Increase In Proteolytic Activity Research Articles

The effect of the probiotic drug "Ecoflor" on the proteolytic activity of the sterlet intestine

The effect of the probiotic preparation "Ecoflor" on proteolytic activity of the intestinal mucosa and chyme of sterlet was studied. This probiotic is a consortium of strains of lacto- and bifidobacteria: B. bifidum, B. longum and L. acidophilus, L. casei, L. plantarum, immobilized on carbon-mineral sorbent SUMS-1. For the experiment, fish were divided into 4 groups: control and three experimental groups. Experimental groups of fish were given feed with probiotic in the amount of 2, 4, 6 g/kg of feed. Sampling was carried out before the beginning of the experiment, on 7, 14, and 21 days. Material for the study was taken from 5 individuals from each group. To obtain enzymatically active preparations, fish intestines were freed from fat and dried on ice bath glass. After incision, the chyme was collected with a spatula and scraper, then the mucous membrane was removed. The total proteinase activity was calculated by the ratio of the difference between the spectrophotometer readings of the sample with substrate and the blank sample to the product of the intestinal weight (g) by the incubation time (min) EA = ΔE425/(g×min). Determinations were performed in three repetitions. Data analysis showed the stimulating effect of the preparation on proteolytic activity. The maximum increase in enzymatic activity in the experimental groups compared to the control group was recorded on the 7th day of the experiment. A more significant increase in proteolytic activity of the intestinal mucosa compared to chyme was found. At the beginning of feeding, the stimulating effect of the preparation was found in all experimental groups compared to the control. In further observation periods the effectiveness of the probiotic significantly decreased.

MMP-7 Levels Decrease in Gingival Epithelium Exposed to Porphyromonas Gingivalis after Curcumin administration

Periodontitis is an irreversible inflammatory disease of the periodontal tissues. The leading cause of periodontitis is Porphyromonas gingivalis bacteria, which secretes virulence factors, namelyLipopolysaccharide (LPS) and proteases, which cause the inflammation of gingival junctional epithelium and loss of attachment, resulting in the formation of a periodontal pocket. The most common type of MMP found in the junctional epithelium is MMP-7. Invasion of Porphyromonas gingivalis bacteria causes an increase in proteolytic activity on the expression and activation of MMP-7, which causes loss of attachment. Efficacious plant extracts, namely irrigated curcumin 1%, have an anti-inflammatory effect. This research is independent research using experimental white rats (Rattus norvegicus) male strain Wistar, 20 weeks old, with a body weight of 325-350 grams. This research is to see the levels of MMP-7 with the ELISA method. Forty-eight periodontitis model rats were divided into two groups. Twenty-four periodontitis model rats as the control group were exposed to Porphyromonas gingivalis, and another twenty-four periodontitis model rats as the treatment group were exposed to Porphyromonas gingivalis and 1% curcumin irrigation. Each group was observed at 24 hours, on days-7 and days-14. The results showed that MMP-7 levels in the treatment group were significantly lower than in the control group on on days-7 and days-14. In the treatment group, MMP-7 levels at 24 hours were significantly lower than on the on days-7 and days-14. In the treatment group, MMP-7 levels did not experience a significant difference between the on days-7 and days-14. The results of this study indicate that 1% curcumin can reduce MMP-7 levels in cases of chronic periodontitis due to Porphyromonas gingivalis bacteria.

Spontaneously fermented ancient wheat sourdoughs in breadmaking: Impact of flour quality on sourdough and bread physico-chemical properties

