Activity of Ca<Superscript>2+</Superscript>-Dependent Neutral Proteases in Tissues of Ground Squirrel during Hibernation and during Self-Warming after Induced Awakening

Cyclic changes in activity of Ca²⁺-dependent neutral protease occur during preparation for hibernation, with an increase in September and November and decrease in October and December. During hibernation proteolytic enzyme activity decreased, while during self-warming after induced awakening, the role of Ca²⁺-dependent processes in the tissues of ground squirrels increased according to the body temperature.     

中国大鲵(Andrias davidianus)7种组织器官蛋白水解酶的种类和活性分析

目的 了解大鲵(Andrias davidianus)脑、心脏、肺、肾脏、眼球、皮肤和肌肉的蛋白水解酶种类和性质。方法 采用蛋白水解酶复性电泳(GPAGE)技术。结果 1．脑的蛋白水解酶在pH4．5时有较弱的活性，在其他pH条件下没有活性；2．心脏和肺的蛋白水解酶在pH4．5和pH7．0时有活性；3．肾脏的蛋白水解酶在pH4．5时有活性；在pH7．0时，蛋白水解酶的活性强、种类多；4．在pH4．5和pH9．5条件下，未检出眼球有蛋白水解酶活性；5．肌肉的蛋白水解酶在pH9．5时活性较强，在pH7．0时，蛋白水解酶活性最强、种类最多；6．皮肤的蛋白水解酶种类与肌肉相似，但活性比肌肉弱。结论 大鲵肾脏和肌肉的蛋白水解酶活性强、种类多；脑、心脏、肺、皮肤的蛋白水解酶活性弱、种类少；眼球几乎无蛋白水解酶活性。脑的蛋白水解酶活性的最适pH为酸性；心、肺和肾的蛋白水解酶活性的最适pH为中性偏酸；皮肤和肌肉的蛋白水解酶活性的最适pH为中性。     

Proteolysis on the body surface of pyrethroid-sensitive and resistant Varroa destructor

The aim of this work was to determine the activity of proteases and protease inhibitors sampled from the body surface of tau-fluvalinate-sensitive and resistant V. destructor. Proteins were isolated from the tau-fluvalinate-sensitive and resistant mites, while mites untreated with tau-fluvalinate constituted the control. Subsequently, the following methodology was applied: protein concentration assay by the Lowry method — as modified by Schacterle and Pollack; assay of proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; identification of proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee and Lin method; identification of acidic, neutral and basic protease activities by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method and for protease inhibitor detection with the Felicioli method. The highest value of protein concentration was found in the tau-fluvalinate-sensitive V. destructor, while the highest activity levels of acidic, neutral and alkaline proteases were observed in the tau-fluvalinate-resistant mites. Aspartic, serine, thiolic and metallic proteases were found in the drug-resistant and drug-sensitive Varroa mites. The control samples were found to contain aspartic and serine proteases. In an acidic and alkaline environment, the results revealed a complete loss of inhibitor activities in the in vitro analyses and electrophoresis. Serine protease inhibitor activities (at pH 7.0) were high, especially in the group of tau-fluvalinate-resistant mites.     

Inhibition of interleukin-1 (IL-1) induced neutral proteases from rabbit articular chondrocytes by WY-46,135 and WY-48,989

The effects of potential anti-osteoarthritic compounds both on the direct inhibition of collagenase and neutral protease activities and on IL-1 induced release of neutral proteases from rabbit articular chondrocytes were investigated. WY-46,135 ((+)-N-[[[(5-chloro-2-benzothiazolyl)thio]phenyl]acetyl]-l-cysteine) directly inhibited collagenase activity (IC₅₀=15.4 M). This inhibition was reversible upon dialysis. WY-46,135 also directly inhibited neutral protease activity (IC₅₀=16.8 M) but did not significantly block bacterial collagenase activity at a concentration of 80 M. In contrast, WY-48,989 (4-[[2-(7-chloro-2-phenyl-2H-pyrazolo[4,3-c]quinolin-4-yl)ethyl]amino]benzonitrile) did not directly inhibit either collagenase (10M) or neutral protease (100 M) activity. Both WY-48,989 and WY-46,135 inhibited IL-1 stimulated release of neutral proteases (IC₅₀=3M). The activities of these compounds represents two potential approaches for the treatment of osteoarthritis. WY-46,135 combines direct metalloprotease inhibitory activity with the inhibition of IL-1 stimulated neutral protease release from articular chondrocytes while WY-48,989 selectively inhibits the IL-1 induced release of metalloproteases.     

