Biochemical and cellular activity of chemically synthesized elastase inhibitor (S-AFUEI) from Aspergillus fumigatus.

PURPOSE: Elastase, produced by Aspergillus fumigatus and A. flavus, is an important pathogenic factor in pulmonary aspergillosis. We investigated the possibility of using A. fumigatus-derived A. fumigatus elastase inhibitor (AFUEI) as a therapeutic agent. As native-AFUEI (N-AFUEI) has an extremely low yield, we generated a synthetic-AFUEI (S-AFUEI) and investigated whether S-AFUEI has a biological activity against A. fumigatus elastase (AFUE) and inhibits cytotoxicity. METHODOLOGY: A. fumigatus was cultured in Yeast Carbon Base (YCB) -elastin culture medium for 3-7 days, and AFUE was purified by chromatography using DE52 cellulose and Sephadex G-75 column. Elastolytic activity was examined using Glt-Ala-Ala-Pro-Leu-pNA (GAAPLNA) as the substrate. The hydrolytic activity of AFUE was determined using the characteristic substrates, fibrinogen and collagen (Type IV), and human cell cytotoxicity was measured colorimetrically. Furthermore, the inhibitory effect of S-AFUEI on these activities was examined. RESULTS: We confirmed that S-AFUEI demonstrated elastase inhibitory activity and heat stability equivalent to that demonstrated by N-AFUEI, and inhibited human collagen hydrolytic activity and human fibrinogen hydrolytic activity. Further, S-AFUEI inhibited cytotoxicity in AFUE human pulmonary artery endothelial cells (HPAEC), human small airway epithelial cells (HSAEC), and human pulmonary alveolar epithelial cells (HPAEpiC). CONCLUSION: As S-AFUEI strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.

Sumatriptan alleviates nitroglycerin-induced mechanical and thermal allodynia in mice.

The association between the clinical use of nitroglycerin (NTG) and headache has led to the examination of NTG as a model trigger for migraine and related headache disorders, both in humans and laboratory animals. In this study in mice, we hypothesized that NTG could trigger behavioural and physiological responses that resemble a common manifestation of migraine in humans. We report that animals exhibit a dose-dependent and prolonged NTG-induced thermal and mechanical allodynia, starting 30-60 min after intraperitoneal injection of NTG at 5-10 mg/kg. NTG administration also induced Fos expression, an anatomical marker of neuronal activity in neurons of the trigeminal nucleus caudalis and cervical spinal cord dorsal horn, suggesting that enhanced nociceptive processing within the spinal cord contributes to the increased nociceptive behaviour. Moreover, sumatriptan, a drug with relative specificity for migraine, alleviated the NTG-induced allodynia. We also tested whether NTG reduces the threshold for cortical spreading depression (CSD), an event considered to be the physiological substrate of the migraine aura. We found that the threshold of CSD was unaffected by NTG, suggesting that NTG stimulates migraine mechanisms that are independent of the regulation of cortical excitability.

Thrombin-like enzyme, flavovilase, with kinin-releasing activity from Trimeresurus flavoviridis (habu) venom.

A thrombin-like enzyme, flavovilase, with kinin-releasing activity was isolated, purified, and characterized from the venom of Trimeresurus flavoviridis (habu) using Sephadex G-100, DEAE-Cellulose, and CM-Cellulose column chromatographies. The final preparation was homogeneous as demonstrated by a single band on polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectric focusing electrophoresis. The enzyme possesses a molecular weight of 26,500, an isoelectric point of 5.0, and consists of 247 total amino acid residues. Specific electrolytic activities of this enzyme on N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE) were determined to be 50.9 and 17.4 micromol/min/mg, respectively. The enzyme was inhibited by p-APMSF (p-amidinophenylmethanesulfonyl fluoride hydrochloride), beta-mercaptoethanol, and N-bromosuccinimide. Additionally, the enzyme was found stable to heat treatment. It was also observed that the enzyme cleaved a kininogen analog with the release of bradykinin.

