Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.
Autophagy is an evolutionarily conserved process that sequestrates and delivers cytoplasmic macromolecules and organelles to the vacuoles or lysosomes for degradation. In plants, autophagy is involved in supplying internal nutrients during starvation and in promoting cell survival during senescence and during biotic and abiotic stresses. Arabidopsis NBR1 is a homolog of mammalian autophagy cargo adaptors P62 and NBR1. Disruption of Arabidopsis NBR1 caused increased sensitivity to a spectrum of abiotic stresses but had no significant effect on plant senescence, responses to carbon starvation, or resistance to a necrotrophic pathogen. NBR1 contains an ubiquitin-binding domain, and the compromised stress tolerance of autophagy mutants was associated with increased accumulation of NBR1 and ubiquitin-positive cellular protein aggregates in the insoluble protein fraction under stress conditions. Based on these results, we propose that NBR1 targets ubiquitinated protein aggregates most likely derived from denatured and otherwise damaged nonnative proteins for autophagic clearance under stress conditions.
Multilevel interactions of the plant hormones ethylene and auxin coordinately and synergistically regulate many aspects of plant growth and development. This study isolated the AUXIN RESISTANT1 (AUX1) allele aux1rcr1 (RCR1 for REVERSING CTR1-10 ROOT1) that suppressed the root growth inhibition conferred by the constitutive ethylene-response constitutive triple response1-10 (ctr1-10) allele. The aux1rcr1 mutation resulted from an L126F substitution at loop 2 of the plasma membrane-associated auxin influx carrier protein AUX1. aux1rcr1 and the T-DNA insertion mutant aux1-T were both defective in auxin transport and many aspects of the auxin response. Unexpectedly, expression of the auxin-response reporter DR5:GUS in the root apex was substantially prevented by the aux1rcr1 but not the aux1-T mutation, even in the presence of the wild-type AUX1 allele. Following treatment with the synthetic auxin 1-naphthaleneacetic acid (NAA), DR5:GUS expression in aux1rcr1 and aux1-T occurred mainly in the root apex and mature zone. NAA-induced DR5:GUS expression in the root apex was markedly prevented by ethylene in genotypes with aux1rcr1 but not in aux1-T genotypes and the wild type. The effect of aux1rcr1 on DR5:GUS expression seemed to be associated with AUX1-expressing domains. Green fluorescence protein-fused aux1rcr1 was localized in the cytoplasm and probably not to the plasma membrane, indicating important roles of the Lys126 residue at loop 2 in AUX1 targeting. The possible effects of aux1rcr1 on DR5:GUS expression are discussed.
AUX1; Arabidopsis; DR5:GUS; auxin; ethylene; root gravitropism
The early detection of major depression in elderly individuals who are at risk of developing the disease is of prime importance when it comes to the prevention of geriatric depression. We used resting-state functional magnetic resonance imaging (fMRI) to examine changes in regional homogeneity (ReHo) of spontaneous activity in late-life subthreshold depression (StD), and we evaluated the sensitivity/specificity performance of these changes. Nineteen elderly individuals with StD and 18 elderly controls underwent a resting-state fMRI scan. The ReHo approach was employed to examine whether StD was related to alterations in resting-state neural activity, in the form of abnormal regional synchronization. Receiver operating characteristic curve analysis and the Fisher stepwise discriminant analysis were used to evaluate the sensitivity/specificity characteristics of the ReHo index in discriminating between the StD subjects and normal controls. The results demonstrated that, compared to controls, StD subjects display lower ReHo in the right orbitofrontal cortex (OFC), left dorsolateral prefrontal cortex (DLPFC), left postcentral gyrus (PCG), and left middle frontal and inferior temporal gyri, as well as higher ReHo in the bilateral insula and right DLPFC. The left PCG and the right DLPFC, OFC, and posterior insula, together reported a predictive accuracy of 91.9%. These results suggest that the regional activity coherence was changed in the resting brain of StD subjects, and that these alterations may serve as potential markers for the early detection of StD in late-life depression.
Objective: Dracorhodin perchlorate (DP) was a synthetic analogue of the antimicrobial anthocyanin red pigment dracorhodin. It was reported that DP could induce apoptosis in human prostate cancer, human gastric tumor cells and human melanoma, but the cytotoxic effect of DP on human breast cancer was not investigated. This study would investigate whether DP was a candidate chemical of anti-human breast cancer.
