Long non-coding RNA MALAT1 promotes angiogenesis and immunosuppressive properties of HCC cells by sponging miR-140.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer in adults. Previous studies in our laboratory found that long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was upregulated in HCC cells, which could affect the metastasis and invasion of HCC. However, the underlying mechanism remains unknown. Herein, we studied the interaction between MALAT1 and miR-140 on the regulation of angiogenesis and immunosuppressive properties. We revealed that the expression of MALAT1 and VEGF-A was significantly increased in HCC cells. Knockdown of MALAT1 in HCC cells suppressed the production of VEGF-A, impaired the angiogenesis of HUVECs, and facilitated the polarization of macrophage toward the M1 subset. Mechanistically, the interaction between MALAT1 and miR-140 or between miR-140 and VEGF-A was confirmed by multiple assays. Besides, a negative correlation between MALAT1 and miR-140 was found in HCC tissues. Furthermore, miR-140 inhibition significantly increased VEGF-A expression, promoted angiogenesis of HUVECs, and redirected the polarization of macrophages toward the M2 subset. In addition, in vivo studies also verified the regulatory network of the MALAT1/miR-140 axis on VEGF-A in HCC progression. In summary, this study revealed the mechanism that MALAT1 worked as a putative HCC promotor via inhibiting miR-140. Therefore, targeting MALAT1 or miR-140 might alleviate the progression of HCC in the future.

Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10.

Antibiotic-producing microorganisms have evolved several self-resistance mechanisms to prevent auto-toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. In this work, two ATP-binding cassette (ABC) transporter-encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5-de-epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up-regulated in response to intracellular accumulation of natamycin and 4,5-de-epoxynatamycin, suggesting a key role in the stress response for self-resistance. This article discusses a novel antibiotic-related efflux and response system in Streptomyces, as well as a self-resistance mechanism in antibiotic-producing strains.

Investigation of associations between ten polymorphisms and the risk of coronary artery disease in Southern Han Chinese.

A large-scale meta-analysis of 14 genome-wide association studies has identified and replicated a series of susceptibility polymorphisms for coronary artery disease (CAD) in European ancestry populations, but evidences for the associations of these loci with CAD in other ethnicities remain lacking. Herein we investigated the associations between ten (rs579459, rs12413409, rs964184, rs4773144, rs2895811, rs3825807, rs216172, rs12936587, rs46522 and rs3798220) of these loci and CAD in Southern Han Chinese (CHS). Genotyping was performed in 1716 CAD patients and 1572 controls using mass spectrography. Both allelic and genotypic associations of rs964184, rs2895811 and rs3798220 with CAD were significant, regardless of adjustment for covariates of gender, age, hypertension, type 2 diabetes, blood lipid profiles and smoking. Significant association of rs12413409 was initially not observed, but after the adjustment for the covariates, both allelic and genotypic associations were identified as significant. Neither allelic nor genotypic association of the other six polymorphisms with CAD was significant regardless of the adjustment. Our results indicated that four loci of the total 10 were associated with CAD in CHS. Therefore, some of the CAD-related loci in European ancestry populations are indeed susceptibility loci for the risk of CAD in Han Chinese.

Transcriptome-guided identification of SprA as a pleiotropic regulator in Streptomyces chattanoogensis.

Quorum sensing molecular γ-butyrolactones (GBL) are widely distributed among the genus Streptomyces. Their cognate receptors have been demonstrated to control secondary metabolism and/or morphological differentiation. ScgA is responsible for the biosynthesis of GBL in Streptomyces chattanoogensis. According to the genome-wide transcriptome analysis of the ΔscgA mutant, we found that the expression of sprA, which encodes a GBL receptor homologue, was shown to be positively regulated by ScgA. Electrophoretic mobility shift assays and DNase I footprinting assays showed that SprA bound to two specific autoregulatory element (ARE) sequences located upstream of the sprA gene, indicating that its expression is self-regulated. SprA was involved in biosynthesis of GBL by repressing the expression of scgA. An Escherichia coli-based luciferase report system demonstrated that SprA directly repressed the expression of scgR, which encodes a GBL receptor. Like deletion of scgA, the disruption of sprA resulted in decreased production of the antibiotic natamycin in liquid culture and retarded morphological differentiation on solid agar. This work indicates that SprA acts as a pleiotropic regulator of both morphogenesis and the production of natamycin.

