Animals ; Aorta ; pathology ; Atherosclerosis ; blood ; etiology ; metabolism ; Chemokine CCL2 ; metabolism ; Cholesterol ; blood ; Cholesterol, Dietary ; administration & dosage ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Dehydroepiandrosterone ; pharmacology ; Diet, Atherogenic ; Immunohistochemistry ; Rabbits ; Random Allocation ; Triglycerides ; blood ; Vascular Cell Adhesion Molecule-1 ; metabolism

Cells, Cultured ; Chemokine CCL4 ; Chemotaxis, Leukocyte ; drug effects ; Endothelial Cells ; cytology ; metabolism ; Homocysteine ; pharmacology ; Humans ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Monocytes ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Umbilical Veins ; cytology

Gastric adenocarcinoma (GC) is one of the most common malignancies in the world and one of the most frequent causes of cancer-related death.Autophagy is a highly regulated catabolic pathway responsible for the degradation of long-lived proteins and damaged intracellular organelles.However,the mechanism and guiding significance of autophagy in the development and progression of GC have remained to be elucidated.This study aimed to explore the clinicopathological significances and prognostic values of autophagy-related proteins AMBRA1 and Beclin-1 in GC.Quantum dots based immunofluorescence histochemistry (QDs-IHC) was performed to observe the expression of AMBRA1 and Beclin-1 proteins in the tissue rmicroarrays including 163 specimens of GC and 20 noncancerous gastric tissues.Simultaneously,AMBRA1 and Beclin-1 proteins were detected by Western blotting in the 10 fresh GC and corresponding normal gastric tissues.The results showed that the expression levels of both AMBRA1 and Beclin-1 proteins were higher in GC tissues than in noncancerous gastric tissues by QDs-IHC and Western blotting (P＜0.05).High AMBRA1 expression was detected in 90 of 163 (55.2％) GCs and high Beclin-1 expression was detected in 83 of 163 (50.9％) GCs.High AMBRA1 expression was closely related to depth of invasion,and lymph nodes metastasis (P＜0.05).High expression of Beclin-1 protein was correlated with tumor grade (P＜0.05).Positive correlation was observed between AMBRA1 and Beclin-1.Survival analysis indicated the high expression of AMBRA1 and Beclin-1 was an independent factor in predicting poor overall survival (OS) of GC patients.These findings suggest the high expression of AMBRA1 and Beclin-1 proteins is significantly correlated with GC progression.High AMBRA1 and Beclin-1 expression heralds worse outcome of GC patients,suggesting a novel candidate prognostic marker and a therapeutic target for GC.

OBJECTIVETo understand whether endotoxin lipopolysaccharide (LPS) is able to induce the expression of macrophage inflammatory protein-1alpha (MIP-1alpha) mRNA and protein in human umbilical vein endothelial cells (HUVECs).

METHODSThe expression of MIP-1alpha mRNA was determined by dot blotting analysis and by in situ hybridization using a digoxigenin-labeled MIP-1alpha cDNA probe after exposure of the cultured HUVECs to LPS at different concentrations. The expression of MIP-1alpha mRNA was determined by RT-PCR as well. In addition, the expression of MIP-1alpha protein was tested by cell enzyme-linked immunosorbent assay (ELISA) using a goat anti-human monoclonal MIP-1alpha antibody.

RESULTSDot blotting showed that the absorbance values of the dots on the nitrocellulose membrane were 1.490 and 3.310 when exposed to LPS at the concentrations of 1 micro g/ml and 10 micro g/ml which were 1.97- and 4.38-fold over that of the control group (0.775), respectively. In situ hybridization revealed that exposure to LPS at a concentration of 1 micro g/ml led to a significant increase in the MIP-1alpha mRNA expression in HUVECs as compared to the control group (F = 142.83, P < 0.01), whereas the MIP-1alpha mRNA in HUVECs was somewhat decreased when exposed to LPS at a concentration of 10 micro g/ml. RT-PCR revealed that the expression of MIP-1alpha mRNA in HUVECs were 1.65-, 2.86- and 1.26-fold over that of the control group when exposed to LPS at the concentrations of 1 micro g/ml, 5 micro g/ml and 10 micro g/ml respectively. Cell ELISA showed that after exposure of the HUVECs to LPS at the concentrations mentioned above, the expression of MIP-1alpha protein was strongly increased, especially in the 5 micro g/ml LPS group. Analysis of variance showed that there was a significant difference between groups (F = 15.36, P < 0.05).

CONCLUSIONSLPS may induce a high level of MIP-1alpha mRNA and protein expression in HUVECs, and it might, hereby, play an important role in the recruitment of the monocytes/macrophages into the arterial intima.

