Effect of alpha1-adrenergic receptor antagonist on the noradrenaline-induced facilitation in respiratory rhythm in newborn rat pons-medulla-spinal cord preparations

We hypothesized that facilitation of respiratory rhythm by noradrenaline (NA) in rat pons-medulla-spinal cord preparations is mediated through alpha1-adrenergic receptors. In 0- to 4-day-old rats, the respiratory frequency (fR) was monitored at the C4 ventral root and trigeminal motor (VMO) outputs. fR at temperature (Te)=23 degrees C was lower than that at a higher Te (27 degrees C) and was increased by NA. At 23 degrees C, lower concentrations of NA were needed to produce the same increases in fR seen at 27 degrees C. With highest NA concentration we tested (50 microM), activity at C4 was maintained in all preparations at both Te, whereas that at VMO was maintained in 50% (27 degrees C) or 88% (23 degrees C) of the preparations. Particularly, tonic activity at C4 appeared in all preparations at both Te, but that at the VMO occurred in 0% (27 degrees C) or 18% (23 degrees C) of the preparations. Based on these results, we used the lower Te (23 degrees C) and applied a low concentration of NA (3 microM) to the preparations. We found that: (1) with the addition of NA, fR was increased without the occurrence of tonic activity and (2) NA-related fR facilitation was inhibited by pre-treatment with the alpha1-adrenergic receptor antagonist prazosin (2 microM). fR was increased by application of the alpha1-adrenergic receptor agonist phenylephrine (4 microM), and this response was inhibited by prazosin (4 microM). At Te=23 degrees C, fR facilitation by NA in newborn rat pons-medulla-spinal cord preparations was obtained by activation of alpha1-adrenergic receptors.     

Purification and characterization of an elastinolytic metalloprotease from Aspergillus fumigatus and immunoelectron microscopic evidence of secretion of this enzyme by the fungus invading the murine lung.

Extracellular proteases have been suggested to be virulence factors in invasive aspergillosis. Since serine protease gene-disrupted mutants retain virulence, other proteases are suspected to be also involved in the degradation of lung structural material. An elastinolytic neutral metalloprotease was purified 320-fold from the extracellular fluid of Aspergillus fumigatus grown on elastin by affinity chromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-75. The molecular mass was determined to be 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No carbohydrate was attached to this metalloprotease, and its first 22 N-terminal amino acids did not show any homology with the known metalloproteases. The enzyme was completely inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. Zn2+ and, to a lesser extent, Co2+ reversed the inhibition caused by 1,10-phenanthroline. The protease hydrolyzed the peptide bonds His-Leu, Ala-Leu, Tyr-Leu, Gly-Phe, and Phe-Phe in the B chain of insulin. Synthetic substrate Abz-Ala-Ala-Phe-Phe-pNA could be used for the fluorimetric assay of the A. fumigatus metalloprotease. This enzyme had maximum activity in the pH range 7.5 to 8.0 and at 60 degrees C. It retained 50% of the protease activity when held at 60 degrees C for 1 h. Zn2+ and Co2+ at 1 mM did not inhibit the protease activity. The metalloprotease was able to hydrolyze elastin, and its elastinolytic activity was comparable to that of the serine protease from this organism. The presence of Zn2+ in the culture medium stimulated the metalloprotease production. Rabbit antibodies prepared against the enzyme severely inhibited the enzyme activity. Immunogold electron microscopy revealed that A. fumigatus invading neutropenic mouse lungs secretes this metalloprotease.     

Purification and properties of hemagglutinin from culture supernatant of Bacteroides gingivalis.

The hemagglutinating factor (hemagglutinin) of Bacteroides gingivalis was prepared from the supernatant of a 5-day diffusate broth culture by ammonium sulfate precipitation and column chromatography with a hydrophobic column of Phenyl-Sepharose CL-4B, DEAE-Sephadex A-50, and Sephadex G-100 gel filtration. The hemagglutinating activity of the preparation was 53.3 times higher than that of ammonium sulfate precipitate. In electron microphotographs, hemagglutinin appears to have a vesicle or tubelike structure. The hemagglutinating activity of intact cells was completely destroyed by heating at 100 degrees C for 10 min, but the activity of extracted hemagglutinin was heat stable. The activity of hemagglutinin was inhibited by L-arginine and L-lysine and partially inhibited by phospholipase D, but it was not affected by proteolytic enzymes, neuraminidase, hyaluronidase, lipase, phospholipase A and C, or sugars. The B. gingivalis hemagglutinin appeared to be comprised mainly of a 40,000-molecular-weight material. The Fab fragment of immunoglobulin G prepared from rabbit antiserum to whole cells of B. gingivalis and monoclonal antibody against the hemagglutinin bound to the cell surface and inhibited the hemagglutinating activity of both the cells and the purified hemagglutinin.     

