A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21 x 10(5) u/L and 7.38 x 10(4) u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39 x 10(5) u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50 degrees C and at a pH range from 3 to 10. However, the specific activity of rLacDx (1761 u/mg) for ABTS was higher than that of rLacDe (1122 u/mg), and the apparent Km value of rLacDx (427 microM) was less than that of rLacDe (604 microM).
Cloning, Molecular ; Fermentation ; Isoenzymes ; biosynthesis ; genetics ; Laccase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Trametes ; enzymology ; genetics

Extracellular ATP (exATP) has been known to be a critical ligand regulating skeletal muscle differentiation and contractibility. ExATP synthesis was greatly increased with the high level of adenylate kinase 1 (AK1) and ATP synthase beta during C2C12 myogenesis. The exATP synthesis was abolished by the knock-down of AK1 but not by that of ATP synthase beta in C2C12 myotubes, suggesting that AK1 is required for exATP synthesis in myotubes. However, membrane-bound AK1beta was not involved in exATP synthesis because its expression level was decreased during myogenesis in spite of its localization in the lipid rafts that contain various kinds of receptors and mediate cell signal transduction, cell migration, and differentiation. Interestingly, cytoplasmic AK1 was secreted from C2C12 myotubes but not from C2C12 myoblasts. Taken together all these data, we can conclude that AK1 secretion is required for the exATP generation in myotubes.
Adenosine Triphosphate/*biosynthesis ; Adenylate Kinase/*metabolism ; Animals ; Cell Line ; Extracellular Space/metabolism ; Isoenzymes/*metabolism ; Mice ; Muscles/cytology/*metabolism

OBJECTIVETo detect expression of p-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and glutathione S-transferase (GST-pi), and to evaluate its clinical significance in neuroblastoma (NB).

METHODSSP immunohistochemical technique was used to investigate expression of P-gp, MRP, and GST-pi in 70 cases of NB.

RESULTSThe frequency of expression of P-gp, MRP, and GST-pi was 61.4%, 38.6%, and 51.4%, respectively. The coexpression rate of P-gp and MRP, P-gp and GST-pi, MRP and GST-pi, P-gp, MRP and GST-pi was 32.9%, 35.7%, 27.1%, and 24.3%, respectively. Significant positive correlation was observed between P-gp and MRP expression (P = 0.001), and between MRP and GST-pi expression (P = 0.012), but no correlation was found between P-gp and GST-pi expression. The expression of P-gp and MRP was higher in tumors from patients over 1 year old compared with those less than 1 year old at diagnosis (P = 0.01, 0.018, respectively). MRP expression was higher in tumors from the metastatic than the non metastatic groups (P = 0.015). All tested proteins showed significant relationship to the differentiation of the tumor (P = 0.006, 0.000, 0.019, respectively), but no correlation was found to the stage of NB or sex of the patients. MRP expression was significantly related to the reduction of both median survival time and the two-year cumulative survival (P = 0.02). In contrast, P-gp and GST-pi expression had no correlation with survival.

CONCLUSIONSThe intrinsic multidrug resistance of NB involves the combined effects of P-gp, MRP, and GST-pi. MRP expression may be an important parameter in predicting the prognosis of patients with NB.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; Adolescent ; Child ; Child, Preschool ; Female ; Gene Expression ; Glutathione S-Transferase pi ; Glutathione Transferase ; biosynthesis ; Humans ; Immunohistochemistry ; Infant ; Isoenzymes ; biosynthesis ; Male ; Multidrug Resistance-Associated Proteins ; biosynthesis ; Neuroblastoma ; metabolism ; mortality ; Survival Rate

OBJECTIVETo study the expression of cyclooxygenase-2 (COX-2) gene in breast cancer and its clinicopathologic characteristics.

METHODSWith beta-actin gene as reference, the COX-2 mRNA was monitored in 30 specimens of breast cancer tissue and adjacent normal breast tissue by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe COX-2 mRNA expression was significantly upregulated in most breast cancer tissues with range of 0.05 - 0.91 (median 0.56), which was rare in normal breast tissue with range of 0 - 0.09 (median 0). The difference of COX-2 mRNA expression between cancer and normal breast tissue was significant (rank sum test, P < 0.05). COX-2 overexpression in breast cancer was related to its lymph node metastasis (P < 0.05) but not to age, tumor size, pathologic grade or pathologic type (P > 0.05).

CONCLUSIONThe level of COX-2 mRNA expression is obviously higher in the breast cancer tissue than that in normal breast tissue. COX-2 overexpression may play a crucial role in the carcinogenesis, development of cancer and lymph node metastasis in breast cancer patients.