Characterization of coagulase from Staphylococcus intermedius.

A protein coagulase was isolated from Staphylococcus intermedius 6131 using bovine prothrombin-Sepharose 4B and Bio-gel P-4 column chromatographies. Homogeneity was demonstrated by the formation of a single band in polyacrylamide gel electrophoresis and isoelectric focusing. The purified preparation possesses a molecular weight of 64,500, an isoelectric point of 4.1, consists of 615 total amino acid residues and demonstrates coagulase activity for human and rabbit fibrinogen, but does not show the activity for rat or guinea pig fibrinogens. This purified protein contains galactose and fucose, and the amino-terminal amino acid sequence was determined. The coagulase activity is inhibited by N-bromosuccinimide (NBS), suggesting that tryptophan is involved in this activity. The coagulase was heat stable to 80 degrees C and stable to pH over the range of 7-9. This is the first report of coagulase from Staphylococcus intermedius.

Hemorrhagic activity and muscle damaging effect of Pseudomonas aeruginosa metalloproteinase (elastase).

Pseudomonas aeruginosa elastase, a Bacillus subtilis thermolysin-like zinc-proteinase was examined for hemorrhagic activity and its effect on muscle and endothelial cells. Subcutaneous and intramuscular injections of elastase into mice caused severe hemorrhage with an acute increase of creatine phosphokinase activity in serum. The elastase also possessed fibrinogenolytic and fibrinolytic activities. The Aalpha and Bbeta chains of fibrinogen were completely hydrolyzed as demonstrated by their electrophoretic disappearance on SDS polyacrylamide gels. The pathological study indicates that elastase induces changes in the structure of the vascular wall and causes leakage of the plasma component and red and white blood cells into the extravascular tissue. This is further supported by results showing injury to cultured endothelial cells and macrophages. These data indicate that P. aeruginosa elastase directly affects endothelial cells and destroys the basement membrane of blood vessels to cause hemorrhage. Since fibrinogenolytic activity is an additional component of this elastase and this activity induces the hemorrhagic tendency, the damage in tissues could become increasingly severe.

Lumbar sympathetic block for pain relief in two patients with interstitial cystitis.

BACKGROUND AND OBJECTIVES: Interstitial cystitis (IC) is characterized clinically by lower abdominal pain, pain during urination, and increased frequency of urination. Treatment of the symptoms in IC remains challenging. We report effective treatment using lumbar sympathetic block for 2 patients with IC. CASE REPORT: A 63-year-old and 78-year-old woman were diagnosed with IC. Medical therapy with nonsteroidal anti-inflammatory drugs (NSAID), anticholinergics, and hydrodistention of the bladder failed to improve their symptoms. Subsequently, a continuous lumbar epidural block using 1% mepivacaine was used in these patients. A transient reduction of the symptoms in both patients was achieved. A lumbar sympathetic block with a neurolytic agent produced almost complete, and long-lasting relief of their symptoms. CONCLUSION: Lumbar sympathetic block using a neurolytic agent produced long-lasting pain relief in 2 patients with IC. Reg Anesth Pain Med 2001;26:271-273.

A new thrombin-like enzyme, flavoviridiobin from the venom of Trimeresurus flavoviridis (habu).

Fibrinopeptide A and B releasing enzyme, flavoviridiobin, was isolated from the venom of Trimeresurus flavoviridis using Q-Sepharose, CM-Cellulose, and Sephadex G-75 column chromatographies. Homogeneity was established by the formation of a single band in polyacrylamide gel electrophoresis, isoelectric focusing, and Ouchterlony immunodiffusion. The enzyme has a molecular weight of 48,000, isoelectric point of 8.1, consists of 237 total amino acid residues, and demonstrates clotting activity. However, no tosyl-L-arginine methyl ester (TAME) hydrolytic and kinin-releasing activities were observed. This clotting enzyme was inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride (p-APMSF), benzamidine, and beta-mercaptoethanol, suggesting that serine, acidic amino acids, and disulfide bonds are involved in the expression of the enzyme's clotting activity. This thrombin-like enzyme hydrolyzes B beta-chain of human fibrinogen at first, followed by hydrolysis A alpha-chain. The enzyme was stable over the pH range of 7-10 and was shown to be heat resistant.