Methods: The MTT assay reflected the number of viable cells through measuring the activity of cellular enzymes. Phase contrast microscopy visualized cell morphology. Fluorescence microscopy detected nuclear fragmentation after Hoechst 33258 staining. Flowcytometric analysis of Annexin V-PI staining and Rodamine 123 staining was used to detect cell apoptosis and mitochondrial membrane potential (MMP). Real time PCR detected mRNA level. Western blot examined protein expression.
Results: DP dose and time-dependently inhibited the growth of MCF-7 cells. DP inhibited MCF-7 cell growth through apoptosis. DP regulated the expression of Bcl-2 and Bax, which were mitochondrial pathway proteins, to decrease MMP, and DP promoted the transcription of Bax and inhibited Bcl-2. Apoptosis-inducing factor (AIF) and cytochrome c which localized in mitochondrial in physiological condition were released into cytoplasm when MMP was decreased. DP activated caspase-9, which was the downstream of mitochondrial pathway. Therefore DP decreased MMP to release AIF and cytochrome c into cytoplasm, further activating caspase 9, lastly led to apoptosis.
Conclusion: Therefore DP was a candidate for anti-breast cancer, DP induced apoptosis of MCF-7 through mitochondrial pathway.
dracorhodin perchlorate; apoptosis; mitochondrial pathway
The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA - random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method.
A novel Getah virus was identified by VIDISCR from suckling mice exposed to mosquitoes (Aedes albopictus) collected in Yunnan Province, China. The non-structural protein gene, nsP3, the structural protein gene, the capsid protein gene, and the 3'-untranslated region (UTR) of the novel Getah virus isolate were cloned and sequenced. Nucleotide sequence identities of each gene were determined to be 97.1–99.3%, 94.9–99.4%, and 93.6–99.9%, respectively, when compared with the genomes of 10 other representative strains of Getah virus.
The VIDISCR method was able to identify known virus isolates and a novel isolate of Getah virus from infected mice. Phylogenetic analysis indicated that the YN08 isolate was more closely related to the Hebei HB0234 strain than the YN0540 strain, and more genetically distinct from the MM2021 Malaysia primitive strain.
Getah virus; Identification; Virus-Discovery; cDNA RAPD
The Hint1 protein, a member of the histidine triad (HIT) family, is highly conserved in diverse species and ubiquitously expressed in mammalian tissues. Previous studies in mice provided evidence that Hint1 may be haplosufficient with respect to its function as a tumor suppressor. In the present study, we investigated the aberrant methylation of Hint1 and explored possible relationships between aberrant methylation and clinicopathological features in hepatocellular carcinoma (HCC). Hypermethylation of Hint1 was evaluated by the methylation specific PCR (MSP) method in 40 patients with HCC (tumor and paired adjacent non-tumor tissues) from Taiwan, 22 cases of normal liver tissue (14 from Taiwan and 8 from the U.S.). HINT1 expression in tissues was detected by immunohistochemistry. The frequencies of hypermethylation of Hint1 in tumor, paired adjacent non-tumor and normal liver tissue were 55.0%, 37.5% and 9.1%, respectively. A statistically significant inverse association was found between Hint1 methylation status and expression of the HINT1 protein in tumor tissues (p<0.003). The relationship between Hint1 methylation status and clinical features and other, previously measured biomarkers was also analyzed. p16 hypermethylation was statistically significantly associated with Hint1 methylation status (p=0.035). There were no correlations between Hint1 methylation and HBV or HCV infection status or AFB1- and PAH-DNA adduct levels. These results suggest that promoter hypermethylation of Hint1 may play a role in hepatocarcinogenesis.
Hint1; HCC; epigenetic changes; promoter hypermethylation; p16; environmental carcinogens
Takayasu arteritis (TA) is a rare form of chronic inflammatory granulomatous arteritis of the aorta and its major branches. Late gadolinium enhancement (LGE) with magnetic resonance imaging (MRI) has demonstrated its value for the detection of vessel wall alterations in TA. The aim of this study was to assess LGE of the coronary artery wall in patients with TA compared to patients with stable CAD.