In this study changes during fermentation of spontaneously fermented modern and ancient (spelt, emmer, and khorasan) wheat sourdoughs and their feasibility to act as leavening agents in breadmaking were evaluated. During 6 h of fermentation, sourdough was characterized for lactic acid bacteria and yeasts counts, pH, total titratable acidity (TTA), proteolytic activity, rheological and electrophoretic profiles. The effects of 25 and 50 g/100g sourdough addition on physico-chemical and sensory properties of refined wheat flour bread were also investigated. Although in all sourdoughs a pH drop, increase in proteolytic activity, gliadin and glutenin degradation with fermentation were recorded, due to different flour composition and microbial activity, tested sourdoughs significantly differed in rheological properties and breadmaking quality. Wheat sourdough being characterized by the highest acidification, increase in proteolytic activity, and the most extensive hydrolysis of gliadins, resulted in bread with the lowest specific volume and hardest crumb texture. Emmer sourdough, having the highest TTA, ash and wet gluten content, exhibited the highest extensibility, the least pronounced changes in proteolytic activity and the electrophoretic pattern which produced bread with the highest volume and softest texture. In general, ancient wheat varieties have shown great potential in sourdough breadmaking in comparison to modern wheat.

Mitigating Apoptotic and Inflammatory Signaling via Global Caspase Inhibition in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractIn most systems, the caspase cascade is activated during cellular stress and results in inflammation and apoptosis. Hibernators experience stressors such as extremely low body temperatures, bradycardia, possible ischemia and reperfusion, and acidosis. However, widespread inflammation and apoptosis would represent an energetic expense that is incompatible with hibernation. To better understand global caspase regulation during hibernation, we employed a systems-level approach and analyzed 11 caspases in ground squirrel liver that are involved in inflammatory (caspases 1, 4, 5, 11, and 12) and apoptotic (caspases 2, 6, 7, 8, 9, and 10) pathways. Western blots revealed liberation of active forms for two inflammatory (caspases 11 and 12) and two apoptotic (caspases 6 and 9) caspases during hibernation (e.g., p15, the most active fragment of caspase 6, increased in interbout-aroused animals). We used specific peptide substrates to interrogate the four seemingly activated caspases and demonstrated no expected increases in proteolytic activity. Specific targets of these four caspases were similarly not cleaved, demonstrating that initiation of caspase activation may occur without concomitant downstream effects. Similarly, we found no evidence for upstream activation for caspase 9 signaling based on permeabilization of the outer mitochondrial membrane. We contend that these caspases are suppressed after seeming activation during hibernation. Incomplete caspase signaling is effectively mitigating the induction of widespread inflammation and apoptosis during hibernation.

Evaluation of disinfectant “Enzidez” according to physical and chemical parameters

The dependence of the national economy on disinfectants is constantly growing through prevention strategies and the development of resistance in microorganisms. Therefore, new disinfectants with different mechanisms of biocidal activity for a wide range of pathogens are appearing on the market. The aim of work has been to determine the physical and chemical parameters of the created disinfectant “Enzidez” active on biofilm forms of bacteria and the possible organic load. The magnitude of the surface tension of disinfectant “Enzidez” at different concentrations of solutions has been determined using a device with a Traube stalagmometer. The wettability of solutions has been determined by the method of measuring the wetting angle with a catheter. Washing properties and stability of foam by the method of washing animal fats, followed by application of indicator liquid. Proteolytic activity has been determined using milk and the effect of Enzidez solutions of different concentrations on it, followed by measurement of the optical density of the solutions spectrophotometrically at a wavelength of 600 nm. It is established that the disinfectant with enzymes – “Enzidez” for disinfection in veterinary clinics for pre-sterilization disinfection and treatment of products and instruments of surgical purpose (plastic, rubber, glass, stainless steel) and objects of veterinary supervision (tables, utensils, walls, floors, doors, windows, etc.) with organic load is well soluble in water and has a pH of 0.25–1.0 % solutions in the range of 8.2–8.0 units. At a solution temperature of +20 ± 1 °С it has had a surface tension not higher than 37.63 ± 0.50 mN/m and a wetting angle of 69.5 ± 0.7 degree. The disinfectant has provided an excellent detergent effect only at solutions with a concentration of 1.0 % and above. When assessing the corrosive action of Enzidez in the concentration of solutions from 0.25 to 1.0 %, the amount of corrosion on stainless steel has been hundreds of times lower than the allowable regulatory limit. The impact on galvanized steel is slightly higher than that of stainless steel, but also several tens below the norm. A study to determine the proteolytic activity of the disinfectant “Enzidez” on milk proteins has revealed that the disinfectant broke down proteins at a concentration of solutions 0.5–1.0 % at a temperature of + 20 °C at 41.3–43.1 %, respectively, for 15 min of exposure. However, increasing the temperature of solutions to +60 °C and exposure for 30 min has provided an increase in proteolytic activity up to 70 %. Thus, disinfectant “Enzidez” is a highly active disinfectant for disinfecting surfaces even in the case of organic contamination.

