Elucidation of the solution structure of cardiotoxin analogue V from the Taiwan cobra (Naja naja atra)--identification of structural features important for the lethal action of snake venom cardiotoxins

The aim of the present study is to understand the structural features responsible for the lethal activity of snake venom cardiotoxins. Comparison of the lethal potency of the five cardiotoxin isoforms isolated from the venom of Taiwan cobra (Naja naja atra) reveals that the lethal potency of CTX I and CTX V are about twice of that exhibited by CTX II, CTX III, and CTX IV. In the present study, the solution structure of CTX V has been determined at high resolution using multidimensional proton NMR spectroscopy and dynamical simulated annealing techniques. Comparison of the high resolution solution structures of CTX V with that of CTX IV reveals that the secondary structural elements in both the toxin isoforms consist of a triple and double-stranded antiparallel beta-sheet domains. Critical examination of the three-dimensional structure of CTX V shows that the residues at the tip of Loop III form a distinct "finger-shaped" projection comprising of nonpolar residues. The occurrence of the nonpolar "finger-shaped" projection leads to the formation of a prominent cleft between the residues located at the tip of Loops II and III. Interestingly, the occurrence of a backbone hydrogen bonding (Val27CO to Leu48NH) in CTX IV is found to distort the "finger-shaped" projection and consequently diminish the cleft formation at the tip of Loops II and III. Comparison of the solution structures and lethal potencies of other cardiotoxin isoforms isolated from the Taiwan cobra (Naja naja atra) venom shows that a strong correlation exists between the lethal potency and occurrence of the nonpolar "finger-shaped" projection at the tip of Loop III. Critical analysis of the structures of the various CTX isoforms from the Taiwan cobra suggest that the degree of exposure of the cationic charge (to the solvent) contributed by the invariant lysine residue at position 44 on the convex side of the CTX molecules could be another crucial factor governing their lethal potency.     

Putative membrane lytic sites of P-type and S-type cardiotoxins from snake venoms as probed by all-atom molecular dynamics simulations

Cardiotoxins (CTXs) belonging to the three-finger toxin superfamily of snake venoms are one of principal toxic components and the protein toxins exhibit membrane lytic activities when the venoms are injected into victims. In the present study, complex formations between CTX VI (a P-type CTX from Naja atra) and CTX1 (an S-type CTX from Naja naja) on zwitterionic POPC bilayers (a major lipid component of cell membranes) have been studied in near physiological conditions for a total dynamic time scale of 1.35 μs using all-atom molecular dynamics (MD) simulations. Comprehensive analyses of the MD data revealed that residues such as Leu1, Lys2, Tyr11, Lys31, Asp57 and Arg58 of CTX VI, and Ala16, Lys30 and Arg58 of CTX1 were crucial for establishing interactions with the POPC bilayer. Moreover, loop I, along with globular head and loop II of CTX VI, and loop II of CTX1 were found to be the structural regions chiefly governing complex formation of the respective proteins with POPC. Rationalizations for the differential binding modes of CTXs and implications of the findings for designing small molecular inhibitors to the toxins are also discussed.

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Graphical Abstract Binding modes of a P-type CTX and an S-type CTX towards the POPC bilayer

     

Colonization of America by Drosophila subobscura: spatial and temporal lethal-gene allelism

About twenty years ago Drosophila subobscura, a western Palearctic species, colonized both North and South America. Lethal genes in the O chromosome has been subject to much research. Lethal gene allelisms between American populations far away have been studied. These allelisms were not negligible, but all cases were due to the lethal gene completely associated to the O5 chromosomal inversion. Here we analyze the lethal genes in a new American population of D. subobscura (Centralia, Washington), located fairly close to a previously studied population (Bellingham, Washington) and separated in space and time with other American populations (Gilroy I and II in California and Santiago de Chile). The frequencies of lethal and semilethal genes of Centralia were 16.9+/-4.6 and 6.2+/-3.0, respectively. The intrapopulational allelism of Centralia was 0.122+/-0.036. Interpopulational allelisms were studied using the lethal genes from the populations separated in space and time from Centralia. The interpopulational allelisms between Centralia and Gilroy I (California) and between Centralia and Bellingham (Washington) were higher than the intrapopulational allelism (0.155+/-0.032 and 0.153+/-0.024, respectively). In all these cases allelism was due to a complete association between a lethal gene and the O5 chromosomal inversion. Accordingly, no other lethal genes are shared in these populations.     