We enrolled 9 patients (8 female, average age 46±13 years) with proven TA. In the CAD group 9 patients participated (8 male, average age 65±10 years). Studies were performed on a commercial 3T whole-body MR imaging system (Achieva; Philips, Best, The Netherlands) using a 3D inversion prepared navigator gated spoiled gradient-echo sequence, which was repeated 34–45 minutes after low-dose gadolinium administration.
No coronary vessel wall enhancement was observed prior to contrast in either group. Post contrast, coronary LGE on IR scans was detected in 28 of 50 segments (56%) seen on T2-Prep scans in TA and in 25 of 57 segments (44%) in CAD patients. LGE quantitative assessment of coronary artery vessel wall CNR post contrast revealed no significant differences between the two groups (CNR in TA: 6.0±2.4 and 7.3±2.5 in CAD; p = 0.474).
Our findings suggest that LGE of the coronary artery wall seems to be common in patients with TA and similarly pronounced as in CAD patients. The observed coronary LGE seems to be rather unspecific, and differentiation between coronary vessel wall fibrosis and inflammation still remains unclear.
The opinion of application of indocyanine green (ICG) in the macular hole surgery was contradictory. Here we conducted a meta-analysis to evaluate the effect of in internal limiting membrane (ILM) peeling for macular hole surgery.
Methods and Findings
We searched electronic databases for comparative studies published before July 2012 of ILM peeling with and without ICG. Twenty-two studies including 1585 eyes were included. Visual acuity (VA) improvement, including the postoperative rate of ≥20/40 VA gained (OR, 0.65; 95% CI, 0.43 to 0.97; P = 0.033) and increased LogMAR (WMD, −0.09; 95% CI, −0.16 to −0.02; P = 0.011), was less in the ICG group. The risk of visual field defects was greater in the ICG group than in the non-ICG group. There was no significant difference in the rate of anatomical outcomes between ILM peeling procedures performed with and without ICG. RPE changes and other postoperative complications were not significantly different between the ICG and non-ICG groups. An additional analysis showed that the VA improvement of the ICG group was less than the non-ICG group only within the first year of follow up. A subgroup analysis showed that the rate of VA improvement was lower in the ICG group than in other adjuncts group. A higher rate of secondary closure and less VA improvement were observed in a high proportion (>0.1%) of the ICG group. A sensitivity analysis after the randomized-controlled trials were excluded from the meta-analysis demonstrated no differences compared with the overall results.
This meta-analysis demonstrated that there is no evidence of clinical superiority in outcomes for ICG-assisted ILM peeling procedure over the non-ICG one. The toxicity of ICG should be considered when choosing the various staining methods.
Brassinosteroids (BRs) are potent regulators of photosynthesis and crop yield in agricultural crops; however, the mechanism by which BRs increase photosynthesis is not fully understood. Here, we show that foliar application of 24-epibrassinolide (EBR) resulted in increases in CO2 assimilation, hydrogen peroxide (H2O2) accumulation, and leaf area in cucumber. H2O2 treatment induced increases in CO2 assimilation whilst inhibition of the H2O2 accumulation by its generation inhibitor or scavenger completely abolished EBR-induced CO2 assimilation. Increases of light harvesting due to larger leaf areas in EBR- and H2O2-treated plants were accompanied by increases in the photochemical efficiency of photosystem II (ΦPSII) and photochemical quenching coefficient (q
P). EBR and H2O2 both activated carboxylation efficiency of ribulose-1,5-bisphosphate oxygenase/carboxylase (Rubisco) from analysis of CO2 response curve and in vitro measurement of Rubisco activities. Moreover, EBR and H2O2 increased contents of total soluble sugar, sucrose, hexose, and starch, followed by enhanced activities of sugar metabolism such as sucrose phosphate synthase, sucrose synthase, and invertase. Interestingly, expression of transcripts of enzymes involved in starch and sugar utilization were inhibited by EBR and H2O2. However, the effects of EBR on carbohydrate metabolisms were reversed by the H2O2 generation inhibitor diphenyleneodonium (DPI) or scavenger dimethylthiourea (DMTU) pretreatment. All of these results indicate that H2O2 functions as a secondary messenger for EBR-induced CO2 assimilation and carbohydrate metabolism in cucumber plants. Our study confirms that H2O2 mediates the regulation of photosynthesis by BRs and suggests that EBR and H2O2 regulate Calvin cycle and sugar metabolism via redox signaling and thus increase the photosynthetic potential and yield of crops.
