As the main source of extracellular matrix proteins in tumor stroma, hepatic stellate cells (HSCs) have a great impact on biological behaviors of hepatocellular carcinoma (HCC). In the present study, we have investigated a mechanism whereby HSCs modulate the chemoresistance of hepatoma cells. We used human HSC line lx-2 and chemotherapeutic agent cisplatin to investigate their effects on human HCC cell line Hep3B. The results showed that cisplatin resistance in Hep3B cells was enhanced with LX-2 CM (cultured medium) exposure in vitro as well as co-injection with LX-2 cells in null mice. Meanwhile, in presence of LX-2 CM, Hep3B cells underwent epithelial to mesenchymal transition (EMT) and upregulation of cancer stem cell (CSC) -like properties. Besides, LX-2 cells synthesized and secreted hepatic growth factor (HGF) into the CM. HGF receptor tyrosine kinase mesenchymal–epithelial transition factor (Met) was activated in Hep3B cells after LX-2 CM exposure. The HGF level of LX-2 CM could be effectively reduced by using HGF neutralizing antibody. Furthermore, depletion of HGF in LX-2 CM abolished its effects on activation of Met as well as promotion of the EMT, CSC-like features and cisplatin resistance in Hep3B cells. Collectively, secreting HGF into tumor milieu, HSCs may decrease hepatoma cells sensitization to chemotherapeutic agents by promoting EMT and CSC-like features via HGF/Met signaling.
Successful viruses have evolved superior strategies to escape host defenses or exploit host biological pathways. Most of the viral immediate-early (ie) genes are essential for viral infection and depend solely on host proteins; however, the molecular mechanisms are poorly understood. In this study, we focused on the modification of viral IE proteins by the crayfish small ubiquitin-related modifier (SUMO) and investigated the role of SUMOylation during the viral life cycle. SUMO and SUMO ubiquitin-conjugating enzyme 9 (UBC9) involved in SUMOylation were identified in red swamp crayfish (Procambarus clarkii). Both SUMO and UBC9 were upregulated in crayfish challenged with white spot syndrome virus (WSSV). Replication of WSSV genes increased in crayfish injected with recombinant SUMO or UBC9, but injection of mutant SUMO or UBC9 protein had no effect. Subsequently, we analyzed the mechanism by which crayfish SUMOylation facilitates WSSV replication. Crayfish UBC9 bound to all three WSSV IE proteins tested, and one of these IE proteins (WSV051) was covalently modified by SUMO in vitro. The expression of viral ie genes was affected and that of late genes was significantly inhibited in UBC9-silenced or SUMO-silenced crayfish, and the inhibition effect was rescued by injection of recombinant SUMO or UBC9. The results of this study demonstrate that viral IE proteins can be modified by crayfish SUMOylation, prompt the expression of viral genes, and ultimately benefit WSSV replication. Understanding of the mechanisms by which viruses exploit host components will greatly improve our knowledge of the virus-host “arms race” and contribute to the development of novel methods against virulent viruses.
Acoustophoresis has been utilized successfully in applications including cell trapping, focusing, and purification. One current limitation of acoustophoresis for cell sorting is the reliance on the inherent physical properties of cells (e.g., compressibility, density) instead of selecting cells based upon biologically relevant surface-presenting antigens. Introducing an acoustophoretic cell sorting approach that allows biochemical specificity may overcome this limitation, thus advancing the value of acoustophoresis approaches for both the basic research and clinical fields.
The results presented herein demonstrate the ability for negative acoustic contrast particles (NACPs) to specifically capture and transport positive acoustic contrast particles (PACPs) to the antinode of an ultrasound standing wave. Emulsification and post curing of pre-polymers, either polydimethylsiloxane (PDMS) or polyvinylmethylsiloxane (PVMS), within aqueous surfactant solution results in the formation of stable NACPs that focus onto pressure antinodes. We used either photochemical reactions with biotin-tetrafluorophenyl azide (biotin-TFPA) or end-functionalization of Pluronic F108 surfactant to biofunctionalize NACPs. These biotinylated NACPs bind specifically to streptavidin polystyrene microparticles (as cell surrogates) and transport them to the pressure antinode within an acoustofluidic chip.
