Trypsin-like Enzyme Activity Research Articles

Metformin as an alternative therapeutic intervention in hemorrhagic shock

Hemorrhagic shock (HS) continues to significantly contribute to civilian and military mortality, underscoring the critical need for new resuscitation strategies. Recent discussions have emphasized the pivotal role of impaired oxygen delivery and cellular metabolism in exacerbating HS-related damage. Metformin, a well-established diabetes medication, exhibits promising potential to enhance cellular metabolism and activate pathways linked to improved oxygen uptake, thereby optimizing cellular energy management. Thus, this study aimed to assess metformin's effcacy in ameliorating outcomes in animals subjected to HS. To achieve this, Wistar rats were surgically prepared with femoral artery and vein catheters. HS was induced by controlled blood withdrawal to maintain a mean arterial pressure (MAP) within the range of 35-40 mmHg for 90 minutes. Throughout the protocol, blood pressure (BP) and heart rate (HR) were assessed. The animals were divided into two groups: Lactated Ringer's solution (LR) and LR treated with Metformin (Met). Metformin was administered in three bolus injections: at the start of resuscitation (10mg), 45 minutes into resuscitation (5mg), and 90 minutes into resuscitation (5mg). After 120 minutes, the animals were euthanized, and blood samples were collected for subsequent analysis, including metabolomic profiling of the tricarboxylic acid (TCA) cycle, amino acids, and lipids. Additionally, trypsin-like enzyme activity in the plasma and markers of brain injury were evaluated. Animals treated with Met showed significantly higher systolic blood pressure after resuscitation (LR: 74±5 mmHg vs. LR+Met: 94±3 mmHg) with no significant changes in HR observed. Brain injury markers such as GFAP (Glial fibrillary acidic protein), S100B (astrocytic protein specific to the central nervous system), Tau (microtubule-associated protein), and NFL (axonal protein neurofilament light) were assessed. Animals reperfused with LR and treated with Met displayed a 22% reduction in GFAP levels compared to those reperfused with LR alone, while other markers remained unchanged. Met treatment also led to a reduction in plasmatic metabolites, including Lactate (30%), Succinate (24%), and Fumarate (22%), along with an increase in Citrate (23%) and α-ketoglutarate (24%)—all constituents of the TCA cycle. Additionally, an increase in amino acid metabolites and a decrease in lipid metabolites were observed. Recent research has suggested that HS results in elevated levels of trypsin-like enzymes in the plasma and that inhibiting these enzymes might improve outcomes during resuscitation. Our study revealed that treatment with Met reduced approximately 60% of trypsin-like enzyme activity in the plasma compared to the LR-only group. In conclusion, our study demonstrates that Met, when administered in conjunction with LR as a resuscitation solution, significantly enhances post-resuscitation BP compared to LR alone. This improved outcome may be associated with alterations in the TCA cycle and the inhibition of trypsin-like enzyme activity. However, further investigations are necessary to elucidate the precise role of these changes in the observed enhancements within this experimental model. This work was supported by National Institutes of Health grant R01HL162120. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Expressions of the trypsin-like-enzyme, AlSP4 and total trypsin-like-enzyme in Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) feeding on different cotton varieties

Apolygus lucorum is currently a main pest of cotton in China. In this study, the expression of trypsin-like enzyme AlSP4 gene and corresponding protein in A. lucorum was confirmed to be significantly higher when it fed on Bt cotton than on conventional cotton and Sauteed green beans (control). Compared with Bt cotton, when A. lucorum fed on conventional cotton, the total trypsin-like enzyme activity and female fertility were reduced overtly. And, when it fed on non-hybrid cotton (Gk19 and Sim3), A. lucorum showed a higher expression of total trypsin-like protease, per female fecundity, hatching rate of eggs and life span than on hybrid cotton (Ezm10 and Sum9). This indicated that the hybrid cotton inhibited the growth and development of A. lucorum better than the non-hybrid cotton. Furthermore, data analysis showed that there were significant positive linear associations between female trypsin-like enzyme activity and key factors of population proliferation, such as female adult life span and per female fecundity. These findings indicated that the total trypsin-like enzyme was a potential target to control A. lucorum, especially the female adults.