Metabolism; Photosynthesis; Reactive oxygen species; Rubisco; Sucrose
A cross-sectional validation study was conducted in several urban and rural communities in Beijing, China, to evaluate the effectiveness of the Beijing version of the Montreal Cognitive Assessment (MoCA-BJ) as a screening tool to detect mild cognitive impairment (MCI) among Chinese older adults.
The MoCA-BJ and the Mini-Mental State Examination (MMSE) were administered to 1001 Chinese elderly community dwellers recruited from three different regions (i.e., newly developed, old down-town, and rural areas) in Beijing. Twenty-one of these participants were diagnosed by experienced psychiatrists as having dementia, 115 participants were diagnosed as MCI, and 865 participants were considered to be cognitively normal. To analyze the effectiveness of the MoCA-BJ, we examined its psychometric properties, conducted item analyses, evaluated the sensitivity and specificity of the scale, and compared the scale with the MMSE. Demographic and regional differences among our subjects were also taken into consideration.
Under the recommended cut-off score of 26, the MoCA-BJ demonstrated an excellent sensitivity of 90.4%, and a fair specificity (31.3%). The MoCA-BJ showed optimal sensitivity (68.7%) and specificity (63.9%) when the cut-off score was lowered to 22. Among all the seven cognitive sub-domains, delayed recall was shown to be the best index to differentiate MCI from the normal controls. Regional differences disappeared when the confounding demographic variables (i.e., age and education) were controlled. Item analysis showed that the internal consistency was relatively low in both naming and sentence repetition tasks, and the diagnostic accuracy was similar between the MoCA-BJ and the MMSE.
In general, the MoCA-BJ is an acceptable tool for MCI screening in both urban and rural regions of Beijing. However, presumably due to the linguistic and cultural differences between the original English version and the Chinese version of the scale, and the lower education level of Chinese older adults, the MoCA-BJ is not much better than the MMSE in detecting MCI, at least for this study sample. Further modifications to several test items of the MoCA-BJ are recommended in order to improve the applicability and effectiveness of the MoCA-BJ in MCI screening among the Chinese population.
MoCA-BJ; MMSE; Mild cognitive impairment; Dementia; Cognitive assessment
The purpose of this study is to evaluate the predictive significance of preoperative serum level of cytokeratin 19 fragments (Cyfra21-1) and squamous cell carcinoma antigen (SCC-Ag) after complete resection in patients with stage II esophageal squamous cell carcinoma (ESCC).
Between 1995 and 2006, a total of 379 patients in stage II ESCC who underwent complete resection were consecutively recruited. Statistical analyses were applied to test the associations between preoperative serum titers of Cyfra21-1 and SCC-Ag, clinicopathological factors and prognoses.
Preoperative high and normal serum level of Cyfra21-1 and SCC-Ag were found in 47.8%, 52.2% and 72.8%, 27.2%, respectively. The 1-, 3-, 5-year overall survival rate for the entire cohort of patients was 95%, 78%, and 56%, respectively. Median overall survival (OS) was 45.3 months longer in patients with low preoperative serum level of Cyfra21-1 (91.9 months) than those with high preoperative serum level of Cyfra21-1 (46.6 months) (P < 0.001). Median OS among patients with SCC-Ag-low level was also longer than those with SCC-Ag-high level (89.7 vs. 63.7 months, P < 0.001), especially for those with stage IIB (P < 0.001). After multivariate analysis, along with pTNM stage, preoperative serum level of Cyfra21-1 and SCC-Ag were independently and significantly predictive factors (P < 0.001, P < 0.001). Furthermore, the five-year survival rate in double-low subset, either-low subset and double-high subset was 100%, 83% and 27%, respectively (P < 0.001).
The preoperative serum level of Cyfra21-1 and SCC-Ag are independently significant predictors which negatively affected the survivals of patients with stage II ESCC.