Adult ; Biomarkers, Tumor ; biosynthesis ; genetics ; Breast Neoplasms ; enzymology ; metabolism ; Cyclooxygenase 2 ; Female ; Gene Expression ; Humans ; Isoenzymes ; biosynthesis ; genetics ; physiology ; Membrane Proteins ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; genetics ; physiology ; RNA, Messenger ; biosynthesis ; RNA, Neoplasm ; analysis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the effect of tetramethylpyrazine (TMP) on lipopolysaccharides (LPS) induced macrophage cyclo-oxidase-2 (COX-2) gene expression and activity in RAW264.7 mice, and to further investigate the effect and mechanism of TMP on LPS induced apoptosis of cardiac myocytes in suckling mice.

METHODSRT-PCR and Western Blot (WB) were used to investigate the macrophage COX-2 gene expression, ELISA was used to measure its activity, fluorescence microscopy was used to determine the apoptosis of murine neonatal cardiac myocyte, and fluorescence spectrophotometry was used to detect the concentration of intracellular calcium ion (Ca2+).

RESULTSTMP of 10(-6) mol/L could significantly reduce the COX-2 mRNA and protein expression (P < 0.05), in 10(-5) mol/L and 10(-4) mol/L could significantly decrease the COX-2 expression (P < 0.01) stimulated by LPS, but couldn't influence the activity of COX-2 by different TMP concentration. TMP in 10(-5) mol/L could significantly lower the concentration of intracellular Ca2+ in cardiac myocyte, and antagonize the LPS induced apoptosis of cardiac myocyte in suckling mice (P < 0.05).

CONCLUSIONTMP has the pharmacological effect in inhibiting LPS induced macrophage COX-2 expression and apoptosis of cardiac myocyte in suckling mice.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Cells, Cultured ; Cyclooxygenase 2 ; Isoenzymes ; biosynthesis ; genetics ; Lipopolysaccharides ; Macrophages ; enzymology ; Mice ; Myocytes, Cardiac ; cytology ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; genetics ; Pyrazines ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe in vitro the effect of Sinomenine, a pure alkaloid extracted from the chinese medical plant Sinomenium acutum on the activity of cyclooxygenase (COX-1 and COX-2) and the expression of COX-1 and COX-2 mRNA.

METHODMononuclear leukocytes were obtained from healthy adults. Isolated mononuclear leucocytes from human peripheral blood (PBMC) were incubated (1 x 10(6).mL-1) with or without sinomenine (or indomethacin), after incubated for 24 hours at 37 degrees C with 5% CO2; the media were assayed for the PGE2 by radioimmunoassay (RIA). LPS was used to stimulate the monocytes at a concentration of 5 micrograms.mL-1. And by RT-PCR, both COX-1 and COX-2 mRNAs were detected in Mononuclear leukocytes after incubation for different hours with drug (sinomenine or indomethacin) or not.

RESULTLPS (stimulated) induced the production of PGE2 in PBMC increasing with high expression of COX-2 mRNA; sinomenine reduced PGE2 production in LPS stimulated human monocytes more than in non-stimulated human monocytes. In comparative experiments, indomethacin, a non selective COX inhibitor, reduced the production of PGE2 equally in both states. Meanwhile, neither sinomenine(0.1-1 mmol.L-1) nor indomethacin(0.5-10 mumol.L-1) inhibited the expression of both COX-1 and COX-2 mRNAs by RT-PCR with beta-actin as reference.

CONCLUSIONIn contrast with indomethacin, Sinomenine shows a preferential inhibitory effect on COX-2 over COX-1, These results suggest that Sinomenine is a selective COX-2 inhibitor, which may be directly related to suppressing cyclooxygenase activity.

Adult ; Cyclooxygenase 1 ; Cyclooxygenase 2 ; Dinoprostone ; blood ; Humans ; Isoenzymes ; biosynthesis ; Leukocytes, Mononuclear ; enzymology ; Membrane Proteins ; Morphinans ; isolation & purification ; pharmacology ; Plants, Medicinal ; chemistry ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; metabolism ; RNA, Messenger ; biosynthesis ; Sinomenium ; chemistry

OBJECTIVETo investigate the changes of the mRNA expression and the intestinal mucosal cyclooxygenase (COXs) activity in scalded rats.

METHODSWistar rats inflicted with 30% TBSA III degree scalding were employed as the model. The changes of COX-1, COX-2 activities were determined by substrate fluorescence analysis and the mRNA expressions of COX-1 and COX-2 by RT-PCR.

RESULTSThe mRNA expressions and the activities of COX-2 in rat intestinal mucosa increased obviously after injury. But those of COX-1 exhibited lower range of change.