Chlorella vulgaris lectin kills Aedes aegypti larvae

Vector-borne diseases are considered public health concerns. As vaccines for some of these diseases are not available or still have serious restrictions, the vector control is an important strategy. Aedes aegypti mosquitoes can transmit dengue, yellow fever, chikungunya and Zika viruses. Chemical compounds are used to control A. aegypti populations which are usually toxic to non-target organisms, and thus the safety of their use is questionable. This work reports the purification and characterization of a lectin from Chlorella vulgaris microalgae (CvL) and its toxicity to the A. aegypti fourth instar larvae (L4). CvL was isolated (purification factor of 8.72; yield of 6.67) from the C. vulgaris aqueous extract (AE) with hemagglutinating activity of 185,130 titre mg−1. The characterization showed that CvL is a 17 kDa protein whose activity was inhibited various carbohydrates, resisted to heating up to 60 °C and was stable over a broad pH range. Additionally, CvL activity was strongly reduced by monovalent and divalent metal ions. AE and CvL were toxic to L4 and the concentrations that killed 50% of larvae after 24 h were 10.62% (v/v) and 164.24 μg mL−1, respectively. CvL inhibited the activity of trypsin-like enzymes from L4 gut and this effect, as well as the larvicidal activity, were abolished when the lectin was denatured by heating or when its carbohydrate-binding site was blocked by fructose or azocasein. These findings points the C. vulgaris biomass as a new source of a biomaterial with potential to control A. aegypti larvae by inhibition of trypsin-like enzymes representing a larvicidal mechanism.

FEATURES OF ACTIVATION OF TRIPSIN-LIKE PLASMA ENZYMES IN STABLE PATIENTS WITH COPD

SESSION TITLE: Obstructive Lung Disease Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: The presence of COPD in patients contributes to an increase in prothrombotic potential and an increased risk of venous thromboembolism. The activity of extracellular proteases of cascade coagulation and fibrinolysis reactions is the main component of the total activity of trypsin-like plasma enzymes (TLE). The aim of the study was to investigate the total activity of TLE (TA TLE) of plasma and to determine the effect of bronchial obstruction, the frequency of exacerbations of COPD, body weight and dyslipidemia in patients with stable COPD on it. METHODS: 54 individuals were included in the study. The I group (G1) included 44 stable patients with COPD A - D who received standard therapy, the II group (G2) included 10 healthy persons. Anthropometry was performed to determine body mass index (BMI), photocolorimetry to determine the TA TLE of plasma, coagulogram, enzymatic colorimetry for determination of total cholesterol (TCh), cholesterol of high density lipoproteins (LHD), cholesterol of low density lipoproteins (LDL), triglycerides (TG), calculation of the level of cholesterol of very low density lipoproteins (VLDL) according to the Friedewald formula, spirometry, for patients with COPD, the frequency of exacerbations of COPD was determined within 1 year. RESULTS: The median age in G1 was (56.0±6.7) years, in G2 was (52.7±4.9) years, BMI in G1 - 26.3 [24.0; 30.3] kg/m2, in G2 – 25.4 [24.3; 26.2] kg/m2. In G1 FEV1 was 51.0 [42.0; 61.0] %, a direct correlation of FEV1 and BMI was found (R=0.36, p=0.016). In G1 the frequency of exacerbations of COPD during the year was (1.9 ± 0.86). The TA TLE of plasma in G1 (76.13 [49.37; 102.18] μmol/l) was significantly higher than in G2 (46.48 [34.17; 50.35] μmol/ /l), p = 0.005. In G1 the TA TLE of plasma was directly associated with an increase in prothrombin index (PTI) (R=0.43, p=0.016), while the PTI in G1 (106.0 [99.0; 109.0] %) was higher than in G2 (98.0 [95.0; 101.0] %), p = 0.48. In patients in G1 the correlation TA TLE with the level of FEV1 has not been established, but a direct correlation has been found with an increase in the frequency of exacerbations of COPD (R = 0.34, p = 0.046), increase in BMI (R = 0.36, p = 0.022) and direct correlation with levels of TCh (R = 0.40, p = 0.014), LDL (R = 0.36, p = 0.029), VLDL (R = 0.43, p = 0.008), TG (R = 0.55, p = 0.0007). CONCLUSIONS: In stable patients with COPD against the background of standard therapy, an increase in the total proteolytic activity of plasma TLE is observed, which is associated with an increase in prothrombotic potential. An increase in TA TLE of plasma is associated with an increase in the frequency of exacerbations of COPD, an increase in BMI and dyslipidemia. CLINICAL IMPLICATIONS: Prevention of thrombotic complications in patients with COPD should include measures to reduce the frequency of exacerbations of COPD, reduce body weight of patients, and correct dyslipidemia. DISCLOSURES: No relevant relationships by Olena Kovalenko, source=Web Response No relevant relationships by Viktoriia Rodionova, source=Web Response

Effects of a Reserve Protein on Spodoptera frugiperda Development: A Biochemical and Molecular Approach to the Entomotoxic Mechanism.

