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International audience; Background: The aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL. Methods: The expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot. Results: We demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax. Conclusion: We identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL.

A 48-year-old male was admitted with severe community-acquired pneumonia after a family visit to turkey. Upon specific questioning he reported having close contact to a tame budgerigar. Sputum PCR for Chlamydia psittaci turned positive. After treatment adaptation to doxycycline he quickly improved. Detailed anamnesis is paramount for establishing correct diagnosis.

Intracellular pH joins the regulatory apparatus of the TMEM16 scramblase module.

In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.

In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.

High transfection efficiency and low cytotoxicity are the two key factors to be considered in the design of gene carriers. Herein, a novel and versatile gene carrier (PLL-RT) was prepared by introducing “molecular string” RT (i.e., p-toluylsulfonyl arginine) onto the polylysine backbone. The introduction of RT string contributed to the formation of multiple interactions between the polycationic gene carriers and cell membrane or DNA, as well as adopting α-helix conformation, all of which would be beneficial to enhance the gene transfection. In addition, RT string grafted onto other polycations such as hyperbranced PEI25k and dendrimer PAMAM could also acquire improved transfection efficiency and low cytotoxicity. Moreover, PLL-RT presented significant tumor inhibition effect in vivo. This work provided an effective strategy for constructing novel gene carriers with high transfection and low cytotoxicity.

Although the uniform initial hydroxylation of methyl tert-butyl ether (MTBE) and other oxygenates during aerobic biodegradation has already been proven by molecular tools, variations in carbon and hydrogen enrichment factors (ε(C) and ε(H)) have still been associated with different reaction mechanisms (McKelvie et al. Environ. Sci. Technol. 2009, 43, 2793-2799). Here, we present new laboratory-derived ε(C) and ε(H) data on the initial degradation mechanisms of MTBE, ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by chemical oxidation (permanganate, Fenton reagents), acid hydrolysis, and aerobic bacteria cultures (species of Aquincola, Methylibium, Gordonia, Mycobacterium, Pseudomonas, and Rhodococcus). Plotting of Δδ(2)H/ Δδ(13)C data from chemical oxidation and hydrolysis of ethers resulted in slopes (Λ values) of 22 ± 4 and between 6 and 12, respectively. With A. tertiaricarbonis L108, R. zopfii IFP 2005, and Gordonia sp. IFP 2009, ε(C) was low (|-1|‰) and ε(H) was insignificant. Fractionation obtained with P. putida GPo1 was similar to acid hydrolysis and M. austroafricanum JOB5 and R. ruber DSM 7511 displayed Λ values previously only ascribed to anaerobic attack. The fractionation patterns rather correlate with the employment of different P450, AlkB, and other monooxygenases, likely catalyzing ether hydroxylation via different transition states. Our data questions the value of 2D-CSIA for a simple distinguishing of oxygenate biotransformation mechanisms, therefore caution and complementary tools are needed for proper interpretation of groundwater plumes at field sites.

Methyl tert-butyl ether (MTBE) and benzene are added to gasoline to improve the combustion process and are found in the urban environment as a consequence of vehicular traffic. Herein we evaluate urinary MTBE (MTBE-U) and benzene (BEN-U) as biomarkers of exposure to urban traffic. Milan urban policemen (130 total) were investigated in May, July, October, and December for a total of 171 work shifts. Personal exposure to airborne benzene and carbon monoxide (CO), and atmospheric data, were measured during the work shift, while personal characteristics were collected by a questionnaire. A time/activity diary was completed by each subject during the work shift. Spot urine samples were obtained for the determination of MTBE-U and BEN-U. Median personal exposure to CO and airborne benzene were 3.3 mg/m3 and 9.6 μg/m3, respectively; median urinary levels in end-of-shift (ES) samples were 147 ng/L (MTBE-U) and 207 ng/L (BEN-U). The time spent on traffic duty at crossing was about 40% of work time. Multiple linear regression models, taking into account within-subject correlations, were applied to investigate the role of urban pollution, atmospheric conditions, job variables and personal characteristics on the level of biomarkers. MTBE-U was influenced by the month of sampling and positively correlated to the time spent in traffic guarding, CO exposure and atmospheric pressure, while negatively correlated to wind speed (R2 for total model 0.63, P

The use of ETBE (ethyl-tert-butylether) as gasoline additive has recently grown rapidly. Contamination of aquatic systems is well documented for MTBE (methyl-tert-butylether), but less for other gasoline additives. Due to their mobility they may easily reach drinking water collection areas. Odour and flavour thresholds of MTBE are known to be low, but for ETBE and TAME (methyl-tert-amylether) hardly information is available. The objective here is to determine these thresholds for MTBE, ETBE and TAME. and relate these to concentrations monitored in thousands of samples from Dutch drinking water collection areas.For ETBE odour and flavour thresholds are low with 1-2 mu g L-1, for MTBE and TAME they range from 7 to 16 mu g L-1.In most groundwater collection areas MTBE concentrations are below 0.1 mu g L-1. In phreatic groundwaters in sandy soils not covered by a protective soil layer, occasionally MTBE occurs at higher concentrations. For surface water collection areas a minority of the locations is free of MTBE. For river bank and dune infiltrates, at a few locations the odour and flavour threshold is exceeded. For ETBE fewer monitoring data are available. ETBE was found in 2 out of 37 groundwater collection areas, in concentrations below 1 mu g L-1. In the surface water collection areas monitored ETBE was found in concentrations near to the odour and flavour thresholds.The low odour and flavour thresholds combined with the high mobility and persistence of these compounds, their high production volumes and their increased use may yield problems with future production of drinking water.

