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Dehydrins are intrinsically disordered (unstructured) proteins that are expressed in plants experiencing stressful conditions such as drought or low temperature. Dehydrins are typically found in the cytosol and nucleus, but also a...
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Dehydrins are intrinsically disordered (unstructured) proteins that are expressed in plants experiencing stressful conditions such as drought or low temperature. Dehydrins are typically found in the cytosol and nucleus, but also associate with chloroplasts, mitochondria, and the plasma membrane. Although their role is not completely understood, it has been suggested that they stabilize proteins or membrane structures during environmental stress, the latter association mediated by formation of amphipathic α-helices by conserved regions called the K-segments. Thellungiella salsuginea is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We have cloned and expressed in Escherichia coli two dehydrins from this plant, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). Here, we show using transmission-Fourier transform infrared (FTIR) spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. Moreover, this induced folding is enhanced at low temperatures, lending credence to the hypothesis that dehydrins stabilize plant outer and organellar membranes in conditions of cold.
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The genetics of gene expression in recombinant inbred lines (RILs) can be mapped as expression quantitative trait loci (eQTLs). So-called "genetical genomics" studies have identified locally acting eQTLs (cis-eQTLs) for genes that...
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The genetics of gene expression in recombinant inbred lines (RILs) can be mapped as expression quantitative trait loci (eQTLs). So-called "genetical genomics" studies have identified locally acting eQTLs (cis-eQTLs) for genes that show differences in steady-state RNA levels. These studies have also identified distantly acting master-modulatory trans-eQTLs that regulate tens or hundreds of transcripts (hotspots or transbands). We expand on these studies by performing genetical genomics experiments in two environments in order to identify trans-eQTL that might be regulated by developmental exposure to the neurotoxin lead. Flies from each of 75 RIL were raised from eggs to adults on either control food (made with 250 microM sodium acetate), or lead-treated food (made with 250 microM lead acetate, PbAc). RNA expression analyses of whole adult male flies (5-10 days old) were performed with Affymetrix DrosII whole genome arrays (18,952 probesets). Among the 1389 genes with cis-eQTL, there were 405 genes unique to control flies and 544 genes unique to lead-treated ones (440 genes had the same cis-eQTLs in both samples). There are 2396 genes with trans-eQTL which mapped to 12 major transbands with greater than 95 genes. Permutation analyses of the strain labels but not the expression data suggests that the total number of eQTL and the number of transbands are more important criteria for validation than the size of the transband. Two transbands, one located on the 2nd chromosome and one on the 3rd chromosome, co-regulate 33 lead-induced genes, many of which are involved in neurodevelopmental processes. For these 33 genes, rather than allelic variation at one locus exerting differential effects in two environments, we found that variation at two different loci are required for optimal effects on lead-induced expression.
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The similarity of an intranasal salmon calcitonin (sCT) employing chlorobutanol as preservative (Calcitonin Salmon Nasal Spray) was compared to the reference listed drug (RLD) employing benzalkonium chloride as preservative (Miaca...
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The similarity of an intranasal salmon calcitonin (sCT) employing chlorobutanol as preservative (Calcitonin Salmon Nasal Spray) was compared to the reference listed drug (RLD) employing benzalkonium chloride as preservative (Miacalcin Nasal Spray). Various orthogonal methods assessed peptide structuring, dynamics, and aggregation state. Mass spectrometry, amino acid analysis, and N-terminal sequencing all demonstrated similarity in primary structure. Near- and far-UV circular dichroism (CD) data supported similarity in secondary and tertiary sCT structure. Nuclear magnetic resonance studies further supported similarity of three-dimensional structure and molecular dynamics of the peptide. Other methods, such as sedimentation velocity and size exclusion chromatography, demonstrated similarity in peptide aggregation state. These latter methods, in addition to reversed phase chromatography, were also employed for monitoring stability under forced degradation, and at the end of recommended shelf storage and patient use conditions. In all cases and for all methodologies employed, similarity to the RLD was observed with respect to extent of aggregation and other degradation processes. Finally, ELISA and bioassay data demonstrated similarity in biological properties. These investigations comprehensively demonstrate physicochemical similarity of Calcitonin Salmon Nasal Spray and the RLD, and should prove a useful illustration to pharmaceutical scientists developing alternative and/or generic peptide or protein products.
