Profile of circulating levels of IL-1Ra, CXCL10/IP-10, CCL4/MIP-1β and CCL2/MCP-1 in dengue fever and parvovirosis

Dengue virus (DENV) and parvovirus B19 (B19V) infections are acute exanthematic febrile illnesses that are not easily differentiated on clinical grounds and affect the paediatric population. Patients with these acute exanthematic diseases were studied. Fever was more frequent in DENV than in B19V-infected patients. Arthritis/arthralgias with DENV infection were shown to be significantly more frequent in adults than in children. The circulating levels of interleukin (IL)-1 receptor antagonist (Ra), CXCL10/inducible protein-10 (IP-10), CCL4/macrophage inflammatory protein-1 beta and CCL2/monocyte chemotactic protein-1 (MCP-1) were determined by multiplex immunoassay in serum samples obtained from B19V (37) and DENV-infected (36) patients and from healthy individuals (7). Forward stepwise logistic regression analysis revealed that circulating CXCL10/IP-10 tends to be associated with DENV infection and that IL-1Ra was significantly associated with DENV infection. Similar analysis showed that circulating CCL2/MCP-1 tends to be associated with B19V infection. In dengue fever, increased circulating IL-1Ra may exert antipyretic actions in an effort to counteract the already increased concentrations of IL-1β, while CXCL10/IP-10 was confirmed as a strong pro-inflammatory marker. Recruitment of monocytes/macrophages and upregulation of the humoral immune response by CCL2/MCP-1 by B19V may be involved in the persistence of the infection. Children with B19V or DENV infections had levels of these cytokines similar to those of adult patients.     

Beneficial effects of quercetin on vincristine-induced liver injury in rats: Modulating the levels of Nrf2/HO-1, NF-kB/STAT3, and SIRT1/PGC-1α

Our experimental objective was to investigate the hepatotoxic effect of vincristine (VCR) administration in rats and determined whether combined therapy with Quercetin (Quer) ensured protection. Five groups with seven rats each were used for this purpose, and experimental groups were formulated as follows: Control group; Quer group; VCR group; VCR plus Quer 25 group; VCR plus Quer 50 group. The results showed that VCR significantly increased the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) enzymes. Besides, VCR caused considerable increases in the malondialdehyde (MDA) contents, along with significant decreases in reduced glutathione levels, superoxide dismutase, catalase, and glutathione peroxidase enzyme activities in the rat livers. Quer treatment in VCR toxicity markedly decreased the activity of ALT, AST, ALP enzymes, and MDA contents and enhanced the activities of antioxidant enzymes. The results also showed that VCR significantly increased the levels of NF-kB, STAT3, and the expression of caspase 3, Bax, and MAP LC3 and decreased the expression of Bcl2 and levels of Nrf2, HO-1, SIRT1, and PGC-1α. Compared to the VCR group, Quer treatment exhibited significantly lower levels of NF-kB, STAT3, and the expression of caspase 3, Bax, and MAP LC3, and higher levels of Nrf2, HO-1, SIRT1, and PGC-1α. In conclusion, our study demonstrated that Quer could alleviate the harmful effects of VCR via activation of NRf2/HO-1 and SIRT1/PGC-1α pathways, and via attenuation of oxidative stress, apoptosis, autophagy, and NF-kB/STAT3 pathways.     

Biosynthesis of 1α,2α,3β-trihydroxy-p-menthane by Fusicoccum amygdali

Measurement of isotope ratios in 1α,2α,3β-trihydroxy-p-menthane, which has been biosynthesized in Fusicoccum amygdali from ³H- and ¹⁴C-labelled mevalonate and in its degradation product diosphenol indicates that: (a) four tritium atoms arising from [5-³H₂, 2-¹⁴C]MVA are retained, one more than suggested from the hydroxylation pattern, (b) menth-2-ene-1-ol is generated from an α-terpinyl cation through a 1,3-hydride shift and (c) trans-cleavage of an α-epoxide by hydrolysis gives 1α,2α,3β-trihydroxy-p-menthane.     

