A proinflammatory role for Fas in joints of mice with collagen-induced arthritis

Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death – Fas (CD95)/FasL – in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor α (TNF-α) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-α and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.     

Potential biochemical markers for somatic embryos ofEurycoma longifolia jack

Biochemical marker is one of the important tools for the early identification and selection of somatic embryogenesis in plants. Studies in developing the biochemical marker for somatic embryogenesis ofEurycoma longifolia disclosed that the regenerated and non-regenerated cotyledons as well as embryogenic and non-embryogenic callus were significantly different in terms of the total protein content as well as the specific activity of peroxidase. The data obtained revealed that embryogenic tissue possess the highest amount of total soluble protein (64.24 mg/g fresh weight) and two protein bands (molecular weight = 25 and 21 kDa) were observed at high intensity. The highest specific activity of peroxidase (578.1 ± 61.6 unit/mg soluble protein) was recorded in embryogenic callus and only 157.1 ± 20.5 unit/mg soluble protein was determined in the non-embryogenic callus. Results obtained also showed that there were variations in the peroxidase banding profiles of the four samples examined. Only two bands were observed in the non-embryogenic callus at the Rf of 0.24 and 0.27, whereas in the embryogenic callus, four bands with high intensity were detected at the Rf of 0.24, 0.41, 0.49 and 0.81.     

Pose estimation in automated visual inspection using genetic algorithm

In this paper, we propose a genetic algorithm based approach to determine the pose of an object in Automated Visual Inspection having three degrees of freedom. We have investigated the effect of noise at 20 dB SNR and also mismatch resulting from incorrect correspondences between the object space points and the image space points, on the estimation of pose parameters. The maximum error in translation parameters is less than 0.45 cm and rotational error is less than 0.2 degree at 20 dB SNR. The error in parameter estimation is insignificant upto 7 pairs of mismatched points out of 24 points in object space and the results skyrockets when 8 or more pairs of points are mismatched. We have compared our result with that obtained by least square technique and it shows that GA based method outperform the gradient based technique when the number of vertices of the object to be inspected is small. These results have clearly established the robustness of GA in estimating the pose of an object with small number of vertices in automated visual inspection.     

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.     

Application of π acceptors to the spectrophotometric and spectrofluorimetric determination of vincamine and naftidrofuryl oxalate in their pharmaceutical preparations

Three different spectrophotometric and two spectrofluorimetric methods have been developed and validated for the determination of vincamine (VN) and naftidrofuryl oxalate (NF) in tablets. The spectrophotometric methods depend on charge transfer complex formation between each of VN and NF with 7,7,8,8‐tetracyano‐quinodimethane (TCNQ), 2,6‐dichloroquinone‐4‐chloroimide (DCQ) and 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ) at 843, 580 and 588 nm, respectively. The spectrofluorimetric methods are based on the formation of charge transfer complex between each of the two drugs and TCNQ, with measurement of the fluorophore formed at 312/375 and 284/612 nm, respectively, or with DDQ at 400/475 and 284/396 nm, respectively. In the spectrophotometric measurements, Beer's law was obeyed at concentration ranges of 1.5–16, 10–180 and 12–140 μg/ml for VN with TCNQ, DCQ, and DDQ, respectively. For NF, the corresponding concentrations were 2–28, 5–75 and 25–150 μg/ml with TCNQ, DCQ, and DDQ, respectively. In the spectrofluorimetric measurements, the ranges for VN were 0.05–0.9 and 0.3–4 μg/ml with TCNQ and DDQ, respectively, whereas for NF the ranges were 0.05–0.85 and 0.5–8 μg/ml with TCNQ and DDQ, respectively. The different experimental parameters affecting the development and stability of the formed color or fluorophore were studied and optimized and the molar ratios of the complexes were calculated. The proposed methods were validated according to ICH guidelines and were successfully applied for the determination of VN and NF in their tablet dosage forms.     

Micelle‐enhanced direct spectrofluorimetric method for the determination of linifanib: Application to stability studies

A new simple stability‐indicating spectrofluorimetric method has been developed and validated for the determination of the tyrosine kinase inhibitor, linifanib (LNF). The proposed method makes use of the native fluorescence characteristics of LNF in a micellar system. Compared with aqueous solutions, the fluorescence intensity of LNF was greatly enhanced upon the addition of Tween‐80. The relative fluorescence intensity of LNF was measured in a diluting solvent composed of 2% Tween‐80: phosphate buffer pH 8.0 (20: 80, v/v) using excitation and emission wavelengths of 290 and 450 nm, respectively. The proposed method was fully validated as per the ICH guidelines. The recorded fluorescence intensity of LNF was rectilinear over a concentration range of 0.3–2 μg/ml with a high correlation coefficient (r = 0.9990) and low limits of detection (0.091 μg/ml) and quantitation (0.275 μg/ml). The applicability of the method was extended to study the inherent stability of LNF under different stress degradation conditions including, alkaline, acidic, oxidative, photolytic and thermal degradation. Moreover, the method was utilized to study the kinetics of the alkaline and oxidative degradation of LNF. The pseudo‐first order rate constants and half‐lives were calculated.     