RAPID PROCEDURE FOR DETECTING CIGUATOXINS IN FISH

Flesh and viscera/gill tissues of six amberjacks (Seriola dumerilii), suspected positive for ciguatoxins, were each extracted and the toxins partially purified. Both flesh and viscera/gill of only five fish were toxic to mice exhibiting ciguatoxins (CTX) symptoms. The methanol extracts of the five fish were pooled and concentrated, the volume of flesh extract was 50.0 mL (129.4 mg toxins/mL) and viscera/gill had 25.0 mL (25.5 mg toxins/mL). Pooled extracts exhibited CTX symptoms in mice but only flesh killed mice in 6 h and the LD₅₀ was 1.72 mg toxins. The lethal potencies of the pooled flesh killed mice in 6 h and the LD₅₀ was 1.72 mg toxins. The lethal potencies of the pooled flesh was 198.17 g fish, equivalent to 58.3 mouse unit. An efficient fractionation and purification procedure was developed for the extracts using an HPTLC and silica gel 60 plate with a chromatographic solvent mixture of chloroform:methanol:water (60:35:8, v/v). The system yielded 10 fractions for flesh and 9 for viscera/gill. Scanned plates were subdivided into three equal zones, each scraped, methanol extracted and tested in mice. The 2nd zone (R_f fractions between 0.40 and 0.66) was very toxic to mice compared to 1st or 3rd zones and the mice had CTX symptoms. The scanner for this 2nd zone had a cluster of minor peaks on both sides of the major one with a sum total area of 62.47% indicating multiplicity of CTX in amber-jack fish. The major peak, at retention time of 1.48 s and a single area of 43.28%, is believed to be the main ciguatoxins present. The HPTLC is a rapid and sensitive procedure for ciguatoxins in fish flesh extracts with a detection limit of 40.0 ± 1.9 picogram toxins.     

Thermodynamics of electron transfer in oxygenic photosynthetic reaction centers: volume change, enthalpy, and entropy of electron-transfer reactions in the intact cells of the cyanobacterium Synechocystis PCC 6803

The volume and enthalpy changes for charge transfer in the 0.1-10 micros time window in photosynthetic reaction centers of the intact cells of Synechocystis PCC 6803 were determined using pulsed, time-resolved photoacoustics. This required invention of a method to correct for the cell artifact at the temperature of maximum density of water caused by the heterogeneous system. Cells grown under either white or red light had different PS I/PS II molar ratios, approximately 3 and approximately 1.7, respectively, but invariable action spectra and effective antenna sizes of the photosystems. In both cultures, the photoacoustic measurements revealed that their thermodynamic parameters differed strongly in the spectral regions of predominant excitation of PS I (680 nm) and PS II (625 nm). On correcting for contribution of the two photosystems at these wavelengths, the volume change was determined to be -27 +/- 3 and -2 +/- 3 A3 for PS I and PS II, respectively. The energy storage on the approximately 1 micros time scale was estimated to be 80 +/- 15% and 45 +/- 10% per trap in PS I and PS II, respectively. These correspond to enthalpies of -0.33 +/- 0.2 and -1 +/- 0.2 eV for the assumed formation of ion radical pairs P700+F(AB-) and Y(Z*)P680Q(A-), respectively. Taking the free energy of the above reactions as the differences of their redox potentials in situ, apparent entropy changes were estimated to be +0.4 +/- 0.2 and -0.2 +/- 0.2 eV for PS I and PS II, respectively. These values are similar to that obtained in vitro for the purified reaction center complexes on the microsecond time scale [Hou et al. (2001) Biochemistry 40, 7109-7116, 7117-7125]. The constancy of these thermodynamic values over a 2-fold change of the ratio of PS I/PS II is support for this method of in vivo analysis. Our pulsed PA method can correct the "cell" or heterogeneous artifact and thus opens a new route for studying the thermodynamics of electron transfer in vivo.     