Sigma factor WhiGch positively regulates natamycin production in Streptomyces chattanoogensis L10.

The roles of many sigma factors are unclear in regulatory mechanism of secondary metabolism in Streptomyces. Here, we report the regulation network of a group 3 sigma factor, WhiGch, from a natamycin industrial strain Streptomyces chattanoogensis L10. WhiGch regulates the growth and morphological differentiation of S. chattanoogensis L10. The whiG ch deletion mutant decreased natamycin production by about 30 % and delayed natamycin production more than 24 h by delaying the growth. Overexpression of the whiG ch gene increased natamycin production in large scale production medium by about 26 %. WhiGch upregulated the transcription of natamycin biosynthetic gene cluster and inhibited the expression of migrastatin and jadomycin analog biosynthetic polyketide synthase genes. WhiGch positively regulated natamycin biosynthetic gene cluster by directly binding to the promoters of scnC and scnD, which were involved in natamycin biosynthesis, and these binding sites adjacent to translation start codon were determined. Thus, this paper further elucidates the high natamycin yield mechanisms of industrial strains and demonstrates that a valuable improvement in the yield of the target metabolites can be achieved through manipulating the transcription regulators.

WblAch, a pivotal activator of natamycin biosynthesis and morphological differentiation in Streptomyces chattanoogensis L10, is positively regulated by AdpAch.

Detailed mechanisms of WhiB-like (Wbl) proteins involved in antibiotic biosynthesis and morphological differentiation are poorly understood. Here, we characterize the role of WblAch, a Streptomyces chattanoogensis L10 protein belonging to this superfamily. Based on DNA microarray data and verified by real-time quantitative PCR (qRT-PCR), the expression of wblAch was shown to be positively regulated by AdpAch. Gel retardation assays and DNase I footprinting experiments showed that AdpAch has specific DNA-binding activity for the promoter region of wblAch. Gene disruption and genetic complementation revealed that WblAch acts in a positive manner to regulate natamycin production. When wblAch was overexpressed in the wild-type strain, the natamycin yield was increased by ∼30%. This provides a strategy to generate improved strains for natamycin production. Moreover, transcriptional analysis showed that the expression levels of whi genes (including whiA, whiB, whiH, and whiI) were severely depressed in the ΔwblAch mutant, suggesting that WblAch plays a part in morphological differentiation by influencing the expression of the whi genes.

Connexin43 and angiotensin II alterations in hearts of rats having undergone an acute exposure to alcohol.

INTRODUCTION: Alcohol-induced heart damage is associated with enzyme and protein alterations. The purpose of this study was to investigate alcohol-induced alterations in cardiac connexin 43 (Cx43) and angiotensin II (Ang II) after acute alcohol administration. METHOD: Male Wistar rats were randomly divided into 2 groups: a control group and an ethanol group. The ethanol group intraperitoneally received 3.8 g/kg ethanol; the controls were given the same amount of saline via the same route. After the righting reflex disappeared, midsternotomy was performed in all animals. Immunohistochemical analysis was performed to evaluate protein expression of Cx43 and Ang II. Sections were analyzed by digital image analysis. RESULT: The expression of Cx43 was significantly reduced after acute ethanol treatment, with the integrated optical density lower when compared with control (P < 0.05). The expression of Ang II was significantly increased after acute ethanol treatment, supported by integrated optical density when compared with control (P < 0.05). CONCLUSIONS: In summary, cardiac protein expression of Cx43 and Ang II were found to be significantly altered after acute ethanol treatment, suggesting that these 2 proteins may be important underlying mechanisms of vulnerability to oxidative injury in the heart during acute ethanol. The present study indicated that acute ethanol toxicity caused different alterations in heart proteins that would be related to oxidative stress.

Reduction in Purkinje fiber number in rats undergone fatal electrocution.