Talisin is a storage protein from Talisia esculenta seeds that presents lectin-like and peptidase inhibitor properties. These characteristics suggest that talisin plays a role in the plant defense process, making it a multifunctional protein. This work aimed to investigate the effects of chronic intake of talisin on fifth instar larvae of Spodoptera frugiperda, considered the main insect pest of maize and the cause of substantial economic losses in several other crops. The chronic intake of talisin presented antinutritional effects on the larvae, reducing their weight and prolonging the total development time of the insects. In addition, talisin-fed larvae also showed a significant reduction in the activity of trypsin-like enzymes. Midgut histology analysis of talisin-fed larvae showed alterations in the intestinal epithelium and rupture of the peritrophic membrane, possibly causing an increase of aminopeptidase activity in the midgut lumen. Talisin also proved to be resistant to degradation by the digestive enzymes of S. frugiperda. The transcription profile of trypsin, chymotrypsin and aminopeptidase genes was also analyzed through qPCR technique. Talisin intake resulted in differential expression of at least two genes from each of these classes of enzymes. Molecular docking studies indicated a higher affinity of talisin for the less expressed enzymes.

PH influences the profiles of midgut extracts in Cnaphalocrocis medinalis (Guenée) and its degradation of activated Cry toxins

Midgut extracts play crucial roles in food digestion and detoxification. We evaluated the effect of pH on the profiles of the midgut extracts in rice leaffolder, Cnaphalocrocis medinalis and the degradation of activated Bt-toxins by the midgut extracts under different pH conditions. Total protease activity increased slightly with the increase with the simulated pH in the midgut extracts and the maximal protease activity was observed at pH 10.5, while an upward trend was observed as the pH of reaction buffer increased. Activity of chymotrypsin-like enzymes increased with pH, both in the buffer and midgut extracts, while the activity of trypsin-like enzyme was unaffected. Degradation of the activated Cry2A by the midgut extracts enhanced as the pH increased. Cry2A was fully degraded into smaller segments at pH 9.0–10.5. Activated Cry1C protein at pH 9.0–10.5 was partially degraded by the midgut extracts. Activated Cry1Aa and Cry1Ac were partially degraded into fragments by the midgut extracts at high pH. These results will facilitate our further understanding of the interactions between C. medinalis and the Cry toxin.

Traffic-derived air pollution compromises skin barrier function and stratum corneum redox status: A population study.

Since air pollution is only one of many environmental stressors that can affect skin, it has been challenging to identify skin appearance or functional features profoundly affected by chronic exposure to traffic-derived air pollution. The current population study focused on taxi drivers working in urban and rural areas in order to take advantage of difference in occupational exposure. The skin conditions of 100 middle-aged male taxi drivers from urban Shanghai and 66 from rural Chongming were measured with facial tape strips were collected for biomarker analyses. Trans-epidermal water loss (TEWL) values before and after tape stripping were considerably higher in urban taxi drivers from Shanghai. Contrary to previous studies, there was no apparent detrimental effect on skin wrinkle or pigmentation from traffic pollution, which might be attributed to the higher than general public level of photo-exposure in this population. At the same time, pollution exposure especially the heavy traffic pollution exposure was found to associate with lower stratum corneum trypsin-like enzyme activity (SCTE), reduced catalase activity and total antioxidant capacity (TAOC) in tape strips. The evidence suggests that traffic-derived air pollution could deteriorate skin's physical and antioxidant barrier, whereas factors like photo-exposure can be overwhelming against appearance aging. Therefore, in addition to photoprotection, skin barrier care should be considered for people with high air pollution exposure.

