Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.

Evidence for involvement of Saccharomyces cerevisiae protein kinase C in glucose induction of HXT genes and derepression of SUC2

The PKC1 gene in the yeast Saccharomyces cerevisiae encodes protein kinase C that is known to control a mitogen-activated protein (MAP) kinase cascade consisting of Bck1, Mkk1 and Mkk2, and Mpk1. This cascade affects the cell wall integrity but the phenotype of Pkc1 mutants suggests additional targets which have not yet been identified. We show that a pkc1Δ mutant, as opposed to mutants in the MAP kinase cascade, displays two major defects in the control of carbon metabolism. It shows a delay in the initiation of fermentation upon addition of glucose and a defect in derepression of SUC2 gene after exhaustion of glucose from the medium. After addition of glucose the production of both ethanol and glycerol started very slowly. The V max of glucose transport dropped considerably and Northern blot analysis showed that induction of the HXT1, HXT2 and HXT4 genes was strongly reduced. Growth of the pkc1Δ mutant was absent on glycerol and poor on galactose and raffinose. Oxygen uptake was barely present. Derepression of invertase activity and SUC2 transcription upon transfer of cells from glucose to raffinose was deficient in the pkc1Δ mutant as opposed to the wild-type. Our results suggest an involvement of Pkc1p in the control of carbon metabolism which is not shared by the downstream MAP kinase cascade. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Structural basis for inhibition of cyclin-dependent kinase 9 by flavopiridol

Flavopiridol has been shown to potently inhibit CDK1 and 2 (cyclin-dependent kinases 1 and 2) and most recently it has been found that it also inhibits CDK9. The complex CDK9-cyclin T1 controls the elongation phase of transcription by RNA polymerase II. The present work describes a molecular model for the binary complex CDK9-flavopiridol. This structural model indicates that the inhibitor strongly binds to the ATP-binding pocket of CDK9 and the structural comparison of the complex CDK2-flavopiridol correlates the structural differences with differences in inhibition of these CDKs by flavopiridol. This structure opens the possibility of testing new inhibitor families, in addition to new substituents for the already known leading structures such as flavones and adenine derivatives. © 2002 Elsevier Science (USA). All rights reserved.

Molecular model of shikimate kinase from Mycobacterium tuberculosis

Tuberculosis (TB) resurged in the late 1980s and now kills approximately 3 million people a year. The reemergence of tuberculosis as a public health threat has created a need to develop new anti-mycobacterial agents. The shikimate pathway is an attractive target for herbicides and anti-microbial agents development because it is essential in algae, higher plants, bacteria, and fungi, but absent from mammals. Homologs to enzymes in the shikimate pathway have been identified in the genome sequence of Mycobacterium tuberculosis. Among them, the shikimate kinase I encoding gene (aroK) was proposed to be present by sequence homology. Accordingly, to pave the way for structural and functional efforts towards anti-mycobacterial agents development, here we describe the molecular modeling of M. tuberculosis shikimate kinase that should provide a structural framework on which the design of specific inhibitors may be based. © 2002 Elsevier Science (USA). All rights reserved.

Putative pyrophosphate phosphofructose 1-kinase genes identified in sugarcane may be getting energy from pyrophosphate

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) has been detected in several types of plant cells, but the gene has not been reported in sugar cane. Using Citrux paradixi PPi-PFK gene (AF095520 and AF095521) sequences to search the sugar cane EST database, we have identified both the α and β subunits of this enzyme. The deduced amino acid sequences showed 76 and 80% similarity with the corresponding α and β subunits of C. paradisi. A high degree of similarity was also observed among the PFK β subunits when the alignment of the sugar cane sequences was compared to those of Ricinus communis and Solanum tuberosum, it appears that α and β are two distinct subunits; they were found at different concentrations in several sugar cane tissues. It remains to be determined if the different gene expression levels have some physiological importance and how they affect sucrose synthesis, export, and storage in vacuoles. A comparison between the amino acid sequences of β PFKs from a variety of organisms allowed us to identify the two critical Asp residues typical of this enzyme's activity site and the other binding sites; these residues are tightly conserved in all members of this protein family. Apparently, there are catalytic residues on the β subunit of the pyrophosphate-dependent enzyme.