To the best of our knowledge, this is the first demonstration of using NACPs as carriers for transport of PACPs in an ultrasound standing wave. By using different silicones (i.e., PDMS, PVMS) and curing chemistries, we demonstrate versatility of silicone materials for NACPs and advance the understanding of useful approaches for preparing NACPs. This bioseparation scheme holds potential for applications requiring rapid, continuous separations such as sorting and analysis of cells and biomolecules.
Cell separation; Continuous cell sorting; Acoustofluidics; Particle synthesis; Ultrasound standing wave
11β-hydroxysteroid dehydrogenases type 2 (11β-HSD2), a key regulator for pre-receptor metabolism of glucocorticoids (GCs) by converting active GC, cortisol, to inactive cortisone, has been shown to be present in a variety of tumors. But its expression and roles have rarely been discussed in hematological malignancies. Proteasome inhibitor bortezomib has been shown to not only possess antitumor effects but also potentiate the activity of other chemotherapeutics. In this study, we demonstrated that 11β-HSD2 was highly expressed in two GC-resistant T-cell leukemic cell lines Jurkat and Molt4. In contrast, no 11β-HSD2 expression was found in two GC-sensitive non-hodgkin lymphoma cell lines Daudi and Raji as well as normal peripheral blood T cells. Inhibition of 11β-HSD2 by 11β-HSD inhibitor 18β-glycyrrhetinic acid or 11β-HSD2 shRNA significantly increased cortisol-induced apoptosis in Jurkat cells. Additionally, pretreatment of Jurkat cells with low-dose bortezomib resulted in increased cellular sensitivity to GC as shown by elevated induction of apoptosis, more cells arrested at G1 stage and up-regulation of GC-induced leucine zipper which is an important mediator of GC action. Furthermore, we clarified that bortezomib could dose-dependently inhibit 11β-HSD2 messenger RNA and protein levels as well as activity (cortisol-cortisone conversion) through p38 mitogen-activated protein kinase signaling pathway. Therefore, we suggest 11β-HSD2 is, at least partially if not all, responsible for impaired GC suppression in Jurkat cells and also indicate a novel mechanism by which proteasome inhibitor bortezomib may influence GC action.
Silkworms are usually susceptible to the infection of Bombyx mori (B. mori) nucleopolyhedrovirus (BmNPV), which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells) of BmNPV-resistant strains (NB and BC8), the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi), the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses.
Persistent and heavy rainfall in the upper and middle Huaihe River of China brought about severe floods during the end of June and July 2007. However, there has been no assessment on the association between the floods and infectious diarrhea. This study aimed to quantify the impact of the floods in 2007 on the burden of disease due to infectious diarrhea in northwest of Anhui Province.
A time-stratified case-crossover analysis was firstly conducted to examine the relationship between daily cases of infectious diarrhea and the 2007 floods in Fuyang and Bozhou of Anhui Province. Odds ratios (ORs) of the flood risk were quantified by conditional logistic regression. The years lived with disability (YLDs) of infectious diarrhea attributable to floods were then estimated based on the WHO framework of the calculating potential impact fraction in the Burden of Disease study.
A total of 197 infectious diarrheas were notified during the exposure and control periods in the two study areas. The strongest effect was shown with a 2-day lag in Fuyang and a 5-day lag in Bozhou. Multivariable analysis showed that floods were significantly associated with an increased risk of the number cases of infectious diarrhea (OR = 3.175, 95%CI: 1.126–8.954 in Fuyang; OR = 6.754, 95%CI: 1.954–23.344 in Bozhou). Attributable YLD per 1000 of infectious diarrhea resulting from the floods was 0.0081 in Fuyang and 0.0209 in Bozhou.