The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition

The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1-31 of St I (StI1-31) or 1-30 of St II (StII1-30) and demonstrated that StII1-30 promotes erythrocyte lysis to a higher extent than StI1-31. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1-10 hydrophobic sequence in StII1-30 for displaying higher binding and activity, in spite of both peptides' abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities.     

Metabolism of angiotensin I by guinea pig aqueous humor

We investigated the degradation of angiotensin I (Ang I) by guinea pig aqueous humor at physiological pH (pH 7.4) and assessed the activity of responsible enzymes using various enzyme inhibitors. The aqueous humor was incubated with Ang I in the presence or absence of an enzyme inhibitor at 37 degrees C for the appropriate time period. The resulting peptides were analyzed by a Beckman HPLC system with a Waters microBondapak C18 analytical column using a 30-min increasing linear gradient of 10 to 40% acetonitrile containing 0.05% trifluoroacetic acid (TFA) and H2O containing 0.05% TFA at a flow rate of 1 mL/min. Detection was done by absorbance at 214 nm. Angiotensin II (Ang II) was a major product (39.3+/-4.10 nmol x h(-1) mL(-1), n = 5) of Ang I hydrolysis. Traces of angiotensin 1-9, angiotensin IV, and angiotensin 1-7 were also produced. Chymostatin (0.05 mmol/L), EDTA (1 mmol/L), enalaprilat (0.1 mmol/L), and ebelacton B (0.01 mmol/L) inhibited generation of Ang II from Ang I by guinea pig aqueous humor by 89+/-4.6, 56+/-7.6, 33+/-5.1, 20+/-6.5%, respectively. Our findings indicate that guinea pig aqueous humor contains several enzymes that can form Ang II. The chymostatin-sensitive type of enzyme was the most active one found in guinea pig aqueous humor. Angiotensin I converting enzyme, carboxypeptidase A, and deamidase may also contribute to angiotensin II formation in guinea pig ocular fluid.     

Trichomonas vaginalis: identification of a phospholipase A-dependent hemolytic activity in a vesicular subcellular fraction

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (530) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3. GT-13. and GT-15), lysed both human and Sprague-Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0. in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 16.71 +/- 0.33 hemolytic units IHU]/mg/hr to 11.60 +/- 0.24 HU/mg/hr) than at pH 8.0 (3.81 +/- 0.30 HU/mg/hr to 5.75 +/- 0.65 HU/mg/hr). and it was greater than that on human red blood cells at pH 6.0 (2.67 +/- 0.19 HU/mg/hr to 4.08 +/- 0.15 HU/mg/hr) or pH 8.0 (2.24 +/- 0.0 9 HU/mg/hr to 2.81 +/- 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60-93% at pH 6.0 and 78-93% at pH 8.0) by the effect of 80 microM Rosenthal's inhibitor, which also inhibited 27-45% and 29-54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.     

Production and properties of theta-toxin of Clostridium perfringens with special reference to lethal activity.

theta-Toxin of Clostridium perfringens produced in a synthetic medium showed high toxicity. The mouse was killed with 5-10 hemolytic units of the toxin. Neutralization experiments showed that lethal and hemolytic activities were due to the same toxic entity. The amount of theta-toxin expressed in lethal activity reached more than 30% that of alpha-toxin in the synthetic medium SM67. Although the activities of alpha-and theta-toxins were not additive in terms of LD50, increase in the ratio of theta-toxin to alpha-toxin resulted in reduction of the survival time of the mouse injected with a lethal dosis of the mixture of the two toxins when compared with alpha-toxin alone. Unlike those produced in other media, the hemolytic and lethal activities of theta-toxin produced in the synthetic medium was not activated by reduction with thioglycollate, even after partial purification. In other respects, the toxin was not different significantly from those reported in the past. The toxic form was detected also in complex medium. It was suggested that theta-toxin may be produced as a nascent entity.     

