Cyclophilin B is involved in p300-mediated degradation of CHOP in tumor cell adaptation to hypoxia.

The regulation of CCAAT/enhancer-binding protein-homologous protein (CHOP), an endoplasmic reticulum (ER) stress-response factor, is key to cellular survival. Hypoxia is a physiologically important stress that induces cell death in the context of the ER, especially in solid tumors. Although our previous studies have suggested that Cyclophilin B (CypB), a molecular chaperone, has a role in ER stress, currently, there is no direct information supporting its mechanism under hypoxia. Here, we demonstrate for the first time that CypB is associated with p300 E4 ligase, induces ubiquitination and regulates the proteasomal turnover of CHOP, one of the well-known pro-apoptotic molecules under hypoxia. Our findings show that CypB physically interacts with the N-terminal α-helix domain of CHOP under hypoxia and cooperates with p300 to modulate the ubiquitination of CHOP. We also show that CypB is transcriptionally induced through ATF6 under hypoxia. Collectively, these findings demonstrate that CypB prevents hypoxia-induced cell death through modulation of ubiquitin-mediated CHOP protein degradation, suggesting that CypB may have an important role in the tight regulation of CHOP under hypoxia.

Tissue integrity: hemidesmosomes and resistance to stress.

How do animal tissues resist the shearing forces to which they are exposed during locomotion or harsh encounters with the environment? Genetic analysis in Caenorhabditis elegans is furthering our understanding of the nature and function of the attachments that preserve tissue integrity.

A cell-based assay system for monitoring NF-kappaB activity in human HaCat transfectant cells.

A cell-based assay system for monitoring NF-kappaB activity was developed to determine the influence of activated NF-kappaB in human HaCaT cells. The pNF-kappaB-SEAP-NPT plasmid that permits expression of the secretory alkaline phosphatase (SEAP) reporter gene in response to the NF-kappaB activity and contains the neomycin phosphotransferase (NPT) gene for geneticin resistance in host cells was constructed and transfected into the human keratinocyte cell line HaCaT. Human HaCaT transfectant cells were demonstrated to secrete the SEAP enzyme into the culture medium in a time-dependent manner until 72 h. NF-kappaB activities were measured by the SEAP reporter gene assay using a fluorescence detection method. HaCaT cell transfectants treated with antioxidants [e.g., N-acetyl-l-cysteine and vitamin C] showed reduction of NF-kappaB activity in a time- and concentration-dependent manner, whereas phorbol 12-myristate 13-acetate known as a stimulator of NF-kappaB expression increased NF-kappaB activity in a time- and concentration-dependent manner. This assay system could be used to determine the quantitative measurement of NF-kappaB activity in the human skin and allow the screening of anti-inflammatory agents for dermatological purpose from various synthetic chemicals and natural products.

Purification and characterization of a plasmin-like protease from Tenodera sinensis (Chinese mantis).

A novel type of protease (mantis egg fibrinolytic enzyme, MEF-2) was isolated from the egg cases of Tenodera sinensis. The protease was homogeneous by SDS-PAGE and its apparent molecular mass was 32,900 Da. The amino acids in the N-terminal region were Ile-Val-Gly-Gly-Glu-Glu-Ala-Val-Ala-Gly-Asp-Phe-Pro-Ile-Val-Ser-Leu-Gln-Glu. The enzyme was inhibited by PMSF, TLCK, aprotinin, benzamidine, soybean trypsin inhibitor and also slightly by elastatinal, EDTA, EGTA, cysteine and beta-mercaptoethanol, but TPCK, iodoacetate and E-64 did not affect the activity. MEF-2 was not sensitive to alpha(1)-antitrypsin but antithrombin III and alpha(2)-antiplasmin inhibited the enzyme. MEF-2 preferentially cleaved the oxidized B-chain of insulin between Arg(22) and Gly(23). Among chromogenic protease substrates, the most susceptible to MEF-2 hydrolysis was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at 30 degrees C and pH 5.0. These results indicate that MEF-2 belongs to the trypsin family. Upon incubation of crosslinked fibrin with MEF-2, a steady increase of D-dimer suggests that the enzyme has a strong fibrinolytic activity. In conclusion, MEF-2 is a new type of proteolytic enzyme and has some potential for practical application in fibrinolysis.

Purification and characterization of calobin II, a second type of thrombin-like enzyme from Agkistrodon caliginosus (Korean viper).