Cells, Cultured ; Chemokine CCL3 ; Chemokine CCL4 ; Endothelial Cells ; drug effects ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation ; drug effects ; Humans ; In Situ Hybridization ; Lipopolysaccharides ; toxicity ; Macrophage Inflammatory Proteins ; analysis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Umbilical Veins ; drug effects

Tetramethylpyrazine (TMP), an effective component of traditional Chinese medicine Chuanxiong, is commonly used to resolve embolism. Its possible therapeutic effect against atherosclerosis has received considerable attention recently. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. The mechanisms of TMP in the proliferation of VSMCs induced by Ang II remain to be defined. The present study was aimed to study the effect of TMP on Ang II-induced VSMC proliferation through detection of nuclear factor-kappaB (NF-kappaB) activity and bone morphogenetic protein-2 (BMP-2) expression. Primary cultured rat aortic smooth muscle cells were divided into the control group, Ang II group, Ang II + TMP group and TMP group. Cells in each group were harvested at different time points (15, 30 and 60 min for detection of NF-kappaB activity; 6, 12 and 24 h for measurement of BMP-2 expression). NF-kappaB activation was identified as nuclear staining by immunohistochemistry. BMP-2 expression was observed through Western blot, immunohistochemistry and in situ hybridization. The results showed that: (1) Ang II stimulated the activation of NF-kappaB. Translocation of NF-kappaB p65 subunit from cytoplasm to nucleus appeared as early as 15 min, peaked at 30 min (P<0.01) and declined after 1 h. (2) TMP inhibited Ang II-induced NF-kappaB activation (P<0.01). (3) Ang II increased BMP-2 expression at 6 h but declined it significantly at 12 and 24 h (P<0.01). (4) BMP-2 expression was also kept at high level at 6 h in Ang II + TMP group but maintained at the normal level at 12 and 24 h. (5) There was no significant difference in NF-kappaB activation and BMP-2 expression between the control group and TMP group. These results indicate that TMP inhibits Ang II-induced VSMC proliferation through repression of NF-kappaB activation and BMP-2 reduction, and BMP-2 expression is independent of the NF-kappaB pathway. In conclusion, TMP has therapeutic potential for the treatment of atherosclerosis.
Angiotensin II ; antagonists & inhibitors ; Animals ; Atherosclerosis ; drug therapy ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; analysis ; antagonists & inhibitors ; Immunohistochemistry ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; NF-kappa B ; analysis ; antagonists & inhibitors ; Pyrazines ; pharmacology ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta ; analysis ; antagonists & inhibitors

A full-length cDNA encoding a MYB-related regulatory gene was isolated from a cDNA library prepared from mRNAs of the red line callus of S. medusa by TD-PCR. The cDNA, designated SmP, is 969 nucleotides long and has an open reading frame of 771 bp with a deduced amino acid sequence of 256 residues. The putative protein of SmP has two typical conversed R2R3-Myb DNA-binding domains in N-terminal and displays a rather high degree of similarity to OsMYB from rice and LBMI from tobacco, showing 73% and 70% identity within the DNA-binding domains. However, the C-terminal domain of the SmP protein does not show obvious similarity to any other known protein sequence. It is rich in hydrophilic amino acids, especially in serine residues (18.38%), partly organized in homopolymeric stretches, a feature often found in activation domain of transcription factors.
Amino Acid Sequence ; Cloning, Molecular ; Molecular Sequence Data ; Phylogeny ; Plant Proteins ; chemistry ; classification ; genetics ; metabolism ; Polymerase Chain Reaction ; Saussurea ; classification ; genetics ; metabolism ; Sequence Homology, Amino Acid ; Transcription Factors ; chemistry ; genetics ; metabolism

OBJECTIVETo investigate the effect of homocysteine (HCY) on activation of nuclear factor (NF-kappaB) and inhibitory factor IkappaB-alpha in human monocyte cell line THP-1, as well as its association with macrophage inflammatory protein (MIP-1alpha) upregulation.

METHODSTHP-1 monocytes were incubated with HCY, with and without NF-kappaB inhibitor pyrolidine dithiocarbamate (PDTC) pretreatment. Northern blot analysis and flow cytometry were used to detect MIP-1alpha mRNA and protein respectively. The nuclear protein NF-kappaB P65 subunit and the inhibitory protein IkappaB-alpha were analyzed by Western blotting.

RESULTSCompared with controls, HCY, at a concentration of 0.1 mmol/L, was able to enhance the expression of MIP-1alpha mRNA (up to 3.69-fold) and protein (1.16-fold) in THP-1 monocytes, as well as enhance NF-kappaB P65 transcription to nuclear proteins. These actions were significantly suppressed after pretreatment with 100 micromol/L PDTC for 30 minutes before HCY incubation; whereas incubation of THP-1 monocytes with PDTC only had no effect on both the expression of MIP-1alpha and nuclear transcription of NF-kappaB P65. Moreover, the level of IkappaB-alpha protein in THP-1 monocytes decreased after a 30-minute incubation with HCY, which gradually increased after 120 minutes.