Esophageal squamous cell carcinoma; Cyfra21-1; SCC-Ag; Prognosis
Ginsenoside Rb1 shows neuroprotective effects in various neurons, including dopaminergic cells. However, the precise mechanisms of action are uncertain. In this paper, we examine whether Rb1 has a neuroprotective effect on MPP+-induced apoptosis and attempt to clarify the signaling pathway in PC12 cells. Apoptosis of PC12 cells was determined by DNA fragmentation assay, the activation of caspase-3, or by the inactivation of Bcl-xL. Rb1 inhibited MPP+-induced caspase-3 activation and DNA fragmentation and activated Bcl-xL in MPP+-treated PC12 cells. These antiapoptotic effect was abrogated in PC12 cells transfected with estrogen receptor siRNA. Levels of DNA fragmentation were increased by wortmannin or PD 98059, while they were decreased by SB 203580 or SP 600125 in MPP+-treated PC12 cells. Rb1 increased phosphorylation levels of ERK1/2 or Akt in MPP+-treated PC12 cells, while it reduced phosphorylated p38 or SAPK/JNK. The increased phosphorylation of ERK/1/2 or Akt by Rb1 was abrogated by estrogen receptor siRNA. Rb1-induced inhibition of SAPK/JNK or p38 phosphorylation was also abolished by estrogen receptor siRNA. These results suggest that ginsenoside Rb1 protects PC12 cells from caspase-3-dependent apoptosis through stimulation of estrogen receptor with consequent activation of ERK1/2 and Akt and inhibition of SAPK/JNK and p38.
Bacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small RNAs (sRNAs). These regulatory RNA molecules are increasingly shown to be able to initiate regulatory cascades and modulate the expression of multiple genes that are involved in or required for survival under environmental challenge. Despite mounting evidence for the importance of sRNAs in stress response, their role upon antibiotic exposure remains unknown. In this study, we sought to determine firstly, whether differential expression of sRNAs occurs upon antibiotic exposure and secondly, whether these sRNAs could be attributed to microbial tolerance to antibiotics.
A small scale sRNA cloning strategy of Salmonella enterica serovar Typhimurium SL1344 challenged with half the minimal inhibitory concentration of tigecycline identified four sRNAs (sYJ5, sYJ20, sYJ75 and sYJ118) which were reproducibly upregulated in the presence of either tigecycline or tetracycline. The coding sequences of the four sRNAs were found to be conserved across a number of species. Genome analysis found that sYJ5 and sYJ118 mapped between the 16S and 23S rRNA encoding genes. sYJ20 (also known as SroA) is encoded upstream of the tbpAyabKyabJ operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. sYJ75 is encoded between genes that are involved in enterobactin transport and metabolism. Additionally we find that the genetic deletion of sYJ20 rendered a reduced viability phenotype in the presence of tigecycline, which was recovered when complemented. The upregulation of some of these sRNAs were also observed when S. Typhimurium was challenged by ampicillin (sYJ5, 75 and 118); or when Klebsiella pneumoniae was challenged by tigecycline (sYJ20 and 118).
Small RNAs are overexpressed as a result of antibiotic exposure in S. Typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. sYJ20, a riboswitch, appears to possess a trans-regulatory sRNA role in antibiotic tolerance. These findings imply that the sRNA mediated response is a component of the bacterial response to antibiotic challenge.
Both p53 tumor suppressor and murine double minute 2 (MDM2) oncoprotein are crucial in carcinogenesis. We hypothesized that MDM2 promoter single nucleotide polymorphism (SNP)309, A2164G, and p53 codon 72 SNP are associated with risk and age at onset of squamous cell carcinoma of head and neck (SCCHN). We genotyped these SNPs in a study of 1,083 Caucasian SCCHN cases and 1,090 cancer-free controls. Although none of these SNPs individually had a significant effect on risk of SCCHN, nor did their combined putative risk genotypes (i.e. MDM2 SNP309 GT + GG, 2164 AA, and p53 codon 72 CC), we found that individuals with 2–3 risk genotypes had significantly increased risk of non-oropharyngeal cancer (OR = 1.42; 95% CI=1.07–1.88). This increased risk was more pronounced among young subjects, men, smokers, and drinkers. In addition, female patients carrying the MDM2 SNP309 GT and GG genotypes showed a 3-year (56.7 years) and 9-year (51.2 years) earlier age at onset of non-oropharyngeal cancer (Ptrend = 0.007), respectively, compared with those carrying the TT genotype (60.1 years). The youngest age (42.5 years) at onset of non-oropharyngeal cancer was observed in female patients with the combined MDM2 SNP309 GG and p53 codon 72 CC genotypes. The findings suggest that MDM2 SNP309, A2164G, and p53 codon 72 SNPs may collectively contribute to non-oropharyngeal cancer risk and that MDM2 SNP309 individually or in combination with p53 codon 72 may accelerate the development of non-oropharyngeal cancer in women. Further studies with large sample sizes are warranted to validate these results.