The Arf-GAP Proteins AoGcs1 and AoGts1 Regulate Mycelial Development, Endocytosis, and Pathogenicity in Arthrobotrys oligospora.

Small GTPases from the ADP-ribosylation factor (Arf) family and their activating proteins (Arf-GAPs) regulate mycelial development, endocytosis, and virulence in fungi. Here, we identified two orthologous Arf-GAP proteins, AoGcs1 and AoGts1, in a typical nematode-trapping fungus Arthrobotrys oligospora. The transcription of Aogcs1 and Aogts1 was highly expressed in the sporulation stage. The deletion of Aogcs1 and Aogts1 caused defects in DNA damage, endocytosis, scavenging of reactive oxygen species, lipid droplet storage, mitochondrial activity, autophagy, serine protease activity, and the response to endoplasmic reticulum stress. The combined effects resulted in slow growth, decreased sporulation capacity, increased susceptibility to chemical stressors and heat shock, and decreased pathogenicity of the mutants compared with the wild-type (WT) strain. Although deletion of Aogcs1 and Aogts1 produced similar phenotfypic traits, their roles varied in conidiation and proteolytic activity. The ΔAogts1 mutant showed a remarkable reduction in conidial yield compared with the WT strain but not in proteolytic activity; in contrast, the ΔAogcs1 mutant showed an increase in proteolytic activity but not in sporulation. In addition, the growth of ΔAogcs1 and ΔAogts1 mutants was promoted by rapamycin, and the ΔAogts1 mutant was sensitive to H-89. Collectively, the ΔAogts1 mutant showed a more remarkable difference compared with the WT strain than the ΔAogcs1 mutant. Our study further illustrates the importance of Arf-GAPs in the growth, development, and pathogenicity of nematode-trapping fungi.

Purification and characterization of actinidin from Actinidia deliciosa and its utilization in inactivation of \u03b1-amylase

BackgroundActinidin is an anionic thiol-proteinase predominant and unique to Chinese gooseberry or kiwifruit, whose strong digestibility enables proteins or enzymes vulnerable to digestion. The arrangement of active cysteine–thiol residues (Cys22-Cys65, Cys56-Cys98, and Cys156-Cys206) stabilizes the catalytic unit, thus allowing an effective Inhibition of α-amylase protein on exposure to the highest concentrations of actinidin under optimum conditions. When starch-rich foods are consumed with kiwifruit, starch digestion may be slowed by the inactivation of α-amylase (digestive enzyme), specifically reducing the blood sugar levels by hindering starch digestion that is helpful in diabetes mellitus. Thus, the study aimed at actinidin purification, optimization for maximal activity, and its demonstration as a potential to degrade α-amylase.ResultsProtease showed a molecular mass of 27 kDa on SDS-PAGE analysis. One factor at a time method was applied for process optimization, increasing the actinidin yield up to 176.03 U/mg. The enzyme was stable at a wide pH range; however, it was most active and stable at pH 7.5. The enzyme possessed half‐life at 35 °C of 5.5 h, at 40 °C of 4.5 h, at 45 °C of 2.5 h, and at 50 °C of 1 h. Lineweaver–Burk plot showed Michaelis–Menten constant (Km: 3.14 mg/ml) and maximal velocity (Vmax: 1.428 mmol/ml/min) using casein. The actinidin activity was enhanced with Ca2+ while it was inhibited by Cd2+ and Hg2+ ions. The α-amylase protein was successfully inactivated upon incubation with actinidin for 30 min; around ~ 85% of the α-amylase activity diminished. IC50 for inhibition of α-amylase was 2.54 mg/ml for crude actinidin and 1.86 mg/ml for purified actinidin.ConclusionsPurified Actinidin showed a 1.28-fold increase in proteolytic activity. The proteinase showed an active pH range of 3.5–8.5 under varied buffer conditions and thermostability up to 50 °C. The results revealed a significant potential utility of actinidin to retard amylase as it effectively degraded the amylolytic enzyme under in vitro conditions and could be beneficial for lowering glycemic response to ingested starch. However, further in vitro as well as in vivo studies need to be conducted under gastrointestinal conditions to establish the hypothesis.

