Spectroscopic properties of the carotenoid 3'-hydroxyechinenone in the orange carotenoid protein from the cyanobacterium Arthrospira maxima

The cyanobacterial water-soluble orange carotenoid binding protein (OCP) is an ideal system for study of the effects of protein environment on photophysical properties of carotenoids. It contains a single pigment, the carotenoid 3'-hydoxyechinenone (hECN). In this study, we focus on spectroscopic properties of hECN in solution and in the OCP, aiming to elucidate the spectroscopic effects of the carotenoid-protein interaction in the context of the function(s) of the OCP. The noncovalent binding of hECN to the OCP causes a conformational change in the hECN, leading to a prolongation of the effective conjugation length. This change is responsible for shortening of the S(1) lifetime from 6.5 ps in solution to 3.3 ps in the OCP. The conformational change and the hydrogen bonding via the carbonyl group of hECN result in stabilization of an intramolecular charge-transfer (ICT) state. No signs of the ICT state were found in hECN in solution, regardless of the solvent polarity; spectral bands in transient absorption spectra of OCP-bound hECN exhibit features typical for the ICT state. Application of global fitting analysis revealed further effects of binding hECN in the OCP. The S(1) state of hECN in the OCP decays with two time constants of 0.9 and 3.3 ps. Modeling of the excited-state processes suggests that these two components are due to two populations of hECN in the OCP that differ in the hydrogen bonding via the carbonyl group. These results support the hypothesis that the OCP functions as a photoprotective shield under excess light. Mechanistically, the broadening of the hECN absorption spectrum upon binding to OCP enhances filtering effect of hECN. Furthermore, the binding-induced conformational change and activation of the ICT state that leads to a shortening of hECN lifetime effectively makes the protein-bound hECN a more effective energy dissipator.     

Conversion of the Escherichia coli cytochrome b562 to an archetype cytochrome b: a mutant with bis-histidine ligation of heme iron

A mutant of the Escherichia coli cytochrome b(562) has been created in which the heme-ligating methionine (Met) at position 7 has been replaced with a histidine (His) (M7H). This protein is a double mutant that also has the His 63 to asparagine (H63N) mutation, which removes a solvent-exposed His. While the H63N mutation has no measurable effect on the cytochrome, the M7H mutation converts the atypical His/Met heme ligation in cytochrome b(562) to the classic cytochrome b-type bis-His ligation. This mutation has little effect on the K(d) of heme binding but significantly reduces the chemical and thermal stability of the mutant cytochrome relative to the wild type (wt). Both proteins have similar absorbance (Abs) and electron paramagnetic resonance (EPR) properties characteristic of 6-coordinate low-spin heme. The Abs spectra of the oxidized and reduced bis-His cytochrome are slightly blue-shifted relative to the wt, and the alpha Abs band of ferrous M7H mutant is unusually split. The M7H mutation decreases the midpoint potential of the bound heme by 260 mV at pH 7 and considerably alters the pH dependence of the E(m), which becomes dominated by a single pK(red) = 6.8.     

TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) is a genetic disease characterized by severe neutrophil-dominated airway inflammation. An important cause of inflammation in CF is Pseudomonas aeruginosa infection. We have evaluated the importance of a number of P. aeruginosa components, namely lipopeptides, LPS, and unmethylated CpG DNA, as proinflammatory stimuli in CF by characterizing the expression and functional activity of their cognate receptors, TLR2/6 or TLR2/1, TLR4, and TLR9, respectively, in a human tracheal epithelial line, CFTE29o(-), which is homozygous for the DeltaF508 CF transmembrane conductance regulator mutation. We also characterized TLR expression and function in a non-CF airway epithelial cell line 16HBE14o(-). Using RT-PCR, we demonstrated TLR mRNA expression. TLR cell surface expression was assessed by fluorescence microscopy. Lipopeptides, LPS, and unmethylated CpG DNA induced IL-8 and IL-6 protein production in a time- and dose-dependent manner. The CF and non-CF cell lines were largely similar in their TLR expression and relative TLR responses. ICAM-1 expression was also up-regulated in CFTE29o(-) cells following stimulation with each agonist. CF bronchoalveolar lavage fluid, which contains LPS, bacterial DNA, and neutrophil elastase (a neutrophil-derived protease that can activate TLR4), up-regulated an NF-kappaB-linked reporter gene and increased IL-8 protein production in CFTE29o(-) cells. This effect was abrogated by expression of dominant-negative versions of MyD88 or Mal, key signal transducers for TLRs, thereby implicating them as potential anti-inflammatory agents for CF.     

