Effect of Ovocystatin on Amyloid ß 1-42 Aggregation-In Vitro Studies.

Amyloid ß peptides (Aß) aggregating in the brain have a potential neurotoxic effect and are believed to be a major cause of Alzheimer's disease (AD) development. Thus, inhibiting amyloid polypeptide aggregation seems to be a promising approach to the therapy and prevention of this neurodegenerative disease. The research presented here is directed at the determination of the inhibitory activity of ovocystatin, the cysteine protease inhibitor isolated from egg white, on Aß42 fibril genesis in vitro. Thioflavin-T (ThT) assays, which determine the degree of aggregation of amyloid peptides based on fluorescence measurement, circular dichroism spectroscopy (CD), and transmission electron microscopy (TEM) have been used to assess the inhibition of amyloid fibril formation by ovocystatin. Amyloid beta 42 oligomer toxicity was measured using the MTT test. The results have shown that ovocystatin possesses Aß42 anti-aggregation activity and inhibits Aß42 oligomer toxicity in PC12 cells. The results of this work may help in the development of potential substances able to prevent or delay the process of beta-amyloid aggregation-one of the main reasons for Alzheimer's disease.

Use of natural cysteine protease inhibitors in limiting SARS-Co-2 fusion into human respiratory cells.

Specific antibodies that humans acquire as a result of disease or after vaccination are needed to effectively suppress infection with a specific variant of SARS CoV-2 virus. The S protein of the D614G variant of coronavirus is used as an antigen in known vaccines to date. It is known that COVID-19 disease resulting from infection with this coronavirus can often be very dangerous to the health and lives of patients. In contrast, vaccines produce antibodies against an older version of the protein S-D614G (January 2020) and therefore have difficulty recognizing new variants of the virus. In our project we propose to obtain specific and precise antibodies by means of so-called controlled infection against specific infectious variants of the SARS-CoV-2 virus "here and now". Currently, several variants of this pathogen have already emerged that threaten the health and lives of patients. We propose to reduce this threat by partially, but not completely, blocking the fusion mechanism of the SARS-CoV-2 virus into human respiratory cells. According to our plan, this can be achieved by inhibiting cathepsin L activity in respiratory cells, after introducing natural and non-toxic cysteine protease inhibitors into this area. We obtain these inhibitors by our own method from natural, "human body friendly" natural resources. We hypothesize that blocking cathepsin L will reduce the number of infecting viruses in cells to such an extent that COVID-19 developing in infected individuals will not threaten their health and life. At the same time, the number of viruses will be sufficient for the body's own immune system to produce precise antibodies against a specific version of this pathogen.

Ovocystatin Induced Changes in Expression of Alzheimer's Disease Relevant Proteins in APP/PS1 Transgenic Mice.

Background: Ovocystatin is marked by structural and biological similarities to human cystatin C, which plays an important role in the course of neurodegenerative diseases. Recently, it has been shown that ovocystatin might prevent aging-related cognitive impairment in rats and reduce memory decline in an APP/PS1 mice model. Thus, this study aimed to assess the effect of ovocystatin on histopathological changes in APP/PS1 mice. Materials and methods: Ovocystatin was administered intraperitoneally for four weeks (40 µg/mouse) to 35-weeks-old transgenic (AD, n = 14) and wild type (NCAR, n = 15) mice (stock B6C3-Tg(APPswe, PSEN1dE9)85Dbo/Mmjax). A histopathological evaluation comprised antibodies directed against ß-amyloid (1:400, SIG-39320-1000, Covance) and Tau (1:4000, AHB0042, Invitrogen). Three regions of the hippocampus­ the dentate gyrus (DG) and the cornu ammonis (CA1 and CA3)­were analyzed by immunohistochemistry in each animal. All differences are expressed as percentage relative to the control group. Results: The main results showed that the percentage of immunoreactive area of ß-amyloid, tau protein deposits in APP/PS1+ovCYS was decreased in DG, CA1, and CA3 regions compared with the APP/PS1 control, respectively (p < 0.05). Conclusions: Ovocystatin caused significant changes in the expression pattern of all investigated proteins in hippocampal tissues both in APP/PS1 and NCAR mice.

Evaluation of Wound Healing Activity of Salvianolic Acid B on In Vitro Experimental Model.