Overexpression of Plasmodium falciparum M1 Aminopeptidase Promotes an Increase in Intracellular Proteolysis and Modifies the Asexual Erythrocytic Cycle Development.

Plasmodium falciparum, the most virulent of the human malaria parasite, is responsible for high mortality rates worldwide. We studied the M1 alanyl-aminopeptidase of this protozoan (PfA-M1), which is involved in the final stages of hemoglobin cleavage, an essential process for parasite survival. Aiming to help in the rational development of drugs against this target, we developed a new strain of P. falciparum overexpressing PfA-M1 without the signal peptide (overPfA-M1). The overPfA-M1 parasites showed a 2.5-fold increase in proteolytic activity toward the fluorogenic substrate alanyl-7-amido-4-methylcoumarin, in relation to the wild-type group. Inhibition studies showed that overPfA-M1 presented a lower sensitivity against the metalloaminopeptidase inhibitor bestatin and to other recombinant PfA-M1 inhibitors, in comparison with the wild-type strain, indicating that PfA-M1 is a target for the in vitro antimalarial activity of these compounds. Moreover, overPfA-M1 parasites present a decreased in vitro growth, showing a reduced number of merozoites per schizont, and also a decrease in the iRBC area occupied by the parasite in trophozoite and schizont forms when compared to the controls. Interestingly, the transgenic parasite displays an increase in the aminopeptidase activity toward Met-, Ala-, Leu- and Arg-7-amido-4-methylcoumarin. We also investigated the potential role of calmodulin and cysteine proteases in PfA-M1 activity. Taken together, our data show that the overexpression of PfA-M1 in the parasite cytosol can be a suitable tool for the screening of antimalarials in specific high-throughput assays and may be used for the identification of intracellular molecular partners that modulate their activity in P. falciparum.

Mechanical Stability and Proteolytic Activity of Resin–dentin Bonds Using the Cross-linked Dry Bonding Technique

To evaluate the mechanical stability and the proteolytic activity of bonds created by a two-step, etch-and-rinse adhesive applied to cross-linked and air-dried etched dentin. Flat dentin surfaces were produced in 64 extracted sound human molars. The dentin was etched with 35% phosphoric acid for 15 seconds, and then the teeth were divided into groups according to the cross-linking solution applied on the etched dentin. Group 1: 5% grape seed extract (GSE), Group 2: 5% glutaraldehyde, Group 3: Gluma Desensitizer, or Group 4: deionized water (control). Solutions were applied for 60 seconds, followed by rinse and blot drying. Then, the teeth were separated into two subgroups where the etched dentin was kept moist or air-dried. The adhesive was applied followed by a composite resin buildup. After 24 hours, the teeth were cut into beams (0.81 mm²) that were tested for microtensile strength immediately or after 12 months of aging in a 37°C saliva-like buffer. Additional teeth (n=32) were bonded as described and cut into 0.5-mm-thick slabs. The slabs were prepared for nanoleakage (scanning electron microscopy) and in situ zymography (EnzChek Protease Assay Kit). Bond strength data were submitted to ANOVA and Tukey tests (α =0.05). Significant reduction in immediate bond strength (ca 65%) and increase in proteolytic activity was seen when the etched dentin was air dried without previous cross-linking biomodification. Conversely, bond strengths did not differ from those produced on wet dentin when collagen was cross-linked before air drying, irrespective of the solution applied. For both moist and air-dried etched dentin, collagen cross-linking resulted in mechanically stable bonds and reduced proteolytic activity after 12 months of storage. Bonds produced by the application of a two-step, etch-and-rinse adhesive to cross-linked, air-dried, etched dentin were mechanically stable and revealed reduced proteolytic activity after 1 year of aging.

