Towards a Quantitative Description of Proteolysis: Contribution of Demasking and Hydrolysis Steps to Proteolysis Kinetics of Milk Proteins.

The hydrolysis of proteins by proteases (proteolysis) plays a significant role in biology and food science. Despite the importance of proteolysis, a universal quantitative model of this phenomenon has not yet been created. This review considers approaches to modeling proteolysis in a batch reactor that take into account differences in the hydrolysis of the individual peptide bonds, as well as the limited accessibility (masking) for the enzymes of some hydrolysis sites in the protein substrate.

mTOR pathway occupies a central role in the emergence of latent cancer cells.

The current focus in oncology research is the translational control of cancer cells as a major mechanism of cellular plasticity. Recent evidence has prompted a reevaluation of the role of the mTOR pathway in cancer development leading to new conclusions. The mechanistic mTOR inhibition is well known to be a tool for generating quiescent stem cells and cancer cells.

Modeling of the Peptide Release during Proteolysis of β-Lactoglobulin by Trypsin with Consideration of Peptide Bond Demasking.

Prospects for predicting the fragmentation of polypeptide chains during their enzymatic hydrolysis using proteolysis models are considered. The opening of the protein substrate during proteolysis and the exposure of its internal peptide bonds for a successful enzymatic attack, the so-called demasking process, were taken into account. The two-step proteolysis model was used, including the parameters of demasking and the rate constants of hydrolysis of enzyme-specific peptide bonds.

Proteolysis of Micellar β-Casein by Trypsin: Secondary Structure Characterization and Kinetic Modeling at Different Enzyme Concentrations.

Tryptic proteolysis of protein micelles was studied using β-casein (β-CN) as an example. Hydrolysis of specific peptide bonds in β-CN leads to the degradation and rearrangement of the original micelles and the formation of new nanoparticles from their fragments. Samples of these nanoparticles dried on a mica surface were characterized by atomic force microscopy (AFM) when the proteolytic reaction had been stopped by tryptic inhibitor or by heating.

Dynamics of the Glycogen β-Particle Number in Rat Hepatocytes during Glucose Refeeding.

Glycogen is an easily accessible source of energy for various processes. In hepatocytes, it can be found in the form of individual molecules (β-particles) and their agglomerates (α-particles). The glycogen content in hepatocytes depends on the physiological state and can vary due to the size and number of the particles.

Modeling of Proteolysis of β-Lactoglobulin and β-Casein by Trypsin with Consideration of Secondary Masking of Intermediate Polypeptides.

The opening of protein substrates during degradation by proteases and the corresponding exposure of their internal peptide bonds for a successful enzymatic attack, the so-called demasking effect, was studied for β-lactoglobulin (β-LG) and β-casein (β-CN) hydrolyzed by trypsin. Demasking was estimated by monitoring the redshift in intrinsic tryptophan fluorescence, characterizing the accessibility of polypeptide chains to aqueous medium. The secondary masking of intermediate polypeptides, giving an inverse effect to demasking, caused a restriction of the substrate opening.

The upregulation of Ulk1-dependent autophagy does not require the p53 activity in mouse embryonic stem cells.

Autophagy is known to play a critical role in the early stages of embryogenesis including the formation of blastocyst. The existence of p53 protein-deficient mice may identify that p53 is not indispensable for the activation of autophagy in pluripotent cells derived from the inner cell mass of the blastocyst. We utilized a p53-knockout (KO) mouse embryonic stem cell (mESC) line to investigate the contribution of p53 in autophagy.

Cancer-testis antigens, semenogelins 1 and 2, exhibit different anti-proliferative effects on human lung adenocarcinoma cells.

Сancer-testis antigens (CTAs) comprise proteins which are aberrantly expressed in various malignancies, yet under normal situation are restricted to only germ cells. Semenogelins 1 and 2 (SEMG1 and 2, respectively) belong to the family of non-X-linked (autosomal) cancer-testis antigens. They are the major protein ingredients of human semen and share 78% of similarity between them on the gene level.

Inducible Ulk1 expression activates the p53 protein in mouse embryonic stem cells.

Defective pluripotent cells are removed from embryos prior to differentiation, presumably due to upregulation of the p53 pathway. However, the mechanism underlying p53 protein activation is still unknown. Embryonic stem cells (ESCs), corresponding to cells of the preimplantation blastocyst, likely have similar mechanisms for abnormal cell elimination.

