Transcriptional activation of MMP-13 by periodontal pathogenic LPS requires p38 MAP kinase

Matrix metal loprotease-13 (MMP-13) is induced by pro-inflammatory cytokines and increased expression is associated with a number of pathological conditions such as tumor metastasis, osteoarthritis, rheumatoid arthritis and periodontal diseases. MMP-13 gene regulation and the signal transduction pathways activated in response to bacterial LPS are largely unknown. In these studies, the role of the mitogen-activated protein kinase (MAPK) pathways in the regulation of MMP-13 induced by lipopolysaccharide was investigated. Lipopolysaccharide from Escherichia coli and Actinobacillus actinomycetemcomitans significantly (P < 0.05) increased MMP-13 steady-state mRNA (average of 27% and 46% increase, respectively) in murine periodontal ligament fibroblasts. MMP-13 mRNA induction was significantly reduced by inhibition of p38 MAP kinase. Immunoblot analysis indicated that p38 signaling was required for LPS-induced MMP-13 expression. Lipopolysaccharide induced proximal promoter reporter (-660/+32 mMMP-13) gene activity required p38 signaling. Collectively, these results indicate that lipopolysaccharide-induced murine MMP-13 is regulated by p38 signaling through a transcriptional mechanism.

Packaging to Preserve 'Aurora-1' Peaches from Two Maturity Stages

The main objective of this research was to evaluate chemical and physical changes in 'Aurora-1' peach harvested at two maturity stages, packed in different types of packaging and kept under refrigeration. Fruit were harvested at the mature green and ripe stages, packed in four different types of packaging (control, PD-900 (TM), PVC and PET) and stored at 6 degrees C. The following variables were evaluated every eight days: coloration, accumulated fresh mass loss, firmness, appearance, acidity, total soluble solids contents, soluble sugars, and percentage of pectin solubilization. We observed that the postharvest life was influenced by packaging and the mature green fruits showed lower disease occurrence. Fresh mass loss was lower in packed fruits. The peel of mature green fruits developed a characteristic ripe peach color at the end of storage, but PD-900 (TM) provided a delay in color change. Packaging also influenced the firmness, allowing for more firmness retention than for the control fruits at both harvest stages. The organic acid content decreased in the packaged fruits and increased in the control fruits. In the packaged fruit, the amount of sugar increased until the eighth day and then decreased until the end of the storage period. The 'Aurora-1' peaches did not show compromised quality by packaging use and exhibited an increase in harvest life to 24 days (compared to 16 days for the control).

The Effects of Piece Size and Storage Temperatures on Minimally Processed 'Aurora-1' Peaches

The production of minimally processed fruit has increased significantly in the last few years due to the high nutritional value, convenience and safety of minimally processed fruit. The objective of the present study was to evaluate the effects of the conservation temperature and processing conditions on the quality and shelf-life of minimally processed peaches. The processing procedure consisted of washing, sanitization, peeling and stone removal. To remove the stone, the fruit was cut longitudinally, and three types of products were obtained, including halves, quarters and one-eighth segments. The fruit pieces were immersed in 2% ascorbic acid for 3 min and were packed in rigid polystyrene trays (Meiwa (c) M-54) coated with 14 mu m PVC film (Omnifilm (TM)) (about 200 g per pack). The pieces were stored at 65% RH at 3, 6 or 9 degrees C for 12 days, and were evaluated every four days. The appearance, fresh mass loss, color, O-2 and CO2 concentration, acidity, total soluble solids, total and soluble pectin content and ascorbic acid concentration were measured. Minimally processed peaches stored at 3 degrees C maintained higher quality due to control of ripening and senescence Moreover, an interaction effect between one-eighth segments and a temperature of 3 degrees C was observed, and fruit with a superior appearance and higher soluble solids content was obtained.

Characterization of glycerol kinase from baker's yeast: Response surface modeling of the enzymatic reaction

The present study describes a methodology of dosage of glycerol kinase (GK) from baker's yeast. The standardization of the activity of the glycerol kinase from baker's yeast was accomplished using the diluted enzymatic preparation containing glycerol phosphate oxidase (GPO) and glycerol kinase. The mixture was incubated at 60 degrees C by 15 min and the reaction was stopped by the SDS solution addition. A first set of experiments was carried out in order to investigate the individual effect of temperature (7), pH and substrate concentration (S), on GK activity and stability. The pH and temperature stability tests showed that the enzyme presented a high stability to pH 6.0-8.0 and the thermal stability were completely maintained up to 50 degrees C during 1 h. The K(m) of the enzyme for glycerol was calculated to be 2 mM and V(max) to be 1.15 U/mL. In addition, modeling and optimization of reaction conditions was attempted by response surface methodology (RSM). Higher activity values will be attained at temperatures between 52 and 56 degrees C, pH around 10.2-10.5 and substrate concentrations from 150 to 170 mM.This low cost method for glycerol kinase dosage in a sequence of reactions is of great importance for many industries, like food, sugar and alcohol. RSM showed to be an adequate approach for modeling the reaction and optimization of reaction conditions to maximize glycerol kinase activity. (C) 2007 Elsevier B.V. All rights reserved.

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.

Mume clonal rootstocks for 'Aurora-1' peach in São Paulo State, Brazil

In spite of significant results achieved with scion genetic improvement in stone fruits, the peach culture in Brazil still needs studies and new technologies regarding the use of rootstocks. A wide research project has being developed at the Faculdade de Ciências Agrárias e Veterinárias (FCAV/UNESP), Jaboticabal, Brazil, dealing with the use of mume clones (Prunus mume) as rootstocks for peach trees, which has produced promising results. In this research, two mume genotypes propagated by herbaceous cuttings were tested as rootstocks for peach cultivar Aurora-1. Three different tree spacing were used: 6 x 2 m, 6 x 3 m and 6 x 4 m. The experiment was carried out at Vista Alegre do Alto (21°10'14 S, 48°37'45 W, 700 m of altitude), São Paulo State, Brazil. Growing field conditions included Hapludalfs soil with medium sandy texture and using micro sprinkler irrigation. The region has an average chilling accumulation 17.9 hours per year. The evaluations were taken in 2005 and 2006 (2nd and 3rd year after planting, respectively). The trunk diameter was evaluated every three months, from the 24th to the 41st month after planting, totalizing seven evaluations. Plants on 'Rigitano' had higher trunk diameter on the 33rd, 39th and 41st month after planting (May/06, November/06 and February/07, respectively). No significant differences were observed in the other evaluations. The diameter at 5 cm above to the graft point was larger than below, but no incompatibility symptoms were observed between rootstocks and scion. Spacing tested did not influence trunk diameter, phenology and flower bud production in 'Aurora-1' scion. In conclusion, 'Rigitano' and 'Clone 15' are recommended for high density plantings of peach 'Aurora-1' in Brazil, and the 6 x 2 m spacing can be recommended, with productivity advantages for peach under low air relative humidity and mild winter conditions. © ISHS 2012.

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.