Cholate-dependent killing of Giardia lamblia by human milk.

We showed previously that nonimmune human milk (NHM) kills Giardia lamblia trophozoites in vitro and presented evidence that killing requires the bile salt-stimulated lipase of milk. Since this enzyme is activated by bile salts, killing should be dependent on the presence of bile salts. We now show that killing by fresh NHM or NHM stored at -70 degrees C is totally dependent on sodium cholate (a bile salt). With less than 0.4 mM cholate, no parasites were killed, whereas with 1 mM cholate, greater than 99.7% were killed by 5% NHM in 30 min. Moreover, killing activity was completely heat labile. The G. lamblia-killing activity of human milk was greatly altered by storage at -10 or -20 degrees C. In less than 23 days, the 50% lethal dose decreased, cholate dependence was lost, and killing activity became heat stable. In contrast, the activity of milk stored at -70 degrees C remained unchanged. Milk lipase activity, like killing activity, became cholate independent during storage at -10 or -20 degrees C. On the basis of these results, we hypothesize that killing of G. lamblia by fresh NHM or NHM stored at -70 degrees C depends on bile salt-stimulated lipase, which must be activated by bile salts. In contrast, NHM stored at -20 degrees C accumulated free fatty acids which kill G. lamblia. In support of this thesis, milk stored at -10 degrees C had a concentration of 18.7 mM free fatty acids compared with only 1.1 mM in an identical sample stored at -70 degrees C.     

The influence of temperature on the effects of acetylcholine and adrenaline on the membrane potential and 86Rb efflux in mouse pancreatic B-cells

Pancreatic mouse islets were used to evaluate the influence of temperature on the B-cell response to acetylcholine and adrenaline. At 20 degrees C, the rate of 86Rb efflux from islet cells was lower, the membrane potential of B-cells was slightly less negative, and glucose-induced electrical activity was characterized by longer slow waves than at 37 degrees C. At 20 degrees C, the acceleration of 86Rb efflux produced by 1 microM-ACh was only reduced by 25%, but its reversibility was slower. Acetylcholine rapidly depolarized the B-cell membrane and increased electrical activity regardless of the temperature. However, this increase was characterized by the appearance of short slow waves of high frequency at 37 degrees C and by continuous spiking at 20 degrees C. Adrenaline (1 microM) inhibited 86Rb efflux at 37 and 20 degrees C, but the amplitude of the inhibition was decreased and its time course and reversibility were altered at the lower temperature. Adrenaline repolarized the B-cell membrane and abolished glucose-induced electrical activity for a longer period at 20 degrees C than at 37 degrees C. In conclusion, no marked decrease in signal transduction occurs at 20 degrees C. This suggests that the difficulty of identifying the currents induced by acetylcholine and adrenaline in patch-clamp experiments performed at room temperature is probably due to the small magnitude of these currents.     

Mechanisms of neutrophil damage to human alveolar extracellular matrix: the role of serine and metalloproteases.

Many syndromes of lung injury are associated with accumulation of neutrophils within the pulmonary parenchyma. These neutrophils have the capacity to produce lung injury by products including proteases and reactive oxygen species (ROS). We examined the ability of activated neutrophils to solubilize human alveolar extracellular matrix (ECM), and by use of scavengers and inhibitors, evaluated the role of ROS and proteases in this process. Supernatants of phorbol myristate acetate-activated neutrophils routinely solubilized 10.2% +/- 0.8% (n = 30) of collagen in human alveolar ECM, as measured by hydroxyproline release. Scavengers of ROS had no significant effect on ECM solubilization. Inhibitors of metalloproteases partially inhibited ECM solubilization (38.5% +/- 4.6% inhibition by ethylenediaminetetraacetic acid [n = 6], and 37.0% +/- 14.7% by 1,10-phenanthroline [n = 6]; p less than 0.05). Inhibitors of the neutrophil serine proteases, elastase and cathepsin G, markedly inhibited ECM solubilization (100.9% +/- 3.7% by alpha 1-protease inhibitor [alpha 1-PI] [n = 6] and 81.9% +/- 0.1% by soybean trypsin inhibitor [n = 6]; p less than 0.01). Since alpha 1-PI completely inhibited solubilization, metalloprotease activity appeared to be related to serine protease activity. This finding was confirmed by the observation that addition of a metalloenzyme activator, p-aminophenylmercuric acetate, in the presence of alpha 1-PI, restored solubilization to the same level as that inhibited by metal chelators. We conclude that human neutrophil metalloproteases and serine proteases directly solubilize human alveolar ECM. Furthermore, neutrophil serine proteases activate latent metalloproteases. However, ROS were not demonstrated to play a major role in ECM solubilization in our system.     