CONCLUSIONSHomocysteine at a pathologic concentration stimulates MIP-1alpha expression in THP-1 monocytes, probably via NF-kappaB activation. Such activation may be caused by enhanced phosphorylation and degradation of the inhibitor protein IkappaB-alpha.

Cell Line, Tumor ; Chemokine CCL3 ; Chemokine CCL4 ; Homocysteine ; pharmacology ; Humans ; I-kappa B Proteins ; metabolism ; Leukemia, Monocytic, Acute ; metabolism ; pathology ; Macrophage Inflammatory Proteins ; biosynthesis ; genetics ; Monocytes ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; Phosphorylation ; Proline ; analogs & derivatives ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Thiocarbamates ; pharmacology ; Transcription Factor RelA ; biosynthesis ; genetics ; Transcription, Genetic

OBJECTIVETo investigate the expression profiles of myocardin gene during the differentiation of bone marrow-derived mesenchymal stem cell to smooth muscle cells in the conditional medium combined with a high concentration of fetal bovine serum (FBS).

METHODSMarrow-derived mesenchymal stem cells were isolated and purified from mouse femoral bone and shinbones using differential adherent methods. Cells at the third passage were induced by 20% FBS in conditioned medium, conditioned medium alone, 20% FBS or 10% FBS alone respectively. Mouse aortic smooth muscle cells were cultured as the positive control. Levels of mRNA and protein expression of myocardin and several smooth muscle cells marker genes were determined by immunofluorescence, RT-PCR and Western blot before and 3, 7, 10, 14 d after the induction. The presence of smooth muscle myofilaments was detected by using transmission electron microscope.

RESULTSNaive bone marrow-derived mesenchymal stem cells displayed multiple morphological forms including fusiform, polygon, oval, and micro-spherical, as compared to the single macro-spindle form after the induction. Typical appearance of peak valley was displayed on the 21st day after induction. At the same time, the expression of smooth muscle marker genes was reinforced along with an up-regulation of myocardin expression. Immunofluorescence study showed that the cells expressing myocardin and smooth muscle marker genes such as alpha-SMA and SM-MHC increased. Fluorescence domain of myocardin translocated from cytoplasm to nucleus and the amounts of double positive cells for myocardin with alpha-SMA or SM-MHC also increased. RT-PCR confirmed that the mRNA expression of myocardin increased gradually and remained stabilized after achieving its peak on the 7th day after induction. The expression of smooth muscle marker genes, alpha-SMA and SM22alpha, remained stable on the 10th day of induction. It was also confirmed by Western blot that the protein expression of both myocardin and alpha-SMA were markedly increased during the induction. Finally, transmission electron microscopy revealed the presence of myofilament on the 21st day after induction.

CONCLUSIONSBone marrow-derived mesenchymal stem cells can be effectively induced into smooth muscle-like cells by conditioned medium combined with 20% FBS. Myocardin plays an important role in the differentiation process of bone marrow-derived mesenchymal stem cells to the peripheral smooth muscle cells.

Animals ; Bone Marrow Cells ; cytology ; physiology ; Cattle ; Cell Differentiation ; physiology ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; genetics ; metabolism ; physiology ; Up-Regulation

Objective To explore the causes of dengue fever resurgence in Guangxi, and to analyze the risk factors of dengue fever. Methods The descriptive epidemiological analysis was conduced based on the dengue fever data reported from 2006 to 2015, and the surveillance results of aedes and antibody levels in health population from 2013 to 2015 in Guangxi. Results Before 2013, dengue fever was imported from foreign country in Guangxi, accounting for 95.35％(42/45), and 75.71％of the imported cases was imported from Southeast Asia. The local outbreak of dengue fever was happened in 2014, accounted for 94.02％(849/903) of the total number of 10 years. From onset to diagnosis, Guangxi dengue fever cases need 0-70 d (median time interval is 6 d). Cases were reported year-round, but the peak season for the onset of dengue fever was from September to November, accounting for 96.46％of all cases (871/903). The number of cases reported in Nanning was the most (83.37％), followed by Wuzhou city (7.44％) and Guilin city (4.81％), and all the three cities had dengue fever outbreaks. The cases were mainly commercial service staff (27.80％) and houseworkers and unemployed people (18.16％). Results of aedes monitoring showed that the density of aedes was high in Guangxi. In more than 50％ of the monitoring seasons the breteau index (BI) stayed greater than 20. However, the antibody positive rate was only 3％ in the healthy residents of Guangxi. Conclusion The risk of dengue fever is high in Guangxi. Therefore, it is essential to emphasizing idea of prevention and control, strengthening immigration surveillance, improving diagnosis ability, enhancing public health education, and expanding monitoring range.

Adult ; Brachytherapy ; methods ; Carcinoma, Hepatocellular ; pathology ; therapy ; Chemoembolization, Therapeutic ; Female ; Humans ; Iodine Radioisotopes ; therapeutic use ; Liver Neoplasms ; pathology ; therapy ; Male ; Middle Aged ; Neoplastic Cells, Circulating ; Portal Vein