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.

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.

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 molecular cloning of calobin, a thrombin-like enzyme from Agkistrodon caliginosus (Korean viper).

A thrombin-like enzyme, calobin, has been purified to homogeneity from the venom of Agkistrodon caliginosus by a procedure involving Bio-Gel P-100, Mono S, and Pro-RPC. The enzyme was identified as a monomer with a molecular weight of 34,000 on SDS-PAGE, and its isoelectric point was 6.2. Calobin acted on fibrinogen to form fibrin with a specific activity of 226 NIH equivalent units, and also exhibited arginine esterase activity. The enzyme predominantly cleaved the alpha-chain of fibrinogen with little degradation of the beta-chain. It contained abundant asparagine/aspartic acid residues, but very few tyrosine or methionine residues. The proteolytic activity of the enzyme with TAME as a substrate was higher than that of thrombin. However, it showed neither lysine esterase nor caseinolytic activity. The enzyme activity was strongly inhibited by PMSF, and moderately by benzamidine and soybean trypsin inhibitor, indicating it is a serine protease. On the other hand, the enzyme activity was not inhibited by hirudin or aprotinin. cDNA (1.6 kb) for calobin has been cloned from an A. caliginosus cDNA library. The cDNA sequence indicates that calobin is synthesized as a pre-zymogen of 262 amino acids, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acid residues. The cDNA sequence encodes a 238-amino acid residue molecule exhibiting strong amino acid sequence homology to those of ancrod, batroxobin, and flavoxobin isolated from other snake venoms. Calobin contains 12 cysteine residues. As judged on alignment of the amino acid sequences of other thrombin-like enzymes (batroxobin, ancrod, and flavoxobin), calobin constitute the formation of six disulfide bridges. Amino acid residues, His43, Asp88, and Ser182, which are thought to be the catalytic active site are highly conserved. As calobin is a glycoprotein, its possible glycosylation site, Asn-X-Thr, is located at amino acid residues 81-83.

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.

Purification and characterization of piscivorase I and II, the fibrinolytic enzymes from eastern cottonmouth moccasin venom (Agkistrodon piscivorus piscivorus).

Fibrinolytic enzymes, piscivorase I and II, were isolated from Agkistrodon piscivorus piscivorus (eastern cottonmouth moccasin) venom using gel filtration on Bio-Gel P-100 and ion-exchange chromatography on CM-Sepharose CL-6B. The mol. wts of these proteases, piscivorase I and II, are 23,400 and 29,000 and isoelectric points are 6.6 and 8.5, respectively. These fibrinolytic enzymes were homogeneous by SDS-polyacrylamide gel electrophoresis. Piscivorase I readily cleaved the A alpha- and B beta-chain of fibrinogen, but piscivorase II cleaved readily the A alpha-chain and more slowly the B beta-chain. These fibrinolytic enzymes were activated by Ca2+, Mg2+ and Ba2+, but inhibited by Zn2+, Cu2+ and Mn2+. Both fibrinolytic enzymes were also inhibited by cysteine, beta-mercaptoethanol, and by metal chelators such as EDTA and EGTA, but not by benzamidine, phenylmethanesulfonyl fluoride (PMSF), soybean trypsin inhibitor and aprotinin. These fibrinolytic enzymes did not act like thrombin, plasmin and kallikrein, using specific chromogenic substrates. Neither fibrinolytic enzyme induced platelet aggregation, and piscivorase I showed low haemorrhagic activity at dosages of 55 micrograms.

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.

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.

Chlorophyll derivatives (CpD) extracted from silk worm excreta are specifically cytotoxic to tumor cells in vitro.

Among chlorophyll derivatives (CpD-A, -B, -C, and -D) extracted from silk worm (Bombyx mori) excretas, CpD-A was extensively studied to clarify its role as a "photosensitizer" for photodynamic therapy (PDT) of tumors in vitro. It was found that CpD-A was photoreactive both in itself and also in its cell bound forms. The cell bound CpD-A produced fluorescent light and singlet oxygen following the exposure to lights of varied wave length. Among them, lights of near 650 nm, which was the maximum absorbance band, efficiently activated CpD-A following the application of only 10 minutes of irradiation. CpD-A was found to have specificity for the human and mouse tumor cells regardless of their species difference. A higher intensity of fluorescence and a larger amount of CpD-A were found in the tumor cells as opposed to the intensity found in normal cells. Only 10 minutes of light irradiation of the CpD-A treated tumor cells resulted in their rapid and complete destruction within 2 hours of irradiation. Simultaneously, more than 80% of the normal human and mouse control cells remained alive after receiving treatment. These findings suggested that CpD-A produced by use of silkworm excreta could be used as a photosensitizer for PDT of tumors by the use of lights of near 650 nm.

Chlorophyll derivatives--a new photosensitizer for photodynamic therapy of cancer in mice.

The in vivo photosensitizing efficacy of chlorophyll derivatives (CpD), which had been developed as a new photosensitizer, was compared with that of hematoporphyrin derivatives (HpD). A murine tumor model implanted subcutaneously with S-180 cells on the abdomen was used. The CpD or HpD was administered by intratumoral injection, and light of appropriate wavelength was irradiated on the tumor areas for 10 minutes at 1h and 24h or 24h and 48h after the injection of photosensitizer. When CpD was injected, the early irradiation group (1h and 24h) showed a 100% tumor cure rate; however, the late irradiation group (24h and 48h) showed a 60% tumor cure rate (p less than 0.01). This showed that the early irradiation with light after injection of CpD was an important factor for obtaining better results. With HpD, there was no difference in tumor cure rate between early (1h and 24h, 80%) and late irradiation (24h and 48h, 80%) groups. Thus, in early irradiation groups, the tumor cure rate using CpD (100%) was superior to that of HpD (80%) (p less than 0.05). However, in late irradiation groups, the tumor cure rate using CpD (60%) was inferior to that of HpD (80%), but this difference was not statistically significant (p greater than 0.1). Pathologic sections of these tumors were made before treatment and 48h and 3 weeks after treatment. These showed geographic necrosis at 48h after treatment and no viable tumor tissue at 3 weeks after treatment. Our results showed that CpD was as effective as HpD as a photosensitizer for in vivo photodynamic therapy.

Evaluation of thein vivo antithrombotic, anticoagulant and fibrinolytic activities ofLumbricus rubellus earthworm powder.

A saline suspension ofLumbricus rubellus earthworm powder (EWP) was administered to rats (1 g/kg/day) orally for 15 days to evaluate an oral effectiveness for thrombotic disorders. Blood was drawn at 2-day interval after the administration. Several parameters for antithrombotic, anticoagulant and fibrinolytic activities were measured, including platelet aggregation, clotting time, plasmin activity and the levels of FDP (fibrin/fibrinogen degradation products), D-dimer, and t-PA antigen. It did not affect platelet aggregation induced by ADP and collagen but anticoagulant activity (aPTT and TT) was gradually increased to two-folds for the first 5 days of administration and back to normal. Fibrinolytic acitivity of euglobulin fraction was highest on the 11th day after the administration. The level of FDP was elevated to be comparable to the positve control (5-10 mug/ml) after 9-day treatment. Oral administration of the EWP could also reduce the formation of venous thrombus induced with viper venom. Complete blood count (CBC) profiles were within normal ranges except for a slight increase in white blood cells after the oral administration for 15 days. These results suggested that the EWP may be valuable for the prevention and/or treatment of thrombotic diseases.

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.

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.

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.