Acute kidney injury (AKI) is a common complication after liver transplantation (LT) and associated with a high mortality. The renal resistive index (RI) is used to assess early renal function impairment in critical care patients. However, limited data are available concerning changes of renal RI and the development of AKI early after reperfusion. We approached to investigate the changes of renal RI and AKI after reperfusion in a rat liver transplantation model.
Rats were randomly divided into sham group or LT group. Ten rats in each group were used for the hemodynamic study and twenty for Doppler measurements during the procedure. Ten rats were sacrificed 30 min or 2 h after the reperfusion. We harvested kidneys, serum and urine for further analysis of the renal function.
The intrarenal RI increased significantly in the anhepatic stage and decreased significantly after the reperfusion in the LT group compared with sham group (P < 0.05). AKI was seen after the reperfusion in the LT group. No correlation was noted between the RI and renal function parameters 30 min after reperfusion.
The intrarenal RI increased significantly during the anhepatic stage, and decreased significantly early after the reperfusion. Intrarenal RI was unable to assess renal function in a rat liver transplantation model.
Liver transplantation; Doppler sonography; Renal function; Resistive index; Cys; NGAL
Dickkopf-1 (DKK-1), a major regulator of the Wnt pathway, plays an important role in cardiovascular disease. However, no study has evaluated the association of DKK-1 and acute coronary syndrome (ACS). We investigated this association and whether the Global Registry of Acute Coronary Events (GRACE) hospital-discharge risk score predicting major adverse cardiac events (MACE) can be improved by adding the DKK-1 value.
We enrolled 291 patients (46 with ST-segment elevation myocardial infarction [STEMI] and 245 with non-ST elevated ACS [NSTE-ACS]) who were divided into groups by tertiles of baseline plasma DKK-1 level measured by ELISA. The GRACE risk score was calculated and predictive value alone and together with DKK-1 and/or high-sensitivity C-reactive protein (hs-CRP) level were assessed, respectively.
Compared with patients with NSTE-ACS, those with STEMI had higher plasma DKK-1 level at baseline (P = 0.006). Plasma DKK-1 level was correlated with hs-CRP level (r = 0.295, P<0.001) and was greater with high than intermediate or low GRACE scores (P = 0.002 and P<0.001, respectively). We found 44 (15.1%) MACEs during a median 2-year follow-up. DKK-1 levels were higher for patients with than without events (P<0.001). The rate of MACE increased with increasing DKK-1 level (P<0.001). The area under the receiver operating characteristic curve for GRACE score with MACE was 0.524 and improved to 0.791 with the addition of hs-CRP level, 0.775 with the addition of DKK-1 level and 0.847 with both values added.
DKK-1 is an independent predictor of long-term MACE of patients with ACS. The long-term predictive ability of post-discharge GRACE score may be enhanced by adding DKK-1 level.
The purposes were to study the role of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α/nuclear factor-κB (NF-κB) signaling in matrix metalloproteinase 9 (MMP9) expression in A549 cells and to investigate the effects of lentivirus-mediated RNAi targeting of the disintegrin and metalloproteinase 17 (ADAM17) gene on LPS-induced MMP9 expression. MMP9 expression induced by LPS in A549 cells was significantly increased in a dose- and time-dependent manner (p<0.05). Pyrrolidine dithiocarbamate (PDTC) and a TNFR1 blocking peptide (TNFR1BP) significantly inhibited LPS-induced MMP9 expression in A549 cells (p<0.05). TNFR1BP significantly inhibited LPS-induced TNF-α production (p<0.05). Both PDTC and TNFR1BP significantly inhibited the phosphorylation of IκBα and expression of phosphorylation p65 protein in response to LPS (p<0.05), and the level of IκBα in the cytoplasm was significantly increased (p<0.05). Lentivirus mediated RNA interference (RNAi) significantly inhibited ADAM17 expression in A549 cells. Lentivirus-mediated RNAi targeting of ADAM17 significantly inhibited TNF-α production in the supernatants (p<0.05), whereas the level of TNF-α in the cells was increased (p<0.05). Lentiviral ADAM17 RNAi inhibited MMP9 expression, IκBα phosphorylation and the expression of phosphorylation p65 protein in response to LPS (p<0.05). PDTC significantly inhibited the expression of MMP9 and the phosphorylation of IκBα, as well as the expression of phosphorylation p65 protein in response to TNF-α (p<0.05). Lentiviral RNAi targeting of ADAM17 down-regulates LPS-induced MMP9 expression in lung epithelial cells via inhibition of TNF-α/NF-κB signaling.