The supramolecular interactions of Mg(ii) and N(alpha)-4-tosyl-l-arginine methyl ester hydrochloride (TAME) with ATP have been investigated using (1)H and (31)P NMR spectra. Furthermore, the hydrolysis of ATP catalyzed by Mg(ii) and TAME has been studied at 60 degrees C and pH 7 using (31)P NMR spectra. In the Mg(ii)-ATP-TAME ternary system, the binding interaction of Mg(2+) with ATP involves not only N1 and N7 in the adenine ring but also beta- and gamma-phosphate of ATP. The binding forces are mainly electrostatic interaction and cation (Mg(2+))-pi interaction. The guanidinium group and the aromatic ring of TAME interacts with ATP by beta and gamma phosphate and the adenine ring of ATP. The binding forces are mainly electrostatic interactions and pi-pi stacking. A significant difference between the binary and the ternary system indicates that TAME is essential to the stablization of the intermediate. Kinetic studies show that the hydrolysis rate constant of ATP is 2.16 x 10(-2) h(-1) at pH 7 in the Mg(ii)-TAME-ATP ternary system. The Mg(ii) ion and TAME can accelerate the ATP hydrolysis process. A possible mechanism has been proposed that the hydrolysis occurs through an addition-elimination, in which the phosphoramidate intermediate was observed at 3.21 ppm in the (31)P NMR of the ternary system. These results provide further information concerning the effect of the key amino acid residue and metal ions as cofactors of ATPase on ATP synthesis/hydrolysis at the molecular level.

Methyl tert-butylether (MTBE) used as fuel oxygenate poses problems for water suppliers since it is persistent in the aquatic environment and the removal efficiency by conventional water treatment methods (aeration or activated carbon filtration) is rather low. Substitution by other ether compounds such as ethyl tert-butylether (ETBE), tert-amylmethylether (TAME) or di-isopropylether (DIPE) is discussed, however, their environmental behaviour is similar to that of MTBE. Experiments investigating the elimination efficiency of AOP were carried out in tap water and water from Lake Constance. The elimination efficiency for all treatment processes was found to follow the order: For all compounds under investigation, neither pure ozonation nor UV irradiation yield a considerable concentration decline. Only the formation of highly reactive OH radicals shows a potential for removing the ethers from water. Therefore the addition of H2O2 in equimolar ratio prior to ozone admixing proved to be quite efficient. The application of combined UV/H2O2 showed good results in all cases; the best concentration decline was achieved with UV/ozone. The rate of elimination of the three substitutes for MTBE (ETBE, TAME and DIPE) is higher in all processes; nevertheless, no complete removal could be achieved. Therefore, from the point of view of water suppliers, the use of other ethers as substitute for MTBE is posing the same problems as MTBE.

Three isoforms of anionic chum salmon trypsin (ST-1, ST-2, and ST-3) were purified from the pyloric caeca of chum salmon (Oncorhynchus keta). The molecular weights of the three isoforms were about 24 kDa as determined by SDS-PAGE. The isoelectric points of ST-1, ST-2, and ST-3 were 5.8, 5.4, and 5.6, respectively. The apparent K(m) values of two isoforms (ST-1 and ST-2) for BAPA (benzoyl-L-arginine-p-nitroanilide) hydrolysis at 5, 15, 25 and 35 degrees C were slightly higher than that of the main isoform ST-3, depending on temperature. The turnover numbers, k(cat), of ST-1 and ST-2 were about twice as high as that of ST-3. Consequently, the catalytic efficiencies (k(cat)/K(m)) of ST-1 and ST-2 were more efficient than ST-3. There were marked differences in both apparent K(m) and k(cat) values of three anionic chum salmon trypsins as compared to bovine cationic trypsin. K(m) values of all chum salmon trypsins were approximately 10 times lower than those of bovine trypsin, depending on the temperature. The k(cat) values of all chum salmon trypsins were about 2- to 5-fold higher than those of bovine trypsin; therefore, the catalytic efficiencies (k(cat)/K(m)) of chum salmon trypsin were 20- to 40-fold more efficient than those of bovine trypsin. On the other hand, k(cat)/K(m) values of ST-1 for TAME (tosyl-L-arginine methyl ester) hydrolysis were lower than those of bovine trypsin, whereas k(cat)/K(m) values of ST-2 and ST-3 were comparable to those of bovine trypsin, depending on the temperature.

Deinagkistrodon (formerly Agkistrodon) actus (Taiwan) snake venom was found to contain at least seven closely related coagulant proteases. One of them, named actibin, was purified to homogeneity by means of four chromatographic steps. Actibin acted on fibrinogen to form fibrin clots with extremely high specific activity of 1630 NIH units/mg and preferentially released fibrinopeptide A. Actibin was an acidic glycoprotein (pI 3.4) with molecular weight of 41,000, which was reduced to 28,800 after deglycosylation with N-glycanase. The kcat/Km values of actibin for hydrolysis of tosyl- l -arginine methyl ester and benzoyl- l -arginine p-nitroanilide were one-third to a half those for thrombin, reflecting a high potency of actibin in fibrinogen clotting. The amidase activities of actibin and its family proteases were inhibited by 3,4-dichloroisocoumarin, a serine protease inhibitor, indicating that actibin and its family proteases are serine proteases. Four cDNAs, named DaP1 and DaP7-DaP9, encoding D. actus coagulant proteases were cloned. All cDNAs contain an open reading frame of 780 bp coding for 260 amino acid residues, including a signal peptide of 24 amino acid residues. Their amino acid sequences predicted are highly homologous to one another with one to five amino acid substitutions. When four D. actus protease cDNAs were compared with the cDNAs coding for Trimeresurus flavoviridis and T. gramineus venom serine proteases, accelerated evolution was clearly observed. Similarity of the nucleotide sequences of four D. actus protease cDNAs with no synonymous and one to five nonsynonymous substitutions seems not to be in direct conformity with accelerated evolution. This possibly suggests that they have evolved to a similar direction to enhance their clotting activity rather than to produce other physiological activities.

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.

Hydrolysis of D-valyl-L-leucyl-L-lysine 4-nitroanilide (1), D-valyl-L-leucyl-L-arginine 4-nitroanilide (2), and N alpha-p-tosyl-L-arginine methyl ester (3) by human tissue kallikrein was studied throughout a wide range of substrate concentrations. At low substrate concentrations, the hydrolysis followed Michaelis-Menten kinetics but, at higher substrate concentrations, a deviation from Michaelis-Menten behavior was observed. With the nitroanilides, a significant increase in hydrolysis rates was observed, while with the ester, a significant decrease in hydrolysis rates was observed. The results for substrates (1) and (3) can be accounted for by a model based on the hypothesis that a second substrate molecule binds to the ES complex to produce a more active or an inactive SES complex. The deviation observed for substrate (2) can be explained as a bimolecular reaction between the enzyme-substrate complex and a free substrate molecule.