Primary structure and functional characterization of bilitoxin-1, a novel dimeric P-II snake venom metalloproteinase from Agkistrodon bilineatus venom.

The amino acid sequence of the hemorrhagic toxin, bilitoxin-1, isolated from the venom of Agkistrodon bilineatus was determined by the Edman sequencing procedure of peptides derived from digests utilizing cyanogen bromide, clostripain, lysyl endopeptidase, and Staphylococcus aureus V8 protease. A molecular mass of 80,000 Da was observed in the nonreduced state and 48,000 Da was observed in the reduced state, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each subunit consists of 291 amino acid residues and has a calculated molecular mass of 32,276 Da. The toxin contains fucose, galactosamine, glucosamine, galactose, mannose, and N-acetylneuraminic acid and three N-linked glycosylation consensus sites. Hydrazinolysis and ESI mass spectrometry revealed that asparagine was the carboxyl-terminal amino acid. The disintegrin-like domain of bilitoxin-1 lacks the RGD cell-binding sequence, which is substituted by the MGD sequence. Under certain conditions, the disintegrin domain is autoproteolytically processed from the native protein. Studies with the bilitoxin disintegrin demonstrated that it lacks platelet aggregation inhibitory activity, probably reflecting the substitution of RGD by MGD. The hemorrhagic activity of the asialobilitoxin-1 was only 25% of bilitoxin-1, while proteolytic activity was unaffected. The three-dimensional structure of this toxin was modeled and was shown to likely possess a structure similar to that of adamalysin II (Gomis-Rüth et al., EMBO J. 12, 151-157 (1993)) and the disintegrin kistrin (Adler et al., Biochemistry 32, 282-289 (1993)). In summary, here we report the first primary structure of a dimeric, P-II snake venom metalloproteinase and the biological role of bilitoxin-1 glycosylation and the disintegrin domain.

Edema factor from the venom of Trimeresurus elegans (Sakishimahabu).

An edema factor was isolated from the venom of Trimeresurus elegans using HW-55, CM-Cellulose, and Mono S column chromatographies. Homogeneity was demonstrated by the formation of a single band in polyacrylamide gel electrophoresis (pH 8.3). The edema factor has a molecular weight of 25,500, an isoelectric point of 7.5, and express edema, proteolytic and capillary permeability-increasing activities. Edema, proteolytic and capillary permeability-increasing activities are inhibited by ethylenediaminetetraacetic acid (EDTA), o-phenanthroline, and N-bromosuccinimide. Additionally, this factor exhibits kinin-releasing activity. The edema factor possesses proteolytic activity as shown by hydrolyzing the Val(3)-Asn(4), His(5)-Leu(6), Ser(9)-His(10), Ala(14)-Leu(15), Leu(15)-Tyr(16), Tyr(16)-Leu(17), and Glu(21)-Arg(22) bonds of oxidized insulin B chain. The A alpha, B beta, and gamma chains of human fibrinogen were also hydrolyzed. The edema factor was found to contain 1 mol of zinc and 2 mols of calcium per mol of protein and the amino-terminal sequence was determined.

[Studies on hemorrhagic toxins from the venoms of Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis and Agkistrodon acutus and arginine ester hydrolases from Trimeresurus mucrosquamatus venom].