Pulmonary artery occlusion is sufficient to increase pulmonary vascular permeability in rabbits.

Unilateral pulmonary artery obstruction (PAO) for 24-48 h, followed by reperfusion, results in pulmonary edema and lung inflammation. We hypothesized that lung injury actually occurred during the period of PAO but, because of low microvascular pressures during the period of occlusion, was not detected until perfusion was reestablished. To test this hypothesis, we studied 14 rabbits divided into three groups: group I rabbits underwent sham occlusion of the left pulmonary artery for 24 h; group II rabbits underwent PAO but were not reperfused; and group III rabbits were subjected to PAO and then reperfused for 4 h. The fluid filtration coefficient measured during a zone 3 no-flow hydrostatic stress (pulmonary arterial pressure = pulmonary venous pressure, both greater than alveolar pressure) in group I lungs was less than that of lungs in either group II or III [0.52 +/- 0.02 (SE) ml.min-1.cmH2O.100 g wet wt-1 vs. 0.94 +/- 0.11 and 0.86 +/- 0.13 for groups II and III, respectively, P less than 0.05]. The wet-to-dry weight ratio of the left lung measured after the zone 3 stress was applied for 20 min was 6.90 +/- 0.09 in group I rabbits and 9.21 +/- 0.75 and 11.75 +/- 0.44 in groups II and III, respectively (P less than 0.05). Radiolabeled microspheres demonstrated that flow to the left lung was diminished after the period of PAO (38 +/- 4, 9 +/- 5, and 2 +/- 1% of cardiac output in groups I, II, and III, respectively; P less than 0.05 for group I vs. groups II and III).(ABSTRACT TRUNCATED AT 250 WORDS)     

Toxic Effect of Staphylococcal Lysins for Goldfish

Goldfish died within 24 hr after intraperitoneal injections of 0.2 ml of Seitz filtrates of hemolytic Staphylococcus aureus cultures grown on Dolman and Wilson medium under increased CO₂ pressure for 72 to 96 hr. Two lethal toxins differing in heat sensitivity, antigenicity, and degree of toxicity were demonstrated. Studies of the relationship between the lethal factors and the hemolysins in the filtrates suggested that α- and β-lysin were responsible for the lethal effects. Filtrates of nonhemolytic staphylococcal cultures were innocuous. Goldfish were suitable animals for detecting toxicity in staphylococcal culture filtrates and for quantitative studies of the toxins. The results were highly reproducible.     

Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski

The Okinawan sea anemone Phyllodiscus semoni is known to cause cases of severe stinging. We isolated P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B; ca. 60 kDa) as the major toxins from the isolated nematocysts of this species for the first time. PsTX-60A and PsTX-60B showed lethal toxicity to the shrimp Palaemon paucidence when administered via intraperitoneal injection (LD(50) values: 800-900 and 800 microg/kg, respectively) and hemolytic activity toward a 0.8% suspension of sheep red blood cells (ED(50) values: 600 and 300 ng/ml, respectively). Furthermore, we sequenced the cDNA encoding PsTX-60A. The deduced amino acid sequence of PsTX-60A did not show any similarity to previously reported proteins. The N-terminal amino acid sequence of PsTX-60B showed homology with that of PsTX-60A. These toxins represent a novel class of cytolytic proteinaceous toxins.     