Our findings confirm that floods have significantly increased the risks of infectious diarrhea in the study areas. In addition, prolonged moderate flood may cause more burdens of infectious diarrheas than severe flood with a shorter duration. More attention should be paid to particular vulnerable groups, including younger children and elderly, in developing public health preparation and intervention programs. Findings have significant implications for developing strategies to prevent and reduce health impact of floods.
Most of the existing multi-recipient signcryption schemes do not take the anonymity of recipients into consideration because the list of the identities of all recipients must be included in the ciphertext as a necessary element for decryption. Although the signer’s anonymity has been taken into account in several alternative schemes, these schemes often suffer from the cross-comparison attack and joint conspiracy attack. That is to say, there are few schemes that can achieve complete anonymity for both the signer and the recipient. However, in many practical applications, such as network conference, both the signer’s and the recipient’s anonymity should be considered carefully. Motivated by these concerns, we propose a novel multi-recipient signcryption scheme with complete anonymity. The new scheme can achieve both the signer’s and the recipient’s anonymity at the same time. Each recipient can easily judge whether the received ciphertext is from an authorized source, but cannot determine the real identity of the sender, and at the same time, each participant can easily check decryption permission, but cannot determine the identity of any other recipient. The scheme also provides a public verification method which enables anyone to publicly verify the validity of the ciphertext. Analyses show that the proposed scheme is more efficient in terms of computation complexity and ciphertext length and possesses more advantages than existing schemes, which makes it suitable for practical applications. The proposed scheme could be used for network conferences, paid-TV or DVD broadcasting applications to solve the secure communication problem without violating the privacy of each participant.
Key words: Multi-recipient signcryption; Signcryption; Complete Anonymity; Public verification.
Degeneration of medium spiny GABA neurons in the basal ganglia underlies motor dysfunction in Huntington’s disease (HD) which presently lacks effective therapy. In this study, we have successfully directed human embryonic stem cells (hESCs) to enriched populations of DARPP32-expressing forebrain GABA neurons. Transplantation of these human forebrain GABA neurons and their progenitors, but not spinal GABA cells, into the striatum of quinolinic acid-lesioned mice results in generation of large populations of DARPP32+ GABA neurons, which project to the substantia nigra as well as receiving glutamatergic and dopaminergic inputs, corresponding to correction of motor deficits. This finding raises hopes for cell therapy for HD.
Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrpy11 mutant embryos or cardia bifida phenotypes in fautm236a mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.
CRISPR; Cas9; genome editing; knockin; conditional knockout
Demyelination contributes to the functional impairment of irradiation injured spinal cord. One potential therapeutic strategy involves replacing the myelin-forming cells. Here, we asked whether transplantation of Olig2+-GFP+-oligodendrocyte precursor cells (OPCs), which are derived from Olig2-GFP-mouse embryonic stem cells (mESCs), could enhance remyelination and functional recovery after spinal cord irradiation injury. We differentiated Olig2-GFP-mESCs into purified Olig2+-GFP+-OPCs and transplanted them into the rats’ cervical 4–5 dorsal spinal cord level at 4 months after irradiation injury. Eight weeks after transplantation, the Olig2+-GFP+-OPCs survived and integrated into the injured spinal cord. Immunofluorescence analysis showed that the grafted Olig2+-GFP+-OPCs primarily differentiated into adenomatous polyposis coli (APC+) oligodendrocytes (54.6±10.5%). The staining with luxol fast blue, hematoxylin & eosin (LFB/H&E) and electron microscopy demonstrated that the engrafted Olig2+-GFP+-OPCs attenuated the demyelination resulted from the irradiation. More importantly, the recovery of forelimb locomotor function was enhanced in animals receiving grafts of Olig2+-GFP+-OPCs. We concluded that OPC transplantation is a feasible therapy to repair the irradiated lesions in the central nervous system (CNS).
To evaluate the promoter methylation status of MUC2 gene and mRNA expression in patients with hepatocellular carcinoma.