Venom samples were corrected from several poisonous snakes, such as Bungarus multicinctus, Trimeresurus mucrosquamatus, T. gramineus, T. flavoviridis, and Agkistrodon acutus, and stored in a desiccator at room temperature for 25 to 31 years. Then they were compared with fresh venoms as to their biological activities. The characteristic local symptoms produced by the bite of venomous snakes of Crotalidae and Viperidae are hemorrhage, necrosis and muscular degeneration. Hemorrhagic toxins were purified from Trimeresurus mucrosquamatus, Crotalus ruber ruber, Vipera aspis aspis, and Agkistrodon acutus venoms and their biological, biochemical, and pathological properties were investigated. Arginine ester hydrolases are present in the venoms of Crotalidae and Viperidae, but are not found in the venoms of Elapidae and Hydrophiidae. In this paper we describe the enzymatic and biological activities of arginine ester hydrolases from a Trimeresurus mucrosquamatus venom.

Amino acid sequence and biological properties of the lectin from the venom of Trimeresurus okinavensis (Himehabu).

A lectin was isolated from the venom of Trimeresurus okinavensis (Himehabu) and the complete amino acid sequence was determined using clostripain, lysyl endopeptidase, and V8 protease. The hemagglutinating activity of this lectin are reported.

Himehabu lectin, a novel inducer of Ca2+-release from the venom of the snake Trimeresurus okinavensis, in sarcoplasmic reticulum.

The lectin himehabu lectin (HHL) has recently been isolated from crude venom of the snake Trimeresurus okinavensis. Ca2+ -electrode and fluorescent Ca2+ -indicator experiments showed that HHL induced release of Ca2+ from the heavy fraction of skeletal muscle sarcoplasmic reticulum (HSR). The release of Ca2+ induced by caffeine from HSR was abolished by ryanodine, Mg2+ and ruthenium red, typical inhibitors of Ca2+ -release channels, whereas that induced by HHL was only partially reduced by these inhibitors. HHL, unlike caffeine, had no effect on [3H]ryanodine binding to HSR. These results suggest that HHL induces release of Ca2+ which is at least partially mediated through Ca2+ -release channels with novel pharmacological properties.

Vascular endothelial growth factor VEGF-like heparin-binding protein from the venom of Vipera aspis aspis (Aspic viper).

The heparin-binding dimeric hypotensive factor (HF) was purified from Vipera aspis aspis (Aspic viper) venom [Komori, Y. and Sugihara, H. (1990) Toxicon 28, 359-369]. In this study, the amino acid sequence, and structure and function of HF, were elucidated. By electrospray ionization mass spectrometry (ESI-MS), the molecular weight of HF was determined to be 25 072.1. The complete amino acid sequence of HF was determined by Edman sequencing of the S-pyridylethylated HF and its peptides derived from enzymatic digestion. The theoretical molecular mass calculated from the primary structure agrees well with the molecular weight determined by ESI-MS. HF consists of two homogeneous monomers bound covalently. The monomer with an N-terminal blocked by pyroglutamic acid contains 110 amino acid residues, including eight cysteine residues, two of which are considered to be involved in intermolecular disulfide bonds. Sequential homology search revealed that the primary structure of HF is similar to that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) with a sequential homology of 45 and 22%, respectively. When injected intradermally into a rat, an increase in capillary permeability was observed with HF or VEGF. On the other hand, only HF exerted a strong hypotensive effect after intravenous injection of samples into a rat. Purified HF has a mitogenic effect on endothelial cells. Through the use of bovine aortic endothelial cells (BAEC), the half-maximal mitogenic concentration of HF was determined to be 5-5. 5 nM (125-138 ng/mL). Similarly, VEGF had a mitogenic concentration at 0.5-1 nM. When incubated with HF and cycloheximide or HF and heparin, the cell growth was inhibited, suggesting that the mechanism of action of HF is similar to that of VEGF.

The effect of an Aspergillus fumigatus derived elastolytic proteinase on human neutrophils.

The effect of elastolytic proteinase on quantitative nitroblue tetrazolium (NBT) dye reduction and chemotaxis of human neutrophil was examined. Elastolytic proteinase is derived from Aspergillus fumigatus 9409. NBT dye reduction was inhibited significantly by 133microEg/ml of elastolytic proteinase (p<0.05). Chemotaxis was also inhibited significantly by 400microEg/ml of the enzyme, compared to the control group (p<0.05). These findings suggest that elastolytic proteinase, depending on its concentration, acts as an inhibitory agent to both NBT dye reduction and chemotaxis activities in the human neutrophil. The findings that this enzyme suppressed neutrophil function in A. fumigatus infection is of importance, because it is now suspected that the enzyme has the potential to cause infection.