Two bacterial strains, E1 and E2, isolated from gasoline-polluted soil completely degraded ethyl tert-butyl ether (ETBE), as the sole source of carbon and energy, at specific rates of about 80 mg g(-1) and 58 mg g(-1) of cell protein day(-1), respectively. On the basis of morphological and phenotypic characteristics, strain E1 was tentatively identified as Comamonas testosteroni and strain E2 as belonging to Centre for Disease Control group A-5. The inhibitory effect of metyrapone on the degradative ability of both strains was the first evidence indicating the involvement of a soluble cytochrome P-450 in the cleavage of the ETBE ether bond. This observation was confirmed by spectrophotometric analysis of reduced cell extracts that gave, in the presence of carbon monoxide, a major absorbance peak at about 450 nm. Both strains were also able to degrade, as the sole source of carbon and energy, ETBE's major metabolic intermediates (tert-butyl alcohol and tert-butyl formate) and other gasoline oxygenates (methyl tert-butyl ether and tert-amyl methyl ether). The degradation rates varied considerably, with both strains exhibiting a preferential activity for ETBE's metabolic intermediates.

Alkylphenols and fuel oxygenates are important environmental pollutants produced by the petrochemical industry. A batch biodegradability test was conducted with selected ortho-substituted alkylphenols (2-cresol, 2,6-dimethylphenol and 2-ethylphenol), fuel oxygenates (methyl tert-butyl ether, ethyl tert-butyl ether and tert-amylmethyl ether) and tert-butyl alcohol (TBA) as model compounds. The ortho-substituted alkylphenols were not biodegraded after 100 days of incubation under methanogenic, sulfate-, or nitrate-reducing conditions. However, biodegradation of 2-cresol and 2-ethylphenol (150 mg l(-1)) was observed in the presence of Mn (IV) as electron acceptor. The biodegradation of these two compounds took place in less than 15 days and more than 90% removal was observed for both compounds. Mineralization was indicated since no UV-absorbing metabolites accumulated after 23 days of incubation. These alkylphenols were also slowly chemically oxidized by Mn (IV). No biodegradation of fuel oxygenates or TBA (1 g l(-1)) was observed after 80 or more days of incubation under methanogenic, Fe (III)-, or Mn (IV)-reducing conditions, suggesting that these compounds are recalcitrant under anaerobic conditions. The fuel oxygenates caused no toxicity towards acetoclastic methanogens activity in anaerobic granular sludge.

Bothrops lanceolatus venom has high caseinolytic, phospholipasic, esterolytic and hemorrhagic activities. In spite of having no coagulant effect on plasma, this venom contains a thrombin-like enzyme. Using gel filtration and ion-exchange chromatographies, we have purified an esterolytic fraction (F-II-1a) from this venom with a protein yield of 4% and a 58% recovery in enzyme activity. SDS-PAGE in the presence of β -mercaptoethanol showed that the enzyme is a single chain polypeptide with a M w =38,100. Immunodiffusion and immunoelectrophoresis of fraction F-II-1a against serum from horses immunized with B. lanceolatus venom and against rabbit antiserum prepared using fraction F-II-1a both showed a single immunoprecipitin line. The K m and V max values for TAME hydrolysis were 0.85 mM and 38.6 μ mol/min/mg, respectively. The esterolytic activity was completely inhibited by PMSF (10 mM) but not by EDTA (20 mM). Fraction F-II-1a hydrolyzed the α and β chains of fibrinogen. Degradation of the α chain occurred within 10 min while that of the β -chain was slower. The enzyme had no effect on the γ -chain even after 4 h of hydrolysis.

The physico-chemical and environmental properties of a new gasoline component tert-amyl methyl ether (TAME, 2-methoxy-2-methylbutane) were determined experimentally and environmental hazards of the new component were thereafter identified. The methodology was based on the chemical regulations of the European Union (EU). On the basis of the experimental results, TAME is a volatile, non-hydrophobic and water-soluble liquid, which is not expected to be adsorbed onto organic matter in soil or sediment. It is not readily biodegradable. TAME has very low acute toxicity to aquatic organisms and is not expected to bioaccumulate.

Solvent-assisted protein digestion involves enzymatic hydrolysis in mixed aqueous–organic solvents. With trypsin, acetonitrile is the modifying solvent of choice, recommended at concentrations from 10 to 80% to improve protein sequence coverage in mass spectrometry. Spectroscopic activity assays employing substrate mimics such as N-benzoyl arginine ethyl ester (BAEE) appear to show a relative enhancement of trypsin activity in mixed solvent systems. However, as reported here, the changing solvent polarity induces bias in the absorbance measurement, lending upward of 35% error in the apparent trypsin activity as the acetonitrile is raised to 70%. Furthermore, time-dependent spectroscopic and mass spectrometric measurements reveal a progressive deactivation of trypsin over a 5- to 10-min period in as little as 30% acetonitrile.

Through three chromatographic steps, a new thrombin-like enzyme (TLE), named TA-2, from the venom of the Chinese white-lipped green pitviper (Trimeresurus albolabris) has been isolated and purified to homogeneity. TA-2 was a single-chain glycoprotein with about 6% sugar, pI 3.9 and a molecular weight of 38.8 kD. Its N-terminal sequence (VVGGDECNIN) showed high sequence conformity with many other TLEs. In vitro, it coagulated bovine fibrinogen (108.6 NIH units/mg) and cleaved the Aα and Bβ chains of bovine fibrinogen-releasing fibrinopeptide A and B, but did not degrade bovine fibrin; displayed high stability at different temperature, pH, and presence of several divalent cations and inhibitors; also exhibited strong activity towards casein (192.3 units/mg) and high esterase activity upon Nα-p-tosyl-L-arginine methyl ester (11 units/mg); and behaved as a promoter to platelet aggregation induced by ADP or collagen. In vivo, TA-2 caused dose-dependent prolongation of bleeding time in mice, but had no hemorrhagic and edema-inducing activities even at high concentrations.