Despite a wide range of bactericides and antiseptics, the treatment of chronic or complicated wounds is still a major challenge for modern medicine. Topical medications are the most sought-after new agents for use as treatment. The therapeutic concentration of their active substances is easy to achieve with the lowest possible burden on the patient's body. This study assesses the effect of salvianolic acid B (Sal B) on the proliferation, migration, and production of collagen type III by fibroblasts, which are the most important processes in wound healing. The study was conducted on human gingival fibroblasts obtained from primary cell culture. The results showed that Sal B at a dose of 75 µg/mL increases the cell viability with significant stimulation of the cell migration as demonstrated in the wound healing assay, as well as an increase in the expression of collagen type III, which has great importance in the initial stages of wound scarring. The results obtained in the conducted studies and previous scientific reports on the antibacterial properties and low toxicity of Sal B indicate its high potential in wound healing.

Renal Handling of Albumin-From Early Findings to Current Concepts.

Albumin is the main protein of blood plasma, lymph, cerebrospinal and interstitial fluid. The protein participates in a variety of important biological functions, such as maintenance of proper colloidal osmotic pressure, transport of important metabolites and antioxidant action. Synthesis of albumin takes place mainly in the liver, and its catabolism occurs mostly in vascular endothelium of muscle, skin and liver, as well as in the kidney tubular epithelium. Long-lasting investigation in this area has delineated the principal route of its catabolism involving glomerular filtration, tubular endocytic uptake via the multiligand scavenger receptor tandem-megalin and cubilin-amnionless complex, as well as lysosomal degradation to amino acids. However, the research of the last few decades indicates that also additional mechanisms may operate in this process to some extent. Direct uptake of albumin in glomerular podocytes via receptor for crystallizable region of immunoglobulins (neonatal FC receptor) was demonstrated. Additionally, luminal recycling of short peptides into the bloodstream and/or back into tubular lumen or transcytosis of whole molecules was suggested. The article discusses the molecular aspects of these processes and presents the major findings and controversies arising in the light of the research concerning the last decade. Their better characterization is essential for further research into pathophysiology of proteinuric renal failure and development of effective therapeutic strategies.

The effects of chicken egg white cystatin and proteinase inhibitor on cysteine peptidase-like activity in the sera of patients with breast cancer.

BACKGROUND: The activity of autogenic proteolytic enzymes is regulated in vivo by autogenic inhibitors. They play important roles in maintaining a balance in many processes in the human body. In pathological conditions, enzymes are overexpressed and the balance is disturbed. Such uncontrolled changes may lead to the development of local or systemic cancer. OBJECTIVES: To evaluate the effects of specific inhibitors, i.e., chicken egg white cystatin (CEWC) and proteinase inhibitor (E-64) on autogenic cysteine peptidases (CPs) in the sera of patients reporting for subsequent stages of treatment after being diagnosed with breast cancer. Cysteine peptidases play a vital role in the basic processes that are associated with cancer progression. MATERIAL AND METHODS: We selected serum samples from 108 patients with a diagnosis of breast cancer (stages IIA-IIIA) who had received no previous treatment. The blood samples were centrifuged, and the resulting serum was placed in liquid nitrogen and stored at -80°C. The biochemical tests were performed at the laboratory of the Department of Physical Chemistry and Microbiology. RESULTS: For CEWC, we found an inhibitory effect in 37 out of 108 samples; for E-64, 14 out of 22 samples displayed an inhibitory effect. In the remaining blood samples, these inhibitors caused an increase in fluorescence. In a parallel test, we added pure cathepsin B to 9 serum samples, and then used CEWC to inhibit the activity of autogenic CPs. Chicken egg white cystatin completely inhibited the cathepsin B that was added to the serum without changing its effect on the autogenic CPs. CONCLUSIONS: The results suggest that there may be a potential difference between the commercially available cathepsin B and its autogenic analogues found in the serum of cancer patients. The increase in fluorescence induced in the reaction between the inhibitors and autogenic CPs is still unexplained. There was no relationship between the observed inhibition/activation of CPs and any of the available indicators of the health of the patients examined.

Beneficial effect of ovocystatin on the cognitive decline in APP/PS1 transgenic mice.

PURPOSE: Cystatin C plays an important role in the course of neurodegenerative diseases and has a beneficial effect through inhibiting cysteine proteases and amyloid-ß aggregation. It also induces proliferation and autophagy. Cystatin isolated from chicken egg white, called ovocystatin, has been widely used in the medical and pharmaceutical research due to its structural and biological similarities to human cystatin C. The aim of this study was to assess the effect of administering ovocystatin on the development of dementia-specific cognitive deficits in APP/PS1 transgenic mice. MATERIALS/METHODS: The study was conducted on transgenic B6C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax mice. Ovocystatin was administered to four-month-old transgenic (AD) and wild type (NCAR) mice in drinking water for 24 weeks (at a dose of 40 and 4 µg/ mouse). The locomotor activity and cognitive functions were determined using an actimeter and the Morris water maze test, respectively. RESULTS: The results of the study indicate that ovocystatin has a beneficial effect on the cognitive functions in APP/PS1 transgenic mice. The strongest effects of ovocystatin were found in the group of AD mice, where ovocystatin was administered in drinking water at a dose of 40 µg/mouse (p < 0.05). Mice from the AD group swam statistically significantly further in the target zone during the trial in the Morris water maze compared to the AD (vehiculum) group (p < 0.05). CONCLUSIONS: The obtained results encourage further research into the protective effect, which may be used as an adjuvant in the treatment of deteriorating cognitive functions.