Discovery of azabenzimidazole derivatives as potent, selective inhibitors of TBK1/IKKε kinases

The design, synthesis and biological evaluation of a series of azabenzimidazole derivatives as TBK1/IKKε kinase inhibitors are described. Starting from a lead compound 1a, iterative design and SAR exploitation of the scaffold led to analogues with nM enzyme potencies against TBK1/IKKε. These compounds also exhibited excellent cellular activity against TBK1. Further structure-based design to improve selectivity over CDK2 and Aurora B resulted in compounds such as 5b-e. These probe compounds will facilitate study of the complex cancer biology of TBK1 and IKKε.     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

Synthesis and bioassay of 3-deoxy-1α-hydroxyvitamin D3, an active analog of 1α,25-dihydroxyvitamin D3

Two synthetic routes to 3-deoxy-1α-hydroxyvitamin D₃, an analog of 1α,25-dihydroxyvitamin D₃, are described. One involved the six-step conversion of 1α,2α-epoxy-6,6-ethylenedioxy-5α-cholestan-3- one to 1α-acetoxycholest-5-ene, whereas, in the second, the same intermediate was prepared from 1α-hydroxycholesterol. Conversion of the Δ⁵-sterol to the required 5,7-diene was accomplished most efficiently via 7-keto and 7-tosylhydrazone intermediates. Bioassay of 3-deoxy-1α-hydroxyvitamin D₃ in the rat establishes that the analog can fulfill all common vitamin D functions including stimulation of intestinal calcium transport, mobilization of calcium and phosphate from bone, stimulation of growth, and calcification of bone. Direct comparison indicates the compound to have $\text{1}\text{20}$ to $\text{1}\text{50}$ of the activity of 1α-hydroxyvitamin D₃, but it acts with a time course indistinguishable from the latter.     

Characterization of ω-secalin genes from rye, triticale, and a wheat 1BL/1RS translocation line

Sixty-two DNA sequences for the coding regions of omega-secalin (ω-secalin) genes have been characterized from rye (Secale cereale L.), hexaploid and octoploid triticale (× Triticosecale Wittmack), and wheat (Triticum aestivum L.) 1BL/1RS translocation line. Only 19 out of the 62 ω-secalin gene sequences were full-length open reading frames (ORFs), which can be expressed into functional proteins. The other 43 DNA sequences were pseudogenes, as their ORFs were interrupted by one or a few stop codons or frameshift mutations. The 19 ω-secalin genes have a typical primary structure, which is different from wheat gliadins. There was no cysteine residue in ω-secalin proteins, and the potential celiac disease (CD) toxic epitope (PQQP) was identified to appear frequently in the repetitive domains. The ω-secalin genes from various cereal species shared high homology in their gene sequences. The ω-secalin gene family has involved fewer variations after the integration of the rye R chromosome or whole genome into the wheat or triticale genome. The higher Ka/Ks ratio (i.e. non-synonymous to synonymous substitutions per site) in ω-secalin pseudogenes than in ω-secalin ORFs indicate that the pseudogenes may be subject to a reduced selection pressure. Based on the conserved sequences of ω-secalin genes, it will be possible to manipulate the expression of this gene family in rye, triticale, or wheat 1BL/1RS translocation lines, to reduce its negative effects on grain quality.     

Involvement of the Akt/NF-κB Pathways in the HTNV-Mediated Increase of IL-6, CCL5, ICAM-1, and VCAM-1 in HUVECs

Background

Hantaan virus (HTNV) infection causes a severe form of HFRS(hemorrhagic fever with renal syndrome)in Asia. Although HTNV has been isolated for nearly forty years, the pathogenesis of HFRS is still unknown, and little is known regarding the signaling pathway that is activated by the virus.

Methodology/Principal Findings

Cardamonin was selected as a NF-κB inhibitor, and indirect immunofluorescence assays were used to detect the effect of cardamonin on HTNV-infected HUVECs. The effect of cardamonin on the HTNV-induced phosphorylation of Akt and DNA-binding activity of NF-κB were determined using Western blot analysis and electrophoretic mobility shift assays (EMSAs), respectively. Then, flow cytometric and quantitative real-time PCR analyses were performed to quantify the expression levels of the adhesion molecules ICAM-1 and VCAM-1, and the concentrations of IL-6, IL-8, and CCL5 in HUVEC supernatants were examined using ELISA. The results showed that cardamonin did not effect the proliferation of HUVECs or the replication of HTNV in HUVECs. Instead, cardamonin inhibited the phosphorylation of Akt and nuclear transduction of NF-κB and further reduced the expression of the adhesion molecules ICAM-1 and VCAM-1 in HTNV-infected HUVECs. Cardamonin also inhibited the secretion of IL-6 and CCL5, but not IL-8.

Conclusion/Significance

HTNV replication may not be dependent upon the ability of the virus to activate NF-κB in HUVECs. The Akt/NF-κB pathways may be involved in the pathogenesis of HFRS; therefore, cardamonin may serve as a potential beneficial agent for HFRS therapy.     