Identification of four new mutations in the short-chain acyl-CoA dehydrogenase (SCAD) gene in two patients: one of the variant alleles, 511C-->T, is present at an unexpectedly high frequency in the general population, as was the case for 625G-->A, together conferring susceptibility to ethylmalonic aciduria

We have shown previously that a variant allele of the short-chain acyl- CoA dehydrogenase ( SCAD ) gene, 625G-->A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote for two mutations, 274G-->T and 529T-->C. These mutations were not present in 98 normal control alleles, but the 529T-->C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C-->T mutation and the 625G- ->A polymorphism in one allele, and a single point mutation, 511C-->T, in the other. The 1147C-->T mutation was not present in 98 normal alleles, but was detected in three alleles of 133 patients with elevated EMA excretion, consistently as a 625A-1147T allele. On the other hand, the 511C-->T mutation was present in 13 of 130 and 15 of 67 625G alleles, respectively, of normal controls and patients with elevated EMA excretion, and was never associated with the 625A variant allele. This over-representation of the haplotype 511T-625G among the common 625G alleles in patients compared with controls was significant ( P < 0.02), suggesting that the allele 511T-625G-like 511C-625A- confers susceptibility to ethylmalonic aciduria. Expression of the variant R147W SCAD protein, encoded by the 511T-625G allele, in COS-7 cells showed 45% activity at 37 degrees C in comparison with the wild- type protein, comparable levels of activity at 26 degrees C, and 13% activity when incubated at 41 degrees C. This temperature profile is different from that observed for the variant G185S SCAD protein, encoded by the 511C-625A allele, where higher than normal activity was found at 26 and 37 degrees C, and 58% activity was present at 41 degrees C. These results corroborate the notion that the 511C-625A variant allele is one of the possible underlying causes of ethylmalonic aciduria, and suggest that the 511C-->T mutation represents a second susceptibility variation in the SCAD gene. We conclude that ethylmalonic aciduria, a commonly detected biochemical phenotype, is a complex multifactorial/polygenic condition where, in addition to the emerging role of SCAD susceptibility alleles, other genetic and environmental factors are involved.      

Oxygen-independent killing of Bacteroides fragilis by granule extracts from human polymorphonuclear leukocytes.

Granule proteins from human neutrophils were prepared by extraction with acetate, and their antibacterial activity against Bacteroides fragilis was determined. Activity was highly dependent on pH; greatest killing occurred at the most acid pH tested (pH 5.0). Optimum activity was observed at physiological ionic strength and low bacterial numbers. Killing was inhibited by incubation temperatures of less than 37 degrees C. Eight times more extract was required to kill 50% of stationary-phase bacteria, compared with those growing in logarithmic phase. The antibacterial effect of granule extract was destroyed by boiling, but some activity was retained after heating to 56 degrees C and 80 degrees C. Granule extract activity was tested under conditions in which oxygen-dependent antibacterial systems were inhibited. The rate and extent of killing was not affected by anaerobiosis, sodium azide, or cysteine hydrochloride. These results suggest that the activity of granule extract is independent of oxidative antibacterial systems, and therefore, under conditions that occur in anaerobic infections, potent leukocyte granule-associated mechanisms exist for the destruction of B. fragilis.     

Purification and characterization of proteases secreted by transforming schistosomula of Schistosoma mansoni

Schistosomula of Schistosoma mansoni which are mechanically transformed at 4 degrees C and are then incubated at 37 degrees C in defined medium spontaneously secrete two proteases, a major one of 28 kDa and a minor one of 60 kDa. These were purified by ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel AcA 54 with yields of 33% and 29%, respectively. Both appeared as single bands by silver staining following sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. The 28 kDa protease is a glycoprotein that has a pI of 11 or higher and an optimal activity around pH 9.0. It cleaves casein, gelatin and human C3 and C3b. It is metal-ion independent and is inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, soy-bean trypsin inhibitor, alpha 1 antitrypsin, Zn2+ ions, sodium dodecyl sulphate and normal human serum. The 60 kDa protease is a glycoprotein with a pI of 9.2. It can also cleave casein and gelatin and its activity is inhibited by phenylmethanesulfonyl fluoride but not by diisopropyl fluorophosphate or sodium dodecyl sulphate. We suggest that these proteases may play a role during cercarial penetration of the skin and in shedding of the cercarial glycocalyx.     