Transport of newly synthesized Rhodopsin upon light stimulation in adult Drosophila photoreceptors is mediated by a Crag/Rab11-dependent vesicular trafficking process.
Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.
Animals sense light through receptors called Rhodopsins. These proteins are typically localized to stacked membranes in photoreceptors. In flies, upon light exposure, Rhodopsin undergoes conformational changes and becomes active as metarhodopsin. Metarhodopsin then initiates a signaling cascade that activates the photoreceptor cell. To deactivate the light response, metarhodopsin is converted back into Rhodopsin by absorption of another photon of light. Under certain conditions, metarhodopsin cannot be converted back to Rhodopsin, and it is then endocytosed and degraded. Rhodopsin then needs to be synthesized and delivered back to the membrane stacks. Here, we show that the Calmodulin-binding protein Crag is required for the delivery of newly made Rhodopsin to the membrane stacks. Loss of Crag leads to the accumulation of Rhodopsin in the cytosol, followed by shrinkage of membrane stack volume, and, eventually, photoreceptor cell degeneration. We also show that Crag activates a target protein, Rab11, which mediates the vesicular transport of Rhodospin to the membrane. Finally, we document that the human homolog of Crag, DENND4A, is able to rescue the loss of Crag in flies, suggesting that DENND4A functions in a similar process in vertebrates.
Summary: Images containing spatial expression patterns illuminate the roles of different genes during embryogenesis. In order to generate initial clues to regulatory interactions, biologists frequently need to know the set of genes expressed at the same time at specific locations in a developing embryo, as well as related research publications. However, text-based mining of image annotations and research articles cannot produce all relevant results, because the primary data are images that exist as graphical objects. We have developed a unique knowledge base (FlyExpress) to facilitate visual mining of images from Drosophila melanogaster embryogenesis. By clicking on specific locations in pictures of fly embryos from different stages of development and different visual projections, users can produce a list of genes and publications instantly. In FlyExpress, each queryable embryo picture is a heat-map that captures the expression patterns of more than 4500 genes and more than 2600 published articles. In addition, one can view spatial patterns for particular genes over time as well as find other genes with similar expression patterns at a given developmental stage. Therefore, FlyExpress is a unique tool for mining spatiotemporal expression patterns in a format readily accessible to the scientific community.
In colorectal cancer (CRC), KRAS and BRAF mutations in primary tumors are associated with resistance to anti-epidermal growth factor receptor (anti-EGFR)-based therapies. However, the correlation between KRAS/BRAF mutation in primary tumors and serum has not been well studied. To evaluate the degree of concordance of KRAS/BRAF mutations between the primary tumors and the matched serum samples in CRC, serum and tumor tissues were collected from 115 patients with CRC and KRAS/BRAF mutations were examined by nested polymerase chain reaction (PCR) and direct sequencing. BRAF mutations were present in 3.5% (4/115) of the primary tumor tissue samples and 0.87% (1/115) of the serum samples. In the 4 primary tumors with BRAF mutations, identical mutations were not observed in the corresponding serum samples (κ=−0.016). KRAS mutations were observed in 32.2% (37/115) of the primary tumors and 11.3% (13/115) of the serum samples. Of the 37 tumor cases with KRAS mutations, 9 had identical mutations in the corresponding serum sample, with a concordance rate of 24.3% (9/37). Discordance was observed in 32 (27.8%) patients. The concordance between KRAS mutations in the primary tumors and KRAS mutations in the matched serums was low (κ=0.231). The results of the present study suggest that the possibility of differences in the mutational status of KRAS/BRAF between primary tumors and matched serum samples should be considered when patients are selected for anti-EGFR-based therapies.
colorectal cancer; cetuximab; KRAS; BRAF; serum
Inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is being pursued as a new therapeutic approach for the treatment of obesity and metabolic syndrome. Therefore, there is an urgent need to determine the effect of 11β-HSD1 inhibitor, which suppresses glucocorticoid action, on adipose tissue inflammation. The purpose of the present study was to examine the effect of BVT.2733, a selective 11β-HSD1 inhibitor, on expression of pro-inflammatory mediators and macrophage infiltration in adipose tissue in C57BL/6J mice.