Characterization of the Cancer-Associated Meprin Βeta Variants G45R and G89R.

Meprin β is a metalloprotease associated with neurodegeneration, inflammation, extracellular matrix homeostasis, transendothelial cell migration, and cancer. In this study, we investigated two melanoma-associated variants of meprin β, both exhibiting a single amino acid exchange, namely, meprin β G45R and G89R. Based on the structural data of meprin β and with regard to the position of the amino acid exchanges, we hypothesized an increase in proteolytic activity in the case of the G45R variant due to the induction of a potential new activation site and a decrease in proteolytic activity from the G89R variant due to structural instability. Indeed, the G89R variant showed, overall, a reduced expression level compared to wild-type meprin β, accompanied by decreased activity and lower cell surface expression but strong accumulation in the endoplasmic reticulum. This was further supported by the analysis of the shedding of the interleukin-6 receptor (IL-6R) by meprin β and its variants. In transfected HEK cells, the G89R variant was found to generate less soluble IL-6R, whereas the expression of meprin β G45R resulted in increased shedding of the IL-6R compared to wild-type meprin β and the G89R variant. A similar tendency of the induced shedding capacity of G45R was seen for the well-described meprin β substrate CD99. Furthermore, employing an assay for cell migration in a collagen IV matrix, we observed that the transfection of wild-type meprin β and the G45R variant resulted in increased migration of HeLa cells, while the G89R variant led to diminished mobility.

Ethephon-Induced Ethylene Enhances Protein Degradation in Source Leaves, but Its High Endogenous Level Inhibits the Development of Regenerative Organs in Brassica napus.

To investigate the regulatory role of ethylene in the source-sink relationship for nitrogen remobilization, short-term effects of treatment with different concentrations (0, 25, 50, and 75 ppm) of ethephon (2-chloroethylphosphonic acid, an ethylene inducing agent) for 10 days (EXP 1) and long-term effects at 20 days (Day 30) after treatment with 100 ppm for 10 days (EXP 2) on protein degradation and amino acid transport in foliar sprayed mature leaves of Brassica napus (cv. Mosa) were determined. In EXP 1, endogenous ethylene concentration gradually increased in response to the treated ethephon concentration, leading to the upregulation of senescence-associated gene 12 (SAG12) expression and downregulation of chlorophyll a/b-binding protein (CAB) expression. Further, the increase in ethylene concentration caused a reduction in protein, Rubisco, and amino acid contents in the mature leaves. However, the activity of protease and expression of amino acid transporter (AAP6), an amino acid transport gene, were not significantly affected or slightly suppressed between the treatments with 50 and 75 ppm. In EXP 2, the enhanced ethylene level reduced photosynthetic pigments, leading to an inhibition of flower development without any pod development. A significant increase in protease activity, confirmed using in-gel staining of protease, was also observed in the ethephon-treated mature leaves. Ethephon application enhanced the expression of four amino acid transporter genes (AAP1, AAP2, AAP4, and AAP6) and the phloem loading of amino acids. Significant correlations between ethylene level, induced by ethephon application, and the descriptive parameters of protein degradation and amino acid transport were revealed. These results indicated that an increase in ethylene upregulated nitrogen remobilization in the mature leaves (source), which was accompanied by an increase in proteolytic activity and amino acid transport, but had no benefit to pod (sink) development.

