Neuronal changes resulting in up-regulation of alpha-1 adrenoceptors after peripheral nerve injury

Under normal conditions, the sympathetic neurotransmitter noradrenaline inhibits the production and release of pro-inflammatory cytokines. However, after peripheral nerve and tissue injury, pro-inflammatory cytokines appear to induce the expression of the alphalA-adreno- ceptor subtype on immune cells and perhaps also on other cells in the injured tissue. In turn, noradrenaline may act on up-regulated alphal-adrenoceptors to increase the production of the pro-inflammatory cytokine interleukin-6. In addition, the release of inflammatory mediators and nerve growth factor from keratinocytes and other cells may augment the expression of alphal-adrenoceptors on peripheral nerve fibers. Consequently, nociceptive afferents acquire an abnormal excitability to adrenergic agents, and inflammatory processes build. These mechanisms could contribute to the development of sympathetically maintained pain in conditions such as post-herpetic neuralgia, cutaneous neuromas, amputation stump pain and complex regional pain syndrome.

Up-regulation of prohibitin 1 is involved in the proliferation and migration of liver cancer cells

Although prohibitin 1(PHB) is known to be associated with tumors,there are few studies regarding the role of PHB in hepatocellular carcinoma.An earlier glycoproteomics study of ours showed that PHB is over-expressed in MHCC97-H cells,a highly metastatic liver cancer cell line,and can be captured by wheat germ agglutinin.In the present study,western blotting and reverse transcription-PCR experiments showed an approximately 2-fold up-regulation in the expression of PHB in MHCC97-H cells.However,PHB was not significantly up-regulated in MHCC97-L cells,a low-metastatic liver cancer cell line.When PHB was over-expressed in MHCC97-L cells,cell proliferation was inhibited by 35% and migration increased about 2-fold.The results of this study show that PHB is up-regulated in MHCC97-H cells and is associated with both proliferation and migration of liver cancer cells.

Up-Regulation of Akt and Nav1.8 in BmK I-Induced Pain

Dear Editor,As the key contributor to the rising phase of action potentials in dorsal root ganglion（DRG）neurons,voltagegated sodium channels（VGSCs）are important in physiological and pathological pain conditions.For instance,abnormal expression of VGSCs in DRG neurons is the

Interleukin-1 beta up-regulates tissue inhibitor of matrix metalloproteinase-1 mRNA and phosphorylation of c-jun N-terminal kinase and p38 in hepatic stellate cells

AIM： To study the relationship between interleukin-lbeta （IL-1β） up-regulating tissue inhibitor of matrix metalloproteinase-1 （TIMMP-1） mRNA expression and phosphorylation of both c-jun N-terminal kinase （INK） and p38 in rat heffatic stellate cells （HSC）. METHODS： RT-PCR was performed to measure the expression of TIMMP-1 mRNA in rat HSC. Western blot was performed to measure IL-1β-induced JNK and p38 activities in rat HSC. RESULTS： TIMMP-1 mRNA expression （1.191± 0.079） was much higher after treatment with IL-1β （10 ng/mL） for 24 h than in control group （0.545±0.091） （P〈0.01）. IL-1β activated INK and p38 in a time-dependent manner. After stimulation with IL-1β for 0, 5, 15, 30, 60 and 120 min, the INK activity was 0.982±0.299, 1.501±0.720, 2.133±0.882, 3.360±0.452, 2.181±0.789, and 1.385 ± 0.368, respectively. There was a significant difference in JNK activity at 15 min （P〈 0.01）, 30 min （P〈 0.01） and 60 min （P〈0.01） in comparison to that at 0 min. The p38 activity was 1.061±0.310, 2.050±0.863, 2.380±0.573, 2.973±0.953, 2.421±0.793, and 1.755 ± 0.433 at the 6 time points （0, 5, 15, 30, 60 and 120 min） respectively. There was a significant difference in p38 activity at 5 min （P〈0.05）, 15 min （P〈0.01）, 30 min （P〈0.01） and 60 min （P〈0.01） compared to that at 0 min. TIMMP-1 mRNA expression trended to decrease in 3 groups pretreated with different concentrations of SP600125 （10 μmol/L, 1.022±0.113; 20 μmol/L, 0.869±0.070; 40 μmol/L, 0.666±0.123）. Their decreases were all significant （P〈0.05, P〈0.01, P〈0.01） in comparison to control group （without SP600125 treatment, 1.163±0.107）. In the other 3 groups pretreated with different concentrations of SB203580 （10 μmol/L, 1.507±0.099; 20 μmol/L, 1.698±0.107; 40 μmol/L, 1.857±0.054）, the expression of TIMMP-1 mRNA increased. Their levels were higher than those in the control group （without SB203580 treatment, 1.027 ± 0.061） with a significant statistical significance （P〈 0.01）. CONCLUSION： IL-1β has a direct action on hepatic fibrosis by up-regulating TIMMP-1 mRNA expression in ratessionin in rate HSC.JNK and p38 mitogen-activated protein kinases （MAPKs） are involved in IL-1β-induced TIMMP-1 gene expression, and play a distinct role in this process, indicating that p38 and .INK pathways cooperatively mediate TIMP-1 mRNA expression in rat HSC.