C57BL/6J mice were fed with a normal chow diet (NC) or high fat diet (HFD). HFD treated mice were then administrated with BVT.2733 (HFD+BVT) or vehicle (HFD) for four weeks. Mice receiving BVT.2733 treatment exhibited decreased body weight and enhanced glucose tolerance and insulin sensitivity compared to control mice. BVT.2733 also down-regulated the expression of inflammation-related genes including monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α) and the number of infiltrated macrophages within the adipose tissue in vivo. Pharmacological inhibition of 11β-HSD1 and RNA interference against 11β-HSD1 reduced the mRNA levels of MCP-1 and interleukin-6 (IL-6) in cultured J774A.1 macrophages and 3T3-L1 preadipocyte in vitro.
These results suggest that BVT.2733 treatment could not only decrease body weight and improve metabolic homeostasis, but also suppress the inflammation of adipose tissue in diet-induced obese mice. 11β-HSD1 may be a very promising therapeutic target for obesity and associated disease.
Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as γ-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB).
Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies.
Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.
Poly(4HB); PHB; Polyhydroxyalkanoates; PhaP; 4-hydroxybutyrate; Escherichia coli; Metabolic engineering; Synthetic biology
AIM: To evaluate the effects of diazoxide on ischemia/reperfusion (I/R)-injured hepatocytes and further elucidate its underlying mechanisms.
METHODS: Male Sprague-Dawley rats were randomized (8 for donor and recipient per group) into five groups: I/R group (4 h of liver cold ischemia followed by 6 h of reperfusion); heme oxygenase-1 (HO-1) small interfering RNA (siRNA) group (injection of siRNA via donor portal vein 48 h prior to harvest); diazoxide (DZ) group (injection of DZ via donor portal vein 10 min prior to harvest); HO-1 siRNA + DZ group; and siRNA control group. Blood and liver samples were collected at 6 h after reperfusion. The mRNA expressions and protein levels of HO-1 were determined by reverse transcription polymerase chain reaction and Western blotting, and tissue morphology was examined by light and transmission electron microscopy. Serum transaminases level and cytokines concentration were also measured.
RESULTS: We observed that a significant reduction of HO-1 mRNA and protein levels in HO-1 siRNA and HO-1 siRNA + DZ group when compared with I/R group, while the increases were prominent in the DZ group. Light and transmission electron microscopy indicated severe disruption of tissue with lobular distortion and mitochondrial cristae damage in the HO-1 siRNA and HO-1 siRNA + DZ groups compared with DZ group. Serum alanine aminotransferase, aspartate transaminase, tumor necrosis factor-α and interleukin-6 levels increased in the HO-1 siRNA and HO-1 siRNA + DZ groups, and decreased in the DZ group.
CONCLUSION: The protective effect of DZ may be induced by upregulation of HO-1. By inhibiting expression of HO-1, this protection pretreated with DZ was abolished.
Ischemia/reperfusion injury; Diazoxide; Heme oxygenase-1; Liver transplantation; Rat
Block polyhydroxyalkanoates (PHA) were reported to be resistant against polymer aging that negatively affects polymer properties. Recently, more and more attempts have been directed to make PHA block copolymers. Diblock copolymers PHB-b-PHHx consisting of poly-3-hydroxybutyrate (PHB) block covalently bonded with poly-3-hydroxyhexanoate (PHHx) block were for the first time produced successfully by a recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum.
The chloroform extracted polymers were characterized by nuclear magnetic resonance (NMR), thermo- and mechanical analysis. NMR confirmed the existence of diblock copolymers consisting of 58 mol% PHB as the short chain length block with 42 mol% PHHx as the medium chain length block. The block copolymers had two glass transition temperatures (Tg) at 2.7°C and −16.4°C, one melting temperature (Tm) at 172.1°C and one cool crystallization temperature (Tc) at 69.1°C as revealed by differential scanning calorimetry (DSC), respectively. This is the first microbial short-chain-length (scl) and medium-chain-length (mcl) PHA block copolymer reported.
It is possible to produce PHA block copolymers of various kinds using the recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. In comparison to a random copolymer poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P(HB-co-HHx)) and a blend sample of PHB and PHHx, the PHB-b-PHHx showed improved structural related mechanical properties.
