Specific or non-specific cytolytic processes of free-living amoebae causing meningoencephalitis have been emphasized and the cytolytic ability related to hydrolases in Entamoeba sp. and Naegleria sp. has also been reported since the latter half of 1970's. However, no information on hydrolase activities in Acanthamoeba sp. is available. Hydrolases in Acanthamoeba culbertsoni, a pathogenic species of free-living amoebae, were assayed and compared with those in a non-pathogenic species, A. royreba. Pathogenicity of these two species was confirmed through experimental infection to BALB/c mice. Hydrolase activities and cytotoxic effects between pathogenic and non-pathogenic species were compared in the trophozoites cultured in CGV media and in CHO cell line, respectively. The results are summarized as follows: The mice infected with A. culbertsoni were all dead 15 days after nasal inoculation, and the mean survival time was 8.5 days. Also the mice infected with this pathogenic species mani fested typical meningoencephalitis, whereas the mice infected with A. royreba did not. Hydrolases detected both in the cell extracts and culture media were acid phosphatase, beta- N-acetyl galactosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase, neutral proteinase and acid proteinase, all of which were detected with remarkably higher rate in A.culbertsoni than in A. royreba. A. culbertsoni revealed strong cytotoxicity for the target CHO cells, whereas A. royreba did not show any specific cytotoxicity. About 80 % of the target cells mixed with A. culbertsoni were dead 48 hours after cultivation, and more than 95% of the target cells were dead 72 hours after cultivation. Hydrolase activities in A. culbertsoni cultured with the target cell line were assayed according to the culture time. The activities of acid phosphatase, beta-N-acetyl glucosaminidase, beta-N-acetyl glucosaminidase, alpha-mannosidase and acid proteinase in this pathogenic amoeba were detected higher in amoeba extracts than in culture media up to 120 hours after cultivation, but after 120 hours of cultivation those activities were detected higher in culture media than in the amoeba lysates. Neutral proteinase activity in A. culbertsoni increased more in EBSS medium than in the lysate specimens although the activity in the extracts was generally steady according to the cultivation time. Summarizing the above results, it is concluded that there were differences in hydrolase activities between pathogenic A. culbertsoni and non-pathogenic A. royreba, and that some hydrolase activities were detected remarkably higher in A. culbertsoni which revealed strong cytotoxicity to the target CHO cell line.
parasitology-protozoa ; Acanthamoeba culbertsoni ; Acanthamoeba royreba ; biochemistry ; hydrolase ; acid phosphatase ; beta-N-acetyl galactosaminidase ; beta-N-acetyl glucosaminidase ; alpha-mannosidase ; neutral proteinase ; acid proteinase ; mouse ; hydrolase ; acid phosphatase ; beta-N-acetyl galactosaminidase ; beta-N-acetyl glucosaminidase ; alpha-mannosidase ; neutral proteinase ; acid proteinase

Proteinase inhibitors are widely distributed in many living organisms and play crucial roles in many biological processes, particularly in regulating the proteinase activity spatially and temporally. However, The Kazal family of serine protease inhibitors is one of the most important and extensively studied protease inhibitor families. This type of protease inhibitor normally consists of one or several domains. Every domain has a highly conserved sequence structure and molecular conformation. It is found that contact residues are hyper variable, which are responsible for the interaction of inhibitors and proteinases. Most of them are in the solvent exposed loop. But P1 residue is the key active site of the interaction between inhibitor and enzyme. The types of the amino acid at P1 site likely play an important role in causing different inhibitory activity. The substitutions at the contact residues cause significant effects on the association constant. By using the Laskowski algorithm, the Ki values of a Kazal domain against six serine proteinases can be predicted from the domain' s sequence alone. At present there are many Kazal proteinase inhibitors found in the organisms, which show important biological functions. This article gives a comprehensive review of the newer developments in the characters and the interaction of the Kazal-type inhibitors.
Amino Acid Sequence ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Serine Proteinase Inhibitors ; chemistry ; physiology