Absence of thrombin-activatable fibrinolysis inhibitor protects against sepsis-induced liver injury in mice

Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as carboxypeptidase R, has been implicated as an important negative regulator of the fibrinolytic system. In addition, TAFI is able to inactivate inflammatory peptides such as complement factors C3a and C5a. To determine the role of TAFI in the hemostatic and innate immune response to abdominal sepsis, TAFI gene-deficient (TAFI-/-) and normal wild-type mice received an i.p. injection with Escherichia coli. Liver TAFI mRNA and TAFI protein concentrations increased during sepsis. In contrast to the presumptive role of TAFI as a natural inhibitor of fibrinolysis, TAFI-/- mice did not show any difference in E. coli-induced activation of coagulation or fibrinolysis, as measured by plasma levels of thrombin-anti-thrombin complexes and D-dimer and the extent of fibrin depositions in lung and liver tissues. However, TAFI-/- mice were protected from liver necrosis as indicated by histopathology and clinical chemistry. Furthermore, TAFI-/- mice displayed an altered immune response to sepsis, as indicated by an increased neutrophil recruitment to the peritoneal cavity and a transiently increased bacterial outgrowth together with higher plasma TNF-alpha and IL-6 levels. These data argue against an important part for TAFI in the regulation of the procoagulant-fibrinolytic balance in sepsis and reveals a thus far unknown role of TAFI in the occurrence of hepatic necrosis.     

The devil in the details: the emerging role of anticitrulline autoimmunity in rheumatoid arthritis

Rheumatoid arthritis is a chronic inflammatory autoimmune disease of unknown cause. The immune response against citrullinated Ags has recently become the prime suspect for disease pathogenesis. Immunity against citrullinated Ags is thought to play a pivotal role in the disease for several reasons: 1) citrullinated Ags are expressed in the target organ, the inflamed joint; 2) anti-citrullinated protein Abs are present before the disease becomes manifest; and 3) these Abs are highly specific for rheumatoid arthritis. In this review, data from clinical, genetic, biochemical, and animal studies is combined to create a profile of this remarkable autoantibody response. Moreover, a model is proposed of how the anti-citrullinated proteins response is generated and how it could eventually lead to chronic inflammation.     

Viral inhibition of IL-1- and neutrophil elastase-induced inflammatory responses in bronchial epithelial cells

Previously, we elucidated the intracellular mechanisms by which neutrophil elastase (NE) up-regulates inflammatory gene expression in bronchial epithelial cells. In this study, we examine the effects of both IL-1 and NE on inflammatory gene expression in 16HBE14o- bronchial epithelial cells and investigate approaches to abrogate these inflammatory responses. IL-1 induced IL-8 protein production in time- and dose-dependent fashions, an important observation given that IL-8 is a potent neutrophil chemoattractant and a key inflammatory mediator. IL-1 and NE were shown to activate the p38 MAPK pathway in 16HBE14o- cells. Western blot analysis demonstrated IL-1R-associated kinase 1 (IRAK-1) degradation in response to stimulation with both IL-1 and NE. In addition, the expression of dominant negative IRAK-1 (IRAK-1delta), IRAK-2delta, or IRAK-4delta inhibited IL-1- and NE-induced NF-kappaB-linked reporter gene expression. Dominant negative versions of the intracellular adaptor proteins MyD88 (MyD88delta) and MyD88 adaptor-like (Mal P/H) abrogated NE-induced NF-kappaB reporter gene expression. In contrast, only MyD88delta was found to inhibit IL-1-induced NF-kappaB reporter activity. We also investigated the vaccinia virus proteins, A46R and A52R, which have been shown to antagonize IL-1 signaling. Transfection with A46R or A52R cDNA inhibited IL-1- and NE-induced NF-kappaB and IL-8R gene expression and IL-8 protein production in primary and transformed bronchial epithelial cells. Furthermore, cytokine array studies demonstrated that IL-1 and NE can up-regulate the expression of IL-6, oncostatin M, epithelial cell-derived neutrophil activating peptide-78, growth-related oncogene family members, vascular endothelial growth factor, and GM-CSF, with induction of these proteins inhibited by the viral proteins. These findings identify vaccinia virus proteins as possible therapeutic agents for the manifestations of several inflammatory lung diseases.     