A coagulant enzyme, okinaxobin I, which was purified from Trimeresurus okinavensis (himehabu snake) venom, releases specifically fibrinopeptide B from fibrinogen to form fibrin clots. In the present study, its isozyme denoted as okinaxobin II has been purified to homogeneity from the same venom by chromatographies on Sephadex G-100, CM-Toyopearl 650M, and FPLC Mono-Q columns. Differently from okinaxobin I, okinaxobin II specifically cleaved fibrinopeptides A and B from fibrinogen similarly as found for α-thrombin. The enzyme acted on fibrinogen with specific activity of 42 NIH units/mg at optimum pH of 8.0. Okinaxobin II was a monomeric glycoprotein with a mol. wt of 37,500 on SDS-PAGE, which was reduced to 29,500 after treatment with N -glycanase. Okinaxobin II was much more basic (pI=8.1) than okinaxobin I (pI=5.4). The N-terminal sequence was highly similar to those of okinazobin I and some other snake venom coagulant enzymes such as flavoxobin ( Trimeresurus flavoviridis ), batroxobin ( Bothrops atrox and Bothrops moojeni ), and catroxobin ( Crotalus atrox ). Okinaxobin II hydrolyzed tosyl-L-arginine methly ester and benzoyl-L-arginine p -nitroanilide. The esterase activity was strongly inhibited by di iso propylfluorophosphate and to a lesser extent by tosyl-L-lysine chloromethly ketone, indicating that the enzyme is a serine protease like α-thrombin. In terms of amino acid composition, okinaxobin II was similar to okinaxobin I and dissimilar to α-thrombin.

We studied antigen-induced platelet activating factor and the 1-0-hexadecyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF) in nasal lavage fluids (NLF) by combined gas chromatography/mass spectrometric analysis (GC/MS). During the early allergic reaction, there was a dramatic increase in the levels of lyso-PAF that peaked at 15 min (2.6 +/- 5.2 ng/ml, mean +/- SEM, n = 6). Increasing doses of antigen produced a dose-dependent increase in the levels of lyso-PAF that peaked at the highest dose. Levels of lyso-PAF correlated strongly with those of N-alpha-tosyl-L-arginine methyl ester (TAME)-esterase activity (rs = 0.82, p = 0.0001) and histamine (rs = 0.57, p = 0.002). There was a no significant increase in the quantity of lyso-PAF found in NLF from allergic individuals challenged with diluent or nonallergic individuals challenged with antigen. In subjects showing a late phase reaction, as indicated by symptoms and histamine release, we detected lyso-PAF along with TAME-esterase activity and histamine during the late phase reaction. In contrast to lyso-PAF, PAF levels were near or below the detection limit of the assay in NLF and remained unchanged after antigen challenge. We also investigated the potential pathways for lyso-PAF generation from 2-acetylated phospholipids. We found that the time required for deacetylation of 50% of [3H]PAF (t1/2) to lyso-PAF was 50 min in baseline secretions and 10 and 22 min in NLF obtained 10 min and 24 h after antigen challenge, respectively. These data suggested that catabolic pathways were present in NLF.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood coagulation systems in cyclostome, elasmobranch, and teleost fish were studied and compared. The plasma of all these fish contained a fibrinogen molecule, capable of being clotted by human thrombin, and a prothrombin molecule, capable of being converted into thrombin which could hydrolyze p-tosyl l-arginine methyl ester. The prothrombin activity could be adsorbed on barium sulfate. Accurate assessment of prothrombin conversion factors is confounded by the "species specificity" of protein-protein interactions, but preliminary observations were made on both the extrinsic and intrinsic clotting schemes. Fish thrombocytes play a central role in the intrinsic conversion of prothrombin to thrombin, and are responsible for clot retraction. The plasma of the smooth dogfish exemplifies a clotting diathesis which can be overcome in vitro by the addition of large amounts of calcium. Under such conditions, other parts of the coagulation scheme become greatly exaggerated, and a very large thrombin generation ensues, subsequently followed by an intense fibrinolysis. A powerful serum inhibition occurs which was shown to be caused by the breakdown products of the fibrinolysis.

A novel thiol-ene "click" strategy for the preparation of monolithic trypsin microreactor was proposed. The hybrid organic-inorganic monolithic capillary column with ene-functionality was fabricated by sol-gel process using tetramethoxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as precursors. The disulfide bonds of trypsin were reduced to form free thiol groups. Then the trypsin containing free thiol groups was attached on the γ-MAPS hybrid monolithic column with ene-functionality via thiol-ene click chemistry to form a trypsin microreactor. The activity of the trypsin microreactor was characterized by detecting the substrate (Nα-p-tosyl-L-arginine methyl ester hydrochloride, TAME) and the product (Nα-p-tosyl-L-arginine, TA) with on-line capillary zone electrophoresis. After investigating various synthesizing conditions, it was found that the microreactor with poly(N,N'-methylenebisacrylamide) as spacer can deliver the highest activity, yielding a rapid reaction rate. After repeatedly sampling and analyzing for 100 times, the monolithic trypsin microreactor still remained 87.5% of its initial activity. It was demonstrated that thiol-ene "click" strategy for the construction of enzyme microreactor is a promising method for the highly selective immobilization of proteins under mild conditions, especially enzymes with free thiol radicals.

