Phosphorylation of p50 NF-kappaB at a single serine residue by DNA-dependent protein kinase is critical for VCAM-1 expression upon TNF treatment

The DNA binding activity of NF-κB is critical for VCAM-1 expression during inflammation. DNA-dependent protein kinase (DNA-PK) is thought to be involved in NF-κB activation. Here we show that DNA-PK is required for VCAM-1 expression in response to TNF. The phosphorylation and subsequent degradation of I-κBα as well as the serine 536 phosphorylation and nuclear translocation of p65 NF-κB were insufficient for VCAM-1 expression in response to TNF. The requirement for p50 NF-κB in TNF-induced VCAM-1 expression may be associated with its interaction with and phosphorylation by DNA-PK, which appears to be dominant over the requirement for p65 NF-κB activation. p50 NF-κB binding to its consensus sequence increased its susceptibility to phosphorylation by DNA-PK. Additionally, DNA-PK activity appeared to increase the association between p50/p50 and p50/p65 NF-κB dimers upon binding to DNA and after binding of p50 NF-κB to the VCAM-1 promoter. Analyses of the p50 NF-κB protein sequence revealed that both serine 20 and serine 227 at the amino terminus of the protein are putative sites for phosphorylation by DNA-PK. Mutation of serine 20 completely eliminated phosphorylation of p50 NF-κB by DNA-PK, suggesting that serine 20 is the only site in p50 NF-κB for phosphorylation by DNA-PK. Re-establishing wild-type p50 NF-κB, but not its serine 20/alanine mutant, in p50 NF-κB(-/-) fibroblasts reversed VCAM-1 expression after TNF treatment, demonstrating the importance of the serine 20 phosphorylation site in the induction of VCAM-1 expression. Together, these results elucidate a novel mechanism for the involvement of DNA-PK in the positive regulation of p50 NF-κB to drive VCAM-1 expression.     

In vivo and in vitro management of Meloidogyne incognita (Tylenchida: Heteroderidae) using rhizosphere bacteria,Pseudomonas spp. and Serratia spp. compared with oxamyl

Biological control using rhizosphere bacteria, Pseudomonas spp. and Serratia spp. is a prospective alternative technique to overcome plant parasitic nematodes infection. So, the current study was conducted in vitro on five egg-masses, 100 free eggs and 100 infective juveniles (IJs) of Meloidogyne incognita as well as greenhouse treatments on Luffa aegyptiaca L. to evaluate the nematicidal potential of six strains belong to Pseudomonas spp. and Serratia spp. as compared to oxamyl.Results showed that the inhibitory effect and juvenile mortality varied according to bacteria species, strains and exposure time. All the tested bacteria significantly (P ≤ 0.05) inhibited egg hatching and increased juvenile mortality in vitro. After 3 days of treatment, Pseudomonas spp. were more effective against eggs (48.31to 55.15%) and IJs (20.98 to 25.30%) than Serratia spp. (44.55 to 49.75% with eggs) and (19.06 to 21.61% with IJs), respectively. In the pot experiment, Luffa aegyptiaca L. treated with Serratia spp. and Pseudomonas spp. displayed significantly higher (P ≤ 0.05) levels of growth (as indicated by root length, fresh roots weight and fresh shoots weight) compared to control plants and significantly (P ≤ 0.05) suppressed galling (number of galls) and reproduction (as indicated by number of egg-masses on roots and number of eggs and juveniles in pot soil). Meanwhile, among the treated plants, Serratia spp. and Pseudomonas spp. gave the best results in shoot weight of pots infected by eggs of M. incognita than those infected with IJs as compared with positive control. While, oxamyl treatment gave the best results in pots infected by eggs and IJs.The lowest galling (gall index), number of eggs and juveniles in soil was observed in the treatment with mixture of Serratia spp. and Pseudomonas spp. as well as, enhanced growth of sponge gourd more than application each of them alone. Pots treated with oxamyl overwhelmed those treated with mixture of Serratia spp. and Pseudomonas spp.     