Backbone resonances assignment of 19 kDa CD1 domain of human mitotic checkpoint serine/threonine-protein kinase, Bub1

Bub1 is an evolutionarily conserved mitotic checkpoint control protein that is present in diverse organisms including yeast and humans. Bub1 is a serine/threonine protein kinase and is required for recruitment of Mad1, Mad2, Bub3, and CENP-E to kinetochores (Sharp-Baker and Chen in J Cell Biol 153:1239–1250, 2001). The evolutionarily conserved amino acid region in the N-terminus has been called as the CD1 domain. To clarify the action mechanism of Bub1 in controlling check point signals, we initiated an NMR structure determination of the Bub1 CD1 domain. Here, we report the sequence-specific backbone resonance assignments of CD1 domain of human Bub1 (hBub1CD1).     

Chemical synthesis of [1β-3H]1α,25-dihydroxyvitamin D3 and [1α-3H]1β,25-dihydroxyvitamin D3: Biological activity of 1β,25-dihydroxyvitamin D3

The simple three-step preparation of [1β-³H]1α,25-dihydroxyvitamin D₃ and [1α-³H]1β,25-dihydroxyvitamin D₃ from 1α,25-dihydroxyvitamin D₃ is described. In the rat, 1β,25-dihydroxyvitamin D₃, when compared with its α-epimer, did not stimulate intestinal calcium transport or bone calcium mobilization at doses 1000-fold higher than the doses of the natural hormone, 1α,25-dihydroxyvitamin D₃.     

Transendothelial Migration of Monocytes in Rat Aorta: Distribution of F-actin, α-Catenin,LFA-1, and PECAM-1

To determine changes in the distribution of cell adhesion molecules during diapedesis of monocytes in situ, we labeled aortic whole mounts from hypercholesterolemic rats with Texas red-phalloidin and antibodies to LFA-1, PECAM-1, or α-catenin, and analyzed them by laser scanning confocal microscopy. Monocytes transmigrated through circular openings (transmigration passages) formed by pseudopodia that penetrated between adjacent en-dothelial cells. Transmigrating monocytes remained spherical above the endothelium, while spreading beneath it. The transmigration passage was lined by F-actin and partially by α-catenin, suggesting cadherin-mediated heterotypic interactions. LFA-1 was present in clusters at the monocyte cell surface throughout diapedesis, but was concentrated at the margin of the transmigration passage. PECAM-1 was enriched in the endothelial contact regions where the monocytes transmigrated. PECAM-1 was barely detectable in monocytes before and after diapedesis, but appeared during diapedesis at the cell surface in the parts of the monocyte located above the endothelium. PECAM-1 was enriched near the endothelial cell-cell junctions, but was not detected in parts that spread beneath the endothelium. Our results suggest a major role for LFA-1 during diapedesis and reveal dynamic changes in the distribution of PECAM-1, the actin cytoskeleton, and α-catenin during monocyte diapedesis in situ.     

Oral formulation of a novel antiviral agent,PG301029, in a mixture of Gelucire 44/14 and DMA (2∶1, wt/wt)

To develop an oral formulation for PG301029, a novel potent agent for the treatment of Hepatitis C virus infection, that not only has very low aqueous solubility but also degrades rapidly in water. The solubility of PG301029 was determined in water, various aqueous media, and several neat organic solvents. The stability of PG301029 was monitored at room temperature in buffess for 4 days, and in several neat organic solvents for up to 8 mo. Drug concentrations were measured by high-performance liquid chromatography (HPLC). Based on solubility and stability data, Gelucire 44/14 and DMA (N,N-dimethylacetamide) at a weight ratio of 2 to 1 were chosen as the formulation vehicle. After the vehicle was prepared, it was maintained in liquid form at ∼40°C until the PG301029 was dissolved. The final formulation product was a semisolid at room temperature. The bioavailability of the formulation was tested on 4 female BALB/c mice. PG301029 is insoluble in all tested aqueous media, while its solubility is promising in DMA. This compound is unstable in aqueous media and some organic solvents; however, it is stable in DMA. This proposed formulation is able to hold up to 10 mg/mL of drug and is stable at 4°C. The shelf life for this formulation stored at 4°C is extrapolated to be greater than 4 years. This formulation dramatically increases the bioavailability of PG301029. This nonaqueous formulation solves the stability, solubility, and bioavailability problems for PG301029. This semisolid formulation can easily be incorporated into soft elastic capsules.     