Purkinje fibers in cardiac conduction tissue during fatal electrocution. A total of 16 Sprague Dawley rats were divided into 2 groups as follows: the electrocution group and the control group.Animals were deeply anesthetized with sodium pentobarbital and, in the electrocution group, all 8 rats underwent a fatal electrical shock (220 v,50 Hz) followed by cervical dislocation. In the control group, all 8 rats underwent execution by cervical dislocation. Following death, hearts were rapidly excised and perfused with 1% paraformaldehyde before tissues of the left ventricular anterior wall (LVAW) were isolated. The microscopic structure of the Purkinje fibers were subsequently analyzed using conventional hematoxylin and eosin staining. A majority of the Purkinje fibers were located in groups among the cardiac muscle of the LVAW. A significant reduction in Purkinje fiber expression was displayed in the electrocution group compared with the control group (P G 0.05).The mean total number of Purkinje fibers for the electrocution and control groups were 59 T 11 and 3287 T 19 cells, respectively (P G 0.05).The estimated number of Purkinje fibers in the LVAW of the control group was significantly greater than observed in the electrocution group(41.09 T 0.24 vs. 0.7375 T 0.14, P G 0.05). The findings of the current study suggest that such a reduction would be reflected in abnormal cardiac conduction and a possible cause of sudden death.

Connexin 43, angiotensin II, endothelin 1, and type III collagen alterations in heart of rats having undergone fatal electrocution.

Death due to accidental electrocution occurs frequently. The aim of this study was to investigate alterations in cardiac connexin 43 (Cx43), angiotensin II (Ang II), endothelin 1 (ET-1), and type III collagen associated with fatal electrocution.Twenty-four Sprague-Dawley rats were divided into control, fatal electrocution (220 V, 50 Hz, 60 seconds), and electrical injury (220 V, 50 Hz, 60 seconds) groups. Animals were deeply anesthetized with sodium pentobarbital before each treatment, with the anode connected to the left foreleg and the cathode to the right hindleg, followed by cervical dislocation. Control animals received cervical dislocation alone. Immunohistochemical analysis was performed to evaluate the cardiac protein expression of Cx43, Ang II, ET-1, and type III collagen. Sections were analyzed by digital image analysis.The expression of Cx43 was significantly reduced after fatal electrocution, with the integrated optical density also lower when compared with control (P < 0.05). Expression of both Ang II and ET-1 was significantly increased after fatal electrocution, supported by integrated optical density when compared with control (P < 0.05). But no significant difference was found in type III collagen expression between the fatal electrocution group and the control group.In summary, cardiac protein expression of Cx43, Ang II, and ET-1 was found to be significantly altered with fatal electrocution, suggesting that these 3 proteins may be important underlying mechanisms of death during fatal electrocution. The current findings indicate that such alterations would be reflected in abnormal cardiac function and a possible cause of sudden death.

[Application of serum total IgE, tryptase and chymase in the identification of death caused by drug anaphylactic shock].

OBJECTIVE: To explore the application value of serum total IgE, tryptase and chymase in the identification of death caused by drug anaphylactic shock. METHODS: The general information from 235 cases of non-drug anaphylactic shock and 32 cases of drug anaphylactic shock were analyzed. The serum IgE level had been detected in the cases. Ten cases caused by coronary disease and 10 cases caused by sudden manhood death syndrome were selected from non-drug anaphylactic shock cases for the control group. Expressions of tryptase and chymase in the lung and heart were detected using immunohistochemistry method. The number and IOD of positive mast cells were counted. RESULTS: In the drug anaphylactic shock group, the IgE value of 18 samples (56.25%) was significantly higher than the normal upper limit of 120 IU/mL. In the non-drug anaphylactic shock group, the IgE value of 67 samples (28.51%) was higher than 120 IU/mL. The expressions of tryptase and chymase were significantly increased in lung and myocardial tissue in drug anaphylactic shock group (P < 0.05). CONCLUSION: Tryptase and chymase are more superior than that of the serum total IgE in the diagnosis of death caused by drug anaphylactic shock, and are more suitable in forensic practice.

[Distribution of human enterovirus 71 in brainstem of infants with brain stem encephalitis and infection mechanism].