Residue analysis of organophosphorus and organochlorine pesticides in fatty matrices by gas chromatography coupled with electron-capture detection

A multiresidue method for the simultaneous determination of 22 organochlorine (OCs) and organophosphorus (Ops) pesticides (including isomers and metabolites), representing a wide range of physicochemical properties, was developed in fatty matrices extracted from meat. Pesticides were extracted from samples with acetonitrile/n-hexane (v:v, 1:1). The analytical screening was performed by gas chromatography coupled with electron-capture detection (ECD). The identification of compounds was based on their retention time and on comparison of the primary and secondary ions. The optimized method was validated by determining accuracy (recovery percentages), precision (repeatability and reproducibility), and sensitivity (detection and quantitation limits) from analyses of samples fortified at 38 to 300 ng/g levels. Correlation coefficients for the 22 extracted pesticide standard curves (linear regression analysis, n = 3) ranged from 0.998 to 1.000. Recovery studies from 2 g samples fortified at 3 levels demonstrated that the GC-ECD method provides 64.4-96.0% recovery for all pesticides except 2,4'-DDE (44.6-50.4%), 4,4'-DDE (51.1-57.5%) and 2,4'-DDT (50.0-51.2%). Both repeatability and reproducibility relative standard deviation values were < 20% for all residues. Detection limits ranged from 0.31 to 1.27 ng/g and quantification limits were between 1.04 and 4.25 ng/g. The proposed analytical method may be used as a simple procedure in routine determinations of OCs and Ops in meat. It can also be applied to the determination of pesticide multi-residues in other animal products such as butter and milk.     

Biodegradation of hydrocarbon contamination by immobilized bacterial cells

This study examined the capacity of immobilized bacteria to degrade petroleum hydrocarbons. A mixture of hydrocarbon-degrading bacterial strains was immobilized in alginate and incubated in crude oil-contaminated artificial seawater (ASW). Analysis of hydrocarbon residues following a 30-day incubation period demonstrated that the biodegradation capacity of the microorganisms was not compromised by the immobilization. Removal of n-alkanes was similar in immobilized cells and control cells. To test reusability, the immobilized bacteria were incubated for sequential increments of 30 days. No decline in biodegradation capacity of the immobilized consortium of bacterial cells was noted over its repeated use. We conclude that immobilized hydrocarbon-degrading bacteria represent a promising application in the bioremediation of hydrocarbon-contaminated areas.     

Biomechanical characterisation of ovine spinal facet joint cartilage

The spinal facet joints are known to be an important component in the kinematics and the load transmission of the spine. The articular cartilage in the facet joint is prone to degenerative changes which lead to back pain and treatments for the condition have had limited long term success. There is currently a lack of information on the basic biomechanical properties of the facet joint cartilage which is needed to develop tissue substitution or regenerative interventions. In the present study, the thickness and biphasic properties of ovine facet cartilage were determined using a combination of indentation tests and computational modelling. The equilibrium biphasic Young's modulus and permeability were derived to be 0.76±0.35 MPa and 1.61±1.10×10^?15 m⁴/(Ns) respectively, which were within the range of cartilage properties characterised from the human synovial joints. The average thickness of the ovine facet cartilage was 0.52±0.10 mm, which was measured using a needle indentation test. These properties could potentially be used for the development of substitution or tissue engineering interventions and for computational modelling of the facet joint. Furthermore, the developed method to characterise the facet cartilage could be used for other animals or human donors.     

Cytochrome b mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression

Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. Malarone(TM)) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Q(o)) site of the mitochondrial cytochrome bc(1) complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Q(o) pocket of cytochrome b, most notably near the conserved Pro(260)-Glu(261)-Trp(262)-Tyr(263) (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (K(i)) for atovaquone with a concomitant reduction in the V(max) of the bc(1) complex of ～40% and a 3-fold increase in the observed K(m) for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc(1) suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc(1) and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc(1) activity.     

Expression of platelet-derived growth factor and its receptors in spontaneous canine hemangiosarcoma and cutaneous hemangioma