Identifcation of up-regulated genes in human uterine leiomyoma by sup-pression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.

Identification of up-regulated genes in human uterine leiomyoma by sup-pression subtractive hybridization

In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.

Using a Suppression Subtractive Library-Based Approach to Identify Non-Heading Chinese Cabbage Genes Up-Regulated in Early Response to Elicitor PB90

Monitoring expression at the transcriptional level is the first essential step for the functional analysis of plant genes. Genes-encoding proteins directly involved in early response to elicitor constitute only a small fraction of all the genes affected by elicitor. Transcriptional responses to various elicitors have been extensively studied in different plants including Nicotiana and Arabidopsis thaliana; however, corresponding data aren＇t available for non-heading Chinese cabbage. To address this problem, we describe a suppression subtractive library-based approach to isolate the plant＇s ESTs up-regulated in the early induction/execution of the HR induced by elicitor PB90 from Phytophthora boehmeriae. According to their putative identification in BLAST searches against the three genome databases, 70 up-regulated genes were classified into 9 parts： some aspect of primary ＇metabolism＇ or ＇energy＇ production; ＇protein synthesis＇ or ＇protein fate＇; cellular communication/signal transduction mechanism; cell fates including Beclin, SPT1, and SPT2; HLA-B and AGO1 which participate in transcription; cellular transport and hypothetical proteins or proteins for which a function has yet to be determined. Seven selected genes such as Beclin, thioredoxin, HLA-B, MAP3K, SPT1, SPT2, and AGO1 were up-regulated induced by PB90, suggesting that the genes may play an important role in PB90-triggered HR.

Cytokeratin 8 is increased in hepatitis C virus cells and its ectopic expression induces apoptosis of SMMC7721 cells

AIM:To investigate cytokeratin 8(CK8)overexpression during hepatitis C virus(HCV)infection and its pathogenesis,and the effect of ectopic CK8 expression on hepatoma cell lines.METHODS:We successfully established an in vitro HCV cell culture system(HCVcc)to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells.The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction(RT-PCR).The expression of CK8 at the protein level was evaluated by Western blotting.We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene.CK8cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFPCK8 was obtained.After confirming the sequence,the recombinant plasmid was transfected into SMMC7721cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy,RTPCR and Western blotting.Besides,we identified biological function of CK8 on SMMC7721 cells,including cell proliferation,cell cycle and apoptosis detection.RESULTS:RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88and 2.95 times higher than in control cells.Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells,respectively.We found that CK8at mRNA and protein levels were both significantly increased in HCVcc.CK8 was up-regulated in SMMC7721cells.CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells.CK8expression in SMMC7721/pEGFP-CK8 cells was 2.69times higher than in SMMC7721 cells,and was 2.64times higher than in SMMC7721/pEGFP-C1 cells.CK8expression at the protein level in SMMC7721/pEGFPCK8 cells was 2.46 times higher than in SMMC7721cells,and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells.Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells.CONCLUSION:CK8 up-regulation might have a functional role in HCV infection and pathogenesis,and could be a promising target for the treatment of HCV infection.