The Rv1712 locus from Mycobacterium tuberculosis H37Rv codes for a functional CMP kinase that preferentially Phosphorylates dCMP

The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of creatine kinase (CK) and aspartate aminotransferase (AST) activities after laparoscopic or conventional ovariectomy in queens

Creatine kinase (CK) and aspartate aminotransferase (AST) are mainly muscle-specific enzymes, which can be associated with muscle tissue damage. The aim of this study was to assess the activities of CK and AST during the postoperative period, after conventional (G1) and videolaparoscopic ovariectomy (G2), in queens. A further group (G3) was subjected to anaesthesia only. Results demonstrate that there were significant differences between groups. The highest levels of CK were recorded in Gl, however at a confidence level of p < 0.05 there was no significant difference between groups during the first 6 hours after surgery. A significant (p < 0.05) increase of CK values was identified between 0h and 3h in both groups (Gl and G2). Regarding AST activity there was no significant variation between groups, but again there was a significant difference between values at 0h and 3h after surgery. In conclusion, ovariectomy performed by videolap-aroscopy seems to cause less muscle damage when compared to the conventional method. © 2009 by Verlag Hans Huber, Hogrefe AG, Bem.

Efeito da suplementação com selênio sobre a concentração sérica de creatina kinase em bovinos

The study, evaluated the addition of different concentrations of Se in mineral mixture affecting creatine kinase (CK) serum concentrations in cattle. 60 male, Nellore cattle, at about 12 months old, were randomly assigned to groups (15 calves/ group), Gc, G3,6, G5,4 or G6,4 (0, 3.6, 5.4, and 6.4 mg Se/bovine/day). The levels of serum CK in the cattle were not affected by neither the interaction selenium concentration x time nor the concentration of supplementation. However, CK levels increased over the experiment irrespective of dietary selenium concentration. In addition, the frequency of animals with CK levels above normal increased (p<0.10) in group G6,4. The concentrations of selenium studied here do not affect serum CK in cattle, but the daily concentration of 6.4 mg selenium is not recommended because it is possibly toxic effect.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates. © 2013 Cruz et al.

Photoprotective and antioxidant effects of Rhubarb: Inhibitory action on tyrosinase and tyrosine kinase activities and TNF-α, IL-1α and α-MSH production in human melanocytes

Background: Exposure to ultraviolet (UV) radiation causes various forms of acute and chronic skin damage, including immunosuppression, inflammation, premature aging and photodamage. Furthermore, it induces the generation of reactive oxygen species, produces proinflammatory cytokines and melanocyte-stimulating hormone (MSH) and increases tyrosinase activity. The aim of this study was to evaluate the potential photoprotective effects of Rheum rhaponticum L. rhizome extract on human UV-stimulated melanocytes.Methods: The effects of Rheum rhaponticum rhizome extract on tyrosine kinase activity, and on interleukin-1α (IL-1α), tumour necrosis factor α (TNF-α), and α-MSH production in human epidermal melanocytes were evaluated under UV-stimulated and non-stimulated conditions. Antioxidant activity was evaluated by lipid peroxidation and 1,1-dyphenyl-2-picryl-hydrazyl (DPPH) assays, while anti-tyrosinase activity was evaluated by the mushroom tyrosinase method.Results: Rheum rhaponticum L. rhizome extract showed in vitro antioxidant properties against lipid peroxidation, free radical scavenging and anti-tyrosinase activities, and inhibited the production of IL-1α, TNF-α, α-MSH, and tyrosine kinase activity in melanocytes subjected to UV radiation.Conclusions: These results support the inclusion of Rheum rhaponticum L. rhizome extract into cosmetic, sunscreen and skin care products for the prevention or reduction of photodamage. © 2013 Silveira et al; licensee BioMed Central Ltd.