Hemangiosarcoma (HSA) is a malignant neoplasia of vascular endothelial cells (ECs). Our previous report on the expression of vascular endothelial growth factor, basic fibroblast growth factor, and their receptors in canine HSA suggested an autocrine/ paracrine mechanism of tumor growth. However, the influence of other angiogenic growth factors in canine HSA was not elucidated; therefore, the expression of platelet-derived growth factor (PDGF) and its receptors was investigated by immunohistochemical analysis. Forty-six canine HSAs and 21 canine cutaneous hemangiomas (HAs) were analyzed. For immunohistochemistry, anti-PDGF-BB, anti-PDGFR-α, and anti-PDGFR-β antibodies were utilized as primary antibodies. Immunoreactivities were scored as strongly positive (>25% positive neoplastic cells), weakly positive (1-25% positive neoplastic cells), and negative if not staining at all. In cutaneous HA, 33.3% and 57.1% of cases were strongly and weakly positive, respectively, and 43.5% and 13.0% of HSAs were strongly and weakly positive for PDGF-BB, respectively. Moreover, 38.1% and 28.6% of cutaneous HAs cases were strongly and weakly positive, respectively, and 23.9% and 4.3% of HSAs cases were strongly and weakly positive, respectively, for PDGFR-α. Thirty-five HSAs cases (76.1%) were strongly positive, and the remaining 11 (23.9%) were weakly positive for PDGFR-β. In contrast, 18 (72.0%) cutaneous HAs were negative, and only 3 cases (12.0%) were weakly positive, for PDGFR-β. The proportion of strongly positive cases of HSAs was significantly higher than that of cutaneous HA for PDGFR-β (P<0.01), while PDGFR-α was highly expressed in cutaneous HA and may be related to pathogenesis of cutaneous HA. Therefore, PDGFR-β may be associated with the malignant nature of canine HSA.     

Physiological and biochemical changes in different sugar beet genotypes infected with root-knot nematode

Ten genotypes of sugar beet plant either monogerm or multigerm seeds were screened under greenhouse conditions for both susceptibility and biochemical reaction to root-knot nematode (RKN) Meloidogyne incognita. All the tested genotypes were susceptible to nematode infection according to the number of root galls and gall indices. All infected genotypes exhibited significant reduction in chlorophyll a, b and carotenoids compared to non-infected ones. The total indole acetic acid and total phenolic compounds contents (mean of both shoot and root) increased significantly in most infected genotypes compared to non-infected genotypes except Disk-01-99 and Monte Rosa as well as LP16 and LP15 genotypes, respectively. Also, total polyamine contents (putrescine, spermidine and spermine) showed significant increases in response to infection with nematodes in all genotypes. The same trend was observed in lipid peroxidation expressed with malondialdehyde content in all tested genotypes. Activities of polyphenol oxidase, peroxidase, superoxide dismutase and catalase enzymes were also induced in most infected genotypes compared with non-infected genotypes. Generally, infection with RKNs induced the appearance of new protein bands at molecular masses 303, 288, 42 and 37?KDa in all infected genotypes. The differentiation in the appearance and/or disappearance of protein bands according to susceptibility to infection reflects the variation between genotypes in defense against infection.     

Biological and cytoselective anticancer properties of copper(II)-polypyridyl complexes modulated by auxiliary methylated glycine ligand

A series of ternary copper(II)-1,10-phenanthroline complexes with glycine and methylated glycine derivatives, [Cu(phen)(aa)(H(2)O)]NO(3)·xH(2)O 1-4 (amino acid (aa): glycine (gly), 1; DL: -alanine (DL: -ala), 2; 2,2-dimethylglycine (C-dmg), 3; sarcosine (sar), 4), were synthesized and characterized by FTIR, elemental analysis, electrospray ionization-mass spectra (ESI-MS), UV-visible spectroscopy and molar conductivity measurement. The determined X-ray crystallographic structures of 2 and 3 show each to consist of distorted square pyramidal [Cu(phen)(aa)(H(2)O)](+) cation, a nitrate counter anion, and with or without lattice water, similar to previously reported structure of [Cu(phen)(gly)(H(2)O)]NO(3)·1?H(2)O. It is found that 1-4 exist as 1:1 electrolytes in aqueous solution, and the cationic copper(II) complexes are at least stable up to 24?h. Positive-ion ESI-MS spectra show existence of only undissociated [Cu(phen)(aa)](+) species. Electron paramagnetic resonance, gel electrophoresis, fluorescence quenching, and restriction enzyme inhibition assay were used to study the binding interaction, binding affinity and selectivity of these complexes for various types of B-form DNA duplexes and G-quadruplex. All complexes can bind selectively to DNA by intercalation and electrostatic forces, and inhibit topoisomerase I. The effect of the methyl substituents of the coordinated amino acid in the above complexes on these biological properties are presented and discussed. The IC(50) values (24?h) of 1-4 for nasopharyngeal cancer cell line HK1 are in the range 2.2-5.2?μM while the corresponding values for normal cell line NP69 are greater than 13.0?μM. All complexes, at 5?μM, induced 41-60?% apoptotic cell death in HK1 cells but no significant cell death in NP69 cells.