Effect of anaerobic and aerobic exercise of equal duration and work expenditure on plasma growth hormone levels

Growth hormone (GH) and lactic acid levels were measured in five normal males before, during and after two different types of exercise of nearly equal total duration and work expenditure. Exercise I (aerobic) consisted of continuous cycling at 100 W for 20 min. Exercise II (anaerobic) was intermittent cycling for one minute at 285 W followed by two minutes of rest, this cycle being repeated seven times. Significant differences (P less than 0.01) were observed in lactic acid levels at the end of exercise protocols (20 min) between the aerobic (I) and anaerobic (II) exercises (1.96 +/- 0.33 mM X 1(-1) vs 9.22 +/- 0.41 mM X 1(-1), respectively). GH levels were higher in anaerobic exercise (II) than in aerobic (I) at the end of the exercise (20 min) (2.65 +/- 0.95 micrograms X 1(-1) vs 0.8 +/- 0.4 micrograms X 1(-1); P less than 0.10) and into the recovery period (30 min) (7.25 +/- 6.20 micrograms X 1(-1) vs 2.5 +/- 2.9 micrograms X 1(-1); P less than 0.05, respectively).     

Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 myotoxins in vivo and in vitro

Previous studies have shown that calcineurin activity plays a critical role in the myotoxic activity induced by crotoxin (CTX), a group II phospholipase A(2) (PLA(2)) with neurotoxic and myotoxic actions. In order to address whether calcineurin is also important for the activity of non-neurotoxic group II PLA(2) myotoxins we have compared the effects of calcineurin inhibition on the myotoxic capacity of CTX and the non-neurotoxic PLA(2)s, myotoxin II (Mt II) and myotoxin III (Mt III) from Bothrops asper venom. Rats were treated with cyclosporin A (CsA) or FK506, calcineurin inhibitors, and received an intramuscular injection of either CTX, Mt II or Mt III into the tibialis anterior. Animals were killed 24 h after injection of toxins. Tibialis anterior was removed and stored in liquid nitrogen. Myofibers in culture were also treated with CsA or FK506 and exposed to CTX, Mt II and Mt III. It was observed that, in contrast to CTX, CsA and FK506 do not attenuate myotoxic effects induced by both Mt II and Mt III in vivo and in vitro. The results of the present study suggest that calcineurin is not essential for the myotoxic activity of Mt II and Mt III, indicating that distinct intracellular pathways might be involved in myonecrosis induced by neurotoxic CTX and non-neurotoxic Bothrops sp. PLA(2) myotoxins. Alternatively, calcineurin dependent fast fiber type shift might render the muscle resistant to the action of CTX, without affecting its susceptibility to Bothrops sp. myotoxins.     

Inactivation of the Pore-Forming Toxin Sticholysin I by Peroxynitrite: Protection by Cys Groups Incorporated in the Toxin

Sea anemones synthesize a variety of toxic peptides and proteins of biological interest. The Caribbean Sea anemone Stichodactyla helianthus, produces two pore-forming toxins, Sticholysin I (St I) and Stichloysin II (St II), with the ability to form oligomeric pores in cell and lipid bilayers characteristically lacking cysteine in their amino acid sequences. Recently, two mutants of a recombinant variant of Sticholysin I (rSt I) have been obtained with a Cys residue in functionally relevant regions for the pore-forming activity of the toxin: r St I F15C (in the amino terminal sequence) and r St I R52C (in the binding site). Aiming at characterizing the effects of oxidants in toxins devoid (r St I) or containing –SH moieties (r St I F15C and r St I R52C), we measured their hemolytic activity and pore forming capacity prior and after their incubation with peroxynitrite (ONOO^?). At low ONOO^?/Toxin ratios, nearly 0.8 Trp groups are modified by each added peroxynitrite molecule, and the toxin activity is reduced in ca. 20 %. On the other hand, in –SH bearing mutants only 0.5 Trp groups are modified by each peroxynitrite molecule and the toxin activity is only reduced in 10 %. The results indicated that Cys is the initial target of the oxidative damage and that Trp residues in Cys-containing toxins were less damaged than those in r St I. This relative protection of Trp groups correlates with a smaller loss of hemolytic activity and permeabilization ability in liposomes and emphasizes the relevance of Trp groups in the pore forming capacity of the toxins.     