Gene cloning and induced expression pattern of IRF4 and IRF10 in the Asian swamp eel(Monopterus albus)

The Asian swamp eel （Monopterus albus） is one of the most economically important freshwater fish in East Asia, but data on the immune genes of M. albus are scarce compared to other commercially important fish. A better understanding of the eel＇s immune responses may help in developing strategies for disease management, potentially improving yields and mitigating losses. In mammals, interferon regulatory factors （IRFs） play a vital role in both the innate and adaptive immune system; though among teleosts IRF4 and IRFIO have seldom been studied. In this study, we characterized IRF4 and IRFIO from M. albus （malRF4 and malRFlO） and found that malRF4 cDNA consists of 1 716 nucleotides encoding a 451 amino acid （aa） protein, while malRFlO consists of 1 744 nucleotides including an open reading frame （ORF） of 1 236 nt encoding 411 aa. The malRFlO gene was constitutively expressed at high levels in a variety of tissues, while malRF4 showed a very limited expression pattern. Expression of malRF4 and malRFlO in head kidney, and spleen tissues was significantly up-regulated from 12 h to 48 h post-stimulation with polyinosinic： polycytidylic acid （poly I：C）, lipopolysaccharide （LPS） and a common pathogenic bacteria Aeromonas hydrophila. These results suggest that IRF4 and IRF10 play roles in immune responses to both viral and bacterial infections in M. albus.

Cardiac reserve mobilization trend during exercise and recovery after exercise

Objective: Little is known about the cardiac contractility recovery after exercise. The objective of the study was to explore a method to evaluate the extent and speed of cardiac function up-regulation during exercise and the recovery course of cardiac contractility and heart rate after exercise. Methods: Ten student athletes and ten student non-athlete voluntarily participated in this controlled study. Three indicators were selected: 1) amplitude ratio of the first to second heart sound (S1/S2);2) heart rate (HR);3) power output (W). Phonocardiogram exercise test (PCGET) was adopted. A four-stage workload increment protocol was used. Phonocardiograms were recorded in the sitting position at rest and immediately after each test stage. The time taken for completing the workloads 1750 J, 3500 J, 5250 J, and 7000 J was recorded, respectively. During recovery heart sound signals were recorded immediately after exercise, and at 1, 5, 10, 15, 20, 25, and 30 minutes after exercise. S1/S2, HR, and W were calculated from the measured data. Cardiac function change trend graphs were constructed. Results: During exercise, HR and S1/S2 ratio increased with the increase in workload from 1750 J to 7000 J;the level and speed of increase in power output and S1/S2 ratio of the athletes were higher than the general students;power done by the general students decreased earlier than the athletes. During recovery course, the recovery course of the general students was slower than the athletes. Conclusion: This method for evaluating cardiac function up-regulation and recovery course is safe, easy, reliable, and effective, which is beneficial for selecting athletes, training, and matchmaking.

Differential Expression of miRNAs in Sorghum bicolor under Drought and Salt Stress

The regulatory mechanisms of drought and salt-associated miRNAs have not been fully understood in Sorghum bicolor. In this study, we investigated the effect of salinity stress (200 and 300 mM NaCl) and drought stress at pre- and post-flowering stages on the expression pattern of small regulatory RNAs in six Sorghum genotypes using semi-quantitative reverse transcriptase PCR (RT-qPCR). The results indicated that both drought and salt stresses altered the expression pattern of miRNAs in a dose-dependent manner. However, each miRNA responded to drought and salt stress in a different pattern among the six sorghum genotypes. miR156, miR167, miR168 and miR399 give different expressions levels compared to other studied miRNAs which may attribute to the adaption of sorghum to drought and salt stress and are good candidates for improving sorghum by transgenic technology.

Lipopolysaccharide upregulates the expression of Toll-like receptor 4 in human vascular endothelial cells

OBJECTIVE: To investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells. METHODS: Total RNA was extracted from ECV304 cells and isolated human umbilical vein endothelial cells (HUVECs) exposed to LPS, respectively. The quantification of TLR2 and TLR4 mRNA in HUVECs and EVC304 cells was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ECV304 cells and HUVECs were able to express TLR2 and TLR4 mRNA, but the expression levels of TLR4 appeared to be stronger than those of TLR2. LPS could upregulate the expression levels of TLR4 obviously, whereas it had no effect on the expression level of TLR2. CONCLUSIONS: Our data indicate that TLR4 may be the LPS signal transducer in endothelial cells and plays important roles in the cell activation of LPS. The ECV304 cell line is a better experimental model than isolated HUVECs in the research of endothelial cells.