We analyzed MUC2 methylation by MSP, and MUC2 mRNA by real-time PCR in 74 HCC.
MUC2 mRNA were lower in HCC tissues (Mean -ΔCt = −4.70) than that in Non-HCC tissues (Mean -ΔCt = −2.98). Expression of MUC2 was elevated in only 23 (31.08%) of the 74 HCC patients. MUC2 promoter was hypermethylated in 62.2% (46/74) of HCCs, and in only 18.9% (14/74) of non-tumor samples. MUC2 mRNA were lower in HCC patients with hypermethylation (Mean -ΔΔCt = −2.25) than those with demethylation (Mean -ΔΔCt = −0.22), and there is a decreased tendency for MUC2 mRNA in HCC patients with promoter hypermethylation (p = 0.011). There was a significantly correlation found between MUC2 mRNA and HBV and AFP in HCC. The loss of MUC2 mRNA and hypermethylation could be poor prognostic factors. After treated by 5-Aza-CdR and TSA, we found that MUC2 mRNA induced significantly in 7721, Huh7 and HepG2 cells.
The results suggested that MUC2 mRNA silenced by promoter hypermethylation is associated with high levels HBV in HCC.
MUC2; Hepatocellular carcinoma; Methylation
The inducible IκB kinase (IKKi/IKKε) is a recently described serine-threonine IKK-related kinase. Previous studies have reported the role of IKKi in infectious diseases and cancer. However, its role in the cardiac response to pressure overload remains elusive. In this study, we investigated the effects of IKKi deficiency on the development of pathological cardiac hypertrophy using in vitro and in vivo models. First, we developed mouse models of pressure overload cardiac hypertrophy induced by pressure overload using aortic banding (AB). Four weeks after AB, cardiac function was then assessed through echocardiographic and hemodynamic measurements. Western blotting, real-time PCR and histological analyses were used to assess the pathological and molecular mechanisms. We observed that IKKi-deficient mice showed significantly enhanced cardiac hypertrophy, cardiac dysfunction, apoptosis and fibrosis compared with WT mice. Furthermore, we recently revealed that the IKKi-deficient mice spontaneously develop cardiac hypertrophy. Moreover, in vivo experiments showed that IKKi deficiency-induced cardiac hypertrophy was associated with the activation of the AKT and NF-κB signaling pathway in response to AB. In cultured cells, IKKi overexpression suppressed the activation of this pathway. In conclusion, we demonstrate that IKKi deficiency exacerbates cardiac hypertrophy by regulating the AKT and NF-κB signaling pathway.
Type 2 diabetes mellitus (T2DM) describes a group of metabolic disorders characterized by defects in insulin secretion and insulin sensitivity. Insulin secretion from pancreatic β-cells is an important factor in the etiology of T2DM, though the complex regulation and mechanisms of insulin secretion from β-cells remains to be fully elucidated. High plasma levels of serotonin (5-hydroxytryptamine; 5-HT) have been reported in T2DM patients, though the potential effect on insulin secretion is unclear. However, it is known that the 5-HT receptor 2C (5-HT2CR) agonist, mCPP, decreases plasma insulin concentration in mice. As such, we aimed to investigate the expression of the 5-HT2CR in pancreatic islets of diabetic mice and the role of 5-HT2CR signaling in insulin secretion from pancreatic β-cells. We found that 5-HT2CR expression was significantly increased in pancreatic islets of db/db mice. Furthermore, treatment with a 5-HT2CR antagonist (SB242084) increased insulin secretion from pancreatic islets isolated from db/db mice in a dose-dependent manner, but had no effect in islets from control mice. The effect of a 5-HT2CR agonist (mCPP) and antagonist (SB242084) were further studied in isolated pancreatic islets from mice and Min-6 cells. We found that mCPP significantly inhibited insulin secretion in Min-6 cells and isolated islets in a dose-dependent manner, which could be reversed by SB242084 or RNA interference against 5-HT2CR. We also treated Min-6 cells with palmitic acid for 24 h, and found that the expression of 5-HT2CR increased in a dose-dependent manner; furthermore, the inhibition of insulin secretion in Min-6 cells induced by palmitic acid could be reversed by SB242084 or RNA interference against 5-HT2CR. Taken together, our data suggests that increased expression of 5-HT2CR in pancreatic β-cells might inhibit insulin secretion. This unique observation increases our understanding of T2DM and suggests new avenues for potential treatment.
