated hyperbilirubinemia. The phenotype of this syndrome is thought to be caused by the impaired expression of the canalicular multispecific organic anion transporter (cMOAT), which transports non-bile salt organic anions into the bile. Recently, a mutation from arginine (Arg) to stop-codon at codon 1066 in the cMOAT gene has been reported in one Caucasian patient with DJS. In this study, we investigated whether this mutation is found in Japanese patients with DJS. Genomic DNAs were extracted from the leukocytes of six Japanese patients and the fragments spanning codon 1066 were amplified by polymerase-chain reaction. The digest of the amplified fragments with a restriction enzyme, Taql, demonstrated that all of six patients did not exhibit an R1066X mutation. No mutation at Arg1066 was also confirmed by direct sequencing of the amplified products. These findings suggested that this R1066X mutation was not a major mutation in Japanese patients with DJS. Further investigation will be required in an attempt to search other mutations in cMOAT gene in Japanese patients with DJS.
The molecular chaperone, GroEL, facilitates correct protein folding and inhibits protein aggregation. The function of GroEL is often, though not invariably, dependent on the co-chaperone, GroES, and ATP. In this study it...The molecular chaperone, GroEL, facilitates correct protein folding and inhibits protein aggregation. The function of GroEL is often, though not invariably, dependent on the co-chaperone, GroES, and ATP. In this study it is shown that GroEL alone substantially reduces the inactivation of purified Ca(++)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum. In the absence of GroEL, the enzyme became completely inactive in about 45-60 hours when kept at 25 degrees C, while in the presence of an equimolar amount of GroEL, the enzyme remained approximately 80% active even after 75 hours. Equimolar amounts of BSA or lysozyme were unable to protect the enzyme from inactivation under identical conditions. Analysis by SDS-PAGE showed GroEL was acting by blocking the aggregation of ATPase at 25 degrees C. GroEL was not as effective in protection at -20 degrees C or 4 degrees C. These results are discussed in the context of current models of the GroEL mechanism.
The human immunodeficiency virus type 1 (HIV-1) and human foamy virus (HFV) integrase proteins were overexpressed in Escherichia coli, and purified to a near homogeneity by one- or two-step purification scheme. The endon...The human immunodeficiency virus type 1 (HIV-1) and human foamy virus (HFV) integrase proteins were overexpressed in Escherichia coli, and purified to a near homogeneity by one- or two-step purification scheme. The endonucleolytic, integration, and disintegration activities for the HIV-1 and HFV integrases were characterized in vitro. The endonucleolytic activities for the HIV-1 and HFV integrases were found only on their own substrates, respectively, indicating that the cognate U5 LTR sequences in the substrates is critical for specific cleavage. However, the integration and disintegration activities showed less specificity on the substrate usage. Our results suggest that the disintegration activity have more preference for substrates based on Y-shaped structure rather than on viral donor DNA sequence.
We reported previously that the arginine-specific ADP-ribosyltransferase in chicken polymorphonuclear leukocytes specifically modified actin, thereby inhibiting actin polymerization in vitro. In the present study, we inv...We reported previously that the arginine-specific ADP-ribosyltransferase in chicken polymorphonuclear leukocytes specifically modified actin, thereby inhibiting actin polymerization in vitro. In the present study, we investigated the effect of ADP-ribosylation on actin polymerization in situ. In the leukocytes, the introduction of NAD inhibited the increase in filamentous actin contents induced by a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, while introduction of NAD together with novobiocin, a specific inhibitor for ADP-ribosyltransferase, did not. These results suggest that ADP-ribosylation regulates the formation of filamentous actin by the covalent modification of the protein in vivo.
After in vivo administration of lead nitrate, functional changes of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthetase have been detected in r...After in vivo administration of lead nitrate, functional changes of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthetase have been detected in rat liver. The rate of citrate transport was greatly reduced in rats during both the proliferative phase (3 days after the lead nitrate administration) and the involutive phase (5 days after the metal injection), which follows hepatic hyperplasia and corresponds to the peak of hepatocyte apoptosis. In both phases, a decrease of the lipogenic enzyme activities has been detected. In treated animals, an alteration of mitochondrial lipid composition has also been found. The modified lipid microenvironment could be responsible for the decreased carrier activity which, in turn, may account for the reduced activities of the lipogenic enzymes.