A novel erythroid differentiation related gene EDRF1 upregulating globin gene expression in HEL cells

OBJECTIVE: To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression. METHODS: By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2. RESULTS: It was shown that when EDRF1 was overexpressed, production of alpha-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of alpha-, gamma-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels compared with control experiments. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression. CONCLUSION: The results suggested that EDRF1 regulated alpha- and gamma-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.

Impacts of Fe2+on 5-aminolevulinic acid(ALA)biosynthesis of Rhodobacter sphaeroides in wastewater treatment by regulating nif gene expression

This study aimed to increase bacterial growth and 5-aminolevulinic acid（ALA） biosynthesis of Rhodobacter sphaeroides in wastewater treatment through adding ferrous ion（ Fe2＋ ）. Results demonstrated that Fe2＋ effectively enhanced the biomass production and ALA yield of R. sphaeroides. Moreover, the optimal Fe2＋ dosage was found to be 400 μmol/L, which was associated with the highest biomass of 4015.3 mg/L and maximum ALA yield of 15.9 mg/g-dry cell weight（mg/g-DCW）. Mechanism analysis revealed that Fe2＋ vastly improved Adenosine Triphosphate（ATP） production by up-regulating the nif gene expression, and increasing ATP enhanced the biomass and ALA yield by supplying energy for bacterial growth and ALA biosynthesis, respectively. Correlation analysis showed that the ALA and ATP yields had positive relation with nifA and nifU gene expression. In addition, the nifA and nifU gene expression displayed high consistency of co-transcription at the optimal Fe2＋ dosage.

Over-transcription of genes in a parathion-resistant strain of mosquito Culex pipiens quinquefasciatus

Insecticide resistance is an evolutionary adaptation that develops quite quickly in mosquitoes because of the high selection pressure of chemical insecticides, rapid generation time and large population size. Identification of genes associated with insecticide resistance is fundamental to understand the complex processes responsible for resistance. We compared the gene transcriptional profiles of parathion-resistant and -susceptible Culex pipiens quinquefasciatus using a combination of suppression subtractive hybridization and complementary DNA （cDNA） microarray techniques. A total of 278 colonies were selected from the resistant-susceptible mosquito subtractive library, 38 of which showed more than two fold stronger immunoblotting signals in the resistant strain than in the susceptible strain using cDNA microarray selection. The sequencing results showed that the 38 colonies can be matched to 12 genes of C. p. quinquefasciatus. Eight genes were confirmed to be overexpressed by more than two fold in the resistant strain. These genes encode chymotrypsin-1, theta glutathione S-transferase, lipase 3, larval serum protein 1β chain, cytochrome b, mitochondrial ribosomal large subunit, 28S rRNA, and a protein with unknown function. This study serves as a preliminary attempt to identify new genes associated with organophosphate resistance in this mosquito species and provides insights into the complicated physiological phenomenon of insecticide resistance.

牛磺酸上调基因1在肾癌组织的表达及临床意义

目的：探讨牛磺酸上调基因1（ TUG1）在肾癌组织及其配对癌旁正常组织中的表达，及其与临床病理特征之间的相关性。方法从46例经手术完整切除的肾癌组织及其配对癌旁正常组织中分别提取获得各自的总RNA，经逆转录得到cDNA，使用RTqPCR检测两者TUG1的表达，分析TUG1表达与患者临床病理特征的相关性。结果 TUG1在肾癌组织中的表达明显低于配对的癌旁正常组织（0．533±0．027、1．000±0．298），差异有统计学意义（t＝－3．350，P＜0．01）。46例肾癌组织中TUG1的△CT值经对数转换后最小值为－5．535，最大值3．085，平均值－0．908，以平均值－0．908为分界线，46例肾癌组织中有25例（54．34％）呈表达下调。 TUG1的表达水平与肾癌患者的年龄、性别、肾癌组织类型、TNM分期以及UICC／AJCC临床分期无显著相关性（ P均＞0．05）。结论肾癌组织中TUG1表达下调，提示其可能与肾癌的发生发展相关。