Immune regulation of bone loss by Th17 cells

A significant macrophage and T-cell infiltrate commonly occurs in inflammatory joint conditions such as rheumatoid arthritis that have significant bone destruction. Cytokines produced by activated macrophages and T cells are implicated in arthritis pathogenesis and are involved in osteoclast-mediated bone resorption. The scope of the present review is to analyze current knowledge and to provide a better understanding of how macrophage-derived factors promote the differentiation of a novel T-helper subset (Th17) that promotes osteoclast formation and activation.     

The Regulation and Function of Histone Methylation

Histones are wrapped around by genomic DNA to form nucleosomes which are the basic units of chromatin. In eukaryotes histones undergo various covalent modifications such as methylation, phosphorylation, acetylation, ubiquitination and ribosylation. Histone modifications play a fundamental role in the epigenetic regulation of gene expression in multicellular eukaryotes. Histone methylation is one of the most important modifications occurring on Lysine (K) and Arginine (R) residues of histones, dynamically regulated by histone methyltransferases and demethylases. Identifications of such histone modification enzymes and to study how they work are the most fundamental questions needs to be answered. Uncovering the regulation and functions of the various histone methylation enzymes will help us to better understand the epigenetic code. This review summarizes the regulation of histone methyltransferases activity, the recruitment of methyltransferases and the distribution patterns and function of histone methylations.     

Independence of stem and leaf hydraulic traits in six Euphorbiaceae tree species with contrasting leaf phenology

Hydraulic traits and hydraulic-related structural properties were examined in three deciduous (Hevea brasiliensis, Macaranga denticulate, and Bischofia javanica) and three evergreen (Drypetes indica, Aleurites moluccana, and Codiaeum variegatum) Euphorbiaceae tree species from a seasonally tropical forest in south-western China. Xylem water potential at 50% loss of stem hydraulic conductivity (P50_stem) was more negative in the evergreen tree, but leaf water potential at 50% loss of leaf hydraulic conductivity (P50_leaf) did not function as P50_stem did. Furthermore, P50_stem was more negative than P50_leaf in the evergreen tree; contrarily, this pattern was not observed in the deciduous tree. Leaf hydraulic conductivity overlapped considerably, but stem hydraulic conductivity diverged between the evergreen and deciduous tree. Correspondingly, structural properties of leaves overlapped substantially; however, structural properties of stem diverged markedly. Consequently, leaf and stem hydraulic traits were closely correlated with leaf and stem structural properties, respectively. Additionally, stem hydraulic efficiency was significantly correlated with stem hydraulic resistance to embolism; nevertheless, such a hydraulic pattern was not found in leaf hydraulics. Thus, these results suggest: (1) that the evergreen and deciduous tree mainly diverge in stem hydraulics, but not in leaf hydraulics, (2) that regardless of leaf or stem, their hydraulic traits result primarily from structural properties, and not from leaf phenology, (3) that leaves are more vulnerable to drought-induced embolism than stem in the evergreen tree, but not always in the deciduous tree and (4) that there exists a trade-off between hydraulic efficiency and safety for stem hydraulics, but not for leaf hydraulics.     

X-Ray Crystal Structure of a Highly Functionalized Thiophene as a New Backbone Amide Linker for Solid-phase Peptide Synthesis. Relationship between Crystal Structure and Reactivity

The development of new linkers (handles) for solid-phase synthesis provides new chemical opportunities for peptide synthesis. To understand the chemical properties of a recently developed backbone amide linker from a structural perspective, the crystal structure of S-((5-formyl-3,4-ethylenedioxy)thiophene-2-yl)-3-thiopropionic acid (T-BAL2) was studied. Specifically, we wished to address whether this highly substituted thiophene retained planarity in the aromatic ring as well as between the aromatic ring and the aldehyde carbonyl. Furthermore, we sought an explanation for the relatively low reactivity in reductive aminations of the thienylaldehyde with amines in solution and on solid phase. Based on the crystal structure of T-BAL2, the thienyl-C (aldehyde) and C–O (aldehyde) bond lengths were applied as measures for the electron-deficiency (electrophilicity) of the aldehyde and compared to similar bond lengths found in previously reported formylated homo- and hetero-aromatic systems, which show significantly higher reactivity towards imine formation. The bond lengths found in the present structure are in accordance with normal C–C single bond and C–O double bond lengths. The high similarity in aldehyde bond lengths in the present system and in the reported systems indicates similar electron distribution in these systems. The lower reactivity of the present system may therefore not be attributed to electronic factors.     