Exchange of a single amino acid switches the substrate properties of RhoA and RhoD toward glucosylating and transglutaminating toxins

Rho GTPases are the preferred targets of various bacterial cytotoxins, including Clostridium difficile toxins A and B, Clostridium sordellii lethal toxin, the cytotoxic necrotizing factors (CNF1) from Escherichia coli, and the dermonecrotizing toxin (DNT) from Bordetella species. The toxins inactivate or activate specific sets of Rho GTPases by mono-O-glucosylation and deamidation/transglutamination, respectively. Here we studied the structural basis of the recognition of RhoA, which is modified by toxin B, CNF1, and DNT, in comparison with RhoD, which is solely a substrate for lethal toxin. We found that a single amino acid residue in RhoA and RhoD defines the substrate specificity for toxin B and lethal toxin. Change of serine 73 to phenylalanine in RhoA turned RhoA into a substrate for lethal toxin. Accordingly, change of the equivalently positioned phenylalanine 85 in RhoD with serine allowed glucosylation by toxin B. Comparable results were achieved with the Rho-activating and transglutaminating enzymes CNF1 and DNT. Here, amino acid glutamate 64 of RhoA and the equivalent aspartate 76 of RhoD define substrate specificity for CNF1 and DNT, respectively. These data indicate that single amino acid residues located in the switch II region of Rho proteins determine enzyme specificity for diverse bacterial toxins.     

Toxic effect of DDT and endosulfan in white shrimp postlarvae Litopenaeus vannamei (Decapoda: Penaeidae) from Chiapas, Mexico

We analized acute toxicity in white shrimp (Litopenaeus vannamei) postlarvae exposed to two chlorinated pesticides, DDT and endosulfan, under laboratory conditions during 168 hours, with controlled temperature (29 +/- 1 degrees C), salinity (3 +/- 1%o) and pH (8 +/- 1). Median lethal concentrations (LC50), "incipient" LC50, median lethal time (LT50) the "maximum acceptable concentration of the toxic compound" (MACT) and "the safety level" (SL) were determined. The concentration of the compounds at which organism growth was reduced by 5 and 50% (EC5 and EC50), as well as changes in oxygen consumption patterns were determined in the surviving postlarvae. They were very sensitive to both compounds and DDT was thrice as toxic as endosulfan. Growth rate decreased 50% and 80% with endosulfan and DDT, respectively, at the experimental pestice concentration. The low resistance of postlarvae to DDT and endosulfan suggests that additional inflow of these pesticides into the aquatic system could affect the rate of shrimp production in the area.     

A comparison of the non-specific acid phosphomonoesterase activity in the larva of Phocanema decipiens (Nematoda) with that of the muscle of its host the codfish (Gadus morhua).

The non-specific phosphomonoesterase (enzyme I) extracted from the larva of the codworm (Phocanema decipiens) is different from the enzyme (enzyme II) from the muscle of its host, the codfish (Gadus morhua). The pH optima were 4.0 and 4.5, and the KM values for p-nitrophenyl phosphate hydrolysis were 1.8 mM and 6.5 mM for enzymes I and II respectively. The specific specific activity in units (0.01 mumol/min) per mg protein was 4.80 +/- 0.85 and 0.54 +/- 0.07 for enzymes I and II respectively. The specific activity from uninfected muscles was only 0.39 (SD +/- 0.017) units per mg of protein. Both enzymes were inhibited by NaF, HgCl2, and cysteine but were stimulated by 2-mercaptoethanol. EDTA and iodoacetamide had no effect on enzyme I but enzyme II was activated by EDTA and inhibited by iodoacetamide. Cadmium ions inhibited both the enzymes but a conspicuous feature with enzyme II was in the increase in percentage inhibition by lowering the concentration of CD2+.     