squamous cell carcinoma of the head and neck; MDM2; p53; polymorphism; risk; age at onset
Brassinosteroids (BRs) and polyamines (PAs) regulate various responses to abiotic stress, but their involvement in the regulation of copper (Cu) homeostasis in plants exposed to toxic levels of Cu is poorly understood. This study provides an analysis of the effects of exogenously applied BRs and PAs on radish (Raphanus sativus) plants exposed to toxic concentrations of Cu. The interaction of 24-epibrassinolide (EBR, an active BR) and spermidine (Spd, an active PA) on gene expression and the physiology of radish plants resulted in enhanced tolerance to Cu stress. Results indicated that the combined application of EBR and Spd modulated the expression of genes encoding PA enzymes and genes that impact the metabolism of indole-3-acetic acid (IAA) and abscisic acid (ABA) resulting in enhanced Cu stress tolerance. Altered expression of genes implicated in Cu homeostasis appeared to be the main effect of EBR and Spd leading to Cu stress alleviation in radish. Ion leakage, in vivo imaging of H2O2, comet assay, and improved tolerance of Cu-sensitive yeast strains provided further evidence for the ability of EBR and Spd to improve Cu tolerance significantly. The study indicates that co-application of EBR and Spd is an effective approach for Cu detoxification and the maintenance of Cu homeostasis in plants. Therefore, the use of these compounds in agricultural production systems should be explored.
Abscisic acid; brassinosteroids; comet assay; copper transporters; Cu homeostasis; Cu-sensitive yeast; indole-3-acetic acid; oxidative stress; polyamines
The fungus of Ganoderma is a traditional medicine in Asia with a variety of pharmacological functions including anti-cancer activities. We have purified an extracellular heteropolysaccharide fraction, PS-F2, from the submerged mycelia culture of G. formosanum and shown that PS-F2 exhibits immunostimulatory activities. In this study, we investigated the molecular mechanisms of immunostimulation by PS-F2.
PS-F2-stimulated TNF-α production in macrophages was significantly reduced in the presence of blocking antibodies for Dectin-1 and complement receptor 3 (CR3), laminarin, or piceatannol (a spleen tyrosine kinase inhibitor), suggesting that PS-F2 recognition by macrophages is mediated by Dectin-1 and CR3 receptors. In addition, the stimulatory effect of PS-F2 was attenuated in the bone marrow-derived macrophages from C3H/HeJ mice which lack functional Toll-like receptor 4 (TLR4). PS-F2 stimulation triggered the phosphorylation of mitogen-activated protein kinases JNK, p38, and ERK, as well as the nuclear translocation of NF-κB, which all played essential roles in activating TNF-α expression.
Our results indicate that the extracellular polysaccharides produced by G. formosanum stimulate macrophages via the engagement of multiple pattern-recognition receptors including Dectin-1, CR3 and TLR4, resulting in the activation of Syk, JNK, p38, ERK, and NK-κB and the production of TNF-α.
Ganoderma formosanum; Polysaccharide; Immunostimulatory; Macrophage; Pattern-recognition receptor
There is accumulating evidence to implicate the importance of EphBs receptors and ephrinBs ligands were involved in modulation of spinal nociceptive information. However, the downstream mechanisms that control this process are not well understood. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K), as the downstream effectors, participates in modulation of spinal nociceptive information related to ephrinBs/EphBs. Intrathecal injection of ephrinB1-Fc produced a dose- and time-dependent thermal and mechanical hyperalgesia, accompanied by the increase of spinal PI3K-p110γ, phosphorylation of AKT (p-AKT) and c-Fos expression. Pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented activation of spinal AKT induced by ephrinB1-Fc. Inhibition of spinal PI3K signaling dose-dependently prevented and reversed pain behaviors and spinal c-Fos protein expression induced by intrathecal injection of ephrinB1-Fc. Inhibition of EphBs receptors by intrathecal injection of EphB1-Fc reduced formalin-induced inflammation and chronic constrictive injury-induced neuropathic pain behaviors accompanied by decreased expression of spinal PI3K,p-AKT and c-Fos protein. Furthermore, pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented ephrinB1-Fc-induced ERK activation in spinal. These data demonstrated that PI3K and PI3K crosstalk to ERK signaling contributed to modulation of spinal nociceptive information related to ephrinBs/EphBs.
