Selective Loss of CDC2 and CDK2 Induction by Tumor Necrosis Factor-α in Senescent Human Diploid Fibroblasts

Normal human diploid fibroblasts undergo a finite number of doublings in culture. This process of senescence is accompanied by a loss in the ability to respond to proliferative stimuli and is therefore distinct from the quiescent state induced by nutrient deprivation. We have studied changes in gene expression induced in these cells following exposure to the cytokine, tumor necrosis factor-α (TNF). We observed that TNF induced CDC2 and CDK2 expression in early-passage quiescent WI-38 fibroblasts. However, as cells approached senescence, their ability to induce CDC2 and CDK2, as well as stimulate DNA synthesis in response to TNF, progressively declined, with minimal to absent induction in senescent cells. This occurred despite the TNF-dependent induction of such proliferation-independent genes as manganese superoxide dismutase and interleukin-6 in senescent and quiescent cells. Serum was similarly unable to induce CDC2 or CDK2 expression in senescent cells. These results demonstrate that senescent cells are selectively deficient in TNF-mediated induction of CDC2 and CDK2, genes crucial to DNA synthesis and mitosis.     

Orbicules in angiosperms: Morphology, function, distribution, and relation with tapetum types

Orbicules, or Ubisch bodies, are sporopollenin particles lining the inner tangential and sometimes also the radial tapetal cell walls. They occur only in species with a secretory tapetum. The surface ornamentation of orbicules and pollen of the same species is often strikingly similar. Although orbicules were discovered more than a century ago, these structures remain enigmatic since their function is still obscure. Proposed hypotheses about their possible function are discussed. We also deal here with topics such as the possible allergenicity of orbicules and their representation in the fossil record. The use of orbicule characters for systematics is reviewed. The distribution of orbicules throughout the angiosperms, based on a literature review from the first report until today, is shown in a list with 314 species from 72 families. Those species found in the literature without orbicules are presented together with their tapetum type. We plotted this information on a dahlgrenogram to visualize the distribution of orbicules. Orbicules occur in all subclasses of the angiosperms. Their occurrence is not correlated with certain modes of pollination or habitats.

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Résumé  Les orbicules, ou corps d’Ubisch, sont des particules de sporopollénine couvrant la surface intérieure tangentiale et parfois la surface radiale des cellules du tapétum. On ne les retrouve que dans les espèces possédant un tapétum sécréteur. L’ornementation superficielle des orbicules et celle du pollen d’une même espèce est souvent remarquablement similaire. Malgré le fait que les orbicules ont été découvert il y a plus d’un siècle, ces structures restent énigmatiques et leur fonction est toujours méconnue. Les hypothèses proposées concernant la fonction éventuelle des orbicules sont commentées dans cet article. Nous avons également traité des sujets tels que les éventuels effets allergènes des orbicules ainsi que leur présence dans les strates fossiles. L’utilisation de caractères orbiculaires dans la systématique est analysée. Nous présentons une liste de 314 espèces appartenant à 72 familles possédant des orbicules, sur base d’une analyse de la litérature à partir de la première observation jusqu’au présent. Pour les espèces rapportées dans la litérature qui ne possèdent pas d’orbicules, nous présentons aussi leur type de tapétum. Nous avons projeté cette information sur un Dahlgrenogramme afin de visualiser la distribution des orbicules. Nous les retrouvons dans toutes les sous-classes des angiospermes. Leur présence n’est pas correlée avec certains modes de pollinisation ou avec divers types d’habitat.