Various protease inhibitors (e.g. phenylmethanesulfonyl fluoride (PMSF), tosyl-phenylalanine chloromethyl ketone (TosPheCH2Cl)) and substrates (e.g., tosyl-arginine methyl ester (TosArgOMe), tryptophan methyl ester (TrpOMe)) inhibit the binding of adrenal and sex steroids to their cognate receptors (Hubbard and Kalimi (1985) Mol. Cell. Biochem. 66, 101–109). Here we extend this finding to the receptor for 3, 5, 3'-triiodothyronine (T3) in rat liver nuclei. We find that PMSF, TosPheCH2Cl and other protease inhibitors as well as TosArgOMe, TrpOMe, tyrosine methyl ester (TyrOMe) and tyrosine ethyl ester (TyrOEt) inhibit binding of 125I-T3 to its receptor in rat liver nuclei. Inhibition by protease substrates appears to be at or close to the hormone binding domain. By analogy with the known mechanism of binding of protease inhibitors and substrates to enzymes, we suggest that the T3 receptor contains a nucleophilic site at or close to the hormone binding domain.

Two serine proteinases, MSP 1 and MSP 2, were isolated from Bothrops moojeni venom by chromatographies on Sephadex G-100, DEAE-Sephacel (pH 7.5) and SP-Sephadex C-50 (pH 7.5). Both enzymes are basic glycoproteins. On sodium dodecyl sulfate-polyacrylamide electrophoresis, MSP 1 presented two close protein bands corresponding to the mol. wts of 34,000 and 32,500. MSP 2 behaved as a single-chain protein with a mol. wt of 38,000. Specific esterolytic activities of MSP 1 and MSP 2 on α- N -tosyl- l -arginine methyl ester (TAME) are 33 υmol min −1 mg −1 and 184 υmol min −1 mg −1 , respectively. The most sensitive substrates for the amidolytic activity of both proteinases were the thrombin substrate d -Phe-pipecolyl(Pip)-Arg-4-nitroanilide(Nan) and the glandular kallikrein substrate d -Val-Leu-Arg-Nan. MSP 1, in a concentration of 10 −8 M, causes platelet aggregation in platelet-rich plasma and washed platelets. It also enhances the ADP-induced platelet aggregation. Prostaglandin E 1 (PGE 1 ), phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA) abolished completely the aggregation induced by MSP 1. Torresea cearensis trypsin inhibitor (TCTI) inhibited both amidolytic ( K i = 1.96 × 10 −7 M) and platelet-aggregating ( K i = 1.66 × 10 −7 M) activities of MSP 1. The esterolytic activity of MSP 1 and MSP 2 was completely abolished by PMSF, only partially by soybean trypsin inhibitor (SBTI) and benzamidine and not affected by Trasylol. MSP 2 was also inhibited by TCTI ( K i = 0.7 × 10 −7 M).

A nasal lavage model and a new filter paper disk model have been used to measure biologic and physiologic responses to antigen challenge in patients with allergic rhinitis. Pretreatment of subjects with cetirizine reduced the number of sneezes induced by antigen challenge but did not significantly reduce levels of histamine or prostaglandin D2 in a double-blind, placebo-controlled trial with the lavage model. Pretreatment with 60 or 300 mg of terfenadine did significantly reduce levels of histamine, kinin, albumin, and TAME-esterase activity in a double-blind, placebo-controlled study with this model. Again with the nasal lavage model, a double-blind, placebo-controlled comparison of pretreatment with 60 mg of terfenadine or 10 mg of loratadine showed that both agents significantly reduced sneezing. Both drugs also lowered levels of antigen-induced histamine and TAME-esterase, but only terfenadine did so significantly. In a double-blind, placebo-controlled study, the new disk method showed that terfenadine reduced sneezing but not nasal congestion in eight patients with allergic rhinitis. Terfenadine significantly reduced the weight of nasal secretions on both sides of the nose and significantly reduced histamine on the ipsilateral side.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-P-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50 % germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyI-L-phenylalanyl-L-valyl-L-arginine-p-nitroaniIid (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors. The possibility that L-BAPNase from B. cereus T might be involved in the initial germination steps was suggested by the similarity of ID50 values for germination and Ki values for inhibition of the enzyme in vitro by TAME and TLCK, and by the fact that both germination and the L-BAPNase were reversibly inhibited by TLCK.

A 'nasal pool' (NP) device, a compressible plastic container with an adapted nozzle, was used to perform a continuous 10-min nasal provocation and lavage. This novel technique brings known concentrations of agents into contact with a large and defined area of the nasal mucosal surface for extended periods of time. Simultaneously, the surface exudations/secretions of the same nasal mucosa are effectively sampled by the NP fluid. A concentration-response study of histamine (80, 400 and 2000 micrograms/ml) was performed in 12 normal subjects on three different occasions. Exudation of plasma albumin into the lavage fluid was measured to quantitate the histamine-induced airway inflammation. The effect of the dwell time on exudation was examined using histamine (400 micrograms/ml) instilled in the nasal cavity for time periods from 10 sec to 10 min. The time course of histamine-induced plasma exudation response was studied by exposing the mucosa to histamine (400 micrograms/ml) for 12 min, with the NP renewed every minute. Allergen-provocations were performed in subjects with hay fever and TAME-esterase activity in the returned lavage fluid was determined to indicate the degree of response. Histamine produced a concentration-dependent increase in albumin levels in the NP fluid; 123.3 +/- 25.6, 213.8 +/- 19.7 and 430.2 +/- 32.0 micrograms/ml (mean +/- s.e.m.), respectively. The time-course study demonstrated that plasma exudation into the lumen occurred promptly and that the exudation response reached a maximum after exposure to histamine for 6-10 min. The dwell-time experiments supported this finding. After 10 min the exudation appeared to decline despite the continued presence of histamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Previously, from the plasma of unrelated haemophilia-B patients, we isolated two non-functional Factor IX variants, namely Los Angeles (IXLA) and Long Beach (IXLB). Both variants could be cleaved to yield Factor IXa-like molecules, but were defective in catalysing the cleavage of Factor X (macromolecular substrate) and in binding to antithrombin III (macromolecular inhibitor). In the present study we have identified the mutation of IXLA by amplifying the exons (including flanking regions) as well as the 5′ end of the gene by polymerase-chain-reaction (PCR) method and sequencing the amplified DNA by the dideoxy chain-termination method. Comparison of the normal IX and IXLA sequences revealed only one base substitution (T----C) in exon VIII of IXLA, with a predicted replacement of Ile-397 to Thr in the mature protein. This mutation is the same as found recently for IXLB. The observation that IXLB and IXLA have the same mutation is an unexpected finding, since, on the basis of their ox brain prothrombin time (PT, a test that measures the ability of the variant Factor IX molecules to inhibit the activation of Factor X by Factor VIIa-tissue factor complex), these variants have been classified into two different groups and were thought to be genetically different. Our observation thus suggests that the ox brain PT does not reflect the locus of mutation in the coding region of the variant molecules. However, our analysis suggests that the ox brain PT is related to Factor IX antigen concentration in the patient's plasma. Importantly, although the mutation in IXLA or IXLB protein is in the catalytic domain, purified IXaLA and IXaLB hydrolyse L-tosylarginine methyl ester at rates very similar to that of normal IXa. These data, in conjunction with our recent data on Factor IXBm Lake Elsinore (Ala-390----Val mutant), strengthen a conclusion that the peptide region containing residues 390-397 of normal Factor IXa plays an essential role in macromolecular substrate catalysis and inhibitor binding. However, the two mutations noted thus far in this region do not distort S1 binding site in the Factor IXa enzyme.

