Mutations conferring streptolydigin resistance ontoEscherichia coli RNA polymerase have been found exclusively in the ␤ subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross, C. A. (1993) J. Biol. Chem. 268, 25369 -25375). We report here the isolation of a streptolydigin-resistant mutation in the E. coli rpoC gene, encoding the ␤ subunit. The mutation is the Phe 793 3 Ser substitution, which occurred in an evolutionarily conserved segment of the ␤ subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring ␣-amanitin resistance. Both streptolydigin and ␣-amanitin are inhibitors of transcription elongation. Thus, the two antibiotics may inhibit transcription in their respective systems by a similar mechanism, despite their very different chemical nature. Streptolydigin (Stl)1 is a 3-acyltetramic acid antibiotic (1), which specifically inhibits bacterial DNA-dependent RNA polymerase (2-6). Stl interacts with RNAP in ternary transcription complexes and inhibits growth of nascent RNA chains during transcription initiation and elongation (4, 6). The binding of RNAP to template DNA is not affected by Stl (2). Thus, the likely target of Stl is either the binding of incoming NTP in the substrate binding site of RNAP or the catalysis of phosphodiester bond formation (4, 6).Transcription by RNAP purified from mutant cells that acquired resistance to the drug is resistant to Stl (3). In Escherichia coli, all Stl-resistant RNAPs studied to date have an altered ␤ subunit (7,8), and all known mutations leading to Stl R RNAP map to the rpoB gene, which codes for the ␤ subunit (8). Substitutions of amino acids in ␤ between 540 and 546 lead to Stl resistance (9 -11). The highest resistance levels in vivo and in vitro were found in the case of substitutions at positions 544 and 545 (10 -11). In the absence of more direct data it has been assumed that these ␤ amino acids participate in Stl binding to RNA polymerase.Since Stl inhibits elongation of nascent RNA, mutations changing RNAP Stl-binding site are likely to change the catalytic properties of the enzyme. However, RNAP with the known ␤ subunit Stl R mutations has unaltered transcription elongation and transcription termination properties in vivo and in vitro (11). Moreover, the site of Stl resistance in the ␤ subunit is dispensable for RNAP function, since mutant RNAPs with deletions spanning amino acids 534 -545 are functional both in vivo and in vitro (10). This apparent discrepancy led Heisler et al. (11) to hypothesize that other site(s) in RNAP may be involved in Stl binding. This hypothesis was substantiated by a report (12) that in RNAP reconstituted in vitro, the ␤Ј subunit from a mutant strain of Bacillus subtilis was responsible for Stl resistance. However, no ␤Ј subunit Stl R mutants have ever been reported in E. coli. Here, we report an isolation and localization of such a mutation. The mutation leads to an amino acid substitution in an evolutionarily conserved region of the ␤Ј subunit. In the ␤Ј homolo...

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Despite numerous studies on the neurobiology of depression, the etiological and pathophysiological mechanisms of this disorder remain poorly understood. A large number of animal models and tests to evaluate depressive-like behavior have been developed. Chronic unpredictable mild stress (CUMS) is the most common and frequently used model of depression, and the sucrose preference test (SPT) is one of the most common tests for assessing anhedonia. However, not all laboratories can reproduce the main effects of CUMS, especially when this refers to a decrease in sucrose preference. It is also unknown how the state of anhedonia, assessed by the SPT, relates to the state of anhedonia in patients with depression. We analyzed the literature available in the PubMed database using keywords relevant to the topic of this narrative review. We hypothesize that the poor reproducibility of the CUMS model may be due to differences in sucrose consumption, which may be influenced by such factors as differences in sucrose preference concentration threshold, water and food deprivation, and differences in animals’ susceptibility to stress. We also believe that comparisons between animal and human states of anhedonia should be made with caution because there are many inconsistencies between the two, including in assessment methods. We also tried to offer some recommendations that should improve the reproducibility of the CUMS model and provide a framework for future research.

Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase ␤ subunit and is nonpermissive for plating of bacteriophage P2. P2 gor mutants productively infect 397c and define a new gene, lysC, encoded by a reading frame that extensively overlaps the P2 lysis accessory gene, lysB. The unusual location of lysC with respect to lysB is reminiscent of the Rz/Rz1 lysis gene pair of phage . Indeed, coexpression of lysB and lysC complemented the growth defect of Rz/Rz1 null mutants, indicating that the LysB/C pair is similar to Rz/Rz1 in both gene arrangement and function. Cells carrying the rpoC397 mutation exhibited an early onset of P2-induced lysis, which was suppressed by the gor mutation in lysC. We propose that changes in host gene expression resulting from the rpoC397 mutation result in changes in the composition of the bacterial cell wall, making the cell more susceptible to P2-mediated lysis and preventing accumulation of progeny phage sufficient for plaque formation.During bacteriophage infection, the machinery for macromolecular synthesis in the cell is recruited to serve the needs of the virus, and systematic changes in viral gene expression take place in a defined sequence. Bacterial DNA-dependent RNA polymerase (RNAP; subunit composition, a 2 ␤␤Ј), the enzyme responsible for most host transcription, is a major target of this regulation (14). Thus, mutations in RNAP rpo genes often specifically prevent bacteriophage development. To date, mutations that interfere with bacteriophage development have been identified in genes coding for all RNAP subunits except the smallest subunit, (reviewed in reference 34). Some of these mutations define RNAP sites that interact directly with viral regulators (25,26,40), while others affect phage gene expression indirectly by altering the properties of RNAP, such as the efficiency of transcription termination (39). The power of phage genetics, allowing isolation of suppressor mutations that overcome the blocks conferred by the changes in RNAP, has been invaluable in the study of these phenomena, and the results have greatly enriched our understanding of the basic processes of transcription (27).From this perspective, the block against plaque formation for phages P2 (10) and N4 (25) conferred by the rpoC397 mutation has long been provocative. This mutation removes 16 bp close to the end of rpoC, resulting in the replacement of the last 50 amino acids of RNAP subunit ␤Ј with 23 incorrect residues (10). In addition to being nonpermissive for the two phages, Escherichia coli strain 397c, carrying rpoC397, has a gross temperature-sensitive (Ts) growth phenotype. Based on biochemical experiments, the N4 block appears to be due to the loss of a contact between N4 SSB (single-strand DNA binding protein) and ␤Ј that is required for activation of viral late transcription (25). However, the mechanism by which the rpoC397 mutation prevents P2 growth has not been determined. Here, we report genetic and physio...

Pigmentation is the result of melanin synthesis, which takes place in melanocytes, and its further distribution. A dysregulation in melanocytes' functionality can result in the loss of pigmentation, the appearance of pigment spots and melanoma development. Tissue engineering and the screening of new skin-lightening drugs require the development of simple and reproducible in vitro models with maintained functional activity. The aim of the study was to obtain and characterize spheroids from normal human melanocytes as a three-dimensional multicellular structure and as a test system for skin-lightening drug screening. Melanocytes are known to lose their ability to synthesize melanin in monolayer culture. When transferred under non-adhesive conditions in agarose multi-well plates, melanocytes aggregated and formed spheroids. As a result, the amount of melanin elevated almost two times within seven days. MelanoDerm TM (MatTek) skin equivalents were used as a comparison system. Cells in spheroids expressed transcription factors that regulate melanogenesis: MITF and Sox10, the marker of developed melanosomes-gp100, as well as tyrosinase (TYR)-the melanogenesis enzyme and melanocortin receptor 1 (MC1R)-the main receptor regulating melanin synthesis. Expression was maintained during 3D culturing. Thus, it can be stated that spheroids maintain melanocytes' functional activity compared to that in the multi-layered MelanoDerm TM skin equivalents. Culturing both spheroids and MelanoDerm TM for seven days in the presence of the skin-lightening agent fucoxanthin resulted in a more significant lowering of melanin levels in spheroids. Significant down-regulation of gp100, MITF, and Sox10 transcription factors, as well as 10-fold down-regulation of TYR expression, was observed in spheroids by day 7 in the presence of fucoxanthin, thus inhibiting the maturation of melanosomes and the synthesis of melanin. MelanoDerm TM samples were characterized by significant down-regulation of only MITF, Sox10 indicating that spheroids formed a more sensitive system allowed for quantitative assays. Collectively, these data illustrate that normal melanocytes can assemble themselves into Zurina et al.Melanocyte-Derived Spheroids as a Test System spheroids-the viable structures that are able to accumulate melanin and maintain the initial functional activity of melanocytes. These spheroids can be used as a more affordable and easy-to-use test system than commercial skin equivalents for drug screening.