Effects of (-)-cubebin (Piper cubeba) on cytotoxicity, mutagenicity and expression of p38 MAP kinase and GSTa2 in a hepatoma cell line

(-)-Cubebin is a lignan extracted from the seeds of the pepper Piper cubeba, a commonly eaten spice with beneficial properties, including trypanocidal, anti-inflammatory, analgesic, anti-proliferative and leishmanicidal activities. Because of its therapeutic potential, we investigated the effects of (-)-cubebin on the cytotoxicity, cell proliferation kinetics, mutagenicity and expression of p38 MAP kinase and glutathione S-transferase a2 (GSTa2) using real-time RT-PCR in Rattus norvegicus hepatoma cells. We found that 280 μM (-)-cubebin was cytotoxic after 24, 48 and 72. h of exposure, but not mutagenic at 0.28 μM, 2.8 μM and 28 μM after 26. h. Similarly, exposure to 0.28 μM, 2.8 μM and 28 μM (-)-cubebin for 24, 48, 72 and 96. h did not alter the cell proliferation kinetics. Cells exposed to 28 μM (-)-cubebin for 24. h did not exhibit changes in p38 MAP kinase and GSTa2 expression, indicating that cellular changes were not induced by extracellular stimuli and that (-)-cubebin is likely not metabolized via this pathway. Our results suggest that high levels of (-)-cubebin should be consumed with caution due to the cytotoxic effect observed at the highest concentration. However, at lower concentrations, no cytotoxic, mutagenic or proliferative effects were observed, providing further evidence of the safety of consuming (-)-cubebin. © 2013 Elsevier Inc.

The Mode of Action of Recombinant Mycobacterium tuberculosis Shikimate Kinase: Kinetics and Thermodynamics Analyses

Tuberculosis remains as one of the main cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. The aroK-encoded M. tuberculosis Shikimate Kinase (MtSK), shown to be essential for survival of bacilli, catalyzes the phosphoryl transfer from ATP to the carbon-3 hydroxyl group of shikimate (SKH), yielding shikimate-3-phosphate and ADP. Here we present purification to homogeneity, and oligomeric state determination of recombinant MtSK. Biochemical and biophysical data suggest that the chemical reaction catalyzed by monomeric MtSK follows a rapid-equilibrium random order of substrate binding, and ordered product release. Isothermal titration calorimetry (ITC) for binding of ligands to MtSK provided thermodynamic signatures of non-covalent interactions to each process. A comparison of steady-state kinetics parameters and equilibrium dissociation constant value determined by ITC showed that ATP binding does not increase the affinity of MtSK for SKH. We suggest that MtSK would more appropriately be described as an aroL-encoded type II shikimate kinase. Our manuscript also gives thermodynamic description of SKH binding to MtSK and data for the number of protons exchanged during this bimolecular interaction. The negative value for the change in constant pressure heat capacity (ΔCp) and molecular homology model building suggest a pronounced contribution of desolvation of non-polar groups upon binary complex formation. Thermodynamic parameters were deconvoluted into hydrophobic and vibrational contributions upon MtSK:SKH binary complex formation. Data for the number of protons exchanged during this bimolecular interaction are interpreted in light of a structural model to try to propose the likely amino acid side chains that are the proton donors to bulk solvent following MtSK:SKH complex formation. © 2013 Rosado et al.

In vitro targeting of Polo-like kinase 1 in bladder carcinoma: Comparative effects of four potent inhibitors

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and preclinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs. © 2013 Landes Bioscience.

Serum activity of creatine kinase and aminotransferase aspartate enzymes of horses submitted to muscle biopsy and incremental jump test

The objective was to evaluate serum activity of the enzymes creatine kinase (CK) and aspartate aminotransferase (AST), which are leakage enzymes responsive to muscle injury, of athletic horses that underwent muscle biopsy and incremental jump test (IJT) involving incremental jumps. The animals were grouped as follows: the first group, horses with history of superior performance (SP); the second, with a history of inferior performance (IP); and lastly, a control group (CG). All groups underwent biopsy of the gluteus medius muscle, while groups SP and IP were also submitted to the incremental jump test (IJT) 24 hours after biopsy. The IJT consisted of three stages with 40 jumps each, where jump height increased progressively, from 40 to 60 and last, 80cm. Blood samples were drawn before biopsy, and 6 and 24 hours after the exercise as well. The levels of CK serum activity increased 6 hours after exercise and decreased 24 hours later in all groups, including CG. AST activity did not increase after biopsy and exercise. There was no increase of both enzyme activities that could be attributed to the exercise, possibly due to exercise short duration and/or low intensity. We conclude that the muscle biopsy was able to show that there was enough stimulus to cause CK enzyme leakage into the plasma, and consequent detection of increased serum activity, while the incremental jump test did not.