Polyhydroxyalkanoates; PHB; Block copolymer; Pseudomonas putida; 3-hydroxybutyrate; 3-hydroxyhexanoate; Synthetic biology
Intravitreal administration of IGFBP-3NB preserves junctional integrity in the presence of VEGF or laser injury by reducing BRB permeability in part by modulating sphingomyelinase levels.
To examine the effect of free insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), independent of the effect of insulin-like growth factors, in modulating retinal vascular permeability.
We assessed the ability of a form of IGFBP-3 that does not bind IGF-1 (IGFBP-3NB), to regulate the blood retinal barrier (BRB) using two distinct experimental mouse models, laser-induced retinal vessel injury and vascular endothelial growth factor (VEGF)-induced retinal vascular permeability. Additionally, in vitro studies were conducted. In the animal models, BRB permeability was quantified by intravenous injection of fluorescein labeled serum albumin followed by digital confocal image analysis of retinal flat-mounts. Claudin-5 and vascular endothelial-cadherin (VE-cadherin) localization at interendothelial junctions was studied by immunofluorescence. In vitro changes in transendothelial electrical resistance (TEER) and flux of fluorescent dextran in bovine retinal endothelial monolayers (BREC) were measured after IGFBP-3NB treatment. Acid (ASMase) and neutral (NSMase) sphingomyelinase mRNA levels and activity were measured in mouse retinas.
Four days postinjury, laser-injured mouse retinas injected with IGFBP-3NB plasmid demonstrated reduced vascular permeability compared with retinas of laser-injured mouse retinas injected with control plasmid. IGFBP-3NB administration resulted in a significant decrease in laser injury-associated increases in ASMase and NSMase mRNA and activity when compared with laser alone treated mice. In vivo, intravitreal injection of IGFBP-3NB reduced vascular leakage associated with intravitreal VEGF injection. IGFBP-3NB partially restored VEGF-induced in vivo permeability and dissociation of claudin-5 and VE-cadherin at junctional complexes. When IGFBP-3NB was applied basally to bovine retinal endothelial cells (BREC) in vitro, TEER increased and macromolecular flux decreased.
Intravitreal administration of IGFBP-3NB preserves junctional integrity in the presence of VEGF or laser injury by reducing BRB permeability in part by modulating sphingomyelinase levels.
The N-myc downstream-regulated gene 2 (NDRG2) is involved in tumor cell differentiation and apoptosis, but its function in tumor angiogenesis remains to be established. Here, we employed adenovirus overexpressing NDRG2 (Ad-NDRG2) to efficiently up-regulate target gene expression in the NDRG2-low-expressing, breast cancer cell line MCF-7. Moreover, VEGF secretion was decreased in MCF-7 cells infected by Ad-NDRG2, and medium conditioned by these infected cells could significantly inhibit the proliferation, tube formation and invasion of human umbilical vein endothelial cells (HUVECs). Further study indicated that the angiogenesis promoting factors VEGF and HIF-1α were down-regulated, whereas the angiogenesis suppressing factors p53 and VHL were up-regulated in MCF-7 cells infected by Ad-NDRG2. Finally, in a nude mouse model, intratumoral injections of Ad-NDRG2 every 3 days for 20 days significantly inhibited the growth and angiogenesis of xenografted MCF-7 tumors. In summary, these data indicate that NDRG2 may be involved in angiogenesis by impacting the expression of angiogenesis related factors. Thus, specific overexpression of NDRG2 by adenovirus represents a promising approach for the treatment of tumor angiogenesis.
Liver fibrosis is a worldwide clinical issue. It has been well established that activated hepatic stellate cells (HSCs) are responsible for excessive extracellular matrix (ECM) deposition in chronically damaged livers. The identification of key elements that control HSCs activation will help to further our understanding of liver fibrosis and improve the outcome of clinical treatment. This study demonstrates that N-Myc downstream-regulated gene 2 (NDRG2) is a potential regulator of liver fibrosis as NDRG2 mRNA and protein levels were reduced during HSCs activation. In addition, enhanced NDRG2 expression reduced Smad3 transcription and phosphorylation, which inhibited HSCs activation by blocking the TGF-β1 signal. Moreover, NDRG2 contributed to an increase in the ratio of matrix metalloproteinase 2 (MMP2) to tissue inhibitor of matrix metalloproteinase 2 (TIMP2), which may facilitate the degradation of the ECM. In dimethylnitrosamine (DMN)-induced fibrotic rat livers, adenovirus-mediated NDRG2 overexpression resulted in decreased ECM deposition and improved liver function compared with controls. In conclusion, the present findings indicate that the modulation of NDRG2 is a promising strategy for the treatment of liver fibrosis.