Background: To investigate the efficacy of TACE combined with CQ, an autophagic inhibitor, in a rabbit VX2 liver tumor model.
Methods: Tumor size was measured. And tumor growth rate was calculated to examine the effect of the combined treatment. Apoptosis was detected by TUNEL assay. Meanwhile, autophagic activity was detected by immunohistochemistry and Western blotting to investigate the mechanism underlying. Liver function was also examined to assess feasibility and safety of the combined therapy.
Results: Tumors in the control grew more than 4 times bigger after 14 days, while that in the group of TACE alone just showed mild growth. But a slight shrinkage was shown after the treatment of CQ+TACE. Growth ratio of TACE alone was 96.45% ± 28.958% while that of CQ+TACE was -28.73% ± 12.265%. Compared with TACE alone, necrosis in CQ+TACE showed no significant difference, however, the apoptosis was much higher. There were only 14.8±3.11% apoptotic cells in TACE, but 33±4.18% in CQ+TACE, which suggests the increased apoptosis in CQ+TACE contributed to the decrease of tumor volume. In terms of autophagic activity, the result is negative when we immunostained sections of the control with LC3 antibody, but positive in TACE alone and CQ+TACE. And the result of Western blot showed that there was just a low level of LC3Ⅱexpressed in the control and CQ alone, but higher in TACE, and much higher in CQ+TACE because CQ inhibited its degradation in autophagy. Compared with control, p62 decreased in TACE, but the decrease was partially reversed in CQ+TACE. In addition, toxicity of CQ+TACE was assessed not higher than TACE alone, which supports the safety of CQ+TACE.
Conclusion: CQ+TACE works better than TACE alone in rabbit VX2 liver tumor model because CQ inhibits autophagy induced by TACE. The inhibited autophagy loses its resistance to apoptosis that apoptosis increased, which contributes to the inhibition of tumor growth. This study indicates CQ may be a promising adjuvant to promote the effect of TACE.
Hepatocellular carcinoma; transcatheter arterial chemoembolization; chloroquine; apoptosis; autophagy.
Intensive research on the molecule structures of the gamma-nminobutyric acid (GABA) receptor in agricultural pests has great significance to the mechanism investigation, resistance prevention, and molecular design of novel pesticides. The GABA receptor a2 (SlGABARα2) subunit gene in Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) was cloned using the technologies of reverse transcription PCR and rapid amplification of cDNA ends. The gemonic DNA sequence of SlGABARα2 has 5164 bp with 8 exons and 7 introns that were in accordance with the GT-AG splicing formula. The complete mRNA sequence of SlGABARα2 was 1965 bp, with an open reading frame of 1500 bp encoding a protein of 499 amino acids. The GABA receptor is highly conserved among insects. The conserved regions include several N-glycosylation, Oglycosylation, and phosphorylation sites, as well as 4 transmembrane domains. The identities that SlGABARα2 shared with the GABA receptor a2 subunit of Spodoptera exigua, Heliothis virescens, Chilo suppressalis, Plutella xylostella, Bombyx mori ranged from 99.2% to 87.2% at the amino acid level. The comparative 3-dimensional model of SlGABARα2 showed that its tertiary structure was composed of 4 major α-helixes located at the 4 putative transmembrane domains on one side, with some β-sheets and 1 small α-helix on the other side. SlGABARα2 may be attached to the membrane by 4 α-helixes that bind ions in other conserved domains to transport them through the membrane. The results of quantitative real time PCR demonstrated that SlGABARα2 was expressed in all developmental stages of S. litura. The relative expression level of SlGABARα2 was the lowest in eggs and increased with larval growth, while it declined slightly in pupae and reached the peak in adults. The expressions of SlGABARα2 in larvae varied among different tissues; it was extremely high in the brain but was low in the midgut, epicuticle, Malpighian tube, and fat body.