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Myocyte enhancer factor 2 (MEF2) regulates specific gene expression in diverse developmental programs and adaptive responses. MEF2 recognizes DNA and interacts with transcription cofactors through a highly conserved N-terminal dom...
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Myocyte enhancer factor 2 (MEF2) regulates specific gene expression in diverse developmental programs and adaptive responses. MEF2 recognizes DNA and interacts with transcription cofactors through a highly conserved N-terminal domain referred to as the MADS-box/MEF2 domain. Here we present the crystal structure of the MADS-box/MEF2 domain of MEF2A bound to DNA. In contrast to previous structural studies showing that the MEF2 domain of MEF2A is partially unstructured, the present study reveals that the MEF2 domain participates with the MADS-box in both dimerization and DNA binding as a single domain. The sequence divergence at and immediately following the C-terminal end of the MEF2 domain may allow different MEF2 dimers to recognize different DNA sequences in the flanking regions. The current structure also suggests that the ligand-binding pocket previously observed in the Cabin1-MEF2B-DNA complex and the HDAC9 (histone deacetylase 9)-MEF2B-DNA complex is not induced by cofactor binding but rather preformed by intrinsic folding. However, the structure of the ligand-binding pocket does undergo subtle but significant conformational changes upon cofactor binding. On the basis of these observations, we generated a homology model of MEF2 bound to a myocardin family protein, MASTR, that acts as a potent coactivator of MEF2-dependent gene expression. The model shows excellent shape and chemical complementarity at the binding interface and is consistent with existing mutagenesis data. The apo structure presented here can also serve as a target for virtual screening and soaking studies of small molecules that can modulate the function of MEF2 as research tools and therapeutic leads.
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Airway and alveolar fluid clearance is mainly governed by vectorial salt movement via apically located rate-limiting Na(+) channels (ENaC) and basolateral Na(+)/K(+)-ATPases. ENaC is regulated by a spectrum of protein kinases, i.e...
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Airway and alveolar fluid clearance is mainly governed by vectorial salt movement via apically located rate-limiting Na(+) channels (ENaC) and basolateral Na(+)/K(+)-ATPases. ENaC is regulated by a spectrum of protein kinases, i.e. protein kinase A (PKA), C (PKC), and G (PKG). However, the molecular mechanisms for the regulation of ENaC by cGMP/PKG remain to be elucidated. In the present study, we studied the pharmacological responses of native epithelial Na(+) channels in human Clara cells and human alphabetagammadelta ENaCs expressed in oocytes to cGMP. 8-pCPT-cGMP increased amiloride-sensitive short-circuit current (I(sc)) across H441 monolayers and heterologously expressed alphabetagammadelta ENaC activity in a dose-dependent manner. Similarly, 8-pCPT-cGMP (a PKGII activator) but not 8-Br-cGMP (a PKGI activator) increased amiloride-sensitive whole cell currents in H441 cells in the presence of CFTRinh-172 and diltiazem. In all cases, the cGMP-activated Na(+) channel activity was inhibited by Rp-8-pCPT-cGMP, a specific PKGII inhibitor. This was substantiated by the evidence that PKGII was the sole isoform expressed in H441 cells at the protein level. Importantly, intratracheal instillation of 8-pCPT-cGMP in BALB/c mice increased amiloride-sensitive alveolar fluid clearance by approximately 30%, consistent with the in vitro results. We therefore conclude that PKGII is an activator of lung epithelial Na(+) channels, which may expedite the resolution of oedematous fluid in alveolar sacs.
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ENMD-2076 is a novel, orally-active molecule that has been shown to have significant activity against aurora and multiple receptor tyrosine kinases. We investigated the activity of ENMD-2076 against multiple myeloma (MM) cells in ...
