[Testing of the Russian-language version of the Thought, Language and Communication Scale]. / Aprobatsiya russkoyazychnogo varianta shkaly Thought, Language, Communication Scale.

OBJECTIVE: To analyze the possibilities of using the Russian version of the Thought, Language and Communication Scale (TLC) for early recognition of thought disorders in patients at clinical high-risk for schizophrenia. MATERIAL AND METHODS: For the main group, the study included 30 adolescent male patients (19.2±2.2 years) hospitalized with the first depressive episode (ICD-10: F32.1, F32.2, F32.28, F32.8), who demonstrated attenuated schizophrenic symptoms (ASS) in the structure of the depression, which made it possible to attribute the patients to the group of clinical high-risk for schizophrenia. The control group consisted of 27 mentally healthy adolescent males (20.0±2.3 years). In both groups, the severity of thought impairment was assessed using the TLC scale. Psychopathological, psychometric and statistical methods were used. RESULTS: The median values of the severity of thought impairment using the TLC scale were 20 points [19.75; 26] in the main group, 10.5 points [9.25; 13] in the control group, with a high degree of statistical significance (p<0.001). The most significant differences (p<0.001) were found in following parameters: Incoherence (2 [1; 3] vs 1 [0; 1]), Tangentiality (2 [2; 2] vs 1 [0; 2]), Derailment (2 [1.25; 2] vs 1 [0.5; 2]), Illogical thinking (2 [2; 2.75] vs 0 [0; 1]), Loss of goal (1 [0; 2] vs 0 [0; 0]) and Blocking (1 [0; 1] vs 0 [0; 0] accordingly). CONCLUSION: Specific, not related to depression, disorders of thinking in patients of the clinical group, which indicates signs of disorganization of thinking and suggests the beginning of the endogenous process of the schizophrenic pole were found. The results show that the TLC scale can be used to detect early cognitive disorders in patients at risk of schizophrenia.

Production of Recombinant Rhomboid Proteases.

Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.

Activity Assays for Rhomboid Proteases.

Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and Vmax to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein.

Localization of non-native D-glyceraldehyde-3-phosphate dehydrogenase in growing and apoptotic HeLa cells.

Monoclonal antibodies that could not bind native tetramers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) but could bind to dimeric, monomeric, or denatured forms of GAPDH were used to investigate its intracellular localization. These antibodies distinctly stained the nucleus in growing HeLa cells. In the cytoplasm, non-native GAPDH was colocalized with actin filaments. Incubation of HeLa cells with tumor necrosis factor α (TNF-α) and the protein synthesis inhibitor emetine led to a drastic increase in the amount of the non-native GAPDH in the nuclei. Overproduction of Bcl-2 protein did not change the non-native GAPDH localization in the growing HeLa cells but prevented the development of apoptosis and the increase in the amount of non-native GAPDH in the nuclei upon incubation with TNF-α.