The elastase inhibitory capacity and the alpha 1-proteinase inhibitor and bronchial inhibitor content of bronchoalveolar lavage fluids from healthy subjects

Pulmonary emphysema is currently thought to be due to an elastase-antielastase imbalance with resultant destruction of alveolar structures. The present study was aimed at testing whether alpha 1-proteinase inhibitor (alpha 1 PI) is the major component of the antielastase screen of the lower respiratory tract of healthy subjects. Bronchoalveolar lavage was performed in 8 nonsmokers (27.8 +/- 3.8 years) and 9 smokers (25 +/- 0.96 years). The lavage fluids were tested for leukocyte and pancreatic elastase inhibitory capacity (LEIC and PEIC) and immunoreactive alpha 1 PI and bronchial inhibitor (brI) content. The mean +/- s.e.m. levels of LEIC, PEIC, alpha 1 PI and brI were 0.16 +/- 0.039, 0.042 +/- 0.006, 0.09 +/- 0.007 and 0.013 +/- 0.002 mol/mol albumin, respectively. Thus, on the average, the molar concentration of brI was about 14% that of alpha 1 PI. The difference between LEIC and alpha 1 PI did not reach statistical significance (P = 0.0503). The PEIC was however significantly lower than the alpha 1 PI levels (P less than 0.05), indicating that the lavage fluids contained both active and inactive alpha 1 PI. Nonsmokers and smokers did not differ in their LEIC, PEIC, alpha 1 PI and brI levels. When the data were examined on an individual basis, the subjects could be divided into 2 groups: group I (n = 9; 3 nonsmokers, 6 smokers) whose LEIC/alpha 1 PI molar ratios were higher than unity and group II (n = 8; 5 nonsmokers, 3 smokers) whose LEIC/alpha 1 PI molar ratios were equal or lower than unity. Group I subjects had significantly higher LEIC values (0.26 +/- 0.05 mol elastase inhibited/mol albumin) than group II individuals (0.055 +/- 0.006; P less than 0.001) but the two groups had similar levels of immunoreactive alpha 1 PI (0.09 and 0.08 mol alpha 1 PI/mol albumin for group I and II, respectively), functionally active alpha 1 PI (percentage of active alpha 1 PI: 53% and 37% for group I and II, respectively) and immunoreactive brI (0.016 and 0.010 mol brI/mol albumin for group I and II, respectively). These results suggested that the lavage fluids from group I contained significant amounts of undefined leukocyte elastase inhibitor(s). Gel filtration of a lavage fluid from group I showed that the undefined elastase inhibitor(s) co-eluted with brI. Most of the lavage fluids were still able to inhibit leukocyte elastase following removal of alpha 1 PI by perchloric acid precipitation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hyperammoniemia during prolonged exercise: an effect of glycogen depletion?

Eight healthy men exercised to exhaustion on a cycle ergometer at a work load of 176 +/- 9 (SE) W corresponding to 67% (range 63-69%) of their maximal O2 uptake (exercise I). Exercise of the same work load was repeated after 75 min of recovery (exercise II). Exercise duration (range) was 65 (50-90) and 21 (14-30) min for exercise I and II, respectively. Femoral venous blood samples were obtained before and during exercise and analyzed for NH3 and lactate. Plasma NH3 was 12 +/- 2 and 19 +/- 6 mumol/l before exercise I and II, respectively and increased during exercise to exhaustion to peak values of 195 +/- 29 (exercise I) and 250 +/- 30 (exercise II) mumol/l, respectively. Plasma NH3 increased faster during exercise II compared with exercise I and at the end of exercise II was threefold higher than the value for the corresponding time of exercise I (P less than 0.001). Blood lactate increased during exercise I and after 20 min of exercise was 3.7 +/- 0.4 mmol/l and remained unchanged until exhaustion. During exercise II blood lactate increased less than during exercise I. It is concluded that long-term exercise to exhaustion results in large increases in plasma NH3 despite relatively low levels of blood lactate. It is suggested that the faster increase in plasma NH3 during exercise II (vs. exercise I) reflects an increased formation in the working muscle that may be caused by low glycogen levels and impairment of the ATP resynthesis.