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ENMD-2076 is a novel, orally-active molecule that has been shown to have significant activity against aurora and multiple receptor tyrosine kinases. We investigated the activity of ENMD-2076 against multiple myeloma (MM) cells in vitro and in vivo. ENMD-2076 showed significant cytotoxicity against MM cell lines and primary cells, with minimal cytotoxicity to haematopoietic progenitors. ENMD-2076 inhibited the phosphoinositide 3-kinase/AKT pathway and downregulated survivin and X-linked inhibitor of apoptosis as early as 6 h after treatment. With longer treatment (24-48 h), ENMD-2076 also inhibited aurora A and B kinases, and induced G(2)/M cell cycle arrest. In non-obese diabetic/severe combined immunodeficient mice implanted with H929 human plasmacytoma xenografts, oral treatment with ENMD-2076 (50, 100, 200 mg/kg per day) resulted in a dose-dependent inhibition of tumour growth. Immunohistochemical staining of excised tumours showed significant reduction in phospho-Histone 3 (pH3), Ki-67, and angiogenesis, and also a significant increase in cleaved caspase-3 at all dose levels compared to tumours from vehicle-treated mice. In addition, a significant reduction in p-FGFR3 was observed on Western blot. ENMD-2076 shows significant activity against MM cells in vitro and in vivo, and acts on several pathways important for myeloma cell growth and survival. These results provide preclinical rationale for clinical investigation of ENMD-2076 in MM.
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RADARADARADARADA (RADA16-I) peptide, consisting of 16 alternating hydrophobic and hydrophilic (also alternating negative and positive charges) amino acids, forms extremely stable beta-pleated sheet structure and then self-assemble...
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RADARADARADARADA (RADA16-I) peptide, consisting of 16 alternating hydrophobic and hydrophilic (also alternating negative and positive charges) amino acids, forms extremely stable beta-pleated sheet structure and then self-assembles into nanofibers to produce high-order interwoven nanofiber scaffold hydrogel. To investigate its therapeutic effects, a burn model of partial thickness-deep dermal injury (the deep second degree burns) was performed at the dorsal skin of female Sprague-Dawley rats with an electrical scalding machine. The wounds treated with either RADA16-I or control materials were carefully examined at morphological, histological and cellular levels. We found that RADA16-I can advance the time of eschar appearance and the time of eschar disappearance both by 3-5 days, and speed up wound contraction by 20-30% compared with contrast groups (chitosan, poly(DL)-lactic acid (PDLA), collagen I and the blank) without obvious edema. Immunohistochemical studies showed that both FGF and EGF were obviously expressed in nascent tissue such as epidermis and glands when wounds were treated with the RADA16-I after injury. When peptide stock solution was diluted from 10 to 0.17 mg/mL, atomic force microscopy (AFM) observation showed that the shape of peptide nanofibers changed from the globular-pieces-clustered filaments with 4.8 +/- 0.38 nm in height, 61.6 +/- 6.10 nm in width and 708 +/- 80.2 nm in length, to general filaments with 1.4 +/- 0.36 nm, 17.5 +/- 1.13 nm and 1108 +/- 184 nm. The nanofiber surface porosity gradually decreased from 49-70% to 12-28%. These characteristics contribute to wound healing by offering an "ideal dressing" moist healing microenvironment and a nanofiber 3D scaffold. These results suggest that the self-assembling peptide might be a promising wound dressing with being simple, effective, and affordable.
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The transcription factor Foxp3 plays a key role in CD4(+)CD25(+) regulatory T (Treg) cell function. A correlation has been shown between survival and the frequency of tumor-infiltrating Foxp3-positive Treg cells in cancer patients...
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The transcription factor Foxp3 plays a key role in CD4(+)CD25(+) regulatory T (Treg) cell function. A correlation has been shown between survival and the frequency of tumor-infiltrating Foxp3-positive Treg cells in cancer patients. However, few studies have characterized the regulation of Foxp3 expression and function in Treg cells, which are known to comprise distinct subsets, with different roles in the complex tumor microenvironment. Here, we show that significantly more Foxp3-positive Treg cells accumulated in gastric tumors. In addition, we found increased expression of Foxp3 protein per cell in tumor-infiltrating Treg cells. Moreover, elevated Foxp3 expression in tumor-infiltrating Treg cells was associated with the TNM stage in gastric cancer patients. Importantly, further investigation within the tumor microenvironment showed that expression of Foxp3 in Treg cells correlated with expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). Furthermore, Treg cells with higher levels of Foxp3 were able to suppress the proliferation of autologous CD4(+)CD25(-) T cells. The suppression of the effector T-cell response was reversed by COX inhibitors and PGE(2) receptor-specific antagonists. Our data demonstrate a mechanism by which tumor-infiltrating Treg cells with increased Foxp3 expression can mediate immune suppression via COX-2/PGE(2) production in the gastric cancer microenvironment. Thus, we provide new insights into overcoming regulatory T-cell activity, which may be beneficial for the treatment of human gastric cancer.