Methyl tert-butyl ether (MTBE) is the most widely used oxygenate in gasoline blending and has become one of the world’s most widespread groundwater and surface water pollutants. Alternative oxygenates to MTBE, namely ethyl tert-butyl ether (ETBE), tert-amyl ether (TAME) and diisopropyl ether (DIPE) have been hardly studied yet. The solubility of these chemicals is a key thermodynamic information for the assessment of the fate and transport of these pollutants. This work reports experimental data of water solubility at the range from 278.15 to 313.15 K and atmospheric pressure of ethers used in fuels (MTBE, ETBE, TAME and DIPE) due to the strong influence of temperature on its trend. From the experimental data, temperature dependent polynomials were fitted, thermodynamic parameters were calculated and theoretical models were used for prediction. Finally, the tert-butyl alcohol (TBA) influence in the solubility of MTBE and ETBE in aqueous media was studied.

Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris-HCl containing 5 mM CaCl2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates L-BApNA and N-alpha-p-tosyl-L-Arg methyl ester (L-TAME). Higher activity was observed at pH 8.5 and 35 degrees C when using L-BApNA as substrate and at pH 8.0 and 30 degrees C when using L-TAME. Maximum enzyme activity against L-BApNA was obtained with 20 mM CaCl2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. KM values obtained were 0.32 mM for L-BApNA and 52.5 microM for L-TAME.

Experimental and occupational inhalational exposure to oxygenate fuel additives in reformulated gasoline has been reported to induce neurological symptoms (e.g., headache, nausea, dizziness). We reported previously that the ether additives (methyl-t-butyl ether (MTBE), t-amyl-methyl ether (TAME) and ethyl-t-butyl ether (ETBE)) and their metabolites (t-amyl alcohol (TAA), t-butyl alcohol (TBA) and ethanol) alter the binding of [3H]t-butylbicycloorthobenzoate ([3H]TBOB), a ligand for the γ-aminobutyric acidA (GABAA) receptor in rat brain membrane preparations. To more directly assess the effects of the ethers and their alcohol precursors on GABAA receptor function, the uptake of 36Cl− was measured in synaptoneurosomes, a preparation of closed membrane sacs comprised of pre- and postsynaptic membranes from adult rat cerebral cortex. Each of the compounds caused a concentration-dependent enhancement of muscimol-stimulated uptake of 36Cl−, which diminished with further increasing concentrations. The potency of the enhancement by the compounds was in the rank order: MTBE=TAME>TAA=ETBE>TBA>ethanol. The half-maximally effective concentration (EC50) for the facilitation of muscimol-stimulated 36Cl− uptake ranged from 0.06 to 3 mM, and that for the higher-dose inhibitory effect (IC50) ranged from 3 to 50 mM. The facilitatory concentrations of the compounds are in the range of the blood concentrations reported in experimental animals after exposures known to induce CNS effects such as ataxia. The results suggest a potential role of the GABAA receptor in some of the reported neurotoxic effects of gasoline additives.

The present study was designed to investigate whether N-alpha-tosyl L-arginine methyl ester [TAME]-esterase activation could be the result of endothelial dysfunction. Thoracic aorta from rats was mounted in an organ bath containing Krebs solution. Intact and endothelium denuded aortic strips were challenged with different concentrations of TAME (10(-15)-10(-1) m). The effects of aspirin, a cyclo-oxygenase pathway inhibitor, were also studied on [TAME]-esterase induced contraction on rat aorta strips. Our results showed that aspirin definitely blocked TAME-esterase induced contractions on rat aortic strips. In conclusion, the present work supported the hypothesis that [TAME]-esterase induced contraction in rat aorta in vitro was mediated through release of prostaglandin(s) as a result of endothelial dysfunction.

Possible pharmacological effects of N-alpha-tosyl L-arginine methyl ester [TAME] were studied on rat aorta strips in vitro. Results showed that [TAME]-esterase was an endothelium dependent component that involved a nitric oxide cyclic-GMP mediated pathway. Furthermore, during activation of Kinin-Kallikrein system, TAME-esterase induced contractions involve degradation of kinins by kininases.