Рання діагностика гіпотиреозу як фактора прогресування остеоартрозу та коморбідних процесів, особливості фітокорекції

Мета: вивчити поширеність і форми гіпотиреозу у хворих на остеоартроз (ОА) та дослідити вплив фітозасобу зобофіт на функціонально-морфологічний стан щитоподібної залози, прояви коморбідних захворювань, оксидантний статус, фібринолітичну і протеолітичну активність крові. Матеріал і методи. У 312 хворих на ОА досліджено прояви коморбідних процесів і серед них — явища гіпотиреозу. Використовували клінічні, біохімічні, радіоімунологічні, ультразвукові методи. Результати. У всіх хворих на ОА після 50 років відзначаються зростаюча кількість і вираженість коморбідних процесів. Серед них у 4,48 % виявлено маніфестний і у 13,78 % — субклінічний гіпотиреоз. Ознаки гіпотиреозу спостерігаються в осіб після 55, особливо 60 років. Субклінічний гіпотиреоз незначно погіршував клінічну симптоматику ОА та коморбідних процесів, але посилював явища оксидативного стресу, зниження фібринолітичної і підвищення протеолітичної активності крові, погіршення швидкості клубочкової фільтрації нирок. Зазначене розглядається як неспецифічна патофізіологічна основа прогресування виявлених хвороб. Тримісячне застосування фітозасобу зобофіт у більшості пацієнтів нормалізувало тиреоїдний гомеостаз, покращило загальні результати лікування основного і коморбідних захворювань, зменшило вираженість метаболічних порушень. Висновки. У хворих на ОА у віці старше 50 років і з високим рівнем коморбідності необхідно здійснювати скринінг на гіпотиреоз. При виявленні ознак субклінічного гіпотиреозу в такого контингенту хворих доцільно застосування полікомпонентних фітозасобів із різноплановою тиреоїдотропною дією.

Farklı protein kaynaklarının turna, Esox lucius Linneaus 1758, jüvenillerinin proteaz aktivitesi üzerine etkisi

The aim of the study is to determine the inhibitory effect of different protein sources on protease activity of Northern pike, E. Lucius, during larval ontogeny. For this purpose, Northern pike were fed from yolk sac absorption until 21 days after hatching (DAH). At that point, larvae were sampled on 7, 14 and 21 DAH days and the activity of enzyme was analyzed in vitro. In the study, two different fish meal, chicken meal, krill meal, corn gluten, soybean protein concentrate, soybean meal and dried distillers grains with solubles were tested. Fish meal-I showed the lowest effect (7.53 %) on 7 DAH larvae. Moreover, chicken meal has the highest inhibitory effect on the proteases in the first week (68.27%). In the following period (DAH 14), although the inhibition ratio dramatically increased in all ingredients, fish meal-I has still the lowest effect on proteolytic activity (55.66%). In the same period, the highest effect was obtained from krill (82.28 %) and chicken meals (86.73 %), respectively. Then, there was no statistical difference between fish meal-I, fish meal-II and corn gluten in the 21 DAH and relatively lower than the others (p>0.05). Additionally, chicken meal again has the highest effect on juveniles with the ratio 89.27 %. As a result, the increase of proteolytic activity was notably increased in 7-14-21 DAH, however, it is concluded that feeding larvae and juveniles with live prey is still suggested to get better result for such a carnivorous species culture.

Increased matrix metalloproteinase 9 activity correlates with flow-mediated intraluminal thrombus deposition and wall degeneration in human abdominal aortic aneurysm

ObjectiveWe have previously demonstrated that human abdominal aortic aneurysm (AAA) rupture occurs in zones of low wall shear stress where flow recirculation and intraluminal thrombus (ILT) deposition are increased. Matrix metalloproteinase-9 (MMP-9) is involved in the pathogenesis of AAA via its lytic effect on collagen and elastin. We hypothesize that flow-mediated ILT deposition promotes increased local inflammatory and MMP-9 activity that leads to AAA wall degeneration. The purpose of this study was to examine the correlation between predicted pulsatile flow dynamics and regional differences in MMP-9, elastin, collagen, and ILT deposition in human AAA. MethodsFull-thickness aortic tissue samples were collected from 24 patients undergoing open AAA repair. Control infrarenal aortic tissue was obtained from 6 patients undergoing aortobifemoral bypass. Full-thickness aortic tissue and ILT were assessed for MMP-9 levels using a cytokine array assay. Histologic and immunohistochemical assessment of inflammation, collagen and elastin content, and MMP-9 levels were also measured. Three-dimensional AAA geometry was generated from computed tomography angiogram (CTA) images using Mimics software and computational fluid dynamics was used to predict pulsatile aortic blood flow. ResultsThe majority of AAA showed eccentric ILT deposition which was correlated with predicted recirculation blood flow (R2 = –0.17; P < .05). The regions of high ILT were associated with significant increases in inflammation and loss of elastin and collagen compared with regions of low ILT, or with control tissue. MMP-9 was significantly higher in areas of high ILT deposition compared with areas devoid of ILT. Tissue MMP-9 was correlated with the thickness of ILT deposition (R2 = 0.46; P < .05), and was also present in high levels in thick compared with thin ILT. ConclusionsWe have shown a correlation between flow-mediated ILT deposition with increased tissue levels of MMP-9 activity, increased inflammatory infiltrate, and decreased elastin and collagen content in stereotactically sampled human AAA, suggesting that ILT deposition is associated with local increases in proteolytic activity that may preferentially weaken and promote rupture at selected regions.