     

Analysis of T-DNA-mediated translational β-glucuronidase gene fusions

Three random translational -glucuronidase (gus) gene fusions were previously obtained in Arabidopsis thaliana, using Agrobacterium-mediated transfer of a gus coding sequence without promoter and ATG initiation site. These were analysed by IPCR amplification of the sequence upstream of gus and nucleotide sequence analysis. In one instance, the gus sequence was fused, in inverse orientation, to the nos promoter sequence of a truncated tandem T-DNA copy and translated from a spurious ATG in this sequence. In the second transgenic line, the gus gene was fused to A. thaliana DNA, 27 bp downstream an ATG. In this line, a large deletion occurred at the target site of the T-DNA. In the third line, gus is fused in frame to a plant DNA sequence after the eighth codon of an open reading frame encoding a protein of 619 amino acids. This protein has significant homology with animal and plant (receptor) serine/threonine protein kinases. The twelve subdomains essential for kinase activity are conserved. The presence of a potential signal peptide and a membrane-spanning domain suggests that it may be a receptor kinase. These data confirm that plant genes can be tagged as functional translational gene fusions.     

The high‐molecular‐weight kininogen Domain 5 is an intrinsically unstructured protein and its interaction with ferritin is metal mediated

High‐molecular‐weight kininogen domain 5 (HK5) is an angiogenic modulator that is capable of inhibiting endothelial cell proliferation, migration, adhesion, and tube formation. Ferritin can bind to a histidine–glycine–lysine‐rich region within HK5 and block its antiangiogenic effects. However, the molecular intricacies of this interaction are not well understood. Analysis of the structure of HK5 using circular dichroism and nuclear magnetic resonance [¹H, ¹⁵N]‐heteronuclear single quantum coherence determined that HK5 is an intrinsically unstructured protein, consistent with secondary structure predictions. Equilibrium binding studies using fluorescence anisotropy were used to study the interaction between ferritin and HK5. The interaction between the two proteins is mediated by metal ions such as Co²⁺, Cd²⁺, and Fe²⁺. This metal‐mediated interaction works independently of the loaded ferrihydrite core of ferritin and is demonstrated to be a surface interaction. Ferritin H and L bind to HK5 with similar affinity in the presence of metals. The ferritin interaction with HK5 is the first biological function shown to occur on the surface of ferritin using its surface‐bound metals.     

Alternative splicing of RyR1 alters the efficacy of skeletal EC coupling

Alternative splicing of ASI residues (Ala³⁴⁸¹–Gln³⁴⁸⁵) in the skeletal muscle ryanodine receptor (RyR1) is developmentally regulated: the residues are present in adult ASI(+)RyR1, but absent in the juvenile ASI(?)RyR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RyR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr³⁴⁷¹–Gly³⁵⁰⁰) in the splice variants. Depolarisation-dependent Ca²⁺ release was enhanced by >50% in myotubes expressing ASI(?)RyR1 compared with ASI(+)RyR1, although DHPR L-type currents and SR Ca²⁺ content were unaltered, while ASI(?)RyR1 channel function was actually depressed. The effect on EC coupling did not depend on changes in ASI domain secondary structure. Probing RyR1 function with peptides possessing the ASI domain sequence indicated that the domain contributes to an inhibitory module in RyR1. The action of the peptide depended on a sequence of basic residues and their alignment in an α-helix adjacent to the ASI splice site. This is the first evidence that the ASI residues contribute to an inhibitory module in RyR1 that influences EC coupling. Implications for development and DM1 are discussed.     

Effects of an alpha-helical ryanodine receptor C-terminal tail peptide on ryanodine receptor activity: modulation by Homer

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM; but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.     

miRNA-148a and miRNA-30c expressions as potential biomarkers in breast cancer patients

BackgroundBreast cancer is an extensively identified malignant tumor and is a prime cause of cancer mortalities in females. It has been shown that alteration of miRNAs expression (up or down regulation) can affect the initiation and progression of many malignancies. We aimed to evaluate the role of circulating miRNA-148a and miRNA-30c in female patients with breast cancer and estimate their usage as potential biomarkers in the diagnosis, prognosis and survival of breast cancer.MethodsThis study included 75 breast cancer female patients.They were compared with 55 apparently healthy female subjects. miRNAs expression analysis was assessed via real-time PCR.ResultsTo discriminate breast cancer patients from controls, miR-30c showed the best performance at a cut off value of ≤20.6 (AUC = 0.998, 97.33% sensitivity, 96.36% specificity, p < 0.001), followed by miR-148a (AUC = 0.995, 94.67% sensitivity, 90.91% specificity, p < 0.001 at a cut off value of ≤0.1), CA 15-3 (AUC = 0.930, 88.0% sensitivity, 81.82% specificity, p < 0.001 at a cut off value of >21.3), and finally CEA (AUC = 0.751, 70.67% sensitivity, 63.64% specificity, p < 0.001 at a cut off value of >2.5).ConclusionmiRNA-148a and miRNA-30c expressions were down regulated in female patients with breast cancer and might be considered as potential blood biomarkers. Both also might have rule in disease treatment and selection of therapeutic targets. Future studies are needed to improve their role in predicting response to treatment and prognosis.     