Excision repair cross-complementation group 4 gene (ERCC4/XPF) plays an important role in nucleotide excision repair and participates in removal of DNA interstrand cross-links and DNA double-strand breaks. Single nucleotide polymorphisms (SNPs) in ERCC4 may impact repair capacity and affect cancer susceptibility.
In this case-control study, we evaluated associations of four selected potentially functional SNPs in ERCC4 with risk of squamous cell carcinoma of the head and neck (SCCHN) in 1,040 non-Hispanic white patients with SCCHN and 1,046 cancer-free matched controls. We found that the variant GG genotype of rs2276466 was significantly associated with a decreased risk of SCCHN (OR = 0.69, 95% CI 0.50–0.96), and that the variant TT genotype of rs3136038 showed a borderline significant decreased risk with SCCHN (OR = 0.76, 95% CI: 0.58–1.01) in the recessive model. Such protective effects were more evident in oropharyngeal cancer (OR = 0.61, 95% CI: 0.40–0.92 for rs2276466; OR = 0.69, 95% CI: 0.48–0.98 for rs3136038). No significant associations were found for the other two SNPs (rs1800067 and rs1799798). In addition, individuals with the rs2276466 GG or with the rs3136038 TT genotypes had higher levels of ERCC4 mRNA expression than those with the corresponding wild-type genotypes in 90 Epstein-Barr virus-transformed lymphoblastoid cell lines derived from Caucasians.
These results suggest that these two SNPs (rs2276466 and rs3136038) in ERCC4 may be functional and contribute to SCCHN susceptibility. However, our findings need to be replicated in further large epidemiological and functional studies.
Continuous and excessive application of insecticides has resulted in the rapid development of insecticide resistance in several mosquito species, including Culex pipiens pallens. Previous studies in our laboratory found that arrestin gene expression was higher in the deltamethrin-resistant (DR) strain than in the deltamethrin-susceptible (DS) strain of Cx. pipiens pallens. Similarly, other studies reported that arrestin was highly expressed in permethrin-resistant Cx. quinquefasciatus and in dichlorodiphenyltrichloroethane (DDT)-resistant Drosophila melanogaster.
Full-length cDNAs of an arrestin gene were cloned from Cx. pipiens pallens via polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE). The mRNA levels of the arrestin gene in the whole life cycle of DR and DS strains of Cx. pipiens pallens were investigated via quantitative real-time PCR. In addition, the relationship between arrestin and deltamethrin (DM) resistance were identified using genetic overexpression strategies and arrestin RNAi in mosquito cells. Cell viability was analyzed with cholecystokinin octapeptide after DM treatment. Moreover, the mRNA levels of cytochrome P450 6A1 (CYP6A1) and opsin in the transfected cells and controls were analyzed.
Complete arrestin gene sequence was cloned and expressed throughout the life cycle of Cx. pipiens pallens. Moreover, arrestin was significantly upregulated in the DR strain, compared with that in the DS strain at the egg, pupae, and adult stages. Arrestin overexpression comparably increased the mosquito cell viability, whereas arrestin knockdown by siRNA decreased mosquito cell viability with deltamethrin (DM) treatment. Meanwhile, the mRNA levels of CYP6A1 and opsin were upregulated in mosquito cells transfected with arrestin and downregulated in mosquito cells with arrestin knockdown.
This study presented the first evidence that arrestin might be associated with insecticide resistance in Cx. pipiens pallens.
Insecticide resistance; Arrestin; Gene cloning; Transfection; SiRNA; Cell viability
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.
To develop and evaluate a practical method for the quantification of signal-to-noise ratio (SNR) on coronary magnetic resonance angiograms (MRA) acquired with parallel imaging.
