Comparative study of the transformation of various thymidine kinase-deficient human and animal cell lines with the thymidine kinase gene of the Herpes simplex virus

The comparative study of transformation of four thymidine kinase deficient cell lines (mouse mammary carcinoma cell line FS tk-; rat cell line Rat-2tk-; mouse cell line Ltk-, clone D1; human cell line 143tk-) with the thymidine kinase cloned gene of Herpes simplex virus 1 was undertaken. The differences in efficiency and optimal conditions of transformation were shown for these cell lines. The advantages and disadvantages of the cell systems examined for the use in experiments for transformation and cotransformation of cultured cells with isolated genes are discussed.     

Binding of K 1-3 and K 4 fragments of plasminogen containing lysine-binding sites with fibrinogen and its fragments

Interaction of plasminogen K 1-3 and K 4 fragments containing lysine binding sites with fibrinogen and its fragments has been investigated. It has been established that K 1-3 fragment binds to fibrinogen and its E and DL fragments. K 4 fragment does not bind to E and DL fragments, but it interacts with fibrinogen. K 4 fragment does not interact with early fibrinogen proteolysis X2 fragment which differs from the native molecule of fibrinogen in the alpha C domain absence. The results obtained indicate that lysine binding sites located at plasminogen K 1-3 and K 4 fragments correspond to different fibrinogen molecule centres. The centre complementary to K 4 fragment lysine binding sites could be located at the fibrinogen alpha C domain.     

Streptokinase immobilization in insoluble carriers, and properties of the immobilized protein

Immobilization of streptokinase was performed by bromine cyan-activated cellulose and by aminoethyl cellulose using glutaric aldehyde and N-cyclohexyl-N'-[2-(4-morpholinyl)-ethyl-carbodiimide. The specific activator activity of the immobilized streptokinase is 70-100% of that of free streptokinase. In multiple application of the immobilized protein preparations streptokinase obtained by bromine cyan-activated cellulose is the most stable: it retains more than 40% of initial activity after 10 repeated applications. The immobilized streptokinase is shown to be more thermostable as compared to the soluble one.     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Peripheral pool of CD8+ T-cells contains lymphocytes with antigen-specific receptors that recognize syngeneic MHC class II molecules

The capacity of T-lymphocytes to recognize "nonself" and tolerating "self" is formed as a result of positive and negative selection in the thymus. While obtaining and testing specificity of T-hybridomas, we demonstrated that the major part of peripheral pool of CD8+ T-lymphocytes carried receptors specific to "self" MHC class II molecules. Such an unexpected specificity of receptors has been found in some T-cell hybridomas produced by fusion of activated peripheral CD8+ T-lymphocytes with a tumor partner transfected by the coreceptor CD4 gene. The reactivity to "self" is not an experimental artifact due to an increased avidity of interaction of the hybridoma cells with antigen-presenting cells. Also, it is not an expression of reactivity of T-cells to superantigens, products of endogenous viruses of mouse breast cancer. The formation of a pool of such T-cells involves both cells with double receptor specificity and cells coexpressing two alpha-chains of T-cell receptor. Their appearance in the periphery can be due to the capacity of thymocytes differentiating in the direction of CD4+ cells to avoid negative selection via change of expression of coreceptor CD4 to CD8.     

Hybrids of human and monkey adenoviruses (adeno-adeno hybrids) that can reproduce in monkey cells: biological and molecular genetic peculiarities

A highly oncogenic monkey adenovirus SA7(C8) facilitates the reproduction of human adenovirus type 2 (Ad2) in monkey cells. Upon mixed infection of monkey cells with both viruses, these viruses recombine producing defective adeno-adeno hybrids Ad2C8 serologically identical to Ad2 and capable of assisting Ad2 to reproduce in monkey cells. Ad2C8 and Ad2 form an intercomplementary pair inseparable in monkey cells. Unlike oncogenic SA7(C8), Ad2C8 is a nononcogenic virus for hamsters but is able to induce tumor antigens of this virus (T and TSTA). Molecular genetic analysis of 68 clones of adeno-adeno hybrids revealed that the left part of their genome consists of Ad2 DNA, and the right part contains no less than 40% of the viral SA7(C8) genome where E2A, E3, and E4 genes are located. Apparently, the products of these genes contribute to the composition of adenoviral tumor antigens, while the E4 gene is involved in complementation of monkey and human adenoviruses and makes a contribution to host range determination of these viruses.     

Role of plasminogen/plasmin in functional activity of blood cells

The article deals with the data concerning structural peculiarities of plasminogen/plasmin molecule, which define the specificity of intermolecular interactions and provide the variety of its biological functions. The main principles of the modern classification of plasminogen receptors and factors, which modulate their expression, have been presented. We have considered the mechanisms regulating both plasmin formation and activity on the surface of cells, fibrin and proteins of extracellular matrix. The data of previous investigators and our own results, concerning the influence of plasminogen/plasmin on platelet aggregation induced by different agonists, have been summarized. The participation of plasminogen/plasmin in atherogenesis and angiogenesis mediated by endotheliocyte receptors has been discussed. Special attention was given to plasminogen/plasmin proinflammatory function, which is realized by regulatory processes of activation, secretion, migration and apoptosis of monocytes and macrophages.     

FE65 as a link between VLDLR and APP to regulate their trafficking and processing

Background

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinson??s disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains.

Results

In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1?/? cells recapitulate the respiratory defect in isolated mitochondria from PINK1?/? mouse brains, suggesting that these PINK1?/? cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1?/? cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1?/? cells, and this genotypic difference between PINK1?/? and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1?/? cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1?/? and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1?/? compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1.

Conclusions

Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1?/? cells.