Characterization of six mutations in five Spanish patients with mitochondrial acetoacetyl-CoA thiolase deficiency: effects of amino acid substitutions on tertiary structure

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine metabolism. We identified and characterized 6 mutations, DelE85, K124R, A127V, Q145E, G152A, and E345V in 5 Spanish T2-deficient patients. Transient expression of mutant cDNAs was done at 37 and at 30 degrees C. Expression of the Q145E mutant cDNA resulted in about 12.5% normal amount at 37 degrees C and it retained 15% residual T2, indicating that specific activity of Q145E mutant protein was almost normal. This mutation reduced the heat stability of T2 activity. Although no significant residual activity was detected in either the G152A and A127V substitution, mutant proteins were detected, at 12.5% the normal amount at 37 degrees C and one-half normal at 30 degrees C for A127V, and 25 % only at 30 degrees C for G152A. Mutant proteins with Q145E, G152A, or A127V accumulated at 30 degrees C expression were stable for 48 h at 37 degrees C after cycloheximide treatment. Expression of DelE85, K124R, and E345V cDNAs gave neither residual T2 protein nor T2 activity. We constructed an improved tertiary structural model of T2 based on the X-ray crystal structure of acetoacetyl-CoA thiolase of Zoogloea ramigera. On the basis of this model, K124, A127, and G152 are located near the active site, mutations of which might affect catalytic function whereas Q145E, De185E, and E345V are distant from the active site with mutants being expected to destabilize the tertiary structure, especially during protein folding and dimerization.     

Long-term storage of peroxidase-labelled immunoglobulins for use in enzyme immunoassay

Optimal conditions for long-term storage of peroxidase-labelled immunoglobulins for use in enzyme immunoassays have been investigated with particular emphasis on the preservation of the immunological and enzyme activity. Immunoglobulin G preparations from different animal species were purified according to conventional methods and labelled with horseradish peroxidase. Conjugates were stored at different temperatures either in liquid form with 50% glycerol, after lyophilization or as ammonium sulphate precipitates. In addition, the conjugates were also frozen and stored at -40 degrees C. The immunological and enzymatic activity of the conjugates was evaluated at regular intervals throughout storage. The best results were obtained with conjugates stored as ammonium sulphate precipitates at 4 degrees C. Under these conditions, the complexes retained after 2 years of storage 92 and 91% of their enzymatic and immunological activity respectively and gave excellent reproducibility in ELISA. The conjugates exhibited a very low degree of self-aggregation. The ammonium sulphate did not interfere in the ELISA. Storage at elevated temperatures resulted in a greater decrease in enzymatic than immunological activity but the results obtained with the ammonium sulphate precipitates were clearly superior to those obtained with alternative storage procedures. The estimated half life of the whole enzymatic-immunological activity of conjugates at 4 degrees C was 9 years. The stability of both activities at 25 degrees C was also reasonably good for short periods. Conjugates stored for 40 days at 37 degrees C lost 60% enzyme activity but retained unaltered their immunological activity and gave rise to high blanks in the ELISA, due to auto-aggregation.     

Heat treatment of normal human sera reveals antibodies to bactericidal permeability-inducing protein (BPI).

Heat treatment of normal sera to 56 degrees C for 30 min, a common procedure for the inactivation of viruses, e.g. HIV, reveals the presence of antibodies to neutrophil cytoplasm antigens (ANCA), as detected by indirect immunofluorescence on ethanol-fixed human neutrophils and by antigen-specific ELISA for BPI. Reactivity was not seen to the other common vasculitis-associated antigens proteinase 3 (PR3) or myeloperoxidase (MPO). The effect of temperature was maximal at 56 degrees C, with substantial antibody demonstrable after only 5 min at this temperature. In experiments using polyethylene glycol (PEG)6000 to remove immune complexes, the effect of heating could be abrogated by preincubation with 8% PEG, which suggested that these anti BPI antibodies might be complexed in sera. After passage of normal plasma over a protein G column, the acid-eluted fraction contained elevated levels of antibodies to BPI but not to other vasculitis-associated antigens such as PR3 or MPO, nor to glomerular basement membrane (GBM), the Goodpasture antigen which is recognized by the pathogenically important human antibodies shown to mediate nephritis in transfer experiments. Moreover the levels of anti-BPI in the IgG fraction could be augmented by preincubation with glycine pH 2.5 for 30 min. This anti-BPI activity could be inhibited by addition of the unbound material from the protein G column and this inhibitory material was not heat-labile at 56 degrees C. The molecular specificity of this autoreactivity was confirmed using recombinant BPI in coincubation experiments and the epitope localized to the C or N terminal moieties by the use of recombinant fusion proteins.     