OBJECTIVE: To explore the mechanism that how human enterovirus 71 (EV71) invades the brainstem and how intercellular adhesion molecules-1 (ICAM-1) participates by analyzing the expression and distribution of human EV71, and ICAM-1 in brainstem of infants with brain stem encephalitis. METHODS: Twenty-two brainstem of infants with brain stem encephalitis were collected as the experimental group and 10 brainstems of fatal congenital heart disease were selected as the control group. The sections with perivascular cuffings were selected to observe EV71-VP1 expression by immunohistochemistry method and ICAM-1 expression was detected for the sections with EV71-VP1 positive expression. The staining image analysis and statistics analysis were performed. The experiment and control groups were compared. RESULTS: (1) EV71-VP1 positive cells in the experimental group were mainly astrocytes in brainstem with [dark]-brown particles, and the control group was negative. (2) ICAM-1 positive cells showed [dark]-brown. The expression in inflammatory cells (around blood vessels of brain stem and in glial nodules) and gliocytes increased. The results showed statistical difference comparing with control group (P &lt; 0.05). CONCLUSION: The brainstem encephalitis can be used to diagnose fatal EV71 infection in infants. EV71 can invade the brainstem via hematogenous route. ICAM-1 may play an important role in the pathogenic process.

Relationships among genetic makeup, active ingredient content, and place of origin of the medicinal plant Gastrodia tuber.

Gastrodia tuber and its component gastrodin have many pharmacological effects. The chemical fingerprints and gastrodin contents of eight Gastrodia populations were determined, and the genomic DNA polymorphism of the populations was investigated. Genetic distance coefficients among the populations were calculated using the DNA polymorphism data. A dendrogram of the genetic similarities between the populations was constructed using the genetic distance coefficients. The results indicated that the genomic DNA of Gastrodia tubers was highly polymorphic; the eight populations clustered into three major groups, and the gastrodin content varied greatly among these groups. There were obvious correlations among genetic makeup, gastrodin content, and place of origin. The ecological environments in Guizhou and Shanxi may be conducive to evolution and to gastrodin biosynthesis, and more suitable for cultivation of Gastrodia tubers. These findings may provide a scientific basis for overall genetic resource management and for the selection of locations for cultivating Gastrodia tubers.

Pulmonary peptidergic innervation remodeling and development of airway hyperresponsiveness induced by RSV persistent infection.

Respiratory syncytial virus (RSV) infection causes bronchiolitis in infants and children, which is an important risk factor for the development of chronic asthma. To probe the underlying mechanisms that RSV infection increases the susceptibility of asthma, this present study was designed to establish a RSV persistent infection animal model by cyclophosphamide (CYP) pretreatment that more closely mimic human RSV infection. CYP is an immunosuppressant, which induced deficiency in cellular and humoral immunity. Pulmonary RSV titers, airway function and peptidergic innervation were measured on 7d, 28 d, 42 d and 60 d postinfection. The results showed that during RSV persistent infection, the lungs of RSV-inoculated animals pretreated with CYP showed higher RSV titers and exhibited obvious chronic inflammation. The results also showed that protein gene product 9.5 (PGP9.5), substance P (SP) and calcitonin gene-related peptide (CGRP)-immunoreactive fibers increased and vasoactive intestinal peptide (VIP)-immunoreactive fibers decreased during RSV persistent infection. These results demonstrate that RSV persistent infection induces significant alterations in the peptidergic innervation in the airways, which may be associated with the development of altered airway function.

Screening and identification of differentially expressed genes in myocardial ischaemia/reperfusion injury using suppression subtractive hybridization.

OBJECTIVE: Myocardial ischaemia/reperfusion (MI/R) injury is characterized by metabolic and ultrastructural changes, which lead to irreversible injury. Several mechanisms have been postulated for the pathogenesis of MI/R injury although little is known regarding the role of myocardial gene expression. METHODS AND RESULTS: In this study, suppression subtractive hybridization (SSH) was employed to systematically isolate and screen the differentially expressed genes in the MI/R injury rat model. The characteristics of these specific genes were analysed using bioinformatics. Our results showed that among the 119 identified genes, 54 genes were expressed at higher levels and 65 genes were at lower levels compared with the control group. CONCLUSIONS: These genes are closely associated with energy metabolism, iron transport, signalling transduction and may provide important clues for the elucidation of the mechanisms of MI/R injury. Our results further indicated that myocardial injury is likely the result of summation of functional impairment of multiple genes rather than the result of damage to a single critical gene.