Bioinformatics tools for cancer metabolomics

It is well known that significant metabolic change take place as cells are transformed from normal to malignant. This review focuses on the use of different bioinformatics tools in cancer metabolomics studies. The article begins by describing different metabolomics technologies and data generation techniques. Overview of the data pre-processing techniques is provided and multivariate data analysis techniques are discussed and illustrated with case studies, including principal component analysis, clustering techniques, self-organizing maps, partial least squares, and discriminant function analysis. Also included is a discussion of available software packages.     

Complement C3 deficiency attenuates chronic hypoxia-induced pulmonary hypertension in mice

Background

Evidence suggests a role of both innate and adaptive immunity in the development of pulmonary arterial hypertension. The complement system is a key sentry of the innate immune system and bridges innate and adaptive immunity. To date there are no studies addressing a role for the complement system in pulmonary arterial hypertension.

Methodology/Principal Findings

Immunofluorescent staining revealed significant C3d deposition in lung sections from IPAH patients and C57Bl6/J wild-type mice exposed to three weeks of chronic hypoxia to induce pulmonary hypertension. Right ventricular systolic pressure and right ventricular hypertrophy were increased in hypoxic vs. normoxic wild-type mice, which were attenuated in C3−/− hypoxic mice. Likewise, pulmonary vascular remodeling was attenuated in the C3−/− mice compared to wild-type mice as determined by the number of muscularized peripheral arterioles and morphometric analysis of vessel wall thickness. The loss of C3 attenuated the increase in interleukin-6 and intracellular adhesion molecule-1 expression in response to chronic hypoxia, but not endothelin-1 levels. In wild-type mice, but not C3−/− mice, chronic hypoxia led to platelet activation as assessed by bleeding time, and flow cytometry of platelets to determine cell surface P-selectin expression. In addition, tissue factor expression and fibrin deposition were increased in the lungs of WT mice in response to chronic hypoxia. These pro-thrombotic effects of hypoxia were abrogated in C3−/− mice.

Conclusions

Herein, we provide compelling genetic evidence that the complement system plays a pathophysiologic role in the development of PAH in mice, promoting pulmonary vascular remodeling and a pro-thrombotic phenotype. In addition we demonstrate C3d deposition in IPAH patients suggesting that complement activation plays a role in the development of PAH in humans.     

Preparation of microcapsules with multi-layers structure stabilized by chitosan and sodium dodecyl sulfate

The microcapsules with oil core and multi-layers shell were developed from poly-cationic chitosan (CS) and anionic SDS in multistep electrostatic layer by layer deposition technique combined with oil in water emulsification process. The net charge of microcapsules determined by zeta potential indicated that microcapsules are highly positive charged because of poly-cationic nature of CS, and charge neutralization of microcapsules occurred after alkali treatment. The granulometry measurement showed increase in average diameter of microcapsules by alkali treatment suggesting swelling or formation of small aggregates. The morphology analysis of microcapsules by optical microscopy corroborated the results of granulometry, and diameter of microcapsules was found to be decreased in multistep process due to tight packing of layers in outer shell of microcapsules. The alkali treatment of microcapsules to solidify outer shell was optimized with 0.02N NaOH to reduce microcapsules aggregation and gel formation by CS chains as found in optical micrographs.