Biophysical studies on molecular mechanism of abortificient action of prostaglandins—IV. Conformation energy calculations on PGA1, PGB1 and PGE1

This paper reports the conformation energy (CE) calculations on three forms of prostaglandins (PGs) PGA₁, PGB₁ and PGE₁ on the basis of the empirical potential energy functions, for the simultaneous rotations around C₇–C₈ (θ), C₁₂–C₁₃ (β) and C₁₄–C₁₅ (β) bonds [Fig. 1(a)]. The isoenergy contours plotted for θβ rotations for the different β values show the existence of two low energy regions for thg equal to about 90° and 240° in all the three cases. The absolute minimum was obtained for thg = 240° and almost coincided with the crystallographic conformation for PGE₁ and PGA₁. In the case of PGB₁ series of low energy conformations were obtained with the thg values equal to about 90° and 270°, but none of them coincided with the observed crystallographic conformation. The paper discusses the comparison of the different low energy conformations in these three molecules, their biological relevance and the cause of disagreement in the case of PGB₁ with the crystallographic data.     

Biochemical and kinetic characterization of the multifunctional β-glucosidase/β-xylosidase/α-arabinosidase,Bgxa1

Functional screening of a metagenomic library constructed with DNA extracted from the rumen contents of a grass/hay-fed dairy cow identified a protein, β-glucosidase/β-xylosidase/α-arabinosidase gene (Bgxa1), with high levels of β-glucosidase activity. Purified Bgxa1 was highly active against p-nitrophenyl-β-d-glucopyranoside (pNPG), cellobiose, p-nitrophenyl-β-d-xylopyranoside (pNPX) and p-nitrophenyl-α-d-arabinofuranoside (pNPAf), suggesting it is a multifunctional β-glucosidase/β-xylosidase/α-arabinosidase. Kinetic analysis of the protein indicated that Bgxa1 has the greatest catalytic activity against pNPG followed by pNPAf and pNPX, respectively. The catalytic efficiency of β-glucosidase activity was 100× greater than β-xylosidase or α-arabinosidase. The pH and temperature optima for the hydrolysis of selected substrates also differed considerably with optima of pH 6.0/45 °C and pH 8.5/40 °C for pNPG and pNPX, respectively. The pH dependence of pNPAf hydrolysis displayed a bimodal distribution with maxima at both pH 6.5 and pH 8.5. The enzyme exhibited substrate-dependent responses to changes in ionic strength. Bgxa1 was highly stable over a broad pH range retaining at least 70 % of its relative catalytic activity from pH 5.0–10.0 with pNPG as a substrate. Homology modelling was employed to probe the structural basis of the unique specificity of Bgxa1 and revealed the deletion of the PA14 domain and insertions in loops adjacent to the active site. This domain has been found to be an important determinant in the substrate specificity of proteins related to Bgxa1. It is postulated that these indels are, in part, responsible for the multifunctional activity of Bgxa1. Bgxa1 acted synergistically with endoxylanase (Xyn10N18) when incubated with birchwood xylan, increasing the release of reducing sugars by 168 % as compared to Xyn10N18 alone. Examination of Bgxa1 and Xyn10N18 synergy with a cellulase for the saccharification of alkali-treated straw revealed that synergism among the three enzymes enhanced sugar release by 180 % as compared to cellulase alone. Our results suggest that Bgxa1 has a number of properties that make it an interesting candidate for the saccharification of lignocellulosic material.     

Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, II: Lifetimes of LFA-1 Bonds Under Force in Leukocyte Signaling

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant α_Lβ₂ immobilized on microspheres and β₂ integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from β₂ integrin throughout the course of each experiment. In our companion article I, we demonstrate the assay using results from tests of a monovalent ICAM-1 probe against recombinant α_Lβ₂ on microspheres in millimolar solutions of divalent cations (Ca²⁺, Mg²⁺, Mn²⁺). In this article, we examine the impact of inside-out and outside-in signaling in neutrophils on the lifetimes and mechanical strengths of ICAM-1 bonds to β₂ integrin on the cell surface. Even though ICAM-1 bonds to recombinant α_Lβ₂ on microspheres in Mg²⁺ or Mn²⁺ can live for long periods of time under slow pulling, here we show that stimulation of neutrophils in Mg²⁺ plus the chemokine IL-8 (i.e., inside-out signaling) induces several-hundred-fold longer lifetimes for ICAM-1 attachments to LFA-1, creating strong bonds at very slow pulling speeds where none are perceived in Mg²⁺ or Mn²⁺ alone. Similar changes are observed with outside-in signaling, i.e., long lifetimes and increased bond strength also occur when neutrophils are bound with the activating (anti-CD18) monoclonal 240Q. Limiting our investigation to rare events using very dilute ICAM-1 probes, we show that although the prolonged lifetimes of cell surface attachments for both inside-out and outside-in signaling exhibit single-bond-like statistics for dissociation under force, they are consistent with a tightly coupled dimeric ICAM-1 interaction with a pair of LFA-1 heterodimers.