The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na+/HCO3− cotransporter in the pH regulation in rat epididymis.
Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit(KH) solution, the intracellular pH (pHi) recovery from NH4Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na+/H+ exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO3− buffered would cause another pHi recovery. The pHi recovery in HCO3− buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2, 2-disulfonic acid (DIDS), the inhibitor of HCO3− transporter or by removal of extracellular Na+. The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH.
The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.
Studies of rice protein expression have increased considerably with the development of rice functional genomics. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. Comparison among the profiling of translation and transcription [expressed sequence tags (EST) and massively parallel signature sequencing (MPSS)] revealed that a correlation existed. Based on the standard curves derived from the antigen–antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.
Antibody-based proteomics; rice (Oryza sativa L.); reference gene; reference protein; western blotting
Shewanella oneidensis is a highly motile organism by virtue of a polar flagellum. Unlike most flagellated bacteria, it contains only one major chromosome segment encoding the components of the flagellum with the exception of the motor proteins. In this region, three genes encode flagellinsaccording to the original genome annotation. However, we find that only flaA and flaB encode functional filament subunits. Although these two genesare under the control of different promoters, they are actively transcribed and subsequently translated, producing a considerable number of flagellin proteins. Additionally, both flagellins are able to interact with their chaperon FliS and are subjected to feedback regulation. Furthermore, FlaA and FlaB are glycosylated by a pathwayinvolving a major glycosylating enzyme,PseB, in spite of the lack of the majority of theconsensus glycosylation sites. In conclusion, flagellar assembly in S. oneidensis has novel features despite the conservation of homologous genes across taxa.
Increased vascular permeability is an inciting event in many vascular complications including diabetic retinopathy. We have previously reported that pigment epithelium-derived factor (PEDF) is able to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis through a novel γ-secretase-dependent pathway. In this study, we asked whether inhibition of VEGF-induced permeability by PEDF is also γ-secretase-mediated and to dissect the potential mechanisms involved. Vascular permeability was assessed in vitro by measuring transendothelial resistance and paracellular permeability to dextran and in vivo by following leakage of intravenous FITC-labelled albumin into the retina in the presence or absence of VEGF and PEDF in varying combinations. Experiments were undertaken in the presence or absence of a γ-secretase inhibitor. To assess junctional integrity immunohistochemistry for the adherens junction (AJ) proteins, VE-cadherin and β-catenin, and the tight junction (TJ) protein, claudin-5 was undertaken using cultured cells and flat mount retinas. Protein expression and the association between AJ proteins, VEGF receptors (VEGFRs) and γ-secretase constituents were determined by immunoprecipitation and Western Blot analysis. In selected experiments the effect of hypoxia on junctional integrity was also assessed. PEDF inhibition of VEGF-induced permeability, both in cultured microvascular endothelial cell monolayers and in vivo in the mouse retinal vasculature, is mediated by γ-secretase. PEDF acted by a) preventing dissociation of AJ and TJ proteins and b) regulating both the association of VEGF receptors with AJ proteins and the subsequent phosphorylation of the AJ proteins, VE-cadherin and β-catenin. Association of γ-secretase with AJ proteins appears to be critical in the regulation of vascular permeability. Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins. In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.
Mutations of the sequence-specific master regulator p53 that alter transactivation function from promoter response elements (REs) could result in changes in the strength of gene activation or spectra of genes regulated. Such mutations in this tumor suppressor might lead to dramatic phenotypic changes and diversification of cell responses to stress. We have determined “functional fingerprints” of sporadic breast cancer-related p53 mutants many of which are also associated with familial cancer proneness such as the Li-Fraumeni Syndrome and germline BRCA1/2 mutant-associated cancers. The ability of p53, wild type and mutants, to transactivate from 11 human target REs has been assessed at variable expression levels using a cellular, isogenomic yeast model system that allows for the rapid analysis of p53 function using a qualitative and a quantitative reporter. Among 50 missense mutants, 29 were classified as loss-of-function. The remaining 21 retained transactivation towards at least one RE. At high levels of galactose induced p53 expression, 12/21 mutants that retain transactivation appeared similar to WT. When the level of galactose was reduced, transactivation defects could be revealed suggesting that some breast cancer related mutants can have subtle changes in transcription. These findings have been compared with clinical data from an ongoing neoadjuvant chemotherapy treatment trial for locally advanced breast tumors. The functional and nonfunctional missense mutations may distinguish tumors in terms of demographics, appearance and relapse, implying that heterogeneity in the functionality of specific p53 mutations could impact clinical behavior and outcome.
