Effects of cigarette smoking, XRCC1 genetic polymorphisms, and age on basal DNA damage in human blood mononuclear cells

The aim of this study was to investigate the effects of smoking, polymorphisms of XRCC1 codons 194 and 399, and age on levels of basal DNA damage (as measured by an alkaline comet assay) on mononuclear cells in 122 healthy Japanese workers. In the whole group of 122 individuals, the tail moment (TM) values of current smokers (P < 0.001) or former smokers (P = 0.03) were significantly higher than those of nonsmokers. Individuals bearing the XRCC1 399Gln variant allele showed significant increases in TM values in all subjects or in referent subgroups stratified by age or smoking status except in the current smokers group; in contrast, the TM values of individuals bearing the XRCC1 194Trp variant allele were significantly lower than those of individuals bearing wild-type Arg/Arg genotypes. Furthermore, older subjects (≥47 years old) had significantly higher TM values than younger subjects (<47 years old) in all subjects (P = 0.008). Multiple regression analysis indicated that smoking habits, polymorphisms of XRCC1 codons 194 and 399, and age were important variables affecting individuals basal DNA damage.     

Tick saliva regulates migration, phagocytosis, and gene expression in the macrophage-like cell line, IC-21

We studied the effects of tick saliva on cell migration, cell signaling, phagocytosis, and gene expression in the murine macrophage cell line, IC-21. Saliva increased both basal- and platelet-derived growth factor (PDGF)-stimulated migration in IC-21 cells. However, saliva did not affect PDGF-stimulated extracellular signal-regulated kinase (ERK) activity. Zymosan-mediated interleukin-1 receptor associated kinase (IRAK) activity increased when cells were pretreated with saliva. Saliva suppressed phagocytosis of zymosan particles by IC-21 cells. An RT² Profiler™ PCR Array revealed that saliva regulates gene expression in a manner consistent with an immune response skewed toward a Th2 reaction, which is characterized by production of anti-inflammatory cytokines IL-4 and IL-10. Our results using IC-21 cells suggest that Dermacentor variabilis has evolved a mechanism for regulating macrophage function, which may contribute to the tick’s ability to modulate immune function.     

Protein kinase C differently regulates quisqualate- and 1S,3R-trans aminocyclopentane dicarboxylate-induced phosphoinositide hydrolysis during in vitro development of hippocampal neurons

Transient peaks of quisqualate (QA)-, but not 1S,3R-1-amino-3-cyclopentane dicarboxylate (1S,3R-ACPD)- and carbachol-induced inositol phosphate formation occur between 2 and 5 days in vitro (DIV) in hippocampal neurons in culture. In order to elucidate the putative origin of such developmental activity differences, the effect of PKC on metabotropic glutamate receptor (mGluR) and muscarinic receptor responses was investigated at 3 and 10 DIV. (i) Stimulation of PKC by phorbol-12,13-dibutyrate inhibited QA, IS,3R-ACPD and carbachol responses at 3 DIV. At 10 DIV, only 1S,3R-ACPD response was still inhibited by phorbol esters. (ii) Inhibition of PKC by staurosporine at 3 DIV potentiated 1S,3R-ACPD-induced inositol phosphate formation, but had no effect on QA and carbachol responses. At 10 DIV, all responses were potentiated by staurosporine. These data strongly suggest that PKC differently modulates 1S,3R-ACPD- and QA-induced inositol phosphate accumulations during in vitro development. The specific activity of mGluRs during development, vs that of muscarinic receptor, and the peculiar modes of regulation by PKC of these two mG1uR activities further suggest their particular involvement in the maturation of neuronal culture.     

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E₂ (PGE₂) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE₂ formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β).     

Evidence of expression variation and allelic imbalance in Crohn's disease susceptibility genes NOD2 and ATG16L1 in human dendritic cells

Human dendritic cells (DCs) play an important role in induction and progression of Crohn's disease (CD). Accumulating evidence suggests that viral infection is required to trigger CD pathogenesis in genetically predisposed individuals. NOD2 and ATG16L1 are among the major CD susceptibility genes implicated in impaired immune response to bacterial infection. In this study, we investigated gene expression and allelic imbalance (AI) of NOD2 and ATG16L1 using common variants in human monocyte-derived DCs. Significant AI was observed in ~ 40% and ~ 70% of NOD2 and ATG16L1 heterozygotes, respectively (p < 0.05). AI of NOD2 was inversely associated with its expression level (p = 0.015). No correlation was detected between gene expression and AI for ATG16L1. When infected with Newcastle Disease Virus (NDV), NOD2 expression in DCs was induced about four-fold (p < 0.001), whereas ATG16L1 expression was not affected (p = 0.88). In addition, NDV infection tended to lower the variance in AI among DC populations for the NOD2 gene (p = 0.05), but not the ATG16L1 gene (p = 0.32). Findings of a simulation study, aimed to verify whether the observed variation in gene expression and AI is a result of sample-to-sample variability or experimental measurement error, suggested that NOD2 AI is likely to result from a deterministic event at a single cell level. Overall, our results present initial evidence that AI of the NOD2 and ATG16L1 genes exists in populations of human DCs. In addition, our findings suggest that viral infection may regulate NOD2 expression.     