ONTOGENETIC CHANGES IN THE ACTIVITY OF TRYPSIN-LIKE ENZYMES IN INDIVIDUALS OF THE LABORATORY POPULATION OF DROSOPHILA MELANOGASTER AND DROSOPHILA VIRILIS

Introduction. The ontogenetic variation of body mass index and activity of trypsin-like enzymes in individuals of laboratory populations of two species of fruit fly: Drosophila melanogaster and Drosophila virilis were studied.The aim of the work was to investigate the level of fluctuation of trypsin-peptide hydrolase activity at different stages of ontogenesis in separate individuals of the laboratory populations of Drosophila virilis and Drosophila melanogaster and to compare the activity indicators of the studied species.Methods. The material of the study was the 3-day-old larvae, pupae and adults of two Drosophila species that were cultivated under standard conditions. In individually prepared homogenates, the total activity of trypsin-like enzymes was determined using the specific substrate N-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BAPNA). The enzymatic hydrolysis of BAPNA was carried out in an alkaline medium in the presence of peptide hydrolases. The dynamics of optical density (ΔE) was determined at λ = 382.5 nm. The calculation of the relative activity of the enzymes was carried out on the basis of the extinction values obtained. The specific activity was found by the formula. The obtained primary data were processed statistically and presented in the form of variation curves and histograms.Results and conclusion. The variation curves of the body mass for larvae, pupae and imago were built by the data of separate individuals. The ontogenetic changes of the individual relative and specific activity of trypsin-like enzymes at the population level were determined. The average body mass index and activity of trypsin-like enzymes for each stage of post-embryonic development of Drosophila were calculated. The ranges of variation of the studied parameters under the stationary conditions of Drosophila cultivation were determined. It was determined that the highest indices of the body weight of individuals and their proteolytic activity (by the data of species melanogaster) coincide with the larval phase of the development. Possible causes of individual differences in the expression of the activity of trypsin-like enzymes in Drosophila are discussed.

Characteristics of hemostasis system and activity of trypsin-like blood enzymes in comorbid patients with chronic obstructive pulmonary disease and hypertension.

This work was aimed at studying the activity of the hemostasis system and trypsin-like blood enzymes (TLE) in patients with chronic obstructive pulmonary disease (COPD) and hypertension (H), depending on anthropometric, clinical features and smoking. The study included 87 patients. I group included 39 patients with COPD A – D, II-IV by GOLD and conco­mitant with H I-II stage, II group – 38 patients with H I - II stage, and K group – 10 apparently healthy persons. The study was conducted on the background of the use of standard basic therapy by patients from the 1st and 2nd groups in a stable period of disease. The degree of thrombocyte (tr) adhesion in I and II groups of patients was higher than in the K group. Both adhesive activity and the amount of tr in the I group was higher than in the II group. With positive status of smoking and increasing nicotine addiction in comorbid patients with COPD and H, the adhesive activity of tr increased. The level of the procoagulantive parameters of the plasma part of hemostasis in the two studied groups was not statistically different, but was higher than in the K group. Increased BMR and dyspnoea by the mMRS scale in group with COPD and H and increased age of patients with H led to the increase in the activity of plasma coagulation factors. In I and II groups, the increase in blood TLE was observed, which increased with the body weight, while in comorbid patients in the subgroup with FEV1≥50%, the incidence of COPD exacerbation additionaly effects the increase of TLE. In I group correlation between reduction of FEV1 with the decrease of TLE was revealed. In patients with COPD and AH activity of thrombocyte and plasma hemostasis, as well as trypsin-like blood enzymes, increases. Smoking, body weight gain and incidence of COPD exacerbation contribute to increased prothrombotic potential.

Activation of the Nrf2 signaling pathway and neuroprotection of nigral dopaminergic neurons by a novel synthetic compound KMS99220

The transcription factor Nrf2 is known to induce gene expression of antioxidant enzymes and proteasome subunits. Because both oxidative stress and protein aggregation have damaging effects on neurons, activation of the Nrf2 signaling should be beneficial against neurodegeneration. In this study, we report a novel synthetic morpholine-containing chalcone KMS99220 that confers neuroprotection. It showed high binding affinity to the Nrf2 inhibitory protein Keap-1 and increased nuclear translocation of Nrf2 and gene expression of the antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and the catalytic and modifier subunits of glutamate-cysteine ligase in dopaminergic CATH.a cells. KMS99220 also increased expression of the proteasome subunits PSMB5, PSMB7, PSMB8 and PSMA1, and the respective chymotrypsin and trypsin-like proteasomal enzyme activities, and reduced α-synuclein aggregate in GFP-α-syn A53T-overexpressing cells. KMS99220 exhibited a favorable pharmacokinetic profile with excellent bioavailability and metabolic stability, did not interfere with activities of the cytochrome p450 isotypes, and showed no apparent in vivo toxicity when administered up to 2000 mg/kg. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, oral administration of KMS99220 prevented degeneration of the nigral dopaminergic neurons, induced the Nrf2 target genes, and effectively prevented the associated motor deficits. These results suggest KMS99220 as a potential candidate for therapy against Parkinson's disease.

Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2.

Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50), (2) its amino acid sequence and (3) its ability to activate calcium signalling and/or ERK1/2 phosphorylation via PAR2. Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signalling. Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signalling via PAR2. We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen sensitization in vivo and may represent attractive therapeutic targets for asthma.

Phenotypic identification of Porphyromonas gingivalis validated with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

ObjectivePorphyromonas gingivalis is a major bacterial pathogen in human periodontitis. This study used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to assess the accuracy of a rapid phenotypic identification scheme for detection of cultivable P. gingivalis in human subgingival plaque biofilms. MethodsA total of 314 fresh cultivable subgingival isolates from 38 adults with chronic periodontitis were presumptively identified on anaerobically-incubated enriched Brucella blood agar primary isolation plates as P. gingivalis based on dark-pigmented colony morphology, lack of a brick-red autofluorescence reaction under long-wave ultraviolet light, and a positive CAAM fluorescence test for trypsin-like enzyme activity. Each presumptive P. gingivalis isolate, and a panel of other human subgingival bacterial species, were subjected to MALDI-TOF mass spectrometry analysis using a benchtop mass spectrometer equipped with software containing mass spectra for P. gingivalis in its reference library of bacterial protein profiles. A MALDI-TOF mass spectrometry log score of ≥1.7 was required for species identification of the subgingival isolates. ResultsAll 314 (100%) presumptive P. gingivalis subgingival isolates were confirmed as P. gingivalis with MALDI-TOF mass spectrometry analysis (Cohen's kappa coefficient = 1.0). MALDI-TOF mass spectrometry log scores between 1.7 and 1.9, and ≥2.0, were found for 92 (29.3%) and 222 (70.7%), respectively, of the presumptive P. gingivalis clinical isolates. No other tested bacterial species was identified as P. gingivalis by MALDI-TOF mass spectrometry. ConclusionsRapid phenotypic identification of cultivable P. gingivalis in human subgingival biofilm specimens was found to be 100% accurate with MALDI-TOF mass spectrometry. These findings provide validation for the continued use of P. gingivalis research data based on this species identification methodology.

The effect of cardioprotectors on protease-іnhіbіtory potentіal in blood and heart of rats with epinephrine-induced myocardial ischemia

Coronary heart disease is one of the major causes of illness and of deaths in Europe, therefore the definition of the signaling pathways involved in cardioprotection represents a challenging goal in order to discover novel pharmacological approaches. One of the mechanisms included in the implementation of signaling methods is proteolysis, so the aim of our study was to study the balance between activity of the proteolytic enzymes and their inhibitors in the experimental myocardial ischemia of rats and the impact of corvitin (C) and doxycycline (D) on these parameters. Ischemia was induced in Wistar rats by the intraperitoneal injection of epinephrine at a dose of 0.2 mg/100 g of weight during 10 days. All animals were divided into 4 groups: 1 – control (8 rats), 2 – rats with epinephrine-induced myocardial ischemia (EIM, n = 10), 3 – rats, which were entered C after completion of epinephrine injections (n = 10), 4 – rats, which accepted D during 6 days after finishing of epinephrine injections (n = 10). The electrical activity of the heart was recorded on an electrocardiogram (ECG). Activities of matrix metalloproteinases 2 and 9 (MMP2, MMP9) were studied by enzyme-zymography, the activity of trypsin-like enzymes (TLE), levels of alpha-1 proteinase inhibitor (IP1) and alpha-2-macroglobulin (α2-МG) in the blood and the heart were determined by spectrophotometric methods. The experimental results suggest that epinephrine injections lead to changes in the ECG typical for ischemic myocardium. The data obtained showed that the protease-іnhіbіtory balance in the blood and in heart was disturbed. Activity of MMP9, proMMP9/2 and level of IP1 increased in blood plasma of rats with EIM, but activities of ММP2, TLE were unchanged in this group. In the heart muscle the activity of MMP2 and level of IP1 increased. Administration of C resulted in the recovery of MMPs level, but disturbed the balance in the system TLE/IP1-α2-МG. Administration of D blocked the activity of MMP9, significantly reduced the activity of MMP2, which testifies the inhibitory effect of this drug on the activity of matrix metalloproteinases. Based on these results, we conclude that definition of protease-inhibitory balance in the blood plasma of patients with coronary artery disease is promising as an additional diagnostic complex and for monitoring of the effectiveness of therapy.

