Protein kinase B phosphorylates AHNAK and regulates its subcellular localization

AHNAK is a ubiquitously expressed giant phosphoprotein that was initially identified as a gene product subject to transcriptional repression in neuroblastoma. AHNAK is predominantly nuclear in cells of nonepithelial origin, but is cytoplasmic or associated with plasma membrane in epithelial cells. In this study we show that the extranuclear localization of AHNAK in epithelial cells depends on the formation of cell–cell contacts. We show that AHNAK is a phosphorylation substrate of protein kinase B (PKB) in vitro and in vivo. Nuclear exclusion of AHNAK is mediated through a nuclear export signal (NES) in a manner that depends on the phosphorylation of serine 5535 of AHNAK by PKB, a process that also plays a major role in determining extranuclear localization of AHNAK. AHNAK is a new PKB substrate whose function, though unknown, is likely to be regulated by its localization, which is in turn regulated by PKB

Application of Spectroscopy Methods for Indication and Identification of Pathogenic Biological Agents

The review presents data on application of UV, IR and optical spectroscopy methods for non-specific indication of pathogenic biological agents, and IR Fourier spectroscopy, and Raman spectroscopy - for their identification. Considered are advantages, disadvantages and prospects of different spectroscopy methods application for monitoring of the environment for the presence of pathogenic biological agents

Solid Tumor-Targeted Infiltrating Cytotoxic T Lymphocytes Retained by a Superantigen Fusion Protein

Successful immune-mediated regression of solid tumors is difficult because of the small number of cytotoxic T lymphocytes (CTLs) that were traffic to the tumor site. Here, the targeting of tumor-specific infiltrating CTLs was dependent on a fusion protein consisting of human epidermal growth factor (EGF) and staphylococcal enterotoxin A (SEA) with the D227A mutation. EGF-SEA strongly restrained the growth of murine solid sarcoma 180 (S180) tumors (control versus EGF-SEA, mean tumor weight: 1.013 versus 0.197 g, difference  = 0.816 g). In mice treated with EGF-SEA, CD4+, CD8+ and SEA-reactive T lymphocytes were enriched around the EGFR expressing tumor cells. The EGF receptors were potentially phosphorylated by EGF-SEA stimulation and the fusion protein promoted T cells to release the tumoricidal cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Intratumoral CTLs secreted cytolytic pore-forming perforins and granzyme B proteins near the surface of carcinomas, causing the death of many tumor cells. We additionally show that labeled EGF-SEA was directly targeted to the tumor tissue after intravenous (i.v.) injection. The findings demonstrate that antibody-like EGF-SEA plays an important role in arresting CTLs in the solid tumor site and has therapeutic potential as a tumor-targeting agent

Differential modulation of the TRAIL receptors and the CD95 receptor in colon carcinoma cell lines

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L) are potent inducers of apoptosis in various tumour cell types. Death receptors DR4 and DR5 can induce and decoy receptors DcR1 and DcR2 can inhibit TRAIL-mediated apoptosis. The study aim was to investigate whether anticancer agents can modulate similarly TRAIL-receptor and CD95 membrane expression and TRAIL and CD95L sensitivity.Three colon carcinoma cell lines (Caco-2, Colo320 and SW948) were treated with 5-fluorouracil (5-FU), cisplatin or interferon-γ. TRAIL-receptor and CD95 membrane expression was determined flow cytometrically. Sensitivity to TRAIL or CD95L agonistic anti-CD95 antibody was determined with cytotoxicity and apoptosis assays. SW948 showed highest TRAIL sensitivity. The protein synthesis inhibitor cycloheximide decreased FLICE-like inhibitory protein levels in all cell lines, and the TRAIL-resistant cell lines Caco-2 and Colo320 became sensitive for TRAIL. Exposure of the cell lines to 5-FU, cisplatin and interferon-γ left TRAIL-receptor membrane expression and TRAIL sensitivity unaffected. CD95 membrane expression and anti-CD95 sensitivity was, however, modulated by the same drugs in all lines. Cisplatin and interferon-γ raised CD95 membrane levels 6–8-fold, interferon-γ also increased anti-CD95 sensitivity. These results indicate that the CD95 and TRAIL pathways use different mechanisms to respond to various anticancer agents. Induced CD95 membrane upregulation was associated with increased anti-CD95 sensitivity, whereas no upregulation of TRAIL-receptor membrane expression or TRAIL sensitisation could be established. For optimal use of TRAIL-mediated apoptosis for cancer therapy in certain tumours, downregulation of intracellular inhibiting factors may be required