Characterization of haptoglobin-binding properties of streptococci of serological group G

Two group G streptococcal cultures (G 10187, G 11122) with surface antigen T4 possess surface receptors for human haptoglobin (Hp). G 10187 additionally interacted with immunoglobulin G and albumin, G 11122 with fibrinogen and fibronectin. Binding of 125I-Hp 2-1 was time-dependent, saturable, reversible in the presence of unlabelled Hp and could be inhibited by unlabelled human-Hp 2-1, -Hp 2-2, -Hp 1-1, Hp-hemoglobin complexes and by Hp preparations from pigs, horses and rabbits. The Hp binding sites could be destroyed by heat treatment (95 degrees C) and by proteolytic treatment of the bacteria. Hp binding sites were solubilized from group G streptococcal surface by heat treatment of the bacteria at acid pH and subsequently isolated by affinity chromatography on Hp 2-1 sepharose. SDS-PAGE and Western blotting of the Hp binding proteins revealed numerous protein bands with 125I-Hp 2-1 binding activity. Specific antibodies against G streptococcal binding proteins prepared in chickens inhibited binding of 125I-Hp to group G and group A streptococci, but not to Actinomyces pyogenes.     

Effects of surface-active agents on neutrophil receptors.

An easily performed assay to identify the C3b and Fc receptors on human neutrophils was developed. Salmonella typhimurium were treated with fluorescein and then incubated in nonimmune fresh human serum, which led to C3b fixation via activation of the alternative pathway. Similarly, type II pneumococci were treated with fluorescein and opsonized with type-specific rabbit antiserum. Neutrophils bearing C3b and Fc receptors formed rosettes with the respective bacteria, which were easily readable because of their bright fluorescence. Incubation of neutrophils at 37 degrees C with C3-coated bacteria generated 54 +/ 4% C3b rosettes, whereas neutrophils incubated with immunoglobulin G-coated bacteria yielded 75 +/ 7% rosettes. Incubation at 4 degrees C inhibited the formation of C3b rosettes but not Fc rosettes. Heat inactivation of the fresh human serum at 56 degrees C for 30 min completely inhibited the formation of the C3b rosettes, and addition of heat-aggregated immunoglobulin G to the polymorphonuclear leukocyte blocked the ability of the polymorphonuclear leukocyte to bind immunoglobulin G-coated bacteria. Addition of 1.0 mM N-ethylmaleimide, 0.1 mg of trypsin per ml, 10 mM H2O2, O2- generated by xanthine-xanthine oxidase, and 8 times 10(-4) M hydrocortisone inhibited the C3b receptor, but did not inhibit the Fc receptor. In neutrophils, the selective effect of the various inhibitors suggests that the Fc and C3b receptors are distinct entities.     

Different antiviral activity and cell specificity of interferon preparations produced by mouse peritoneal cells at 37 degrees C and at 26 degrees C

Three sublines of mouse L cells and mouse embryo fibroblasts were used for determination of the antiviral activity of mouse interferons produced by nonadherent peritoneal exudate cells incubated either at 37 degrees C or at 26 degrees C. IFN produced at 37 degrees C or at 26 degrees C had the same antiviral activity in L Borgen, L929 cells. However, in MEC IFN-37 degrees had relatively higher activity than IFN-26 degrees. Of the interferon investigated only IFN-37 degrees exhibited antiviral activity in the established line of rat kidney cells. The IFN preparations showed no activity in the human and chicken cells. The studies on the sensitivity of viruses to both forms of IFN revealed that EMC and VSV viruses were equally sensitive to IFN-26 degrees C. However, the replication of EMC virus was more strongly inhibited by IFN-37 degrees than the multiplication of VSV virus.     