[Effects of serum from crush injury rats on vascular endothelial cell apoptosis and their potential mechanism].

OBJECTIVE: To investigate the effects of serum from crush injury rats on vascular endothelial cell apoptosis and their potential mechanism. METHODS: Bovine aorta endothelial cells were cultured in vitro and the effects of serum from crush injury rats on cell apoptosis and intracellular calcium concentration ([Ca2+]i) were observed. Meanwhile, the levels of rat blood plasma endothelin-1 (ET-1) and atrial natriuretic peptide(ANP) were measured. RESULTS: Compared with normal rat serum treatment, the cell apoptosis rate decreased from (8.26+/-1.75)% to (2.75+/-0.90)%, while the concentration of [Ca2+]i increased from (96.98+/-3.95) to (118.79+/-3.22) nmol/L in serum from crush injury rats, respectively. The concentration of ET-1 and ANP increased significantly in crush injury rat serum. CONCLUSION: Serum from crush injury rats could inhibit apoptosis of the vascular endothelial cells. These effects may be related to increased level of [Ca2+]i mediated by ET-1 and ANP.

[Apoptosis of cultured cortical neurons of rat's brain induced by heroin].

OBJECTIVE: To investigate whether heroin can directly induce apoptosis in primary cultured cortical neurons of rat's brain. METHODS: Cultured primary neurons cultures were obtained from cerebral cortex of embryo rats. After 7 days, the cells were incubated with different concentrations of heroin (purity-80%) for 24 hours. The neuronal survival was assessed by cell viability counting with fluorescent diacetate (FDA) staining. The morphological and biochemical changes were observed with Hoechst 33258 fluorescent staining and then analyzed by agarose gel electrophoresis, respectively. RESULTS: After treatment with different concentrations of heroin, the neurons showed a decreased survival rate in a dose dependent manner, and there was a significant difference in the survival rate between the heroin group and the control group (P < 0.05). When exposed to different concentrations of heroin, neurons exhibited the morphological and biochemical features of apoptosis, including cell shrinkage, neurite degeneration, network disappearance, condensation and aggregation of nuclear chromatin, and the formation of DNA ladders. With the increase of heroin concentration of rat's brain more apoptotic bodies were seen. CONCLUSION: Heroin can directly induce apoptosis in primary cultured cortical neurons in rat's brain.

[The effects of heroin on intracellular free Ca2+ of rat myocardium].

OBJECTIVE: To observe the effects of heroin on intracellular free Ca2+ in rat myocardium. METHODS: The effects of heroin on intracellular free Ca2+ were observed in cultured neonatal rat myocardium by measuring intracellular free Ca2+ concentration using calcium fluorescent probe Flou-3/AM and laser scanning confocal microscope. RESULTS: Different doses and concentrations of heroin appeared to have different effects on intracellular free Ca2+ concentrations, with a dosage dependent short linear increase in the fluorescence intensity (i.e., Ca2+ concentration) leading to [Ca2+]i peak. CONCLUSION: Heroin could affect concentrations of [Ca2+]i in myocardium and its dosage related effect needs further investigation.

[Influence of human cytomegalovirus infection on cell cycle and replication licensing factor Cdt1 in human embryonic lung fibroblastic cells].