CONCLUSIONThe pathological mechanism of rat intestinal mucosa injury after scalding might be closely related to the COXs participation by different styles between the two enzymes.

Animals ; Burns ; enzymology ; genetics ; pathology ; Cyclooxygenase 1 ; Cyclooxygenase 2 ; Female ; Gene Expression ; Intestinal Mucosa ; enzymology ; Isoenzymes ; biosynthesis ; genetics ; metabolism ; Male ; Membrane Proteins ; Prostaglandin-Endoperoxide Synthases ; biosynthesis ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Wistar

OBJECTIVETo explore the correlation of chemotherapy efficacy in tongue squamous cell carcinoma(SCC) with expression level of P-glycoprotein(Pgp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathiones-tranferase (GST-pi), DNA topo-isomerase II alpha (Topo II alpha).

METHODSThe expression patterns of Pgp, MRP, LRP, GST-pi and Topo II alpha in 40 patients (pre and post-chemotherapy, respectively) with tongue SCC were examined by immunohistochemically labelled streptavidin bioein method (LsAB).

RESULTSThe expression ratios of Pgp, MRP, LRP, GST-pi and Topo II alpha in pre-chemotherapy cases were 47.5%, 50%, 35%, 45%, 82.5%, respectively. No relations between expression of Pgp, MRP, LRP, GST-pi, Topo II alpha and clinic indexes were established (P > 0.05). Expression ratios of Pgp, MRP in post-chemotherapy cases were higher than that in pre-chemotherapy cases (P < 0.05). Expression of Pgp and MRP showed relevance with drug resistance (P < 0.05). The co-expression was common, the ratios of co-expression of Pgp, MRP, GST-pi and MRP, GST-pi in chemotherapy non-responders were 40% and 50%, respectively, but 0 in responders.

CONCLUSIONThe intrinsic multidrug resistance of tongue SCC is relevant to the effects of Pgp, MRP, GST-pi.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Adult ; Antigens, Neoplasm ; Carcinoma, Squamous Cell ; metabolism ; DNA Topoisomerases, Type II ; biosynthesis ; genetics ; DNA-Binding Proteins ; Female ; Glutathione S-Transferase pi ; Glutathione Transferase ; biosynthesis ; genetics ; Humans ; Isoenzymes ; biosynthesis ; genetics ; Male ; Middle Aged ; Multidrug Resistance-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Random Allocation ; Tongue Neoplasms ; metabolism ; Vault Ribonucleoprotein Particles ; biosynthesis ; genetics

To examine the involvement of phospholipase D (PLD)isozymes in postnatal testis development, the expression ofPLD1 and PLD2 was examined in the mouse testis atpostnatal weeks 1, 2, 4, and 8 using Western blot analysisand immunohistochemistry. The expression of both PLD1and PLD2 increased gradually with development frompostnatal week 1 to 8. Immunohistochemically, PLDimmunoreactivity was detected in some germ cells in thetestis and interstitial Leydig cells at postnatal week 1.PLD was mainly detected in the spermatocytes andresidual bodies of spermatids in the testis after 8 weeksafter birth. The intense immunostaining of PLD in Leydigcells remained unchanged by postnatal week 8. Thesefindings suggest that PLD isozymes are involved in thespermatogenesis of the mouse testis.
Animals ; Blotting, Western ; Female ; Immunohistochemistry ; Isoenzymes ; Male ; Mice ; Mice, Inbred BALB C ; Phospholipase D/biosynthesis/*metabolism ; Spermatogenesis/physiology ; Testis/*enzymology/growth & development

OBJECTIVESTo construct a prokaryotic recombinant vector for mouse lactate dehydrogenase-C and to detect its expression in BL21.

METHODSThe coding sequence of mouse lactate dehydrogenase subunit C was amplified from mouse testis RNA with specific primers, and cloned into pGEX-2T after the restriction digestion with BamH I and EcoR I. GST fusion protein was expressed after induction with IPTG.

RESULTSSequencing and restriction digestion of the recombinant plasmid revealed the existence of coding sequence for mouse lactate dehydrogenase subunit C. A protein band of about 60,000 could be induced by IPTG in the recombinant plasmid.

CONCLUSIONSThe coding sequence of mouse lactate dehydrogenase subunit C was introduced into the pGEX-2T plasmid and a GST-fused protein could be induced at a high level.

Animals ; Escherichia coli ; genetics ; Glutathione Transferase ; genetics ; Isoenzymes ; genetics ; L-Lactate Dehydrogenase ; genetics ; Male ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; Spermatozoa ; enzymology