Rheumatoid Arthritis: Applicability of Ready-to-Use Human Cartilaginous Cells for Screening of Compounds with TNF-Alpha Inhibitory Activity

In the context of modern drug discovery, there is an obvious advantage to designing phenotypic bioassays based on human disease-relevant cells that express disease-relevant markers. The specific aim of the study was to develop a convenient and reliable method for screening compounds with Tumor Necrosis Factor-alpha (TNF-α) inhibitory activity. This assay was developed using cryopreserved ready-to-use cartilage-derived cells isolated from juvenile donors diagnosed with polydactyly. It has been demonstrated that all donor (10 donors) cells were able to respond to TNF-α treatment by increased secretion of pro-inflammatory cytokine IL-6 into subcultural medium. Inhibition of TNF-α using commercially available TNF-α inhibitor etanercept resulted in a dose-dependent decrease in IL-6 production which was measured by Enzyme-Linked Immunosorbent Assay (ELISA). TNF-α dependent IL-6 production was detected in the cells after both their prolonged cultivation in vitro (≥20 passages) and cryopreservation. This phenotypic bioassay based on ready-to-use primary human cells was developed for detection of novel TNF-α inhibitory compounds and profiling of biosimilar drugs

Morphometric Assessment of the Influence of <i>Vibrio cholerae</i> Strains with Different Set of Virulence Determinants on Experimental Animal Organism

Comparison of clinical and morphological analysis data and the results of morphometric assessment of changes in suckling rabbits infected intra-intestinally with cholera vibrios with different set of virulence determinants proved the presence of ctxA gene in Vibrio cholerae genome to be the necessary condition for the development of choleragen reaction. Experimental infection in this case is followed by the excess of the considered morphometric parameters in kidneys and liver in comparison with those of the intact animals. Changes in suckling rabbits infected with ctxA- strains are mainly of the adaptive and compensatory character and are determined by ZOT and ACE toxins of Vibrio cholerae to some extent. The reaction of the suckling rabbits` intestinal apudocytes depends on the genotype of V. cholerae strains used for challenge and correlates with the intensity of morphological changes in the intestine and internal organs of experimental animals

Biosensors: Current State and Prospects of Applying in Laboratory Diagnostics of Particularly Dangerous Infectious Diseases

The review presents the functional characteristics of the main types of biosensors: electrochemical, piezoelectric and optical. Shown are the examples of biosensors application for pathogenic biological agents detection. The prospects of biosensors development for laboratory diagnostics of particularly dangerous infectious diseases are discussed

3D Bioprinting of Hyaline Articular Cartilage: Biopolymers, Hydrogels, and Bioinks

The musculoskeletal system, consisting of bones and cartilage of various types, muscles, ligaments, and tendons, is the basis of the human body. However, many pathological conditions caused by aging, lifestyle, disease, or trauma can damage its elements and lead to severe disfunction and significant worsening in the quality of life. Due to its structure and function, articular (hyaline) cartilage is the most susceptible to damage. Articular cartilage is a non-vascular tissue with constrained self-regeneration capabilities. Additionally, treatment methods, which have proven efficacy in stopping its degradation and promoting regeneration, still do not exist. Conservative treatment and physical therapy only relieve the symptoms associated with cartilage destruction, and traditional surgical interventions to repair defects or endoprosthetics are not without serious drawbacks. Thus, articular cartilage damage remains an urgent and actual problem requiring the development of new treatment approaches. The emergence of biofabrication technologies, including three-dimensional (3D) bioprinting, at the end of the 20th century, allowed reconstructive interventions to get a second wind. Three-dimensional bioprinting creates volume constraints that mimic the structure and function of natural tissue due to the combinations of biomaterials, living cells, and signal molecules to create. In our case—hyaline cartilage. Several approaches to articular cartilage biofabrication have been developed to date, including the promising technology of 3D bioprinting. This review represents the main achievements of such research direction and describes the technological processes and the necessary biomaterials, cell cultures, and signal molecules. Special attention is given to the basic materials for 3D bioprinting—hydrogels and bioinks, as well as the biopolymers underlying the indicated products