Регуляція тіаміном активності трипсиноподібних ферментів в тканинах білих щурів

The effect of intramuscular thiamine injection on the activity of trypsin-like enzymes in the liver, kidneys, stomach and small intestine of white rats has been researched. In the organs of intact rats, the maximum activity of trypsin-like proteases has been established in the small intestine, and the minimum one in the liver. Thiamine parenteral administration leads to the decrease of trypsin-like protease activity in the liver, kidney and stomach, and the increase of the enzyme activity in the small intestine that suggests the possibility of non-coenzyme thiamine effect on the trypsin-like enzyme activity.

Melanin production through novel processing of proopiomelanocortin in the extracellular compartment of the auricular skin of C57BL/6 mice after UV-irradiation.

The production of melanin is regulated by α-melanocyte-stimulating hormone (α-MSH), which is produced from proopiomelanocortin (POMC). Keratinocytes release POMC along with lower levels of α-MSH and ACTH. To clarify the mechanism of melanogenesis after ultraviolet (UV)-irradiation, this study focused on the expression of POMC and POMC-derived peptides after UV-irradiation. Western blot analysis and immunoassays indicated that both POMC and α-MSH-like immunoreactivity (α-MSH-LI) increased after UV-irradiation. However, other POMC-derived products were very low. In hypophysectomized mice, α-MSH-LI increased to the same level as in control mice after UV-irradiation. Structural analysis revealed that the major α-MSH-LI product was ACTH(1–8). Furthermore, ACTH(1–8) competed with [125I]-α-MSH for receptor binding and increased melanin production via a melanocortin-1 receptor. These results suggested that melanin was produced through ACTH(1–8) after UV-irradiation. Trypsin-like enzymatic activity, which is responsible for POMC activation, increased after UV-irradiation and was identified as tryptase. In mast cell-deficient mice, which do not produce tryptase, α-MSH-LI levels were unchanged after UV-irradiation. The present study demonstrates the production of ACTH(1–8) from POMC by tryptase, which is a novel peptide-processing mechanism in the extracellular compartment of the skin.

Enhancement of trypsin-like enzymes by A23187 ionophore is crucial for sperm penetration through the egg vestment of the giant freshwater prawn.

We report the presence of trypsin-like enzymes preferring Boc-QAR-MCA substrate in sperm collected from different portions of male reproductive tracts of the giant freshwater prawn, Macrobrachium rosenbergii and compare enzyme activities before and after an A23187 calcium ionophore treatment. Fluorogenic enzyme assays revealed that testicular sperm lysates showed high trypsin-like enzyme activity but the activity was relatively low in vas deferens sperm lysates as well as in the live sperm. Upon sperm treatment with A23187, trypsin-like activity was greatly enhanced in distal vas deferens sperm. Substrate- and inhibitor-based localization studies indicated that the sperm trypsin-like enzymes were not of a soluble type but were rather of a membrane-borne type, localized at the anterior spike and upper part of the main body. Notable structural changes were also evident in A23187-induced sperm including extensive ruffling of the sperm membrane structure at the base of the main body thereby supporting the acrosome reaction response in this species. We further proved by substrate inhibition assays that the enhanced trypsin-like enzyme activity participates in sperm penetration through the vitelline envelope, a novel sperm-egg penetration mechanism that is unique in this species.