BackgroundFluvoxamine therapy is used for treatment of patients with depressive disorder, but it is often ineffective, and some patients suffer from dose-dependent undesirable side effects such as vertigo, headache, indigestion, xerostomia, increased anxiety, etc. CYP2D6 is involved in the biotransformation of fluvoxamine. Meanwhile, the genes encoding these isoenzymes have a high level of polymorphism, which may affect the protein synthesis.ObjectiveThe primary objective of our study was to investigate the effects of CYP2D6 genetic polymorphisms on the efficacy and safety of fluvoxamine in patients with depressive disorder and comorbid alcohol use disorder, in order to develop the algorithms of optimization of fluvoxamine therapy for reducing the risk of dose-dependent undesirable side effects and pharmacoresistance.MethodsThe study involved 45 male patients (average age: 36.44±9.96 years) with depressive disorder and comorbid alcohol use disorder. A series of psychometric scales was used in the research. Genotyping of CYP2D6 (1846G>A) was performed using real-time polymerase chain reaction.ResultsAccording to results of Mann–Whitney U-test, statistically significant differences between the efficacy and safety of fluvoxamine were obtained on 9th and 16th days of therapy in patients with GG and GA genotypes (The Hamilton Rating Scale for Depression: 10.0 [10.0; 23.0] vs 25.0 [24.0; 16.0] (P<0.001) on the 9th day and 4.0 [2.0; 5.0] vs 6.0 [6.0; 7.0] on the 16th day; The UKU Side Effect Rating Scale: 6.0 [4.0; 6.0] vs 9.0 [9.0; 10.0] (P<0.001) on the 9th day and 5.0 [1.0; 9.0] vs 19.0 [18.0; 22.0] on the 16th day).ConclusionThis study demonstrated the lower efficacy and safety of fluvoxamine in patients with depressive disorder and comorbid alcohol use disorders with GA genotype in CYP2D6 1846G>A polymorphic marker.

The study demonstrates that CYP2D6 genetic polymorphism (1846G>A) can affect haloperidol concentration levels in patients with alcohol use disorder.

Major depressive disorder (MDD) is one of the most common mood disorders worldwide. A lack of understanding of the exact neurobiological mechanisms of depression complicates the search for new effective drugs. Animal models are an important tool in the search for new approaches to the treatment of this disorder. All animal models of depression have certain advantages and disadvantages. We often hear that the main drawback of the chronic unpredictable mild stress (CUMS) model of depression is its poor reproducibility, but rarely does anyone try to find the real causes and sources of such poor reproducibility. Analyzing the articles available in the PubMed database, we tried to identify the factors that may be the sources of the poor reproducibility of CUMS. Among such factors, there may be chronic sleep deprivation, painful stressors, social stress, the difference in sex and age of animals, different stress susceptibility of different animal strains, handling quality, habituation to stressful factors, various combinations of physical and psychological stressors in the CUMS protocol, the influence of olfactory and auditory stimuli on animals, as well as the possible influence of various other factors that are rarely taken into account by researchers. We assume that careful inspection of these factors will increase the reproducibility of the CUMS model between laboratories and allow to make the interpretation of the obtained results and their comparison between laboratories to be more adequate.