Materials and Methods
To quantify the spatially varying noise due to parallel imaging reconstruction, a new method has been implemented incorporating image data acquisition followed by a fast noise scan during which radiofrequency pulses, cardiac triggering and navigator gating are disabled. The performance of this method was evaluated in a phantom study where SNR measurements were compared to those of a reference standard (multiple repetitions). Subsequently, SNR of myocardium and posterior skeletal muscle was determined on in vivo human coronary MRA.
In a phantom, the SNR measured using the proposed method deviated less than 10.1% from the reference method for small geometry factors (<=2). In-vivo, the noise scan for a 10 minutes coronary MRA acquisition was acquired in 30s. Higher signal and lower SNR, due to spatially varying noise, were found in myocardium compared to posterior skeletal muscle.
SNR quantification based on a fast noise scan is a validated and easy-to-use method when applied to 3D coronary MRA obtained with parallel imaging as long as the geometry factor remains low.
SNR measurement; parallel imaging; coronary MRA; phased array coils; image noise
The hydrothermal reaction of Zn(CH3COO)2, NaOH and l-cysteic acid produced the title compound, [Na2Zn(C3H5NO5S)2]n. The ZnII cation is situated on an inversion centre and is in a distorted octahedral environment, being chelated by two deprotoned l-cysteic acid ligands through two amino N atoms and two carboxylic O atoms, with the two axial positions occupied by two carboxylic O atoms from two other l-cysteic acid ligands. Each l-cysteic acid ligand bridges five NaI ions via its sulfonate group and two ZnII ions via its carboxyl group, forming a three-dimensional framework. Weak N—H⋯O hydrogen bonding is observed in the crystal structure.
The purpose of this study was to determine whether compound zhi zhu xiang (CZZX) exerts anxiolytic-like effects in rats. The animals were orally administered CZZX (0.75, 1.5, and 3 g/kg daily) for 10 days and tested in the elevated plus maze (EPM), Vogel conflict test (VCT), and open field. Repeated treatment with CZZX (3 g/kg/day, p.o.) significantly increased the percentage of both entries into and time spent on the open arms of the EPM compared with saline controls. In the VCT, repeated treatment with CZZX (1.5 and 3 g/kg/day, p.o.) significantly increased the number of punished licks. The drug did not change the total entries into the open arms of the EPM or interfere with water consumption or nociceptive threshold, discarding potential confounding factors in the two tests. In the open field, locomotion was not reduced, discarding the possible sedative effect of CZZX. In the binding assay, the binding of [3H] Ro 15-1788 (flumazenil) to the benzodiazepine binding site in washed crude synaptosomal membranes from rat cerebral cortex was affected by CZZX. These data indicate an anxiolytic-like profile of action for CZZX without sedative side effects, and this activity may be mediated by benzodiazepine binding site modulation at γ-aminobutyric acid-A receptors.
AIM: To determine global DNA methylation in paired hepatocellular carcinoma (HCC) samples using several different assays and explore the correlations between hypomethylation and clinical parameters and biomarkers, including that of aflatoxin B1 exposure.
METHODS: Using the radio labeled methyl acceptance assay as a measure of global hypomethylation, as well as two repetitive elements, including satellite 2 (Sat2) by MethyLight and long interspersed nucleotide elements (LINE1), by pyrosequencing.
RESULTS: By all three assays, mean methylation levels in tumor tissues were significantly lower than that in adjacent tissues. Methyl acceptance assay log (mean ± SD) disintegrations/min/ng DNA are 70.0 ± 54.8 and 32.4 ± 15.6, respectively, P = 0.040; percent methylation of Sat2 42.2 ± 55.1 and 117.9 ± 88.8, respectively, P < 0.0001 and percent methylation LINE1 48.6 ± 14.8 and 71.7 ± 1.4, respectively, P < 0.0001. Aflatoxin B1-albumin (AFB1-Alb) adducts, a measure of exposure to this dietary carcinogen, were inversely correlated with LINE1 methylation (r = -0.36, P = 0.034).
CONCLUSION: Consistent hypomethylation in tumor compared to adjacent tissue was found by the three different methods. AFB1 exposure is associated with DNA global hypomethylation, suggesting that chemical carcinogens may influence epigenetic changes in humans.
Hepatocellular carcinoma; Epigenetics; Hypomethylation; [3H]-methyl acceptance assay; Satellite 2; Long interspersed nucleotide element-1; Aflatoxin B1