The biotransformation of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) was studied in humans and in rats after inhalation of 4 and 40 ppm of MTBE, ETBE, and TAME, respectively, for 4 hours, and the biotransformation of MTBE and TAME was studied after ingestion exposure in humans to 5 and 15 mg in water. tert-Butyl alcohol (TBA), a TBA conjugate, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate were found to be metabolites of MTBE and ETBE. tert-Amyl alcohol (TAA), free and glucuronidated 2-methyl-2,3-butanediol (a glucuronide of TAA), 2-hydroxy-2-methyl butyrate, and 3-hydroxy-3-methyl butyrate were found to be metabolites of TAME. After inhalation, MTBE, ETBE, and TAME were rapidly taken up by both rats and humans; after termination of exposure, clearance from blood of the ethers by exhalation and biotransformation to urinary metabolites occurred with half-times of less than 7 hours in rats and humans. Biotransformation of MTBE and ETBE was similar in humans and rats after inhalation exposure. 2-Hydroxyisobutyrate was recovered as a major product in urine. All metabolites of MTBE and ETBE excreted with urine were eliminated with half-times of less than 20 hours. Biotransformation of TAME was qualitatively similar in rats and humans, but the metabolic pathways were different. In humans, 2-methyl-2,3-butanediol, 2-hydroxy-2-methyl butyrate, and 3-hydroxy-3methyl butyrate were recovered as major urinary products. In rats, however, 2-methyl-2,3-butanediol and its glucuronide were major TAME metabolites recovered in urine. After ingestion of MTBE and TAME, both compounds were rapidly absorbed from the gastrointestinal tract. Hepatic first-pass metabolism of these ethers was not observed, and a significant part of the administered dose was transferred into blood and cleared by exhalation. Metabolic pathways for MTBE and TAME and kinetics of excretion were identical after ingestion and inhalation exposures. Results of studies presented here suggest (1) that excretion of MTBE, ETBE, and TAME in rats and humans is rapid, (2) that biotransformation and excretion of MTBE and ETBE are identical in rats, and (3) that biotransformation and excretion of TAME is quantitatively different in rats and humans.

Aerobic degradation of ethyl tert-butyl ether (ETBE), Methyl tert-butyl ether (MTBE) and tert- amyl methyl ether (TAME), as tertiary-substrates, was studied in a continuous upflow fixed-bed reactor (UFBR) using an external oxygenator and sintered glass rings as biomass carriers. The UFBR has been shown to be an effective system for the simultaneous and continuous long-term degradation of the three- oxygenates mixture as sole source of carbon and energy. Therefore, the oxygenates feed concentration must be related in conjunction with the hydraulic retention time ''HRT'' and vice versa. The permissible feed concentration of both MTBE and TAME to achieve more than 99% removal efficiency is about 80 mg L � 1 at a constant HRT of 24 h. The same performance can be obtained if the HRT kept at a value equal or above to 15 h for a feed concentration of about 80 mg L � 1 of both MTBE and TAME. However, the ETBE removal efficiency was always greater than 99% whatever the ETBE concentration feed (between 10 and 100 mg L � 1 at a constant HRT of 24 h) and the HRT (between 24 and 13 h at a constant concentration feed of about 80 mg L � 1 ) tested in this study. The highest ETBE, MTBE and TAME removal rates achieved throughout the UFBR runs, with efficiency better than 99%, were 140 � 5, 132 � 2 and 135 � 2m g L � 1 d � 1 , respectively. No metabolic intermediates including tert-butyl alcohol (TBA), tert- butyl formate (TBF) and tert-amyl alcohol (TAA) were detected in the effluent during all the reactor runs. Furthermore, based on the chemical oxygen demand balance, all the removed oxygenates were completely metabolized. The results of this study suggest that the higher resistance to biodegradation exhibited by the MTBE and the TAME is probably due to the steric hindrance for the attacking enzyme(s); and the major limiting step to the oxygenate degradation maybe the accessibility and the cleavage of the ether bond, but not the assimilation of their major metabolites such as TBA, TBF and TAA. These results were concomitant with the batch tests using the reactor's immobilized biomass as inoculum. # 2001 Elsevier Science Ltd. All rights reserved

Two enzyme inhibitors namely L-NAME, a nitric oxide synthase (NOS) inhibitor and methylene blue, a guanylate cyclase inhibitor, were used to elucidate whether N-alpha-tosyl L-arginine methyl ester (TAME)-induced contractions in toad intestinal rings in vitro are mediated through a nitric oxide (NO)- cyclic GMP (c-GMP) pathway. Moreover, a NO precursor, L-arginine was also used to investigate its effect on TAME-induced contractions. Our findings provide evidence that TAME-induced contractions have both an endothelium-dependent and an endothelium-independent component. Based on our findings we now propose that TAME induced contraction involves an endothelium-dependent component mediated through NO and c-GMP.

At present the physiological role of most oviductal proteins remains unknown. In this work, we present evidence that the oviductal secretion as well as the crude oviductal tissue-extract show proteolytic-like esterase and amidase activity. The proteolytic activity of the oviductal enzymes was higher in the oviducts of superovulated hamster females than in those of normal ones, indicating that gonadotrophic hormones would stimulate the synthesis and secretion of these enzymes. Some of their properties were analyzed in the 15,600-g supernatant of both oviductal tissue extracts (OE) and oviductal fluid (OF). The enzymatic activity toward the synthetic substrates p-tosyl-l-arginine methyl ester-HCl (TAME) and alpha-N-benzoyl-dl-arginine-p-nitroanilide HCl (BAPNA) was activated by calcium ions, reached a maximum at pH 7.5, and was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-l-lysine chloromethyl ketone HCl (TLCK), phenyl methyl sulfonyl fluoride (PMSF), and benzamidine. The OE glycoprotein fraction recognized by WGA-Sepharose affinity columns (37% total proteins) showed proteolytic activity with properties similar to the OE and OF enzymes. The protease activity could be ascribed to a plasminogen activator (PA) detected in the Triton X-100 treated tissue crude membrane fraction (Triton-CMF) and in the oviductal secretion of the superovulated females. In the Triton-CMF fraction, 100% of the proteolytic activity was plasminogen-dependent. The use of amiloride, a selective urokinase-type plasminogen activator (uPA) inhibitor, shows that 90% of this activity was due to a tissue-type plasminogen activator (tPA) and 10% to uPA whereas in the uterus 100% of the activity was tPA. Only a small percentage of the OF proteolytic activity was plasminogen-dependent, probably due to the presence of PA inhibitors in this medium.

Under in vitro conditions N-alpha-tosyl L-arginine methyl ester (TAME) induced a concentration dependent contractile response on ileal strips with EC50 of 4.3 x 10(-5) M as compared to acetylcholine which induced sustainable contractions with EC50 of 3.2 x 10(-6) M. The present study is the first to demonstrate that TAME is a potent constrictor of non-airway smooth muscle.