Proteolytic activity of Aeromonas caviae

Aeromonas strains produce a variety of virulence factors including proteases. Studies on the kinetics of growth of Aeromonas caviae NRRL B-966 and its proteases suggest that the proteolytic activities are produced throughout the growth phase, with peak level occurring at stationary phase. A. caviae synthesize both intracellular and extracellular proteases with the latter account for major portion of the total activity. Optimum pH for the A. caviae proteolytic activity is at 7.0. A. caviae produces a thermoresistant protease, whose activity is dependent on Mg⁺⁺ and Ca⁺⁺ ions. Inhibition of proteolytic activity by phenyl methyl sulfonyl fluoride suggest the presence of a serine protease in A. caviae. Nitrogenous compounds enhance the proteolytic activity while carbohydrates tested in this study inhibit the activity.     

Digestion of the fifth component of complement by leukocyte enzymes. Sequential generation of chemotactic activities for leukocytes and for tumor cells.

Leukocytes contain within their lysosomal granules enzymatic activity that will generate from C5 chemotactic activity for leukocytes (neutrophils) and tumor (Walker carcinosarcoma) cells. Similar activity has been found in phagocytic supernatant fluids from neutrophils and in purified preparations of the leukocyte neutral proteases elastase and cathepsin G. White leukotactic activities can be generated from either the third (C3) or the fifth (C5) components of complement, only C5 serves as a source for generation of the chemotactic activity for tumor cells. As has been previously shown with trypsin, the C5-related chemotactic activities generated by leukocyte proteases are time-dependent: leukotactic activity appears early, then disappears, and is replaced by chemotactic activity for tumor cells. The generation of these chemotactic activities from C5 is blocked by prior treatment of leukocyte preparations with the neutral protease inhibitor Trasylol. The demonstration that enzyme activities from leukocytes have the ability to generate tumor cell chemotactic factors from C5 suggests a possible mechanism by which the development of metastatic lesions may be promoted at sites of tissue injury or inflammation.     

Influence of ACTH on activity of some granulocytic proteases in patients with multiple sclerosis.

The effect of treatment with ACTH on activity of granulocytic acid and neutral proteases in patients suffering from multiple sclerosis was studied. Protease activity was distinctly higher in granulocytes from patients. ACTH normalized the activities investigated in patients with exacerbating course multiple sclerosis, but had no influence on hydrolases in the progressive form of the disease.     

Annual skeletal changes in the little brown bat,Myotis lucifugus lucifugus,with particular reference to pregnancy and lactation

Studies of bone from summer-active little brown bats, Myotis lucifugus lucifugus, have demonstrated sex differences in the renewal of skeletal mineral reserves following spring-arousal from hibernation. Patterns of bone remodeling in both sexes of bats indicate that new bone formation does not occur during hibernation: All new bone formation occurs during the summer-active season. Results show that a short period of time elapses after hibernation before the initial demands of a large fetus and rapidly growing neonate are expressed on maternal skeletal reserves. Bone loss in summer-active females was associated with pregnancy and lactation, whereas summer-active males did not show evidence of bone loss but, instead, uninterrupted bone accretion throughout the summer-active season. Osteoclasts and bone-forming osteoblasts, absent during the hibernation period, reappeared on bone surfaces following spring-arousal from hibernation. There was no apparent increase in osteoclast numbers or activity during lactation but resorption cavities were found in deep cortical lamellae distant from bone surfaces. The increase in bone resorption in lactating bats appeared to be by osteocytic osteolysis, suggesting that it might be a significant mechanism of bone/calcium regulation in this hibernating mammal throughout the year.     