Effect of foliar disease on the epiphytic yeast communities of creeping bentgrass and tall fescue

The effect of mechanical wounding or foliar diseases caused by Sclerotinia homoeocarpa or Rhizoctonia solani on the epiphytic yeast communities on creeping bentgrass and tall fescue were determined by leaf washing and dilution plating. Total yeast communities on healthy bentgrass and tall fescue leaves ranged from 7.9 x 103 to 1.4 x 105 CFU.cm-2 and from 2.4 x 103 to 1.6 x 104 CFU.cm-2, respectively. Mechanically wounded leaves (1 of 2 trials) and leaves with disease lesions (11 of 12 trials) supported significantly larger communities of phylloplane yeasts. Total yeast communities on S. homoeocarpa infected or R. solani infected bentgrass leaves were 3.6-10.2 times and 6.2-6.4 times larger, respectively, than the communities on healthy leaves. In general, healthy and diseased bentgrass leaves supported larger yeast communities than healthy or diseased tall fescue leaves. We categorized the majority of yeasts as white-pigmented species, including Cryptococcus laurentii, Cryptococcus flavus, Pseudozyma antarctica, Pseudozyma aphidis, and Pseudozyma parantarctica. The percentage of pink yeasts in the total yeast community ranged from 2.6% to 9.9% on healthy leaves and increased to 32.0%-44.7% on S. homoeocarpa infected leaves. Pink-pigmented yeasts included Rhodotorula glutinis, Rhodotorula mucilaginosa, Sakaguchia dacryoidea, and Sporidiobolus pararoseus. Foliar disease significantly affected community size and composition of epiphytic yeasts on bentgrass and tall fescue.     

Spatial genome organization

The linear sequence of genomes exists within the three-dimensional space of the cell nucleus. The spatial arrangement of genes and chromosomes within the interphase nucleus is nonrandom and gives rise to specific patterns. While recent work has begun to describe some of the positioning patterns of chromosomes and gene loci, the structural constraints that are responsible for nonrandom positioning and the relevance of spatial genome organization for genome expression are unclear. Here we discuss potential functional consequences of spatial genome organization and we speculate on the possible molecular mechanisms of how genomes are organized within the space of the mammalian cell nucleus.     

The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs

The formation of genomic RNA dimers during the retroviral life cycle is essential for optimal viral replication and infectivity. The sequences and RNA structures responsible for this interaction are located in the untranslated 5' leader RNA, along with other cis-acting signals. Dimer formation occurs by specific interaction between identical structural motifs. It is believed that an initial kissing hairpin forms following self-recognition by autocomplementary RNA loops, leading to formation of an extended stable duplex. The dimerization initiation site (DIS) of the deltaretrovirus human T-cell lymphotropic virus type-I (HTLV-I) has been previously localized to a 14-nucleotide sequence predicted to contain an RNA stem loop. Biochemical probing of the monomeric RNA structure using RNAse T1, RNAse V1, RNAse U2, lead acetate, and dimethyl sulfate has led to the generation of the first structural map of the HTLV-I DIS. A comprehensive data set of individual nucleotide modifications reveals that the structural motif responsible for HTLV-I RNA dimerization forms a trinucleotide RNA loop, unlike any previously characterized retroviral dimerization motif. Molecular modeling demonstrates that this can be formed by an unusual C:synG base pair closing the loop. Comparative phylogeny indicates that such a motif may also exist in other deltaretroviruses.