Subcellular localization of EGFR in esophageal carcinoma cell lines

Background: The EGF receptor is a therapeutic target in cancer cells, whereby mutations of EGFR and/or signalling members act as predictive markers. EGFR however also exhibits dynamic changes of subcellular localization, leading to STAT5 complex formation, nuclear translocation and induction of Aurora-A expression in squamous cancer cells. We previously described high EGFR and Aurora-A expression in esophageal cancer cells. Here, we investigated subcellular localization of EGFR and STAT5 in esophageal cancer cells. Results: Quantitative immunofluorescence analyses of four esophageal cancer cell lines reflecting esophageal squamous cell carcinomas (ESCC) and esophageal adenocarcinomas (EAC) revealed that the subcellular localization of EGFR was shifted from a membranous to cytoplasmic localization upon EGF-stimulation in OE21 (ESCC) cells. Thereby, EGFR in part co-localized with E-Cadherin. In parallel, phosphorylated STAT5-Tyr694 appeared to increase in the nucleus and to decrease at the cell membrane. In three additional cell lines, EGFR was only marginally (Kyse-410/ESCC; OE19/EAC) and weakly (OE33, EAC) detectable at the cell membrane. Partial co-localization of EGFR and E-Cadherin occurred in OE33 cells. Post EGF-stimulation, EGFR was detected in the cytoplasm, resembling endosomal compartments. Furthermore, OE19 and OE33 exhibited nuclear STAT5-Tyr694 phosphorylation upon EGF-stimulation. None of the four cell lines showed nuclear EGFR expression and localization. Conclusion: In contrast to other (squamous) cancer cells, activation of EGFR in esophageal squamous cancer cells does not result in nuclear translocation of EGFR. Still, the subcellular localization of EGFR may influence STAT5-associated signaling pathways in esophageal cancer cells and hence possibly also the responses to ErbB, respective EGFR-targeted therapies.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Comparison of enzyme-linked immunomagnetic chemiluminescence with U.S. Food and Drug Administration's Bacteriological Analytical Manual method for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7, a major foodborne pathogen, has been associated with numerous cases of foodborne illnesses. Rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. Unfortunately, many rapid methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. The previously developed method, enzyme-linked immunomagnetic chemiluminescence (ELIMCL), has been improved upon to allow for fewer incidences of false positives when used to detect E. coli O157:H7 in the presence of mixed cultures. The key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format. This work presents application of a newly semi-automated version of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline yielding detection limits of approximately 1 x 10(5) to 1 x 10(6) of live cells/mL. ELIMCL was further demonstrated to detect E. coli O157:H7 inoculated into artificially contaminated ground beef at ca. 400 CFU/g after a 5 h enrichment and about 1.5 h assay time for a total detection time of about 6.5 h. Finally, ELIMCL was compared with USFDA's Bacteriological Analytical Manual method for E. coli O157:H7 in a double-blind study. Using McNemar's treatment, the two methods were determined to be statistically similar for the detection of E. coli O157:H7 in ground beef inoculated with mixed cultures of select bacteria.     

Generation of <Emphasis Type="Italic">Vibrio anguillarum</Emphasis> Ghost by Coexpression of PhiX 174 Lysis <Emphasis Type="Italic">E</Emphasis> gene and Staphylococcal Nuclease <Emphasis Type="Italic">A</Emphasis> Gene

Vibrio anguillarum ghosts (VAG) were generated, for the first time, using a conjugation vector containing a ghost bacteria inducing cassette, pRK-λP_R-cI-Elysis, in which the expression of PhiX174 lysis gene E was controlled by the P _R /cI regulatory system of lambda phage. By scanning electron microscopy, holes ranging 80–200 nm in diameter were observed in the VAG. To avoid the presence of bacterial genomic DNA and an antibiotic resistance gene in the final VAG product, we constructed a new dual vector, pRK-λP_R-cI-E-SNA, containing the E-mediated lysis cassette and the staphylococcal nuclease A (SNA)-mediated DNA degradation cassette, and generated safety-enhanced VAG for use as a fish vaccine.     

Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to <Emphasis Type="Italic">Zucchini yellow mosaic virus</Emphasis>

Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar ‘New Hampshire Midget’ (‘NHM’) showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F₂ and 114 BC_1R progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F₂ and BC_1R populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.