developmental expression; GABA receptor; gene structure; three-dimensional model; tissue distribution
The endotoxin level in the portal and peripheral veins of hepatocellular carcinoma (HCC) patients is higher and lipopolysaccharide (LPS), a cell wall constituent of gram-negative bacteria, has been reported to inhibit tumor growth. However, in this study, we found that LPS-induced toll-like receptor 4 (TLR4) signaling was involved in tumor invasion and survival, and the molecular mechanism was investigated,
Four HCC cell lines and a splenic vein metastasis of the nude mouse model were used to study the invasion ability of LPS-induced HCC cells and the epithelia-mesenchymal transition (EMT) in vitro and in vivo. A total of 106 clinical samples from HCC patients were used to evaluate TLR4 expression and analyze its association with clinicopathological characteristics
The in vitro and in vivo experiments demonstrated that LPS could significantly enhance the invasive potential and induce EMT in HCC cells with TLR4 dependent. Further studies showed that LPS could directly activate nuclear factor kappa B (NF-κB) signaling through TLR4 in HCC cells. Interestingly, blocking NF-κB signaling significantly inhibited transcription factor Snail expression and thereby inhibited EMT occurrence. High expression of TLR4 in HCC tissues was strongly associated with both poor cancer-free survival and overall survival in patients.
Our results indicate that TLR4 signaling is required for LPS-induced EMT, tumor cell invasion and metastasis, which provide molecular insights for LPS-related pathogenesis and a basis for developing new strategies against metastasis in HCC.
Toll-like receptor 4; Epithelial-mesenchymal transition; Lipopolysaccharide; Human hepatocellular carcinoma
In response to inflammation, mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodeling and wound healing. Tumors apply persistent mechanical and pathological stress to tissues and causes continual infiltration of MSCs. Here, we demonstrate that MSCs promote human hepatocellular carcinoma (HCC) metastasis under the influence of inflammation. The metastasis promoting effect could be imitated with the supernatant of MSCs pretreated with IFNγ and TNFα. Interestingly, treatment of HCC cells with the supernatant leads to epithelial-mesenchymal transition (EMT), an effect related to the production of TGFβ by cytokines stimulated MSCs. Importantly, the levels of MSCs expressing SSEA4 in clinical HCC samples significantly correlated with poor prognosis of HCC, and EMT of HCC was strongly associated with a shorter cancer-free interval (CFI) and a worse overall survival (OS). Therefore, our results suggest that MSCs in tumor inflammatory microenvironment could promote tumor metastasis through TGFβ-induced EMT.
Hypobaric intermittent hypoxia (HIH) produces many favorable effects in the cardiovascular system such as anti-hypertensive effect. In this study, we showed that HIH significantly attenuated a depressor response induced by acute hypoxia.
Sprague-Dawley rats received HIH in a hypobaric chamber simulating an altitude of 5000 m. The artery blood pressure (ABP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were recorded in anesthetized control rats and rats received HIH. The baseline ABP, HR and RSNA were not different between HIH and control rats. Acute hypoxia-induced decrease in ABP was significantly attenuated in HIH rat compared with control rats. However, acute hypoxia-induced increases in HR and RSNA were greater in HIH rat than in control rats. After removal of bilateral ascending depressor nerves, acute hypoxia-induced depressor and sympathoexcitatory responses were comparable in control and HIH rats. Furthermore, acute hypoxia-induced depressor and sympathoexcitatory responses did not differ between control and HIH groups after blocking ATP-dependent K+ channels by glibenclamide. The baroreflex function evaluated by intravenous injection of phenylephrine and sodium nitroprusside was markedly augmented in HIH rats compared with control rats. The pressor and sympathoexcitatory responses evoked by intravenous injection of cyanide potassium were also significantly greater in HIH rats than in control rats.