Genetic diversity of Gallibacterium anatis isolates from different chicken flocks

Amplified fragment length polymorphisms (AFLPs) were used to characterize the genotypic diversity of a total of 114 Gallibacterium anatis isolates originating from a reference collection representing 15 biovars from four countries and isolates obtained from tracheal and cloacal swab samples of chickens from an organic, egg-producing flock and a layer parent flock. A subset of strains was also characterized by pulsed-field gel electrophoresis and biotyping. The organic flock isolates were characterized by more than 94% genetic similarity, indicating that only a single clone was apparent in the flock. The layer parent flock isolates were grouped into two subclusters, each with similarity above 90%. One subcluster contained only tracheal isolates, while the other primarily included cloacal isolates. In conclusion, we show that AFLP analysis enables fingerprinting of G. anatis, which seems to have a clonal population structure within natural populations. There was further evidence of clonal lineages, which may have adapted to different sites within the same animal.     

A cobra venom factor (CVF)-induced C3 convertase activity in the hemolymph of Galleria mellonella

A cobra venom factor (CVF)-induced C3 convertase has been generated from the hemolymph of Galleria mellonella. CVF was immobilized on Sepharose 4B and treated with cell-free hemolymph obtained from either unvaccinated G. mellonella larvae or larvae immunized with formalized Pseudomonas aeruginosa. The C3-cleaving activity was detected by the ability to cleave the alpha-chain of bovine C3 in a manner analogous to the CVF-induced mammalian C3 convertase, CVF,Bb. The insect-derived C3 convertase formed at 28 degrees C but not at 37 degrees C, then once formed was active at both 28 degrees C and 37 degrees C. EDTA did not inhibit the formation and action of the insect derived C3 cleaving activity.     

Allergenicity and lymphocyte-stimulating property of rice protein.

Two protein fractions of rice, grain, glutelin and globulin, were prepared by dilute alkali and salt extraction, respectively. The globulin fraction was separated into G1-1, G1-2, and G1-3 fractions by Sephadex G-200 column chromatography. The allergenic activities and lymphocyte-stimulating properties of these fractions were investigated by the radioallergosorbent test (RAST) with sera from 6 individuals who showed immediate skin reaction to soluble rice extract and by 3H-thymidine incorporation tests with 5 subjects with indurated skin reaction of delayed onset. All fractions were found to be reactive with specific IgE antibody, and G1-1 and G-2 revealed lymphocyte-stimulating activity. RAST inhibition revealed considerable cross-reactivity of IgE antibody with the glutelin and globulin fractions. When the glutelin and globulin fractions were heated at 60 degrees C for 1 hr, 100 degrees C for 2 min, or 100 degrees C for 10 min, RAST activities were reduced to 40%-70% of native. On the other hand, lymphocyte-stimulating activities of the globulin fraction heated at 60 degrees C for 1 hr or 100 degrees C for 2 min were enhanced up to 6 times of native activities, while those of identically treated glutelin fractions remained unchanged.     

Novel thermostable serine collagenase from Thermoactinomyces sp. 21E: purification and some properties

A thermophilic actinomycete strain Thermoactinomyces sp. 21E producing a highly thermostable serine collagenase was isolated from Bulgarian soil. The collagenase, produced extracellular by Thermoactinomyces sp. 21E, was purified to homogeneity by heat treatment, ultrafiltration, saturation with ammonium sulfate and gel filtration chromatography with a 101-fold increase in specific activity and 58% recovery. The collagenase has a relative molecular mass of 50000 by SDS-PAGE. The optimum temperature for the enzyme activity was 60-65 degrees C in the absence of Ca(2+) and 70-75 degrees C in the presence of Ca(2+). About 40% of the original activity remaining after incubation at 85 degrees C for 30 min in the presence of Ca(2+). The optimum pH for the enzyme activity was 9.0-9.5 and the enzyme was stable for 1h at 70 degrees C in the pH range from 7.5 to 12.5. The collagenase was strongly inhibited by active-site inhibitors of serine protease PMSF and DFP, which indicated that the enzyme is serine protease. The enzyme activity was completely inhibited by Hg(2+), Cu(2+) and Fe(2+). However, Ca(2+ )strongly activated the collagenase activity. The collagenase from Thermoactinomyces sp. 21E showed high activity toward type I collagen, acid-soluble collagen, gelatin and Pz-PLGPR. However, elastin for collagenase was inert as substrate. The properties of the collagenase from strain 21E suggest that this enzyme is a new collagenolytic protease that differs from the collagenases and serine proteases reported so far.