We have previously shown virus particles encapsulated in aqueous spermine alginate constructs retain immunogenicity and infectivity both in vitro and in vivo. However, because virions are complex structures with multiple reinforcing components, it was uncertain if isolated single proteins would retain functional integrity when similarly encapsulated. To examine this question trypsin, used as a model protein, was blended with aqueous sodium alginate and the blend was dispersed as fine droplets in aqueous spermine hydrochloride to generate self-assembling, trypsin-containing microcapsules. Trypsin was assayed spectrophotometrically for retention of enzymatic activity using N-alpha-p-tosyl-L-arginine methyl ester as substrate. Neither of the encapsulating reagents alone inhibited enzyme activity. Enzyme that escaped capture was assayed directly in the manufacturing supernatant. In mass balance studies we found that about 20-30% of activity was retained in intact capsules with the remainder resident in the aqueous manufacturing supernatant and washes. However, we found that the capsule wall appeared to inhibit enzyme activity by retarding substrate diffusion into and product diffusion out from the capsules, as evidenced by an increase in activity on lysis. Thus, it is clear that a single protein, as represented by trypsin, can retain functional integrity when encapsulated in this all aqueous system.

Under in vitro conditions atrial natriuretic peptide (ANP) caused specific relation of bronchoconstriction induced by tosyl arginine methyl ester (TAME) on rat trachea. This supports the hypothesis that ANP is a relaxant of airway as well as vascular smooth muscle and since lung tissue contains ANP and ANP receptors, it is hypothesized that the lungs may be a target organ for ANP. It is concluded that ANP had definite bronchodilating properties on rat trachea mounted in vitro and could be a possible antagonist of TAME.

Tosyl arginine methyl ester induced bronchoconstriction in rabbit bronchial rings mounted in vitro with a calculated EC50 of 5.6 x 10(-5) M as compared with acetylcholine which induced sustainable constriction with an EC50 of 2.5 x 10(-6) M. Tosyl arginine methyl ester, however, had definite bronchoconstricting properties though less potent than acetylcholine. We concluded that our data support the hypothesis that tosyl arginine methyl ester could be a possible biochemical mediator of airway contraction.

A study was performed in 43 workers exposed to methyltetrahydrophthalic anhydride (MTHPA) used as a hardener in an epoxy resin system. Ten workers sensitized to MTHPA (group SS; presence of serum IgE antibodies against a conjugate of MTHPA and human serum albumin (HSA) detected by RAST) had significantly higher levels of tryptase in nasal lavage fluid than 19 nonsensitized workers with work-related nasal symptoms (group NS) and 14 nonsensitized workers without nasal symptoms (group NN). This suggests an ongoing mast-cell-mediated reaction in the sensitized group. No statistically significant differences were found in the three groups concerning eosinophil cationic protein (ECP) and TAME-activity in lavage fluid. However, there was a significant increase in serum ECP in the SS group, as compared with a group of unexposed controls. Nasal challenge with MTHPA-HSA, performed in a subsequent study in seven workers from the SS group, six from the NS group, and seven from the NN, caused a larger increase of symptom score and a more pronounced decrease in nasal inspiratory peak flow in the SS group than in the other two groups. No significant rise was recorded for tryptase and ECP in lavage fluid in any of the three groups after challenge. The combined results of the two studies indicate that specific IgE antibodies play a pathogenetic role in at least some of the cases of work-related nasal symptoms associated with MTHPA exposure.

Anesthetic agents may modify the tissue content of low molecular weight proteinase inhibitors (mol wt less than 50,000) and affect the colonization and proliferation of B16-F10 melanoma cells in lungs. Lungs of female mice exposed to halothane in oxygen had significantly greater low molecular weight proteinase inhibitor activity than those from unexposed female mice. The amount of activity in male mice similarly exposed did not differ from unexposed controls. Lungs of exposed female mice also had greater activity as compared with males similarly exposed. No differences occurred in identical experiments with ketamine. High-performance liquid chromatography revealed that the inhibitor in all groups of both sexes shared a major peak with a molecular weight similar to that of the trypsin inhibitor aprotinin. Female mice bearing B16-F10 melanoma cells and exposed to halothane in oxygen had significantly more small lung tumor nodules than tumor-bearing female mice injected with ketamine. However, the total number of nodules did not differ between the two groups. These observations support the hypothesis that stimulation of proteinase inhibitory activity by halothane in oxygen may be responsible for an inhibition of tumor cell proliferation, resulting in smaller tumor nodules and no effect on the incidence of colonization.

LPS stimulation of human monocytes in vitro induced release of the 17-kDa mature IL-1 beta (mIL-1 beta) but did not result in release of precursor IL-1 beta (pIL-1 beta). In contrast, the presence of a serine protease inhibitor, N alpha-(p-toluene sulfonyl)-L-arginine methyl ester (TAME; 10 mM) for 6 or 18 h was associated with the LPS-stimulated release of the 33-kDa pIL-1 beta as well. These effects were initially discerned from observations that the fraction of the total IL-1 beta produced (as detected by ELISA) that was released from monocytes increased in the presence of TAME, and immunoblot assays confirmed that this fraction was predominantly 33-kDa IL-1 beta. A global decrease in monocyte protein synthesis was also observed after prolonged (18-h) exposure to TAME and was associated with a decrease in IL-1 beta synthesis, predominantly affecting 31-kDa pIL-1 beta, and a dose-dependent inhibition of TNF-alpha production. Parallel examination of lactate dehydrogenase (LDH) release indicated that pIL-1 beta release was unrelated to cell lysis. These results demonstrate that TAME-inhibitable serine proteases are probably involved in the production and eventual proteolysis of the 33-kDa pIL-1 beta in situ but are probably not mechanistically related to either maturation of the IL-1 beta molecule or signaling of IL-1 beta release. IL-1 beta release appears to be dependent on the amount of total IL-1 beta synthesized. Serine proteolysis may constitute a degradative pathway for excess precursor, which, if interfered with, could result in release of the higher-molecular-weight forms of IL-1 beta.