Inhibitor-2 induced M-phase arrest in <Emphasis Type="Italic">Xenopus</Emphasis> cycling egg extracts is dependent on MAPK activation

The evolutionarily-conserved protein phosphatase 1 (PP1) plays a central role in dephosphorylation of phosphoproteins during the M phase of the cell cycle. We demonstrate here that the PP1 inhibitor inhibitor-2 protein (Inh-2) induces an M-phase arrest in Xenopus cycling egg extracts. Interestingly, the characteristics of this M-phase arrest are similar to those of mitogen-activated protein kinase (p42MAPK)-induced M-phase arrest. This prompted us to investigate whether Inh-2-induced M-phase arrest was dependent on activation of the p42MAPK pathway. We demonstrate here that MAPK activity is required for Inh-2-induced M-phase arrest, as inhibition of MAPK by PD98059 allowed cycling extracts to exit M phase, despite the presence of Inh-2. We next investigated whether Inh-2 phosphorylation by the MAPK pathway was required to induce an M-phase arrest. We discovered that while p90Rsk (a MAPK protein required for M-phase arrest) is able to phosphorylate Inh-2, this phosphorylation is not required for Inh-2 function. Overall, our results suggest a novel mechanism linking p42MAPK and PP1 pathways during M phase of the cell cycle.     

The effect of salt stress on lipid peroxidation and antioxidative enzymes in callus of two <Emphasis Type="Italic">Acanthophyllum</Emphasis> species

The changes in lipid peroxidation and the involvement of the antioxidant system in relation to salt stress tolerance were investigated in the callus of Acanthophyllum glandulosum and Acanthophyllum sordidum. The callus was subjected to NaCl stress (50–200 mM) for 40 d. The callus of A. glandulosum was less sensitive to NaCl stress than that of A. sordidum. Increasing concentrations of NaCl from 50 to 200 mM correlated to increased proline content in A. glandulosum. Total protein content was higher in extracts of A. glandulosum than in extracts of A. sordidum under both control and salinity treatments. Compared with A. sordidum, lipid peroxidation and H₂O₂ content were lower and the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase, and glutathione reductase were higher in A. glandulosum under salt stress. Activity staining of antioxidant enzymes separated by native polyacrylamide gel electrophoresis (PAGE) revealed that callus of A. sordidum had five Fe-SOD isoforms and one Mn-SOD isoform, all of which were reduced by salinity. In A. glandulosum, two Mn-SOD, three Fe-SOD, and one Cu/Zn-SOD isoforms were detected. Out of these six SOD isoforms, expression of the Mn-SOD and Fe-SOD isoforms was enhanced at 100 mM and higher NaCl concentrations. Two and six GPX isoforms were detected in A. sordidum and A. glandulosum, respectively. Expression of the single CAT isoform in A. sordidum was preferentially reduced by salinity. In A. glandulosum, the two CAT isoforms showed differential down regulation under NaCl stress, with the CAT2 isoform detected only under control condition. These results suggest that A. glandulosum callus is better protected against salinity-induced oxidative damage by maintaining higher activities of antioxidant enzymes than the callus of A. sordidum.     

HLA-DRB1 shared epitope genotyping using the revised classification and its association with circulating autoantibodies,acute phase reactants,cytokines and clinical indices of disease activity in a cohort of South African rheumatoid arthritis patients

Introduction  

The revised shared epitope (SE) concept in rheumatoid arthritis (RA) is based on the presence (S) or absence (X) of the SE RAA amino acid motif at positions 72 to 74 of the third hypervariable region of the various human leucocyte antigen (HLA)-DRB1 alleles. The purpose of this study was to investigate SE subtypes on the basis of the American College of Rheumatology 1987 revised criteria for the classification of RA in a cohort of South African RA patients (n = 143) and their association with clinical and circulating biomarkers of disease activity (autoantibodies, acute phase reactants and cytokines).     

Cysteine-3 and cysteine-4 are essential for the thioredoxin-like oxidoreductase and antioxidant activities of Plasmodium falciparum macrophage migration inhibitory factor

Plasmodium falciparum macrophage migration inhibitory factor (PfMIF) exhibits thioredoxin (Trx)-like oxidoreductase activity but the active site for this activity and its function have not been evaluated. A bioinformatics search revealed that the conserved CXXC motif, which is responsible for Trx-like oxidoreductase activity, is absent from PfMIF. In contrast, the adjacent N-terminal Cys-3 and Cys-4 are conserved in MIF across species of malarial parasites. Mutation of either vicinal Cys-3 or Cys-4 of PfMIF abolished the Trx-like activity, whereas the mutation of the remaining Cys-59 or Cys-103 did not affect it. PfMIF has an antioxidant function. It prevents reactive oxygen species-mediated lipid peroxidation and oxidative damage of DNA as evident from DNA nicking assay. Interestingly, chemical modification of the vicinal cysteines by phenylarsine oxide (PAO), a specific vicinal thiol modifier, significantly prevented this antioxidant activity. Modification of Cys-3 and Cys-4 was confirmed by MALDI-TOF mass spectroscopy of peptide fragments obtained after cyanogen bromide digestion of PAO-modified PfMIF. Furthermore, mutation of either Cys-3 or Cys-4 of PfMIF resulted in the loss of both Trx-like oxidoreductase and antioxidant activities of PfMIF. Altogether, our results suggest that the vicinal Cys-3 and Cys-4 play a critical role in the Trx-like oxidoreductase activity and antioxidant property of PfMIF.     