Primary structure and biological activity of snake venom lectin (APL) from Agkistrodon p. piscivorus (Eastern cottonmouth).

A lectin (APL) was purified from the venom of Agkistrodon piscivorus piscivorus (Eastern cottonmouth moccasin). APL is a disulfide-linked, homodimeric protein consisting of identical monomers of molecular weight 16,200. Native rabbit and human erythrocytes were agglutinated by APL and the activity was found to be calcium-dependent. Galactose, lactose, rhamnose and EGTA strongly inhibited the hemagglutination activity of APL. The complete amino acid sequence determined by Edman sequencing of the S-pyridylethylated derivative and its peptides derived from enzymatic digestion indicate the structure of APL to be highly homologous with lectins and the platelet glycoprotein Ib (GPIb)-binding proteins isolated from other snake venoms. These results suggest that APL belongs to the C-type beta-galactoside binding lectin family which possess structural similarities with the C-terminal carbohydrate-recognition domain (CRD) of animal membrane lectins.

Partial amino acid sequence and biological characterization of elegatoxin, hemorrhagic toxin from Trimeresurus elegans (Sakishimahabu) venom.

A hemorrhagic toxin, designated Elegatoxin, was isolated from the venom of Trimeresurus elegans using HW-55, DEAE-Sephacel, CM-Cellulose and Mono S column chromatographies. The purified toxin was shown to be homogeneous by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, isoelectric electrophoresis, and Ouchterlony immunodiffusion. Elegatoxin has a molecular weight of 26,000 with an isoelectric point of 8.6. The toxin demonstrated both hemorrhagic and proteolytic activities. Hemorrhagic activity was inhibited by ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis-(2-amino-ethylether)N,N'-tetraacetic acid (EGTA), o-phenanthroline, and N-bromosuccinimide, but not by amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF). The minimum hemorrhagic dose was found to be 0.8 microgram/mouse. Elegatoxin possesses proteolytic activity as evidenced by hydrolyzing type IV collagen, actin and the A alpha, B beta, and gamma chains of bovine fibrinogen. This purified toxin contains 1 mol of zinc and 2 mols of calcium per mol of protein and a partial amino acid sequence was determined. The pathological and biochemical properties of Elegatoxin were investigated, and these results are reported in this paper.

[The role of virulence factor of elastolytic proteinase from Aspergillus fumigatus].

The role of virulence factor of elastolytic proteinase produced by Aspergillus fumigatus was investigated in mice immunocompromised with cyclophosphamide (CY) to block neutrophil and lymphocyte functions or with carrageenan (CA) to block the phagocytic activity of macrophages. These mice were infected through inhalation with the spores of elastolytic proteinase producing or nonproducing strains of A. fumigatus, and then were observed daily for 15 days or until death. Compared to CY-immunocompromised mice treated with the spores of proteinase nonproducing strains, CY-immunocompromised mice treated with the spores of proteinase producing strains had a significantly shorter survival rate. Furthermore, when treated with the spores of proteinase producing strains, CA-immunocompromised mice survived significantly longer than did CY-immunocompromised mice. Assessment of the effect of elastolytic proteinase on rat lung showed that this enzyme induced hemorrhage of the lung, alveolar septal edema and alveolar hypertrophy due to blood neutrophil infiltration. Neither fusion nor denaturation of the elastic fiber was observed, however. These studies have suggested that when infected with A. fumigatus, a severe lesion in bronchioli or alveoli is induced by the proteinase producing strains, and that these strains are more virulent than the proteinase nonproducing strains.

Brain dysfunction associated with an induction of nitric oxide synthase following an intracerebral injection of lipopolysaccharide in rats.

