Proteolytic Processing Activity Research Articles

Extracellular Matrix Proteins and Transcription of Matrix-Associated Genes in Mesenchymal Stromal Cells during Modeling of the Effects of Microgravity.

We studied the effects of simulated microgravity (10 days) on the production of extracellular matrix proteins and expression of extracellular matrix-associated genes in human mesenchymal stem cells. A decrease in collagen production, reduced expression of TIMP-1, TIMP-3, and MMP-11 genes, and enhanced expression of tenascin and laminin subunit were revealed. The results attest to activation of proteolytic processes in the matrix of mesenchymal stromal cells and weakening of cell adhesion to extracellular matrix under conditions of simulated microgravity.

Efficacy of nanoceria for periodontal tissues alteration in glutamate-induced obese rats\u2014multidisciplinary considerations for personalized dentistry and prevention

BackgroundNowadays, we face the global epidemic of obesity, that is known to contribute to the development of many diseases, such as the oral cavity pathologies. Dental and oral pathologies are frequently caused by and overlapped with systemic multifactorial diseases such as obesity being its early indicators and risk factors.The aim was to study the influence of nanoceria on periodontal tissues alteration in glutamate (MSG)-induced obese rats.MethodsWe included 52 Wistar rats of both genders and divided into four groups: newborn rats in group 1 (control) received subcutaneously 8 μl/g saline. Group 2 received 3 to 4 mg/g MSG subcutaneously on the second, fourth, sixth, eighth, and tenth day of life; group 3—intragastric administration of nanocrystalline cerium dioxide at a dose of 1 mg/kg volume of 2.9 ml/kg against the background of glutamate-induced obesity; the fourth group of animals was treated with a solution of sodium citrate intragastric volume of 2.9 ml/kg (solvent of nanocrystalline cerium). We determined the total proteolytic activity, the total antitrypsin activity, the content-free fucose and glycosaminoglycanes (GAG), content of TBA-active of products, the content of oxidation-modified proteins (OMB), and catalase activity in the homogenate of soft periodontal tissues of rats.ResultsIntragastric injection of nanoceria prevents activation of proteolytic processes, reducing the catabolism of glycoproteins and proteoglycans of periodontal tissue in MSG-induced obese rats. Injection of nanoceria prevents activation of proteolytic processes, significantly decreases the total proteolytic activity, and inhibits the activation of free radical oxidation in periodontal tissues of rats compared with MSG-induced obesity model without corrections. Further, it significantly increases the total antitrypsin activity in periodontal tissues by 1.7 times, TBA-reagents by 1.7 times, and content of OMB by 1.4 times compared with glutamate-induced obese animals.ConclusionsMSG-induced obesity triggers periodontal tissue alterations in the rat model. Nanoceria contributes to the corrections of pathological changes in periodontal tissues in glutamate-induced obese rats via balancing protein-inhibitory capacity and reducing the depolymerization of fucosylated proteins and proteoglycans and antioxidative activity.

Experience of the optimum exposition of enterosgel establishment for treatment of the periodontal disease at patients with vesicular disease

Abstract: Based on clinical studies it was found that the optimum mode enterosgelya exposure to the area affected by peri -odontal pockets is 2 hours. During this period it was observed decrease in the total microbial contamination gingival fluid in more than 25,000 times; restores the balance of gram-negative and gram-positive microorganisms. Keywords: Enterosgel, periodontal disease, vesicular disease, gram-negative and gram-positive microorganisms. From the clinical point of view vesicular disease belongs to difficult multifactorial diseases and is bound to development and persistence of processes of endogenic intoxication which is shown by essential augmentation in a blood of molecules of average weight and activation of proteolytic processes [4, 22–25; 6, 28].It is known that emergence and advance of a periodontal dis-ease is caused by activation of constantly present microorganisms, development of clinically defined inflammatory reaction which ap-pointment consists in neutralizing the toxins and enzymes blasting tissues is a consequence of that, and also to destroy and microbial cells.At synchronization of an inflammation in tissues in a large number the substances which are initially protecting, and as a re-sult actively striking these tissues accumulate. Accumulation of the toxins formed by infectious agents aggravates metabolic dis-orders, leads to development of various pathological states. As a result in liquid of the parodontal pockets (PP) and other biological liquids (stomatic liquid, a blood) products of a destruction of tissues collect, causing intoxication syndrome. Among bonds of this sort the important role belongs to middlemolecular pep-tides (MP) [2, 52–54; 5, 22].In complex treatment of generalized periodontitis (GP) as an agent of detoxication therapy the sorbent of “Enterosgel” is widely applied. Enterosgel — a silicon organic compound — is made by JSC Silma (Russia). It is used inside, in a dose of 15 g. 2–3 times a day between meals, washing down with a glass of water. It is shown that reception of enterosgel in the specified dosage leads to depres -sion at sick with GP of symptoms and indicators of endogenic in-toxication [3, 22–25].It is obvious that local application of sorbents for treatment of diseases of a parodont especially significantly at patients with vasicular disease at whom endogenic intoxication is one of the main pathogenetic mechanisms of development of a basic disease.