Rat Left Ventricular Cardiomyocytes Characterization in the Process of Postinfarction Myocardial Remodeling.

Ischemic lesions of the heart, including myocardial infarction, are the most common pathologies of human cardiovascular system. Despite all the research and achievements of medicine in this field, the mortality from this disease remains heavy. Therefore, studying of processes occurring in the myocardium in the early and late postinfarction periods remains important.

Spatial Structure of Glycogen Molecules in Cells.

Glycogen is a strongly branched polymer of α-D-glucose, with glucose residues in the linear chains linked by 1→4-bonds (~93% of the total number of bonds) and with branching after every 4-8 residues formed by 1→6-glycosidic bonds (~7% of the total number of bonds). It is thought currently that a fully formed glycogen molecule (β-particle) with the self-glycosylating protein glycogenin in the center has a spherical shape with diameter of ~42 nm and contains ~ 55,000 glucose residues. The glycogen molecule also includes numerous proteins involved in its synthesis and degradation, as well as proteins performing a carcass function.

Kinetic modeling of demasking and hydrolysis of peptide bonds during proteolysis of β-lactoglobulin by trypsin.

Proteolysis of β-lactoglobulin by trypsin was studied with fluorescence spectroscopy and an empirical exponential model was engaged to describe the peptide bond hydrolysis kinetics. The shift in the fluorescence maximum of tryptophan residues, from 342 to 352 nm, in the course of β-lactoglobulin degradation was used as an indicator of the transition of masked peptide bonds to the demasked ones, which were accessible for the enzyme action. A simple equation with only two parameters was suggested to link together the degree of demasking of peptide bonds and the degree of their hydrolysis, allowing the kinetic description of proteolysis.

Proteolytically-induced changes of secondary structural protein conformation of bovine serum albumin monitored by Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy.

Enzymatically-induced degradation of bovine serum albumin (BSA) by serine proteases (trypsin and α-chymotrypsin) in various concentrations was monitored by means of Fourier transform infrared (FT-IR) and ultraviolet circular dichroism (UV-CD) spectroscopy. In this study, the applicability of both spectroscopies to monitor the proteolysis process in real time has been proven, by tracking the spectral changes together with secondary structure analysis of BSA as proteolysis proceeds. On the basis of the FTIR spectra and the changes in the amide I band region, we suggest the progression of proteolysis process via conversion of α-helices (1654 cm(-1)) into unordered structures and an increase in the concentration of free carboxylates (absorption of 1593 and 1402 cm(-1)).

Glycogen content in hepatocytes is related with their size in normal rat liver but not in cirrhotic one.

Background & Aims: Hepatocytes differ from one another by the degree of the ploidy, size, position in the liver lobule, and level of the DNA-synthetic processes. It is believed, that the cell size exerts substantial influence on the metabolism of the hepatocytes and the glycogen content in them. The aim of the present study was to test this hypothesis.

A straightforward kinetic evidence for coexistence of "induced fit" and "selected fit" in the reaction mechanism of a mutant tryptophan indole lyase Y72F from Proteus vulgaris.

The interaction of the mutant tryptophan indole-lyase (TIL) from Proteus vulgaris Y72F with the transition state analogue, oxindolyl-l-alanine (OIA), with the natural substrate, l-tryptophan, and with a substrate S-ethyl-l-cysteine was examined. In the case of wild-type enzyme these reactions are described by the same kinetic scheme where binding of holoenzyme with an amino acid, leading to reversible formation of an external aldimine, proceeds very fast, while following transformations, leading finally to reversible formation of a quinonoid intermediate proceed with measureable rates. Principally the same scheme ("induced fit") is realized in the case of mutant Y72F enzyme reaction with OIA.

[A comparison of a neuroprotective effects of hypoxic postconditioning and cerebrolysin in the experimental model].

Hypoxic postconditioning using episodes of mild hypobaric hypoxia is a new neuroprotective technique. We compared the neuroprotective efficacy of hypoxic postconditioning and cerebrolysin in a model of posthypoxic pathology in rats. Animals that survived the severe hypoxia (180 Torr, 3 h) were exposed to hypoxic postconditioning or received cerebrolysin.

[Hypoxic postconditioning corrects behavioral abnormalities in a model of post-traumatic stress disorder in rats].