PURPOSE: Recent studies have indicated that cytokines and inflammatory responses are related to hemorrhagic shock-(HS) induced acute lung injury. Novel, synthetic, broad-acting serine protease inhibitors that protect a wide range of animals from lethal shock have been evaluated as potential immunoresuscitation modulators. The aim of this study was to test the hypothesis that a test modulator could decrease serum and tissue pro-inflammatory mediator levels, prevent HS-induced acute lung injury, and suppress activation of the inflammatory cascade. METHODS: This HS model consisted of four phases: Phase I, initiation of HS (from 15~30 min) with a volume-controlled hemorrhage of 2.7 mL/100 g over 15 min; Phase II, maintenance of HS (HS Phase, from 30~90 min), with maintenance of shock without resuscitation; Phase III, resuscitation (RT phase, from 90~150 min), with reinfusion of 1.5 mL/100 g of blood and Ringer's lactate fluid; and Phase IV, observation and post-resuscitation (OB phase, from 150~270 min). The test rats were randomized into two groups of 15: group 1 with fluid resuscitation (control group) and group 2 with fluid and 0.5mg/kg nafamostat mesilateinfusion (treated group). RESULTS: The mean arterial pressure (MAP) of the treated group increased significantly during the observation and post-resuscitation period (Phase IV, OB 90 min). The heart rate of the control group increased significantly during the maintenance of shock (Phase II, HS 60 min), resuscitation (Phase III, RT 30 and 60 min), and observation periods (Phase IV, OB 120 min). The serum concentrations for IL-6 and IL-10 did not differ significantly between the treated and control groups. The TNF-alpha levels for the treated group were significantly lower than those of the control group (p<0.05). At the end of the observation period (OB 120 min), the treated group had significantly lower concentrations of IL-8 in the bronchoalveolar lavage fluid (BALF) than the control group (676.7+/-791.9 vs. 1062.5+/-609.9, p=0.013). CONCLUSION: We conclude that the tested serine protease inhibitor improves hemodynamic parameters, prevents acute lung injury after HS, and attenuates a robust proinflammatory cytokine response in rats.
Acute Lung Injury ; Animals ; Arterial Pressure ; Bronchoalveolar Lavage Fluid ; Cytokines ; Guanidines ; Heart Rate ; Hemodynamics ; Hemorrhage ; Interleukin-10 ; Interleukin-6 ; Interleukin-8 ; Isotonic Solutions ; Lactic Acid ; Lung ; Rats ; Resuscitation ; Serine Proteases ; Serine Proteinase Inhibitors ; Shock ; Shock, Hemorrhagic ; Tumor Necrosis Factor-alpha

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.
Taenia solium/*enzymology ; Serum Albumin, Bovine/metabolism ; Leucine/analogs & derivatives/pharmacology ; Iodoacetic Acid/pharmacology ; Immunoglobulin G/metabolism ; Humans ; Cysteine Proteinase Inhibitors/pharmacology ; Cysteine Endopeptidases/chemistry/*isolation & purification/metabolism ; Collagen/metabolism ; Chromatography, Ion Exchange ; Chromatography, Gel ; Animals

OBJECTIVETo investigate the effect of interleukin-6 (IL-6) on the apoptosis of annulus fibrosus (AF) cell induced by interleukin-1beta (IL-1beta).

METHODSCultured AF cells were divided into 6 groups and treated with no drug, 10 ng/mL IL-6, 10 ng/mL IL-1beta, 10 ng/mL IL-1beta and Z-VAD-FMK (a caspase-9 inhibitor), 10 ng/mL IL-1beta and 10 ng/mL IL-6, 10 ng/mL IL-1beta and 100 ng/mL IL-6, respectively. After three days of culture, the apoptosis rate, the positive rates of caspase-3, -8, and -9 of AF cells were detected with flow cytometry.

RESULTSThe apoptosis rates of cells in group 1 to 6 were 2.67% +/- 1.08%, 2.71% +/- 0.53%, 20.37% +/- 1.57%, 11.34% +/- 0.67%, 18.17% +/- 0.74%, and 9.42% +/- 1.08%, respectively. There was no significant difference between group 1 and 2, while the apoptosis rates of group 4, 5, and 6 were significantly lower than group 3 (P = 0.001, P = 0.172, and P = 0.001, respectively). Positive rates of caspase-3 in group 5 (12.35% +/- 0.64%) and 6 (9.26% +/- 0.36%) were significantly lower than group 3 (17.14% +/- 0.72%; P = 0.001 and P < 0.001, respectively). And positive rates of caspase-9 in group 5 (15.13% +/- 1.45%) and 6 (10.17% +/- 2.50%) were significantly lower than group 3 (19.4% +/- 0.98% ; P = 0.014 and P = 0.004, respectively). But there was not obvious change of caspase-8 activity after IL-6 was added.

CONCLUSIONIL-6 is capable of protecting AF cells from IL-1beta induced apoptosis in vitro. Mechanism of the protection is related with the inhibition of caspase-3 and -9 activities.

Amino Acid Chloromethyl Ketones ; pharmacology ; Animals ; Apoptosis ; drug effects ; Caspase Inhibitors ; Cells, Cultured ; Cysteine Proteinase Inhibitors ; pharmacology ; Interleukin-1beta ; pharmacology ; Interleukin-6 ; pharmacology ; Intervertebral Disc ; cytology ; drug effects ; enzymology ; Rabbits

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.
Amino Acid Chloromethyl Ketones/pharmacology ; Antineoplastic Agents/*pharmacology ; Caspases/antagonists & inhibitors/metabolism ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Cysteine Proteinase Inhibitors/pharmacology ; Cytochrome c Group/metabolism ; Drug Resistance, Neoplasm/genetics ; Humans ; Neoplasms/therapy ; RNA, Small Interfering ; Telomerase/*antagonists & inhibitors/genetics/metabolism ; bcl-2-Associated X Protein/metabolism