Protease activity of Blastocystis hominis

Parasite-derived proteases are important for the parasite life cycle and the pathogenesis of the disease they produce. Proteases of intestinal protozoan parasite Blastocystis hominis were studied for the first time with azocasein assays and gelatin SDS-PAGE analysis. Parasitic lysates were found to have high protease activity and nine protease bands of low (20–33 kDa) and high (44–75 kDa) molecular weights were reported. Proteases were found to be pH-dependent and highest proteolytic activity was observed at neutral pH. Inhibition studies showed that B. hominis isolate B, like many other protozoan parasites, contains mainly cysteine proteases.     

Proteolytic activities in cultures of selected white-rot fungi

The presence of multiple intracellular and extracellular proteolytic activities in trophophasic (nutrientrich) and idiophasic (carbon-or nitrogen-starved) cultures of the white-rot fungi Trametes versicolor and Phlebia radiata was demonstrated by electrophoresis on polyacrylamide gels containing denatured haemoglobin as a substrate. In the trophophasic cultures of T. versicolor, seven electrophoretically distinguishable proteases were defined using mycelial extracts and six (three clear and three less intensive) of secreted proteases. For P. radiata eight bands of intracellular and five bands (one distinct and four less active) of extracellular proteolytic activities were detected. Gel electrophoresis revealed changes in patterns of secreted and mycelial proteinases upon carbon or nitrogen deprivation. The changes were seen both as an increase in activity of certain bands and as the appearance of new proteolytic bands. Specific activities of extracellular proteinases, assayed under idiophasic (—C or —N) conditions, increased 2—3 fold as compared to those upon nutrient sufficiency. These changes accompanied a shift to secondary metabolism and onset of ligninolytic activity.     

Screening of different classes of proteases in microfilarial and adult stages of Setaria cervi

Many of the filarial proteases involved in critical physiological functions are expressed in stage-specific manner and belong to various mechanistic classes. Setaria cervi, a bovine filarial parasite express different classes of proteases. This parasite shows strong antigenic cross-reactivity with human filarial parasites Wuchereria bancrofti and Brugia malayi. Somatic extracts of S. cervi microfilariae (mf) and adult stages as well as their excretory–secretory (ES) products were screened for the presence of different classes of proteases using general (casein, bovine hemoglobin) and class specific substrates. Detergent-soluble extracts of male and female worms were also screened. Significant enzyme activity was detected in ES products both at pH 5.0 and 7.0 with casein. Cathepsin B-like activity was found to be much higher in membrane-bound extract than in the crude-soluble extract. However, it was also found to be actively secreted by both mf and adult worms. Cathepsin D-like activity assayed at pH 3.0 was very low both in somatic extract as well as in ES products. Collagenase activity at neutral pH showed higher levels, both in somatic extract and ES products. Cathepsin L-like activity was detected only in crude-soluble extract but was below detectable limit in ES products. Leucine aminopeptidase activity was significant both in crude-soluble extract and ES products. This study, thus, might be helpful for a better understanding of host–parasite interaction and identification of appropriate virulence factors that may be targeted as vaccine and/or drug targets against lymphatic filariasis.     

Effects of cold exposure and hibernation on renal Na,K-ATPase of the jerboa Jaculus orientalis

Changes in activity and abundance of renal Na,K-ATPase were evaluated during cold exposure and hibernation of the jerboa Jaculus orientalis by measuring the hydrolytic activity, the number of units and the transport activity of Na,K-ATPase in isolated nephron segments. As compared to controls, jerboas exposed to cold (6 °C) for 4–5 weeks displayed mild diuresis, decreased urinary osmolality and increased kaliuresis. In cold-exposed jerboas, Na,K-ATPase hydrolytic activity was reduced in the medullary thick ascending limb and enhanced in the cortical and outer medullary collecting duct, whereas it was not altered in other nephron segments. The number of Na,K-ATPase units and the activity of Na,K-pump, determined by [³H]-ouabain binding and by ouabain-sensitive rubidium uptake respectively, changed in parallel with the hydrolytic activity in the medullary thick ascending limb and cortical collecting duct. The maximal rate of activity (V _max) of Na,K-ATPase was not modified further during hibernation. Thus, cold exposure, but not the onset of hibernation, induces segment-specific changes in the abundance and activity of Na,K-ATPase units which are likely to be related to the entry into hibernation, but not to the maintenance of some renal functions during deep hibernation.     