p53; breast cancer; altered-function mutations
Choroidal neovascularization (CNV) is the common pathological basis of
irreversible visual impairment encountered in a variety of chorioretinal
diseases; the pathogenesis of its development is complicated and still
imperfectly understood. Recent studies indicated that delta-like ligand 4
(Dll4), one of the Notch family ligands might participate in the HIF-1α-VEGF
pathway to regulate CNV angiogenesis. But little is known about the influence
and potential mechanism of Dll4/Notch signals on CNV angiogenesis. Real-time
RT-PCR, Western blotting were used to analyze the expression alteration of Dll4,
VEGF and HIF-1α in hypoxic RF/6A cells. Immunofluorescence staining, a
laser-induced rat CNV model and intravitreal injection techniques were used to
confirm the relationships among these molecules in vitro and
in vivo. RPE-RF/6A cell co-culture systems were used to
investigate the effects of Dll4/Notch signals on CNV angiogenesis. We found that
the Dll4 was involved in hypoxia signaling in CNV angiogenesis. Results from the
co-culture system showed that the enhancement of Dll4 expression in RF/6A cells
led to the significantly faster proliferation and stronger tube forming ability,
but inhibited cells migration and invasion across a monolayer of RPE cells in
hypoxic environment, while siRNA-mediated Dll4 silencing caused the opposite
effects. Pharmacological disruption of Notch signaling using gamma-secretase
inhibitor (GSI) produced similar, but not identical effects, to that caused by
the Dll4 siRNA. In addition, the expression of several key molecules involved in
the angiogenesis of CNV was altered in RF/6A cells showing constitutively active
Dll4 expression. These results suggest that Dll4 play an important role in CNV
angiogenesis, which appears to be regulated by HIF-1α and VEGF during the
progression of CNV under hypoxic conditions. Targeting Dll4/Notch signaling may
facilitate further understanding of the mechanisms that underlie CNV
This study was carried out to evaluate the protective effect of anthocyanins extract of blueberry on trinitrobenzene sulfonic acid (TNBS)-induced inflammatory bowel disease (IBD) model of mice. The study employed female C57BL/6 mice (n = 50), and colitis was induced by intracolonic injection of 0.5 mg of TNBS dissolved in 50% ethanol–phosphate buffered solution. The mice were divided into five groups (n = 10): vehicle, TNBS control and anthocyanins groups that received different doses of anthocyanins extract (10, 20 and 40 mg kg−1) daily for 6 days. Both increase in body weight and diarrhea symptoms were monitored each day. After 6 days, the animals were killed, and the following parameters were assessed: colon length, morphological score, histological score and biochemical assay (NO, myeloperoxidase (MPO), interleukin (IL)-12, IL-10, tumor necrosis factor (TNF)-α and interferon (IFN)-γ). The results showed that the anthocyanins extract of blueberry rendered strong protection against TNBS-induced colonic damage at a dosage of 40 mg kg−1. When compared with the control, anthocyanins extract significantly prevented loss of body weight and ameliorated the scores of diarrhea, morphology and histology. Treatment with anthocyanins extract restored IL-10 excretion, as well as caused reduction in the levels of NO, MPO, IL-12, TNF-α and IFN-γ. Our research revealed the protective effect of anthocyanins extract from blueberry on TNBS-induced experimental colitis in mice, as well as examined whether high levels of dietary blueberries would lower the risk or have protective effects on human IBD, which may require further investigation.
Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.
In the title compound, C15H12O4·H2O, the two benzene rings are not coplanar, making a dihedral angle of 7.24 (16)°. An intramolecular hydroxy–carbonyl O—H⋯O hydrogen bond occurs. In the crystal, four intermolecular O—H⋯O hydrogen bonds involving the hydroxy residues, the carbonyl group and the water molecule lead to the formation of a three-dimensional network. The supramolecular structure is further stabilized by weak C—H⋯O interactions.