Modulating infestation rate of white fly (<Emphasis Type="Italic">Bemicia tabaci</Emphasis>) on okra (<Emphasis Type="Italic">Hibiscus esculentus</Emphasis> L.) by nitrogen application

Pests are major factor affecting the crop yield, quality, and esthetic value. Although pest resistance is genetically controlled, it is mediated through physiological and biochemical processes interrelated with the nutritional status of the plant. Nutrition of plant may determine its resistance or susceptibility to pests. An experiment was conducted to determine whether and up to what extent varying concentrations of N modulate the infesting rate of whitefly on Okra (Hibiscus esculentus). Twenty-one-day-old okra plants were subjected to five concentrations of 4, 8, 12, 16, and 20 mM N for 30 days. Number of eggs, nymph were counted on each plant after every 4 days. Growth and yield of okra plants increased with increasing supply of N but it decreased slightly at the highest level of N (20 mM). Leaf soluble proteins, amino acids, and soluble sugars were determined on fresh weight basis. From these results, it is clear that increasing N level increased the biosynthesis or accumulation of protein, free amino acid, and sugars that might have attracted more whitefly for feeding.     

Regulation of microsomal triglyceride transfer protein by apolipoprotein A-IV in newborn swine intestinal epithelial cells

Apolipoprotein (apo) A-IV overexpression enhances chylomicron (CM) assembly and secretion in newborn swine intestinal epithelial cells by producing larger particles (Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW, Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg RB, Black DD. J Biol Chem 281: 3473-3483, 2006). To determine the impact of apo A-IV on microsomal triglyceride transfer protein (MTTP), IPEC-1 cell lines containing a tetracycline-regulatable expression system were used to overexpress native swine apo A-IV and "piglike" human apo A-IV, a mutant human apo A-IV with deletion of the EQQQ-rich COOH-terminus, previously shown to upregulate basolateral triglyceride (TG) secretion 5-fold and 25-fold, respectively. Cells were incubated 24 h with and without doxycycline and oleic acid (OA, 0.8 mM). Overexpression of the native swine apo A-IV and piglike human apo A-IV increased MTTP lipid transfer activity by 39.7% (P = 0.006) and 53.6% (P = 0.0001), respectively, compared with controls. Changes in mRNA and protein levels generally paralleled changes in activity. Interestingly, native swine apo A-IV overexpression also increased MTTP large subunit mRNA, protein levels, and lipid transfer activity in the absence of OA, suggesting a mechanism not mediated by lipid absorption. Overexpression of piglike human apo A-IV significantly increased partitioning of radiolabeled OA from endoplasmic reticulum (ER) membrane to lumen, suggesting increased net transfer of membrane TG to luminal particles. These results suggest that the increased packaging of TG into nascent CMs in the ER lumen, induced by apo A-IV, is associated with upregulation of MTTP activity at the pretranslational level. Thus MTTP is regulated by apo A-IV in a manner to promote increased packaging of TG into the CM core, which may be important in neonatal fat absorption.     

Kinesin-73 is a processive motor that localizes to Rab5-containing organelles

Drosophila Kinesin-73 (Khc-73), which plays a role in mitotic spindle polarity in neuroblasts, is a metazoan-specific member of the Kinesin-3 family of motors, which includes mammalian KIF1A and Caenorhabditis elegans Unc-104. The mechanism of Kinesin-3 motors has been controversial because some studies have reported that they transport cargo as monomers whereas other studies have suggested a dimer mechanism. Here, we have performed single-molecule motility and cell biological studies of Khc-73. We find that constructs containing the motor and the conserved short stretches of putative coiled-coil-forming regions are predominantly monomeric in vitro, but that dimerization allows for fast, processive movement and high force production (7 piconewtons). In Drosophila cell lines, we present evidence that Khc-73 can dimerize in vivo. We also show that Khc-73 is recruited specifically to Rab5-containing endosomes through its "tail" domain. Our results suggest that the N-terminal half of Khc-73 can undergo a monomer-dimer transition to produce a fast processive motor and that its C-terminal half possesses a specific Rab5-vesicle binding domain.     

CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection

Hematopoietic cells constitutively express CD31/PECAM1, a signaling adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4(+) T cells, its function on these cells is unclear. To address this, we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends on CD4(+) T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4(+) T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4(+) T cells exhibiting an activated phenotype and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis postinfection compared with wild-type T cells. Using adoptive transfer experiments, we show that loss of CD31 on CD4(+) T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation, because in its absence, T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory, and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis.     

Mitigation of lung injury after accidental exposure to radiation

There is a serious need to develop effective mitigators against accidental radiation exposures. In radiation accidents, many people may receive nonuniform whole-body or partial-body irradiation. The lung is one of the more radiosensitive organs, demonstrating pneumonitis and fibrosis that are believed to develop at least partially because of radiation-induced chronic inflammation. Here we addressed the crucial questions of how damage to the lung can be mitigated and whether the response is affected by irradiation to the rest of the body. We examined the widely used dietary supplement genistein given at two dietary levels (750 or 3750 mg/kg) to Fischer rats irradiated with 12 Gy to the lung or 8 Gy to the lung + 4 Gy to the whole body excluding the head and tail (whole torso). We found that genistein had promising mitigating effects on oxidative damage, pneumonitis and fibrosis even at late times (36 weeks) when drug treatment was initiated 1 week after irradiation and stopped at 28 weeks postirradiation. The higher dose of genistein showed no greater beneficial effect. Combined lung and whole-torso irradiation caused more lung-related severe morbidity resulting in euthanasia of the animals than lung irradiation alone.