Expression of cysteine protease inhibitors in human gliomas and meningiomas

Increased levels of human cysteine proteases have been implicated in the progression of tumors from the premalignant to the malignant state. The physiological activities of these proteases are regulated by their interactions with specific inhibitors. To our knowledge there have been no previous reports about the cysteine protease inhibitors (CPIs) in human brain tumors. In the study reported here, we determined CPI activity during glioma progression and compared that with normal human brain tissue. We also determined CPI activities in meningioma and glioblastoma cell lines in vitro. This activity was significantly higher in normal brain tissue and low-grade glioma than in anaplastic astrocytoma and glioblastoma. CPI activity was significantly higher in benign and atypical meningioma cell extracts in comparison with those from malignant meningiomas and with those from glioblastoma cell lines. After several passages, one benign meningioma cell line showed reduced levels of CPI and increased levels of cathepsin. Our results suggest that decreases in the activities of CPI may contribute to the malignant properties of brain tumors.     

Collagenolytic protease complex from hepatopancreas of kamchatka crab: Enzyme activity of individual components

We determined primary substrate specificity and proteolytic activity with respect to polypeptides and short synthetic substrates for 9 individual proteases, components of collagenolytic complex of Kamchatka crab, and their dependence on temperature and pH. It was shown that the enzyme complex containing at least one metalloprotease and two serine proteases exhibits a variety of enzyme activities acting synergistically, which is of great importance for medical and cosmetological applications. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 10, pp. 391–396, October, 1999     

Mechanism of action of proteinases of Pseudomonas aeruginosa

Of the two proteases produced by Pseudomonas aeruginosa, one whose optimal pH is neutral, exhibits elastolytic activity. This elastase is produced as a prepropeptide, subsequently modified by proteolysis, and finally secreted as an active enzyme. Both proteases act mainly on hydrophobic aminoacids. The most important site for the elastase seems to be the P'1 site where Phe, Tyr and Leu residues enhance hydrolysis. The alkaline protease is less specific of this site. At least four aminoacids are needed to obtain measurable rates of hydrolysis. A synthetic substrate, Ala-Ala-Phe-Ala, is proposed for conductimetric measurements of Pseudomonas aeruginosa elastase activities in the nanomolar range. A review of recent studies shows that a very wide range of proteins can be hydrolyzed by the two Pseudomonas aeruginosa proteases, a fact which may explain why these enzymes are major determinants of the bacteria's infectivity.     

Membrane-bound proteinases of Halobacterium halobium

Cell envelopes of Halobacterium halobium contain proteolytic activities, which cleave ¹²⁵I-insulin and ¹⁴C-casein with pH-optima in the alkaline range. About 50% of the intracellular proteases are strongly attached to the cell envelopes and cannot be removed by washing with 25% sodium chloride solution. This high amount of membrane-bound proteases is independent of the kind of cultivation. The main part of the envelope-bound proteolytic activity is attributed to metalloproteases, with EDTA and o-phenanthroline causing inhibition. Serine and cysteine protease inhibitors had no influence. In density gradients (consisting of Ficoll and 20% sodium chloride) proteolytic activities could be detected in two distinct bands, a minor band at a density of 1.165 and the main proteolytic activity at 1.145 g/cm³. When the cell wall was removed from the other cell envelope constituents by EDTA treatment, a single band with proteolytic activity in the density gradient at 1.145 g/cm³ was detectable. Lubrol, Brij 35, and octyl glucoside were used for the solubilization of the membrane-bound proteases. The greatest part of them could be solubilized using 0.2% octyl glucoside. ¹⁴C-labelled bacteriorhodopsin was not cleaved by any of the intracellular or extracellular proteinases.     