Inhibition of Advanced Glycation End-Product Formation and Antioxidant Activity by Extracts and Polyphenols from Scutellaria alpina L. and S. altissima L.

Methanolic extracts from the aerial parts and roots of two Scutellaria species, S. alpina and S. altissima, and five polyphenols from these plants demonstrated a significant ability to inhibit the formation of advanced glycation end-products (AGE) in vitro. S. alpina, which is richer in polyphenolic compounds, had strong antiglycation properties. These extracts demonstrated also high activity in the FRAP (ferric-reducing antioxidant power), antiradical (DPPH) and lipid peroxidation inhibition assays. Among the pure compounds, baicalin was the strongest glycation inhibitor (90.4% inhibition at 100 µg/mL), followed by luteolin (85.4%). Two other flavone glycosides had about half of this activity. Verbascoside was similar to the reference drug aminoguanidine (71.2% and 75.9%, respectively). The strong correlation observed between AGE inhibition and total flavonoid content indicated that flavonoids contribute significantly to antiglycation properties. A positive correlation was also observed between antiglycative and antioxidant activities. The studied skullcap species can be considered as a potential source of therapeutic agents for hyperglycemia-related disorders.

[Clathrin-independent endocytosis - role in disease processes and pharmaceutical aspects]. / Endocytoza niezalezna od klatryny-rola w patomechanizmie chorób i aspekty farmaceutyczne.

Clathrin-independent endocytosis (CIE) is the process of cellular uptake of various particles, including pathogens, without the coat protein clathrin. It occurs commonly in mammalian cells and is regulated by protein-lipid composition of the cell membranes. Understanding of different routes of CIE allowed the identification of novel molecular mechanisms involved in uptake of molecules and cell signaling and explained their role in pathological processes. In this paper we characterize diseases associated with genetic defects of proteins involved in CIE and the relationship between expression of these proteins and pathology of atherosclerosis, hypercholesterolemia, diabetes and neoplasia. The role of CIE in bacterial, viral, fungal, and protozoal infections is also presented. In the second part we describe the plausible use of clathrin-independent endocytosis in increasing drug absorption, their penetration through biological membranes, and the design of specific nanocarriers for selective cell uptake.

Antipapain activity in the serum of patients with breast cancer.

BACKGROUND: Complement information about the share the role of antipapain activity in serum people with breast cancer. OBJECTIVE: We measured the activity of cysteine peptidase inhibitors in the sera of 150 patients with breast cancer. Patients were divided into four groups depending on the cancer type and treatment method. We also analysed the control group. The activity of cysteine peptidase inhibitors was defined as a 'defensiveness' marker. METHODS: The activity of cysteine peptidase inhibitors was measured against papain using the colorimetric method and the BANA substrate. RESULTS: The highest activity of enzymes was found in the group of patients with BC and hereditary predisposition to it, and the lowest activity was found in patients after surgical treatment. CONCLUSION: The activity of cysteine peptidase inhibitors in serum was measured against papain. We found that the activity levels were correlated with the cancer stage and treatment method. The lowest activity was found in patients after surgical treatment; the highest in women with active cancer and a hereditary predisposition to it.

Evaluation of the antibacterial activity of cystatin against selected strains of Escherichia coli.

The aim of this study was to analyze the antibacterial activity of hen egg white cystatin against selected Escherichia coli strains. We used a monomeric solution of hen egg white cystatin in bovine serum albumin (BSA) with added phosphate buffered saline (PBS), and three test strains: Escherichia coli ATCC 23811, Escherichia coli ATCC 8739 and Escherichia coli ATCC 25922. The effect of cystatin against the tested strains was determined on the basis of minimal inhibitory concentration (MIC) and survival curves of the microorganisms in a cystatin-containing environment during incubation at various temperatures. Our study confirmed the activity of cystatin against the analyzed Escherichia coli strains. taining environment, as compared to control samples incubated in a ovocystatin-deficient medium. Depending on the incubation temperature (20 degrees C or 37 degrees C) the reduction persisted up to 12 hours after incubation.