We investigated the pathophysiological role of nitric oxide synthesized by inducible nitric oxide synthase in the brain, by injecting lipopolysaccharide directly into the rat cerebral cortex/hippocampus. The levels of nitric oxide metabolites, nitrite and nitrate, began to increase in a dose-dependent manner with a 3-h lag, and reached approximately seven-fold the basal levels 8 h after the direct injection of lipopolysaccharide (5 microg). The lipopolysaccharide-induced increase in nitrite and nitrate levels was inhibited by treatment with the specific inducible nitric oxide synthase inhibitor aminoguanidine. The protein synthesis inhibitor cycloheximide delayed the onset of the increase in nitric oxide metabolite levels, and reduced the peak levels. Lipopolysaccharide increased Ca2+-independent, but not Ca2+-dependent, nitric oxide synthase activity in the brain. Intense nicotinamide adenine dinucleotide phosphate-diaphorase activity was observed in round cells in the vicinity of the site of injection of lipopolysaccharide 8 h after the injection. Neuronal death was observed seven days after the injection of lipopolysaccharide. Spatial memory, as assessed by performance in a water maze task and spontaneous alternation behavior in a Y-maze, was significantly impaired in rats which had had previous bilateral injections of lipopolysaccharide into the hippocampus. The lipopolysaccharide-induced neuronal death and spatial memory impairments were prevented by aminoguanidine. These results suggest that direct injection of lipopolysaccharide into the brain causes an induction of inducible nitric oxide synthase in vivo. Furthermore, it is suggested that nitric oxide produced by inducible nitric oxide synthase is responsible for the lipopolysaccharide-induced brain dysfunction.

Biological properties of kinin-releasing enzyme from Trimeresurus okinavensis (himehabu) venom.

A kinin-releasing enzyme was isolated and characterized from the venom of Trimeresurus okinavensis (himehabu) using Sephadex G-100, DEAE-Cellulose, and CM-Cellulose column chromatographies. The kinin-releasing enzyme was homogeneous as demonstrated by a single band on polyacrylamide gel electrophoresis, and isoelectric focusing. The enzyme possesses a molecular weight of 31,000 Da and isoelectric point of 8.2 and consists of 312 total amino acid residues. Specific esterolytic activities of the kinin-releasing enzyme on N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethylester (BAEE) were determined to be 235.3 and 111.3 mumol/min/mg, respectively. The enzyme was inhibited by p-APMSF (p-amidinophenylmethanesulfonyl fluoride hydrochloride) and benzamidine. Additionally, the enzyme was found stable to heat treatment. The enzyme cleaved a kininogen analog with the release of bradykinin, resulting in an immediate drop in blood pressure, and contractions of the rat uterus were also observed.

Comparison of the lethal components in Vipera aspis aspis and Vipera aspis zinnikeri venom.

Biological activities in Vipera a. aspis and V. a. zinnikeri venom were investigated and compared. Phospholipase A2 and lethal activities were found to be much higher in V. a. zinnikeri venom; the LD50 values for V. a. aspis and V. a. zinnikeri crude venom were 0.55 and 0.35 microgram/g, while PLA2 activities were 25.4 and 41.8 unit/mg, respectively. Other enzymatic and pharmacological activities investigated were similar in both venoms, suggesting that PLA2s might be responsible for the higher toxicity of V. a. zinnikeri venom. PLA2s contained in both venoms were compared immunologically, and the highly lethal phospholipase A2 (PLA2-I) purified from V. a. zinnikeri venom was not found in V. a. aspis venom as determined by antiserum for purified PLA2-I. The toxic effect of V. a. zinnikeri venom was inhibited by anti-PLA2-I, while the same antiserum could not prevent lethality by V. a. aspis venom. These results indicate that PLA2-I in V. a. zinnikeri venom possesses an important role in the lethal activity of this venom. Although V. a. zinnikeri is found in a defined area of France, it is possible that its lethal venom component developed differently than that of V. a. aspis.