Altered balance between proteolysis and antiproteolysis in oncohematologic diseases

Destruction of malignant cell clones and search for markers of the effectiveness of chemotherapy in patients with hematologic malignancies is an urgent direction of research. The purpose of this paper was to study plasma of patients with various forms of blood tumor, the activity of trypsin-like enzymes, matrix metalloproteinases MMP2 and MMP9, content of α1-proteinase inhibitor and α2-macroglobulin before and after the cytotoxic therapy by anthracycline antibiotics, doxorubicin and daunorubicin. It was established that concentration of inhibitors and activity of trypsin-like enzymes increased in patients with acute myeloid leukemia and multiple myeloma against the backdrop of multidirectional changes of proMMP9. Activity of the latter enzyme was reduced to 0,03±0,01 rel. u. in acute myeloid leukemia, and after chemotherapy it increased 7 times. Reducing level of inhibitors and increasing activity of MMP9 were found in chronic lymphocytic leukemia. The presence of the correlation between the α2-macroglobulin and gelatinase activity in the treatment indicates that the inhibitor is an important mechanism for storing of gelatinоlytic potential. Balance between proteolysis and antiproteolysis depends on the type of proliferating cells and the course of the disease. Indicators of tissue destruction may be an additional criterion for monitoring the stage of blood tumors and efficacy of treatment.

Activity of trypsin-like enzymes and gelatinases in rats with doxorubicin cardiomyopathy

Activity of trypsin-like enzymes (ATLE) and gelatinases A and B were studied in the blood plasma and extracts from cardiac muscle, cerebral cortex and cerebellum of rats with cardiomyopathy caused by anthracycline antibiotic doxorubicin against the background of preventive application of corvitin and α-ketoglutarate. ATLE significantly increased in blood plasma and extracts from cerebral cortex but decreased in extracts from cardiac muscle and cerebellum in doxorubicin cardiomyopathy (DCMP). In addition, a significant increase of activity of both gelatinases in plasma and tissue extracts was observed. Preventive administration of corvitin and α-ketoglutarate resulted in differently directed changes of activity of the above mentioned enzymes in heart and brain tissues. Obtained data confirm the hypothesis about activation of proteolysis under the influence of anthracycline antibiotics and testify to selective effect of corvitin and α-ketoglutarate on ATLE and gelatinases.

Effects of Fraxinellone on the midgut enzyme activities of the 5th Instar Larvae of Oriental Armyworm, Mythimna separata walker.

Isolated from Dictamnus dasycarpus Turcz., fraxinellone exhibited multiple bioactivities against insects. In the present paper, the changes of digestive enzymes and detoxification enzymes of Mythimna separata Walker (5th instar larvae), treated with fraxinellone, were investigated. Compared with those of the control, the α-amylase activity of the fraxinellone-treated 5th instar larvae was inhibited, whereas the level of their protease activity was increased. Based upon further studies on the specific proteases, the levels of the active alkaline trypsin-like enzyme (BApNA as the substrate) and the chymotrypsin-like enzyme (BTEE as the substrate) activities of the treated larvae were declined; however, the level of activity of the weak alkaline trypsin-like enzyme (TAME as the substrate) of the treated ones was increased. Meanwhile, the activities of two detoxification enzymes, such as carboxylesterase (CarE) and glutathione S-transferase (GST), of the treated larvae were increased to some extent, but the activities of NADPH-P450 reductase and O-demethylase of the treated ones declined. Therefore, protease (especially the weak alkaline trypsin-like enzyme), CarE and GST played important roles in the metabolism of fraxinellone in the midgut of Mythimna separata (M. separata).

Spatial characterization of proteolytic enzyme activity in the foregut region of the adult necrophagous fly, Protophormia terraenovae

The spatial distribution of proteolytic enzymes in the adult foregut of Protophormia terraenovae was studied in the context of protein digestion and regurgitation. Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin. Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop. Pepsin-like activity was only evident in crop fluids and tissues. The activity of all three enzymes was low or undetectable (pepsin) in the fluids and tissue homogenates derived from the esophagus and cardia of any of the adults assayed. Fed adult females displayed higher enzyme activities than fed males, and the activity of all three enzymes were much more prevalent in fed adults than starved. The pH optimum of the trypsin-like enzyme was between pH 7.0 and 8.0; chymotrypsin was near pH 8.0; and maximal pepsin-like activity occurred between pH 1.0 and 2.0. Regurgitate from fed adult females displayed enzyme activity consistent with the proteolytic enzymes detected in crop gut fluids. Enzymes in regurgitate were not derived from food sources based on assays of bovine liver samples. These latter observations suggest that adult flies release fluids from foregut when encountering dry foods, potentially as a means to initiate extra-oral digestion.