Influence of different type of toothbrushes on gingival fluid proteolytic potential during orthodontic treatment

To study the effect of toothbrushes of various types on the accumulation of plaque and the proteolytic potential of gingival fluid as a pathogenic factors in long-term orthodontic treatment of patients with a distal permanent bite. We examined 63 patients of both sexes aged 11-18 years. Orthodontic treatment was carried out for the close position of the teeth with a distal permanent bite (K07.2) and cleft palate and lips (Q37.1). Patients were divided into three groups depending on the type of toothbrush used for individual oral hygiene: group 1 (n=21) - an orthodontic manual toothbrush (Professor Persin, «Spazzolificio Piave», Italy); Group 2 (n=22) - an ultrasonic toothbrush (Emmi-dent, EMAG AG, Germany) and group 3 (n=20) - an electric toothbrush ("Oral-B Pro-Expert, «Procter & Gamble», USA). The amount of soft plaque was assessed by the Silness-Loe hygiene index in the subgingival region. The activity of proteolytic enzymes - elastase, collagenase, and a α-inhibitor of proteinases was determined by the enzymatic method using a XL 200 biochemical analyzer («Erba Lachema», Czech Republic). The accumulation of plaque in the gingival region on both jaws during the entire period of orthodontic treatment is most pronounced when using a manual brush compared to ultrasonic and electric brushes. An increase in proteolytic activity in the gingival fluid with a simultaneous decrease in antiprotease potential with long-term orthodontic treatment of patients was observed when using a manual orthodontic brush, which can be regarded as an increase in the activity of the pathogenic factors for the development of local inflammatory and destructive changes. To prevent inflammatory and destructive changes during prolonged orthodontic treatment of patients, the use of ultrasonic and electric toothbrushes in daily oral hygiene is justified.