Autoimmune hepatitis (AIH) is a chronic necroinflammatory disease of the liver with a poorly understood etiology. Detection of non-organ-specific and liver-related autoantibodies using immunoserological approaches has been widely used for diagnosis and prognosis. However, unambiguous and accurate detection of the disease requires the identification and characterization of disease-specific autoantigens. In the present study, we have profiled the autoantigen repertoire of patients with AIH versus those with other liver diseases, identifying and validating three novel and highly specific biomarkers for AIH. In Phase I we fabricated a human protein chip of 5,011 non-redundant proteins and used it to quickly identify 11 candidate autoantigens with relative small serum collection. In Phase II we fabricated an AIH-specific protein chip and obtained autoimmunogenic profiles of serum samples from 44 AIH patients, 50 healthy controls, and 184 additional patients suffering from hepatitis B, hepatitis C, systemic lupus erythematosus, primary Sjögren’s syndrome, rheumatoid arthritis, or primary biliary cirrhosis. Using this two-phase approach, we identified three new antigens, RPS20, Alba-like, and dUTPase, as highly AIH-specific biomarkers, with sensitivities of 47.5% (RPS20), 45.5% (Alba-like), and 22.7% (dUTPase). These potential biomarkers were further validated with additional AIH samples in a double-blind design. Finally, we demonstrated that these new biomarkers could be readily applied to ELISA-based assays for use in clinical diagnosis/prognosis.
AIH; biomarker; human protein chip; autoantigens; serum; autoimmune; human liver; clinical proteomics
Although solid surface-associated biofilm development of S. oneidensis has been extensively studied in recent years, pellicles formed at the air-liquid interface are largely overlooked. The goal of this work was to understand basic requirements and mechanism of pellicle formation in S. oneidensis.
We demonstrated that pellicle formation can be completed when oxygen and certain cations were present. Ca(II), Mn(II), Cu(II), and Zn(II) were essential for the process evidenced by fully rescuing pellicle formation of S. oneidensis from the EDTA treatment while Mg (II), Fe(II), and Fe(III) were much less effective. Proteins rather than DNA were crucial in pellicle formation and the major exopolysaccharides may be rich in mannose. Mutational analysis revealed that flagella were not required for pellicle formation but flagellum-less mutants delayed pellicle development substantially, likely due to reduced growth in static media. The analysis also demonstrated that AggA type I secretion system was essential in formation of pellicles but not of solid surface-associated biofilms in S. oneidensis.
This systematic characterization of pellicle formation shed lights on our understanding of biofilm formation in S. oneidensis and indicated that the pellicle may serve as a good research model for studying bacterial communities.
Studies on the relationship between obesity and bone have recently become widespread. The aim of this study was to investigate the effect of obesity on bone, utilizing a diet-induced obese mouse model, and to explore the role of free fatty acids (FFAs) in the osteogenesis/adipogenesis of mouse bone marrow-derived mesenchymal stem cells (BMSCs). An obese mouse model was established by a high-fat diet (HFD). Proximal femurs were collected at sacrifice, and bone mineral density (BMD) in the proximal femurs was measured by dual-energy X-ray absorptiometry. Bone histomorphometry was performed using undecalcified sections of the proximal femurs. The effect of obesity on the differentiation of mouse BMSCs was assessed by colony formation assays and gene expression analysis. In vitro, various osteogenic and adipogenic genes were determined by real-time quantitative PCR in mouse BMSCs after exposure to conditioned medium (CM) from FFA-treated 3T3-L1 adipocytes. Western blotting was further performed to analyze the representative protein expression of PPARγ and Runx2. BMD and trabecular thickness were significantly greater in the HFD mice than in the control mice. CFU-osteo assay showed significantly increased osteogenesis of BMSCs. The mRNA level of Runx2 was significantly higher, while PPARγ and Pref-1 were significantly lower in BMSCs from the HFD mice compared to the control mice. In mouse BMSCs, the Sox9 and Runx2 genes were significantly up-regulated after exposure to CM from FFA-treated adipocytes, while PPARγ and CEBP-α were significantly down-regulated. Osteogenesis was significantly increased, while adipogenesis was significantly decreased. In conclusion, HFD-induced obesity may play a protective role in bone formation by concomitantly promoting osteogenic and suppressing adipogenic differentiation of BMSCs through factors secreted by FFA-treated adipocytes.
mesenchymal stem cells; free fatty acids; obesity; osteogenesis; adipogenesis