Ovocystatin affects actin cytoskeleton organization and induces proapoptotic activity.

Ovocystatin is a chicken egg white protein, generally known for its inhibitory activity against cysteine proteases. However, biological activity of ovocystatin does not seem to be well recognized in respect to other possible cellular effects. Our attention has been focused on ovocystatin cytotoxic effects in relation to its influence on actin cytoskeleton organization and apoptosis induction. In vitro studies with human melanoma A375, human cervix HeLa cancer cells and normal human fibroblasts - NHDF were done. Cytotoxic activity of ovocystatin was seen in respect to apoptosis induction - manifested by cell shape changes, phosphatydylserine translocation and actin cytoskeleton reorganization. Normal human fibroblasts have shown lower sensitivity to ovocystatin as compared with human melanoma A375 and human cervix HeLa cancer cells. In conclusion, ovocystatin affects actin cytoskeleton organization and displays proapoptotic activity towards applied cell lines. This implicates its application as a potential anticancer drug. However, its adverse effects on normal cells should be taken into consideration.

Influence of aminoglycoside antibiotics on chicken cystatin binding to renal brush-border membranes.

OBJECTIVES: Drug-induced kidney injury is a serious adverse event which needs to be monitored during aminoglycoside therapy. Urine cystatin C is considered an early and sensitive marker of nephrotoxicity. Cystatin C, a low-molecular-weight serum protein, and basic drugs have a common transport system expressed in the apical membrane of renal proximal tubular cells. The aim of this study was to investigate whether aminoglycoside antibiotics influenced cystatin C binding to the renal brush-border membrane. METHODS: The binding study was performed using a rapid filtration technique and affinity column displacement method. KEY FINDINGS: Concentration-dependent inhibition of chicken cystatin binding to brush-border membranes by gentamicin was observed. The gentamicin interaction with brush-border membranes was of relatively low affinity (Ki = 32 µm) in comparison with the chicken cystatin affinity to the binding sites (Kd = 3.6 µm). Amikacin and gentamicin were only able to displace chicken cystatin from the chromatographic affinity column in concentrations several times higher than normally found in the tubular fluid during standard aminoglycoside therapy. CONCLUSION: Cystatin reabsorption in the proximal tubule cannot be significantly affected by aminoglycoside antibiotics because of their relatively low affinity to common binding sites on the brush-border membrane.

Binding of glycated ovocystatin to rat renal brush border membranes.

Glycated proteins are considered as one of the factors involved in the pathogenesis of diabetic complications, including nephropathy. These proteins are formed endogenously under conditions of hyperglycemia, as well as being provided with food containing sugars, which was subjected to high temperature. Examples are egg products. One of the proteins found in eggs in a relatively high concentration is chicken cystatin (ovocystatin). It is now believed that some proteins can passage the intestinal epithelium by transcytosis directly into the bloodstream. Thus, glycated protein present in food can be an additional source of glycotoxins. The aim of this study was to compare the affinity of native and glycated cystatin to the brush border membranes of rat kidney. Kinetic analysis was performed with surface plasmon resonance technique using sensor chip L1. Dissociation constants for native and glycated cystatin (Kd ) were 2.76 µmol/L and 3.82 µmol/L, respectively. The results of our study indicate that glycation only slightly affects binding of cystatin to brush border membranes. This suggests that glycated cystatin and other glycated proteins may also be efficiently taken up in the kidney proximal tubule. The observation may be important for understanding the mechanisms involved in the development of diabetic nephropathy.

[Renal catabolism of albumin - current views and controversies]. / Nerkowy katabolizm albuminy ­ aktualne poglady i kontrowersje.

Albumin is the main protein of blood plasma, lymph, cerebrospinal fluid and interstitial fluid. The protein assists in many important body functions, including maintenance of proper colloidal osmotic pressure, transport of important metabolites and antioxidant action. Synthesis of albumin takes place mainly in the liver, and its catabolism occurs mostly in vascular endothelium of muscle, skin and liver as well as in the kidney tubular epithelium. Renal catabolism of albumin consists of glomerular filtration and tubular reabsorption. The tubular processes include endocytosis via the multiligand scavenger receptor tandem megalin and cubilin-amnionless complex. Possible ways of further catabolism of this protein are lysosomal proteolysis to amino acids and short peptides, recycling of degradation products into the bloodstream and tubular lumen or transcytosis of whole molecules. The article discusses the molecular aspects of these processes and presents the controversies arising in the light of the last decade of research.

[Serum chitotriosidase activity as a biomarker of pulmonary sarcoidosis]. / Aktywnosc chitotriozydazy jako biomarker plucnej postaci sarkoidozy.