OBJECTIVE: To study the influence of human cytomegalovirus (HCMV) infection on cell cycle and the expression of replication licensing factor Cdt1 in human embryonic lung fibroblastic (HEL) cells and to explore the pathogenesis of HCMV infection. METHODS: HEL cells were synchronized in the G0/G1 phase by the serum starvation method. The synchronized HEL cells were infected with HCMV, and those that were not subjected to HCMV infection were used as the control group. The HEL cells were harvested at 12, 24, 48, 72 and 96 hrs of HCMV infection. The cell cycle of HEL cells was detected by the flow cytometry. The expression of Cdt1 mRNA in HEL cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The cells in the G1 phase in the control group was significantly more than in the HCMV-infected group 12 and 24 hrs after infection (P < 0.01). The expression of Cdt1 mRNA in the HCMV-infected group was significantly lower 12 and 24 hrs after infection but increased significantly 48 hrs after infection compared with the control group (P < 0.05). The expression of Cdt1 mRNA reached a peak at 12 hrs of infection in the control group, but at 48 hrs of infection in the HCMV-infected group, which markedly lagged behind the control group. CONCLUSIONS: HCMV infection arrests the cell cycle of HEL cells at the G1 phase. HCMV infection makes Cdt1 expression delay. HCMV infection can interfere cell cycle of HEL cells possibly through affecting the expression of Cdt1.

Downregulation of Cavin-1 Expression via Increasing Caveolin-1 Degradation Prompts the Proliferation and Migration of Vascular Smooth Muscle Cells in Balloon Injury-Induced Neointimal Hyperplasia.

BACKGROUND: Percutaneous coronary intervention has been widely used in the treatment of ischemic heart disease, but vascular restenosis is a main limitation of percutaneous coronary intervention. Our previous work reported that caveolin-1 had a key functional role in intimal hyperplasia, whereas whether Cavin-1 (another important caveolae-related protein) was involved is still unknown. Therefore, we will investigate the effect of Cavin-1 on neointimal formation. METHODS AND RESULTS: Balloon injury markedly reduced Cavin-1 protein and enhanced ubiquitin protein expression accompanied with neointimal hyperplasia in injured carotid arteries, whereas Cavin-1 mRNA had no change. In cultured vascular smooth muscle cells (VSMCs), Cavin-1 was downregulated after inhibition of protein synthesis by cycloheximide, which was distinctly prevented by pretreatment with proteasome inhibitor MG132 but not by lysosomal inhibitor chloroquine, suggesting that proteasomal degradation resulted in Cavin-1 downregulation. Knockdown of Cavin-1 by local injection of Cavin-1 short hairpin RNA (shRNA) into balloon-injured carotid arteries in vivo promoted neointimal formation. In addition, inhibition or overexpression of Cavin-1 in cultured VSMCs in vitro prompted or suppressed VSMC proliferation and migration via increasing or decreasing extracellular signal-regulated kinase phosphorylation and matrix-degrading metalloproteinases-9 activity, respectively. However, under basic conditions, the effect of Cavin-1 on VSMC migration was stronger than on proliferation. Moreover, our results indicated that Cavin-1 regulated caveolin-1 expression via lysosomal degradation pathway. CONCLUSIONS: Our study revealed the role and the mechanisms of Cavin-1 downregulation in neointimal formation by promoting VSMC proliferation, migration, and synchronously enhancing caveolin-1 lysosomal degradation. Cavin-1 may be a potential therapeutic target for the treatment of postinjury vascular remodeling.

[Cellular mechanism of heart injury in the early stage of crush injury in rats].

OBJECTIVE: To study cellular mechanism of cardiomyocytes injury in the early stage of crush injury by observing some effects of crush injury rat sera on cultured neonatal rat cardiomyocytes. METHODS: One to three days old neonatal rat cardiomyocytes were cultured in vitro and some effects of crush injury rat sera on beating rate, cell surface area, total protein content, 3H-Leu incorporation, intracellular calcium concentration ([Ca2+]i) and Fos protein expression were observed in cultured rat cardiomyocytes. RESULTS: Compared with normal rat serum group, crush injury rat sera decreased beating rate(beats/min) of cardiomyocytes from 88.3 to 26.4, cell surface area, total protein content, 3H-Leu incorporation, [Ca2+]i (nmol/L) and PI of Fos protein expression were increased. CONCLUSION: Crush injury rat sera suppress cell beating, increase intracellular calcium, induce Fos protein synthesis and cause cell hypertrophy, which may cause cardiac injury in the early stage of rush injury.