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BACKGROUND: Because of the rapid change in lifestyle in China, there is concern that diabetes may become epidemic. We conducted a national study from June 2007 through May 2008 to estimate the prevalence of diabetes among Chinese ...
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BACKGROUND: Because of the rapid change in lifestyle in China, there is concern that diabetes may become epidemic. We conducted a national study from June 2007 through May 2008 to estimate the prevalence of diabetes among Chinese adults. METHODS: A nationally representative sample of 46,239 adults, 20 years of age or older, from 14 provinces and municipalities participated in the study. After an overnight fast, participants underwent an oral glucose-tolerance test, and fasting and 2-hour glucose levels were measured to identify undiagnosed diabetes and prediabetes (i.e., impaired fasting glucose or impaired glucose tolerance). Previously diagnosed diabetes was determined on the basis of self-report. RESULTS: The age-standardized prevalences of total diabetes (which included both previously diagnosed diabetes and previously undiagnosed diabetes) and prediabetes were 9.7% (10.6% among men and 8.8% among women) and 15.5% (16.1% among men and 14.9% among women), respectively, accounting for 92.4 million adults with diabetes (50.2 million men and 42.2 million women) and 148.2 million adults with prediabetes (76.1 million men and 72.1 million women). The prevalence of diabetes increased with increasing age (3.2%, 11.5%, and 20.4% among persons who were 20 to 39, 40 to 59, and > or = 60 years of age, respectively) and with increasing weight (4.5%, 7.6%, 12.8%, and 18.5% among persons with a body-mass index [the weight in kilograms divided by the square of the height in meters] of < 18.5, 18.5 to 24.9, 25.0 to 29.9, and > or = 30.0, respectively). The prevalence of diabetes was higher among urban residents than among rural residents (11.4% vs. 8.2%). The prevalence of isolated impaired glucose tolerance was higher than that of isolated impaired fasting glucose (11.0% vs. 3.2% among men and 10.9% vs. 2.2% among women). CONCLUSIONS: These results indicate that diabetes has become a major public health problem in China and that strategies aimed at the prevention and treatment of diabetes are needed.
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Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mediate the development of numerous inflammatory lung diseases. Since IL-1beta is typically activated in situations where TNF-alpha is produced, it was hypot...
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Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mediate the development of numerous inflammatory lung diseases. Since IL-1beta is typically activated in situations where TNF-alpha is produced, it was hypothesized that IL-1beta alters TNF-alpha-induced proinflammatory epithelial cell function by altering TNF receptor shedding and surface abundance. In this study, the impact of IL-1beta on TNF-alpha-mediated chemokine production as well as TNF receptor surface expression and shedding were investigated from mouse pulmonary epithelial cells (MLE-15). Interleukin-1beta rapidly and persistently enhanced soluble and surface TNFR2. These effects were dependent on TNFR1 expression. TNFR2 small-interfering RNA (siRNA) shifted IL-1beta responses, significantly increasing surface and shed TNFR1 implying IL-1beta selectively modifies TNF receptors depending on cellular receptor composition. mRNA expression of both receptors was unaltered by IL-1beta up to 24 h or in combination with TNF-alpha indicating effects were post-transcriptional. Interleukin-1beta pretreatment enhanced TNF-alpha-induced macrophage inflammatory protein (MIP)-2 and KC mRNA expression as well as MIP-2 and KC protein levels at the same time point analyzed. Experiments utilizing siRNA against the TNF receptors and a TNFR1 neutralizing antibody demonstrated TNF-alpha induced MIP-2 through TNFR1, whereas both receptors may have contributed to KC production. These data suggest IL-1beta modulates TNF-alpha-mediated inflammatory lung diseases by enhancing epithelial cell TNF receptor surface expression.
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