Catabolism of Lipid-Free Recombinant Apolipoprotein Serum Amyloid A by Mouse Macrophages In Vitro Results in Removal of the Amyloid Fibril-Forming Amino Terminus

Serum amyloid A fibrils are formed when the normally rapid catabolism of the acute-phase reactant apolipoprotein serum amyloid A (apoSAA) is incomplete; thus amyloidosis may be viewed as a condition of dysregulated proteolysis. There is evidence that apoSAA is dissociated from plasma high-density lipoprotein (HDL) prior to fibril formation. The objective of this study was to investigate degradation of lipid-free apoSAA by tissue macrophages derived from amyloid-susceptible CBA/J mice in vitro . Peritoneal macrophages derived from untreated (normal) mice converted apoSAA (12 kDa) to a single 4 kDa C-terminal peptide while splenic macrophages converted apoSAA to 10, 7 and 4 kDa C-terminal peptides and a 4 kDa peptide that lacked the C- and N-terminal regions. Similar patterns of proteolysis occurred when peritoneal and splenic macrophages from amyloidotic CBA/J mice were used. Conditioned medium prepared from peritoneal, but not splenic macrophages, degraded apoSAA. Specific sites of cleavage indicated activity of cathepsin G- and elastase-like neutral proteases. The data indicate that lipid-free apoSAA can be degraded by secreted or cell-associated neutral proteases that are generated by macrophages to yield peptides that lack fibrillogenic potential.     

Endurance training decreases the alkaline proteolytic activity in mouse skeletal muscles

Summary Alkaline and myofibrillar protease activities of rectus femoris, soleus, and tibialis anterior muscles and the pooled sample of gastrocnemius and plantaris muscles were analyzed in male NMRI-mice during a running-training program of 3, 10, or 20 daily 1-h sessions. The activity of citrate synthase increased during the endurance training, reflecting the increased oxidative capacity of skeletal muscles. The activities of alkaline and myofibrillar proteases continually decreased in the course of the training program in all muscles studied. Instead, the activity of-glucuronidase (a marker of lysosomal hydrolases) increased in all muscles. The highest activities were observed at the beginning of the training program. Present results, together with our earlier observations, show that the type of training, running as opposed to swimming, modulates the training responses in alkaline protease activities. Further, diverse adaptations in the activities of alkaline proteases and a lysosomal hydrolase suggest differences in the function of different proteolytic systems.This study was supported by the Academy of Finland and the Research Council for Physical Education and Sport (Ministry of Education, Finland)     

Morphohistochemical changes of the blood cells in the hibernating frog (Rana esculenta L.)

Some morphological features (degree of nuclear segmentation in neutrophils and eosinophils), histochemical patterns (DHFR, LDH, G6PDH) and, in addition, the nucleic acid distribution (DNA, RNA with acridine orange) in the peripheral blood cells of Rana esculenta, during the hibernation phase were investigated. All the observed parameters varied significantly in the frog during hibernation in comparison with the active period. The most evident changes were an increase in the nuclear segmentation of the neutrophils and a lower activity of the histochemically demonstrable DHFR and LDH, probably due to cell ageing. These findings suggest that, in hibernating frogs, there is an increase in the life span of the peripheral blood cells as a consequence of a reduced metabolic activity and a slowing of haematopoiesis.     

Destructive potential of the aspartyl protease cathepsin D in MHC class II-restricted antigen processing

Whether specific proteases influence MHC class II antigen presentation is still not clearly defined. Cathepsin D, one of the most abundant lysosomal proteases, is thought to be dispensable for MHC class II antigen presentation, yet in vitro digestions of antigen substrates with endosomes/lysosomes from antigen-presenting cells sometimes reveal a dominant role for pepstatin-sensitive aspartyl proteases of which cathepsin D is the major representative. We tested whether the aspartyl protease substrate myoglobin requires cathepsin D activity for presentation to T cells. Surprisingly, in dendritic cells (DC) lacking cathepsin D, presentation of two different myoglobin T cell epitopes was enhanced rather than hindered. This paradox is resolved by the finding that pepstatin-sensitive myoglobin processing activity persists in lysosomes from cathepsin D-null DC and that this reduced activity, most likely due to cathepsin E, is closer to the optimum level required for myoglobin antigen presentation. Our results indicate redundancy among lysosomal aspartyl proteases and show that while processing activities can be productive for MHC class II T cell epitope generation at one level, they can become destructive above an optimal level.