Host derived inflammatory phospholipids regulate rahU (PA0122) gene, protein, and biofilm formation in Pseudomonas aeruginosa

This study describes the role of “inflammatory” oxidized (Ox) phospholipids in regulation of rahU (PA0122) expression and biofilm formation in Pseudomonas aeruginosa (383) wild type (rahU⁺) and rahU mutant (rahU⁻) strains. Functional analysis of RahU protein from P. aeruginosa in presence of Ox-phospholipids show: (a) LysoPC modulates RahU gene/and protein expression in rahU⁺ cells; (b) rahU promoter activity is increased by lysoPC and inhibited by PAPC, Ox-PAPC and arachidonic acid; the latter inhibitory effect can be reversed by lysoPC, which was enzymatically derived from PAPC; (c) biofilm formation increased in rahU⁻ cells as compared to rahU⁺; and (d) inhibition of rahU promoter activity by PAPC and AA (but not lysoPC) showed significantly augmented biofilm formation in rahU⁺ but not in rahU⁻ cells. This study shows that host derived Ox-phospholipids affect P. aeruginosa-rahU gene and protein expression, which in turn modulates biofilm formation. The accompanying paper describes the role of RahU protein in eukaryotic-host cells.     

Mechanism of vitamin D3-induced transcription of phospholipase D1 in HaCat human keratinocytes

1α,25-Dihydroxyvitamin D₃ (VitD₃) increases protein and gene expression of phospholipase D1 (PLD1), but not PLD2, in HaCaT human keratinocytes. We show that VitD₃ increases PLD1 gene expression through a vitamin D responsive element (VDRE) on the 5′ PLD1 promoter (−260 bp to −246 bp from exon 1). Similar results were obtained by transfecting VitD₃ receptor (VDR) into HEK293 cells, which are originally VitD₃-unresponsive. Electrophoresis mobility shift assays (EMSA) and chromatin immunoprecipitation (CHIP) assays showed that the complex of VitD₃, VDR and retinoid X receptor α (RXRα) binds to the VDRE and increases PLD1 gene expression in HaCaT cells.     

Parallel expression of alternate forms of psbA2 gene provides evidence for the existence of a targeted D1 repair mechanism in Synechocystis sp. PCC 6803

The D1 protein of Photosystem II (PSII) is recognized as the main target of photoinhibitory damage and exhibits a high turnover rate due to its degradation and replacement during the PSII repair cycle. Damaged D1 is replaced by newly synthesized D1 and, although reasonable, there is no direct evidence for selective replacement of damaged D1. Instead, it remains possible that increased turnover of D1 subunits occurs in a non-selective manner due for example, to a general up-regulation of proteolytic activity triggered during damaging environmental conditions, such as high light. To determine if D1 degradation is targeted to damaged D1 or generalized to all D1, we developed a genetic system involving simultaneous dual expression of wild type and mutant versions of D1 protein. Dual D1 strains (nS345P:eWT and nD170A:eWT) expressed a wild type (WT) D1 from ectopic and a damage prone mutant (D1-S345P, D1-D170A) from native locus on the chromosome. Characterization of strains showed that all dual D1 strains restore WT like phenotype with high PSII activity. Higher PSII activity indicates increased population of PSII reaction centers with WT D1. Analysis of steady state levels of D1 in nS345P:eWT by immunoblot showed an accumulation of WT D1 only. But, in vivo pulse labeling confirmed the synthesis of both S345P (exists as iD1) and WT D1 in the dual strain. Expression of nS345P:eWT in FtsH2 knockout background showed accumulation of both iD1 and D1 proteins. This demonstrates that dual D1 strains express both forms of D1, yet only damage prone PSII complexes are selected for repair providing evidence that the D1 degradation process is targeted towards damaged PSII complexes. Since the N-terminus has been previously shown to be important for the degradation of damaged D1, the possibility that the highly conserved cysteine 18 residue situated in the N-terminal domain of D1 is involved in the targeted repair process was tested by examining site directed mutants of this and the other cysteines of the D1 protein. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.