Our findings suggest that HIH suppresses acute hypoxia-induced depressor response through enhancement of baroreflex and chemoreflex function, which involves activation of ATP-dependent K+ channels. This study provides new information and underlying mechanism on the beneficiary effect of HIH on maintaining cardiovascular homeostasis.
The indigenous small non-coding RNAs, known as microRNAs (miRNAs), are important regulators of gene expression and many of them are evolutionarily conserved. Whether stem-loop RT-PCR, as a sensitive method, could be utilized to clone conserved miRNAs from non-model insects lacks information. Here, three miRNAs, sli-miR-14, sli-miR-2a and sli-bantam, were cloned from Spodoptera litura by stem-loop RT-PCR. Two groups of primers were designed, and one of them performed especially well and proved stable. The sequences of two highly conserved miRNAs, sli-miR-14 and sli-miR-2a were identical to those in Drosophila melanogaster. To validate the reliability of this strategy, pre-miR-14 and pre-miR-2a in S. litura as representatives were given as well; this shared high homology with those in D. melanogaster and Bombyx mori, and both mature sequences of sli-miR-14 and sli-miR-2a in their precursors shared 100% identity to the results shown by stem-loop RT-PCR. Moreover, expression patterns of these miRNAs were investigated by real-time quantitative PCR. Sli-miR-14 and sli-miR-2a could be detected successfully and their expression patterns showed similar characteristics with those in model insects, further suggesting stem-loop RT-PCR technology can be used for identification of highly conserved miRNAs in non-model insects. These results provide a simplified and efficient strategy for studying the structure and function of highly conserved miRNAs, especially some critical miRNAs in non-model insects.
microRNA; precursor; identification; stem-loop RT-PCR; non-model insect; sequence conservation; Spodoptera litura
Background: To evaluate the promoter methylation status of RECK gene and mRNA expression in patients with hepatocellular carcinoma (HCC).
Methods: We analyzed RECK methylation by MSP, and RECK mRNA by real-time PCR in 74 HCC. The liver cell lines (7721, Chang and Hep-G2) were treated with 5-Aza-CdR and TSA.
Results: RECK mRNA were lower in HCC tissues (Mean -∆Ct = -3.29) than that in Non-Hcc tissues (Mean -∆Ct = -2.42). Expression of RECK was elevated in only 24 (32.43%) of the 74 HCC patients but decreased (-∆∆Ct<0) in 50 (67.57%) of the patients. RECK promoter was hypermethylated in 55.4% (41/74) of HCCs, and in only 17.6% (13/74) of Non-Hcc samples. RECK mRNA were lower in HCC patients with hypermethylation (∆MI>=0.5) (Mean -∆∆Ct = -1.75) than those with demethylation (∆MI<0.5) (Mean -∆∆Ct = 0.05), and there is a decreased tendency for RECK mRNA in HCC patients with promoter hypermethylation (p = 0.002). There was a significantly correlation found between RECK mRNA and poor survival after surgery. After treated by 5-Aza-CdR and TSA, we found that RECK mRNA induced different changes in 7721, Chang and Hep-G2 cells. And RECK demethylation also induced by epigenetic inhibitors.
Conclusion: The results suggested that the hypermethylation may lead to promoter silencing of RECK mRNA and associated with poor survival in HCC.
RECK; hepatocellular carcinoma; methylation.
Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H2S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H2S produced locally in the control of uterine contractility during labour is unknown.
Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principle enzymes responsible for H2S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H2S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10−7–10−2 mol/L), a precursor of H2S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10−3 mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (KATP) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.
H2S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H2S synthetic enzymes is down-regulated during labour, suggesting that H2S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.