Antioxidant response and proteomic modulations in Indian mustard grown under salt stress

Productivity of Indian mustard (Brassica juncea L. Czern. and Coss.) is markedly reduced by salt stress. To develop salt tolerance in this important oilseed crop is a need of the hour. This study, based on analysis of growth parameters and antioxidant profile of fourteen Indian mustard genotypes treated with 50, 100, 150 and 200 mM of sodium chloride, was performed to identify the salt-sensitive and salt-tolerant genotypes. Salinity stress inhibited biomass accumulation and reduced the protein and chlorophyll contents in a dose-dependent manner. The reduction was the highest in genotype Pusa Agrani and lowest in CS-54, depicting their contrasting sensitivity to salt stress. Salt treatments triggered a concentration-dependent overproduction of reactive-oxygen species and a concurrent upregulation of the expression of different antioxidants. Genotype CS-54 showed the least damage and maintained a high antioxidant level with almost each salt treatment, exhibiting its competence to withstand the damage provoked by salinity stress. Genotype Pusa Agrani, on the contrary, depicted a salt-sensitive nature by way of its very high lipid peroxidation and low intensity of antioxidants. These two genotypes were further investigated through gel-based proteomic approach, which resulted in the identification and quantification of 42 salinity-responsive proteins related to different metabolic modifications. Molecular processes, including photosynthesis, redox homeostasis, nitrogen metabolism, ATP synthesis, protein synthesis and degradation, signal transduction and respiratory pathways, have exhibited significant changes. The identified stress-responsive proteins could pave the way to develop salt tolerance in Indian mustard plant, thus sustaining its productivity under salinity.     

Adsorption of Cd(II) ions from aqueous solutions using activated carbon prepared from olive stone by ZnCl2 activation

This study is aimed to remove Cd(II) ions from aqueous solutions by adsorption. As adsorbent, activated carbon prepared from olive stone, an agricultural solid by-product was used. Different activating agent (ZnCl(2)) amounts and adsorbent particle size were studied to optimize adsorbent surface area. The adsorption experiments were conducted at different parameters such as, adsorbent dose, temperature, equilibrium time and pH. According to the experiments results, the equilibrium time, optimum pH, adsorbent dosage were found 60 min, pH > 6 and 1.0 g/50 ml respectively. The kinetic data supports pseudo-second order model and intra-particle model but shows very poor fit for pseudo-first order model. Adsorption isotherms were obtained from three different temperatures. These adsorption data were fitted with the Langmuir and Freundlich isotherms. In addition, the thermodynamic parameters, standard free energy (DeltaG(0)), standard enthalpy (DeltaH(0)), standard entropy (DeltaS(0)) of the adsorption process were calculated. To reveal the adsorptive characteristics of the produced active carbon, BET surface area measurements were made. Structural analysis was performed using SEM-EDS. The resulting activated carbons with 20% ZnCl(2) solution was the best sample of the produced activated carbons from olive stone with the specific surface area of 790.25 m(2)g(-1). The results show that the produced activated carbon from olive stone is an alternative low-cost adsorbent for removing Cd(II).     

Association of angiotensin-converting enzyme (ACE) gene insertion–deletion polymorphism with spondylarthropathies

Summary Low back pain (LBP) is a common medical problem. Interaction between genetic and environmental factors predisposes individuals to LBP even at an early age. Inflammatory back pain or spondylarthropathies include ankylosing spondylitis (AS), psoriatic arthritis (PSA), reactive arthritis enteropathic and undifferentiated arthropathies. Angiotensin-converting enzyme (ACE) plays an important role in circulatory homeostasis, physiology of vasculature and inflammation. The insertion–deletion (I/D) polymorphism of the ACE gene has been shown to determine the plasma and tissue levels of ACE especially in the synovial fluid. The aim of this study was to investigate an association between ACE gene I/D polymorphism and inflammatory back pain (spondylarthropathies) secondary to ankylosing spondylitis (AS), psoriatic arthritis, inflammatory bowel disease and undifferentiated spondylarthropathies. The prevalence of ACE gene I/D polymorphism genotypes was determined in 63 patients with inflammatory back pain by polymerase chain reaction (PCR) and compared with that in 111 healthy controls. Of the 63 patients studied, 45 (71.4%) were with AS, 13 (20.6%) were with PSA, 4 (6.3%) were with reactive arthropathy and 1 (1.6%) manifested undifferentiated arthropathy. There were 43 males and 20 females. Mean age of patients was 39.0 ± 11.36 years, age at onset of spondylarthropathy was 27.7 ± 7.49 years and disease duration was 10.3 ± 7.74 months. The controls were selected to match with the patients group in terms of gender ratio, age and ethnicity. The ACE gene polymorphism showed an overall significant difference between patients and controls (p = 0.050). When the ID and II genotype frequency was combined and compared with that for DD genotype amongst patient and control groups, a considerably higher incidence was detected for ID and II genotypes than the DD genotype in spondylarthropathy patients compared to that in the controls (p = 0.036). This study showed a significant association of the I-allele of ACE gene I/D polymorphism with spondylarthropathy in Kuwaiti Arabs.     

A weakly antimalarial biflavanone from Rhus retinorrhoea

The biflavanone (2S,2"S)-7,7"-di-O-methyltetrahydroamentoflavone and five known flavonoids, 7-O-methylnaringenin, 7,3'-O-dimethylquercetin, 7-O-methylapigenin, 7-O-methylluteolin, and eriodictyol were isolated from the leaves of Rhus retinorrhoea Steud, Ex Olive. The biflavanone exhibited moderate antimalarial activity with IC50 0.98 microg/ml against Plasmodium falciparum (W2 Clone) and weak activity against P. falciparum (D6 Clone) with IC50 2.8 microg/ml. Nevertheless, it did not display any cytotoxicity. 7-O-Methylnaringenin showed weak antimicrobial activity against Candida albicans, C. krusei, Staphylococcus aureus, Mycobacterium smegmatis, M. intracellulare, and M. xenopi with MIC approximately 100 microg/ml. Characterization of each compound was based on spectral analysis and comparison with reported data.