Cellular effects of functional unloading and passive strain of soleus muscle in dystrophin-deficient mice

There is a suggestion that dystrophin, a subsarcolemmal protein communicating fiber cytoskeleton to extracellular matrix, participates in signal transduction reflecting the mechanical state of skeletal muscle (mechanotransduction). Recent works indicate the possible signaling role of this protein in the prevention of the activation of proteolytic processes accompanying development of muscle fiber atrophy and in realization of anabolic effects of muscle passive stretching. To assess the role of dystrophin in these processes, the experiment was carried out on two-month old C57 black and mdx (dystrophin-deficient) mice subjected to hind-limb suspension with stretching and without it. Passive stretching results in the partial prevention of atrophy in two muscle fiber types of both C57 black and mdx mice; at the same time, in mdx mice, the slow-to-fast transformation of the soleus muscle fiber type was not observed. Proliferative activity in soleus muscle decreased as a result of hind-limb suspension, but markedly increased during muscle passive stretching. We have found no correlation between the altered dystrophin synthesis and proliferative activity of satellite cells during hind-limb suspension and hind-limb suspension with stretching. Hence, the disturbed dystrophin synthesis retards the atrophy of slow muscle fibers and practically does not affect the stretching preventive action.

Lithium down-regulates tau in cultured cortical neurons: A possible mechanism of neuroprotection

In tauopathies such as Alzheimer's disease (AD), the moleccular mechanisms of tau protein agregation into neurofibrillary tangles (NFTs) and their contribution to neurodegeneration are not fully understood. Recent studies indirectly demonstrated that tau, regardless of its aggregation, might represent a key mediator of neurodegeneration, especially that induced by the amyloid (Abeta) pathology. Lithium is a medication for bipolar mood disorders. Its therapeutic mechanism of action remains unclear, in part because of the large number of biochemical effects attributed to lithium. Since lithium directly inhibits glycogen synthase kinase-3beta (GSK3beta), a key enzyme involved in tau phosphorylation, it was suggested that the therapeutic use of lithium could be expanded from mood disorders to neurodegenerative conditions. Lithium has been also reported to protect cultured neurons against Abeta toxicity, and to prevent NFTs accumulation and cognitive impairments in transgenic models of tauopathies. However, the exact mechanism of neuroprotection provided by lithium remains unknown. Here, we show that exposure of cultured cortical neurons to lithium decreased tau protein levels. This decrease was not linked to the activation of proteolytic processes including calpains, caspases and proteasome or to neuronal loss, but was rather associated with a reduction in tau mRNA levels. Moreover, prior exposure to lithium, at concentrations effective in reducing tau protein levels, markedly reduced pre-aggregated Abeta-induced neuronal apoptosis. Our findings raise the possibility that lithium could exert its neuroprotective effect against Abeta toxicity through the downregulation of tau proteins and that, at least, by acting at the level of tau mRNA.

A bacteriophage lambda-based genetic screen for characterization of the activity and phenotype of the human immunodeficiency virus type 1 protease.

Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors.

Pre- and Post-translational Regulation of Lysyl Oxidase by Transforming Growth Factor-β1 in Osteoblastic MC3T3-E1 Cells

The final enzymatic step required for collagen cross-linking is the extracellular oxidative deamination of peptidyl-lysine and -hydroxylysine residues by lysyl oxidase. A cross-linked collagenous extracellular matrix is required for bone formation. The goals of this study were to compare the transforming growth factor (TGF)-beta 1 regulation of lysyl oxidase enzyme activity and steady state mRNA levels to changes in COL1A1 mRNA levels in MC3T3-E1 osteoblastic cells. TGF-beta 1 increased steady state lysyl oxidase and COL1A1 mRNA levels in a dose- and time-dependent manner. The increase in lysyl oxidase mRNA levels was transient, peaking at 12 h and 8.8 times controls in cells treated with 400 pM TGF-beta 1. COL1A1 steady state mRNA levels increased maximally to 3.5-fold of controls. Development of increased lysyl oxidase enzyme activity was delayed and was of slightly lower magnitude than the increase in its mRNA levels. This suggested limiting post-translational processing of lysyl oxidase proenzyme. Pulse-labeling/immunoprecipitation studies demonstrated slow proenzyme secretion and proteolytic processing. Development and application of an independent assay for lysyl oxidase proenzyme proteolytic processing activity verified its proportionately lower stimulation by 400 pM TGF-beta 1. Thus, lysyl oxidase regulation by TGF-beta 1 in osteoblastic cell cultures occurs at both pre- and post-translational levels. This regulation is consistent with increased production of a collagenous extracellular matrix.

A kinetic model for comparing proteolytic processing activity and inhibitor resistance potential of mutant HIV-1 proteases

A kinetic model is presented for the comparison of potential proteolytic processing activities of wild-type and mutant human immunodeficiency virus proteases in the presence and absence of protease inhibitors. The protease processing of gag substrate in the immature virions is assessed by the kinetic parameters, k cat, K m and K i. The relationship of the estimated potential processing activities to the viability of the HIV mutants and their tendencies to resist inhibitors also are discussed. A fully developed model has the potential to simulate the results of inhibitor resistance either in vivo or in cell culture.

Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components.

The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCl procedures can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added IgG light chain mRNA so as to segregate the translation product in a proteolysis-resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed. Taken together, these results establish unequivocally that the information for segregation of a translation product is encoded in the mRNA itself, not in the protein-synthesizing apparatus; this provides strong evidence in support of the signal hypothesis.