In our previous report, we purified and cloned the gene of a thrombin-like enzyme, calobin, from the venom of Agkistrodon caliginosus (Hahn, B.S., Yang, K.Y., Park, E.M., Chang, I.M., Kim, Y. S., 1996. Purification and molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper). J. Biochem. 119, 835-843.). During the purification of calobin, a second type of thrombin-like protease was found and it was purified using Affi-Gel Blue and Mono-S cation-exchange chromatography. It was identified as a serine protease with a molecular weight of 41, 000 on SDS-PAGE and its isoelectric point was determined to be 7.4 by isoelectric focusing. It showed little azocaseinolytic and fibrinolytic activity. However, this enzyme acted on fibrinogen to form fibrin with a specific activity of 7,587 NIH equivalent units and also exhibited arginine esterase activity. Amino acid sequencing of the N-terminal region established a primary structure composed of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Arg-Phe-Leu-Val-Ala-X -Tyr. This sequence was entirely consistent with that of calobin and showed a high homology with other thrombin-like enzymes, such as ancrod, batroxobin and gyroxin. Based on the biochemical properties such as molecular weight and isoelectric point, we can demonstrate a second thrombin-like protein showing a high potent clotting activity from the venom of Korean viper.

Purification and characterization of a serine protease with fibrinolytic activity from Tenodera sinensis (praying mantis).

Mantis egg fibrolase (MEF) was purified from the egg cases of Tenodera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The protease was assessed homogeneous by SDS-polyacrylamide gel electrophoresis and has a molecular mass of 31500 Da. An isoelectric point of 6.1 was determined by isoelectric focusing. Amino acid sequencing of the N-terminal region established a primary structure composed of Ala-Asp-Val-Val-Gln-Gly-Asp-Ala-Pro-Ser. MEF readily digested the Aalpha- and Bbeta-chains of fibrinogen and more slowly the gamma-chain. The nonspecific action of the enzyme results in extensive hydrolysis of fibrinogen and fibrin releasing a variety of fibrinopeptide. The enzyme is inactivated by Cu2+ and Zn2+ and inhibited by PMSF and chymostatin, yet elastinal, aprotinin, TLCK, TPCK, EDTA, EGTA, cysteine, beta-mercaptoethanol, iodoacetate, E64, benzamidine and soybean trypsin inhibitor do not affect activity. Antiplasmin was not sensitive to MEF but antithrombin III inhibited the enzymatic activity of MEF. Among chromogenic protease substrates, the most sensitive to MEF hydrolysis was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 30 degrees C. MEF preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. D-Dimer concentrations increased on incubation of cross-linked fibrin with MEF, indicating the enzyme has a strong fibrinolytic activity.

Molecular cloning of capillary permeability-increasing enzyme-2 from Agkistrodon caliginosus (Korean viper).

The gene of capillary permeability-increasing enzyme-2 (CPI enzyme-2) was cloned from the cDNA library of Agkistrodon caliginosus and its nucleotide sequence was determined. Its sequence indicates that CPI enzyme-2 is synthesized as a pre-zymogen of 258 amino acid residues, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acids. The amino terminal sequence deduced from the cDNA sequence was exactly consistent with that of CPI enzyme-2 except for the substitution of an amino acid (Gly27-->Ser). The open reading frame is very similar to those of plasminogen activator and thrombin-like proteases cloned from other snakes. The clone encoding CPI enzyme-2 belongs to the serine protease family. The active site of the enzyme is highly conserved at His41, Asp86 and Ser180. Its possible glycosylation sites, Asn-X-Thr/Ser, are located at amino acid residues 20-22, 55-57, 79-81 and 97-99.

Antiplatelet and antithrombotic effects of a combination of ticlopidine and ginkgo biloba ext (EGb 761).

The antiplatelet and antithrombotic effects of the oral combination treatment of ticlopidine and Ginkgo biloba extract (EGb 761) were studied in normal and thrombosis-induced rats. The ex vivo inhibitory effect on ADP-induced platelet aggregation of a small dose of ticlopidine (50 mg/kg/day) in combination with EGb 761 (40 mg/kg/day) was comparable to a larger dose of only ticlopidine (200 mg/kg/day). Bleeding time was also prolonged by 150%. Thrombus weight was also consistently decreased by a combination of ticlopidine and EGb 761 in an arterio-venous shunt model at two doses of ticlopidine (50 mg/kg) plus EGb 761 (20 mg/kg) and ticlopidine (50 mg/kg) plus EGb 761 (40 mg/kg). A combinatory treatment in acute thrombosis model in mice also showed a higher recovery than a single treatment.

Dose dependency of earthworm powder on antithrombotic and fibrinolytic effects.

The freeze-dried powder of Lumbricus rubellus earthworm was administered orally to rats and its fibrinolytic and antithrombotic effects were investigated. The fibrinolytic activity of plasma was determined by measuring the plasmin activity of the euglobulin fraction and was increased to two-folds of the control at a dose of 0.5 g/kg/day and five times with 1 g/kg/day after 4-day administration. The antithrombotic effect was studied in an arterio-venous shunt model of rats. The thrombus weight decreased significantly from 43.2 mg to 32.4 mg at a dose of 0.5 g/kg/day after 8-day treatment. The level of fibrinogen/fibrin degradation product (FDP) in serum was elevated in a dose-dependent manner during the treatment period. On the 8th day after administration, the FDP value was increased to 7.7 micrograms/ml compared with the control value of 3.3 micrograms/ml. These results support that earthworm powder is valuable for the prevention and/or treatment of thrombotic conditions.