Sarcoidosis is a multisystemic granulomatous disorder of unknown etiology, which is characterized by a variable clinical presentation and course. The diagnosis of this disease is usually supported by the three elements: compatible clinical and radiologic findings, tissue biopsy specimen that reveals noncaseating epithelioid granulomas and the absence of known granulomagenic agent. During the last years the new diagnostic methods have been discovered, but serum markers of the sarcoidosis are still under studying. Among the potential markers of sarcoidosis, a recently proposed indicator is chitotriosidase, a chitinase produced by chronically activated macrophages. The review of the literature showed that chitotriosidase activity is only a surrogate biomarker to confirm diagnosis, but is a useful marker for disease activity monitoring and prognosis. The correlation with the radiological stages of disease suggest that determination of chitotriosidase activity could decrease the number of X-ray examination.

[Serum activity of chitotriosidase, lysozyme and cathepsin H in patients with lung cancer and patients with inflammatory exudate (preliminary report)]. / Aktywnosc chitotriozydazy oraz lizozymu i katepsyny H w surowicy chorych na raka pluca i chorych z wysiekiem zapalnym (doniesienie wstepne).

UNLABELLED: Lung cancer remains the leading cause of cancer death over the world. Although new diagnostic methods have been discovered, new biomarkers of the cancer are still under studying. A human chitinolytic enzyme called chitotriosidase hydrolyzes chitin and chitotrioside substrates. It is specifically expressed by activating macrophages and seems to play a role in the defense against chitinous human pathogens. Recently it has been shown that chitotriosidase may also attend to the inflammatory process. The aim of the study was to determine chitotriosidase activity in serum of patients with lung cancer and patients with inflammatory exudate. We studied the usefulness of the above parameter determination in differentiation between lung cancer and inflammation. In addition, serum activity of lysozyme and cathepsin H was determined. MATERIALS AND METHODS: The study included 17 patients with inflammatory pleural exudate--group 1., 40 lung cancer patients with malignant pleural effusion--group 2. and 37 healthy subjects. All the patients of group 2. were divided into 2 subgroups: 2A without metastases (n = 23) and 2B with metastases (n = 17). Chitotriosidase and cathepsin H activity was determined in serum by a fluorometric methods. Serum lysozyme activity was measured by turbidimetric method with Micrococcus luteus as substrate. RESULTS: We observed an increase of the chitotriosidase activity in serum patients of both studied group in comparison with the control. The activity of the chitotriosidase in lung cancer patients was significantly higher than in the control (36.7 vs 68.1 nmol/ml/h; p < 0.01). There were no significant differences in serum lysozyme and cathepsin H activity in patients in comparison to healthy subjects. CONCLUSION: The results suggest that activity of the chitotriosidase can not be used to differentiation between inflammation and cancer in lung.

Characterization of chicken cystatin binding to rat renal brush-border membranes.

Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (K(d)=3.67-4.07 microM) and high capacity (B(max)=2.32-2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (K(i)=0.7 microM) among other megalin/cubilin ligands tested. The presence of Ca(+2) ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells.

[Cathepsin B activity and concentration of elastase and alpha-1 proteinase inhibitor complex in non-small-cell lung cancer: 2 year follow-up study]. / Aktywnosc katepsyny B i stezenie kompleksu elastazy z inhibitorem alpha 1-proteinaz w niedrobnokomórkowym raku pluca--obserwacja dwuletnia.

In 21 patients with non-small-cell lung cancer subjected to radical surgery followed by 3 cycles of chemotherapy, serum cathepsin B activity and plasma E-alpha 1IP concentration in peripheral blood and tumour arterial and venous blood were studied. Cathepsin B activity was determined by a fluorometric assay. E-alpha 1IP concentration was measured with an ELISA kit. The measurements were performed before surgery, before each chemotherapy cycle and every 60 days after chemotherapy completion, for 2 years. All the patients (n = 21) were divided into 2 subgroups: without metastases n = 16 and with metastases n = 5. There was no significant difference between preoperative serum cathepsin B activity and E-alpha 1IP plasma values in peripheral blood and blood coming from tumour artery and vein. The surgery and chemotherapy caused a statistically significant decrease of serum cathepsin B activity and plasma E-alpha 1IP concentration both in the whole group and in the subgroup without metastases. A significant increase of cathepsin B activity in comparison to initial values was observed 2,5-4 months before cerebral metastasis appeared in the subgroup with metastases. The elevation of E-alpha 1IP concentration preceded the increase of cathepsin B activity in this subgroup. It was not statistically significant. A decrease of cathepsin B and E-alpha 1IP values was observed after cerebral metastasis excision.