СТАН СИСТЕМИ ПРОТЕЇНАЗИ/ІНГІБІТОРИ ПРОТЕЇНАЗ У ЩУРІВ У ДИНАМІЦІ ІММОБІЛІЗАЦІЙНОГО СТРЕСУ НА ТЛІ ГІПОТИРЕОЗУ

Вступ. У патогенезі багатьох патологічних станів важливе місце посідають порушення протеїназо-інгібіторної системи. При відсутності належного контролю за протеолізом розвиваються патологічні стани, що супроводжуються виникненням деструктивних, запальних та імунних реакцій. Ряд дослідників встановив захисну дію йодовмісних тиреоїдних гормонів при стресі, які реалізуються в результаті їх взаємодії з клітинним геномом, що призводить до стимуляції локальних стрес-лімітуючих систем.&#x0D; Мета дослідження – встановити особливості функціонування системи протеїнази/інгібітори протеїназ за умов іммобілізаційного стресу на тлі зниження рівня йодовмісних гормонів щитоподібної залози.&#x0D; Методи дослідження. Гіпотиреоз моделювали, щоденно вводячи тваринам per os тиреостатик мерказоліл (“Здоров’я”, Україна) у дозі 25 мг/кг протягом 21-ї доби. Гострий іммобілізаційний стрес моделювали шляхом прив’язування піддослідних щурів у положенні на спині за 4 кінцівки без обмеження рухо­мості голови тривалістю 3 год.Для дослідження концентрації йодовмісних гормонів щитоподібної залози, активності протеїназної системи і вмісту інгібіторів протеолізу використано спектрофотометричні та імуноферментні методи.&#x0D; Результати й обговорення. Досліджено вплив іммобілізаційного стресу в щурів з попередньо змодельованим гіпотиреозом на показники протеїназо-інгібіторної системи крові. На стадії тривоги розвитку стрес-реакції в евтиреоїдних тварин встановлено підвищення протеолітичної активності крові на тлі збільшення вмісту α1-інгібітора протеаз та α2-макроглобуліну, що вказує на посилення антипротеолітичного потенціалу для стримування розвитку деструкції.На стадії резистентності відбувалася стабілізація протеолітичної активності крові. Однак при тривалому стресі (стадія виснаження) вона знову достовірно зростала на тлі зниження активності інгібіторів протеаз, що свідчить про виснаження захисного інгібіторного резерву. У тварин з гіпотиреозом на всіх стадіях розвитку стрес-реакції мало місце достовірне підвищення протеолітичної активності крові, причому показники інгібіторів протеаз достовірно зменшувалися. Це призводило до різкого зростання індексу протеолізу, що відбувалося на тлі збільшення проникності лізосомальних мембран.&#x0D; Висновок. На тлі гіпотиреозу спостерігають інтенсивніше, ніж у евтиреоїдних тварин, зростання протеолітичної активності крові на фоні пригнічення антипротеазної активності та збільшення проникності лізосомальних мембран.

Activities of Vacuolar Cysteine Proteases in Plant Senescence.

Plant senescence is accompanied by a marked increase in proteolytic activities, and cysteine proteases (Cys-protease) represent the prevailing class among the responsible proteases. Cys-proteases predominantly locate to lytic compartments, i.e., to the central vacuole (CV) and to senescence-associated vacuoles (SAVs), the latter being specific to the photosynthetic cells of senescing leaves. Cellular fractionation of vacuolar compartments may facilitate Cys-proteases purification and their concentration for further analysis. Active Cys-proteases may be analyzed by different, albeit complementary approaches: (1) in vivo examination of proteolytic activity by fluorescence microscopy using specific substrates which become fluorescent upon cleavage by Cys-proteases, (2) protease labeling with specific probes that react irreversibly with the active enzymes, and (3) zymography, whereby protease activities are detected in polyacrylamide gels copolymerized with a substrate for proteases. Here we describe the three methods mentioned above for detection of active Cys-proteases and a cellular fractionation technique to isolate SAVs.

Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria.

Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites.

Improvement in extracellular protease production by the marine antarctic yeast Rhodotorula mucilaginosa L7

Microorganisms from extreme and restrictive eco systems, such as the Antarctic continent, are of great interest due to their ability to synthesize products of commercial value. Among these, enzymes from psychrotolerant and psychrophilic microorganisms offer potential economical benefits due to their high activity at low and moderate temperatures. The cold adapted yeast Rhodotorula mucilaginosa L7 was selected out of 97 yeasts isolated from Antarctica as having the highest extracellular proteolytic activity in preliminary tests. The present study was aimed at evaluating the effects of nutrient composition (peptone, rice bran extract, ammonium sulfate, sodium chloride) and physicochemical parameters (temperature and pH) on its proteolytic activity. A 26−2 fractional factorial design experiment followed by a central composite design (CCD 23) was performed to optimize the culture conditions and improve the extracellular proteolytic activity. The results indicated that the presence of peptone in the medium was the most influential factor in protease production. Enzymatic activity was enhanced by the interaction between low glucose and peptone concentrations. The optimization of culture conditions with the aid of mathematical modeling enabled a c. 45% increase in proteolytic activity and at the same time reduced the amount of glucose and peptone required for the culture. Thus culture conditions established in this work may be employed in the biotechnological production of this protease.

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors.

The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.