Protective effects of the novel technique of hypoxic postconditioning with a hypobaric hypoxia paradigm were studied in "stress-restress" model ofposttraumatic stress disorder in rats. It was shown that repeated (3 times) exposure of rats that survived after severe traumatic stress to mild hypobaric hypoxia (postconditioning mode) efficiently abolished the development of stress-induced anxiety state. Postconditioning had a clear anxiolytic effect both when it was delivered after traumatic stress and after restress, but the intensity of this effect depended on the period ofpathogenesis of the posttraumatic stress disorder, when postconditioning was given.

[Changes in intensity of hypoxic brain damage in rats induced by hypoxic postconditioning].

The present study has been aimed to estimate a neuroprotective effect of postconditioning (PostC) by using mild hypobaric hypoxia (360 mm Hg, 2 h) in a model of severe hypoxic brain injury (180 mm Hg, 3 h) in rats. PostC was performed by three trials of mild hypoxia with 24 h intervals, according to two different protocols--PostC was started 3 h (early PostC) or 24 h (delayed PostC) following severe hypoxia. Using histological methods and computer image analysis, loss of neurons in hippocampus and neocortex was analyzed 7 days after severe hypoxia.

Real time observation of proteolysis with Fourier transform infrared (FT-IR) and UV-circular dichroism spectroscopy: watching a protease eat a protein.

Fourier transform infrared (FT-IR)- and UV-circular dichroism (UV-CD) spectroscopy have been used to study real-time proteolytic digestion of β-lactoglobulin (β-LG) and β-casein (β-CN) by trypsin at various substrate/enzyme ratios in D(2)O-buffer at 37°C. Both techniques confirm that protein substrate looses its secondary structure upon conversion to the peptide fragments. This perturbation alters the backbone of the protein chain resulting in conformational changes and degrading of the intact protein.

Methionine gamma-lyase: mechanistic deductions from the kinetic pH-effects. The role of the ionic state of a substrate in the enzymatic activity.

We have studied and compared the pH-dependencies of the main kinetic parameters for the alpha,gamma-elimination reactions of methionine gamma-lyase (MGL) of Citrobacter intermedius with natural substrate, l-methionine, with its phosphinic analogue, and for alpha,beta-elimination reaction with S-methyl-l-cysteine. From the pH-dependency of k(cat)/K(m) for the reaction with l-methionine we have concluded that MGL is selective with respect to the zwitterionic form of its natural substrate. For the reaction of MGL with 1-amino-3-methylthiopropylphosphinic acid the pK(a) of the substrate's amino group, equal to 7.

Hydration characterization of hydrophobically modified polymers by dielectric measurements in the millimeter range.

Bound water molecules, the fraction of water molecules that are present in the hydration shells of polymers, lose their rotational mobility in the time scale of 10-50 ps, leading to additional ordering compared with unperturbed water. The amount of bound water per number of carbons in the nonpolar groups as measured by the method of waveguide dielectric resonance increased in the following order: hydrophobically modified chitosan < globular protein < hydrophobically modified polyacrylamide. The hydrophobic modification of acrylamide and chitosan polymers gave smaller variation in hydration than did both the coil-to-globule transition of poly(N-isopropylacrylamide) and the masking of nonpolar groups within the protein globule.

[Comparative evaluation of the motor-evacuation function of the stomach after functional operations for complicated duodenal ulcers].

Motor-evacuatory disorders (MED) of the stomach in 114 patients who have undergone functional operations for duodenal ulcers were analyzed. Clinical picture, barium contrast and ultrasonic examinations, fibrogastroduodenoscopy, pH-monitoring were used for evaluation. Many patients have MED of different degree in early postoperative period.

[Classification of enterprises according to degree of accident and occupational disease risk].

The authors suggested approach to classification of enterprises and economy branches according to occupational conditions. Suggestion is to classify by means of quantitative methods based on claster analysis.

Kinetics of beta-casein hydrolysis by wild-type and engineered trypsin.

Apparent rate constants of tryptic hydrolysis of amide bonds containing Arg and Lys residues in beta-casein were determined by the analysis of kinetics of accumulation of 17 major peptide components revealed by high performance liquid chromatography. When studying pH influence on Arg/Lys bond cleavage preference, averaged rate constants over several Arg&bond;X and Lys&bond;X bonds were used for analysis of kinetics of wild-type trypsin, K188H, K188F, K188Y, K188W, and of K188D/D189K mutants. The pK(a1) value of 6.