The hMSH2 gene, a member of the mismatch repair (MMR) pathway, plays a key role in the maintenance of genomic integrity. The common sequence variation in hMSH2, IVS12-6 T>C, has been implicated in cancer risk. However, the results of published studies on this polymorphism remain conflicting. Hence, we conducted a meta-analysis to clarify the role of the hMSH2 IVS12-6 T>C polymorphism in cancer. We performed a comprehensive literature search updated to March 2011 of studies on the associations between the hMSH2 IVS12-6 T>C polymorphism and cancer risk. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the associations. Thirteen studies involving 7,527 patients and 8,762 control subjects were included in this meta-analysis. The overall results indicated no major influence of the polymorphism on cancer risk. However, stratified analysis by cancer types showed that the hMSH2 IVS12-6 polymorphism increased the risk for non-Hodgkin's lymphomas (heterozygote comparison: OR=1.62; 95% CI 1.06–2.47). When stratified by the source of controls, significant associations were observed in hospital-based populations (heterozygote comparison: OR=1.28; 95% CI 1.02–1.61). These results indicate that the polymorphism of hMSH2, IVS12-6, may cause a different effect in different types of cancers. To draw more comprehensive conclusions, further prospective studies with larger numbers of participants worldwide are required to examine the associations between this polymorphism and cancer risk.
mismatch repair; hMSH2; polymorphism; meta-analysis
Potassium channels play critical roles in the regulation of cell membrane potential, which is central to the excitability of myometrium. The ATP-sensitive potassium (KATP) channel is one of the most abundant potassium channels in myometrium. The objectives of this study were to investigate the protein expression of KATP channel in human myometrium and determine the levels of KATP channel in lower and upper segmental myometrium before and after onset of labour.
Both lower segmental (LS) and upper segmental (US) myometrial biopsies were collected at cesarean section from pregnant women not-in-labour (TNL) or in-labour (TL) at term. Protein expression level and cellular localization of four KATP channel subunits in US and LS myometrium were determined by Western blot analysis and immunohistochemistry, respectively. The contractile activity of myometrial strip was measured under isometric conditions.
Four KATP channel subunits, namely Kir6.1, Kir6.2, SUR1 and SUR2B were identified in pregnant myometrium. While found in vascular myocytes, these subunits appear to be preferentially expressed in myometrial myocytes. Diazoxide, a KATP channel opener, inhibited the spontaneous contractility of pregnant myometrium, suggesting that the KATP channels are functional in human pregnant myometrium. Diazoxide was less potent in TL strips than that in TNL strips. Interestingly, expression of SUR1 was greater in TL than TNL tissues, although no differences were found for SUR2B in these two tissues. For both lower and upper segmental myometrium, Kir6.1 and Kir6.2 were less in TL compared with TNL tissues.
Functional KATP channels are expressed in human pregnant myometrium. Down-regulation of Kir6.1 and Kir6.2 expression in myometrium may contribute to the enhanced uterine contractility associated with the onset of labour.
Sustained high concentration of glucose has been verified toxic to β-cells. Glucose augments Ca2+-stimulated insulin release in pancreatic β-cells, but chronic high concentration of glucose could induce a sustained level of Ca2+ in β-cells, which leads to cell apoptosis. However, the mechanism of high glucose-induced β-cell apoptosis remains unclear. In this study, we use a calcium channel blocker, nifedipine, to investigate whether the inhibition of intracellular Ca2+ concentration could protect β-cells from chronic high glucose-induced apoptosis. It was found that in a concentration of 33.3 mM, chronic stimulation of glucose could induce INS-1 β-cells apoptosis at least through the endoplasmic reticulum stress pathway and 10 μM nifedipine inhibited Ca2+ release to protect β-cells from high glucose-induced endoplasmic reticulum stress and apoptosis. These results indicated that inhibition of Ca2+ over-accumulation might provide benefit to attenuate islet β-cell decompensation in a high glucose environment.
nifedipine; Ca2+ homeostasis; β-cell; endoplasmic reticulum stress; apoptosis; high glucose