A hybrid cDNA tu-pa, which contains Ser1-Thr263 of tissue-type plasminogen activator (t-PA) and Ser138-Leu411 of pro-urokinase (pro-UK) was constructed and expressed in the Sf9-AcNPV system. The expression level was appr...A hybrid cDNA tu-pa, which contains Ser1-Thr263 of tissue-type plasminogen activator (t-PA) and Ser138-Leu411 of pro-urokinase (pro-UK) was constructed and expressed in the Sf9-AcNPV system. The expression level was approximate 2.5 mg/L. Tu-PA was purified via one-step affinity column conjugated with monoclonal antibody against the B chain of pro-UK, which showed a single band of approximate 60 kDa in SDS-PAGE. The specific activity of the chimeric protein on fibrin plate was 200,000 IU/mg protein. Tu-PA had a higher selectivity for fibrin than UK and pro-UK. Its activity can be promoted by CNBr degraded fibrin fragments as t-PA.
Ansamycin antibiotic, geldanamycin has a unique pharmacological effect to bind to heat shock protein 90 (hsp90) and deplete hsp90 substrates. We investigated the immunopharmacological effects of geldanamycin. Geldanamyci...Ansamycin antibiotic, geldanamycin has a unique pharmacological effect to bind to heat shock protein 90 (hsp90) and deplete hsp90 substrates. We investigated the immunopharmacological effects of geldanamycin. Geldanamycin depleted cellular Raf-1 of rat splenic cells without affecting the steady state levels of hsp90 and downstream mitogen activated protein (MAP) kinases, ERK1 and ERK2. In parallel, it inhibited mitogen-induced nuclear factor-kappa B (NF-kappa B) activation in these cells. Geldanamycin also had a potent suppressive effect on recall antigen-induced T cell proliferation, with an IC50 value of 1 nM. In vivo, geldanamycin suppressed the progression of adjuvant-induced arthritis dose-dependently. These results suggest that geldanamycin exerts an immunosuppressive effect partly through destabilizing Raf-1, and raise a new strategy for the prevention of inflammatory diseases.
Novel gelatinolytic activities in both latent and active forms were detected in the normal organs of rat by gelatin zymography. Multiple active bands were detected in the extracts from the skin, jejunum, muscle, and kidn...Novel gelatinolytic activities in both latent and active forms were detected in the normal organs of rat by gelatin zymography. Multiple active bands were detected in the extracts from the skin, jejunum, muscle, and kidney without any activation. These activities were inhibited by 1,10-phenanthroline or leupeptin, nor by E64, suggesting that these activities were derived from metallo-proteinases or serine-proteinases. Some gelatinolytic active bands were newly induced or enhanced by p-aminophenylmercuric acetate. These results suggest that matrix degrading activities due to metallo- and serine-proteinases were constitutively expressed in various rat normal organs.
The role of phosphorylation of the C-terminal tail of endothelin B receptor (ETBR) in agonist-induced desensitization was investigated, using a mutant lacking C-terminal 40 amino acids (delta 40 ETBR). In cells expressin...The role of phosphorylation of the C-terminal tail of endothelin B receptor (ETBR) in agonist-induced desensitization was investigated, using a mutant lacking C-terminal 40 amino acids (delta 40 ETBR). In cells expressing the wild type or delta 40 ETBR, ET-1 caused rapid desensitization of calcium responses. The wild type ETBR was phosphorylated by biotinylated ET-1, and the phosphorylation was markedly enhanced by coexpression with G protein-coupled receptor kinase 2 (GRK2). However, delta 40 ETBR was not phosphorylated regardless of coexpression with GRK2. On the other hand, ET-1-induced IP3 formation in these cells was decreased by coexpression with GRK2 or catalytically inactive Lys220Arg GRK2 to the similar extent. The present study demonstrates the presence of phosphorylation-independent desensitization mechanism in delta 40 ETBR and suggests that GRK2 might play a role other than that as a kinase.
To examine whether two DEAD box genes, DDX1 and DDX6, would have some roles in the progression of tumors, we investigated the correlation of the expression of these genes with that of MYCN in neuroblastomas either with o...To examine whether two DEAD box genes, DDX1 and DDX6, would have some roles in the progression of tumors, we investigated the correlation of the expression of these genes with that of MYCN in neuroblastomas either with or without MYCN amplification. The mRNA of MYCN was observed only in the cell lines with amplification of MYCN. The mRNAs of DDX1 and DDX6 were found in all the cell lines examined, but the correlation between the mRNA levels of DDX1 or DDX6 and MYCN was poor. These findings suggest that the expression of neither DEAD box gene is correlated with the gene expression of MYCN.