OBJECTIVETo study the effect of ultrafiltration-membrane extracts of Radix Rehmanniae Praeparata (UMERRP) on theproliferation and genetic stability of bone marrow-derived mesenchymal stem cells (BMSCs) induced by cadmium chloride (CdCl2).

METHODSProtective effects on the proliferation, micronuclear rates, chromosome aberration rates, and apoptosis rates were observed by micronuclei test, karyotype analysis, and flow cytometry.

RESULTSCompared with the CdCl2 group, UMERRP with different molecular weights at 0. 8 g/L could obviously promote the proliferation (P <0. 05). Compared with the control group, micronuclear rates, chromosome aberration rates, and apoptosis rates were obviously enhanced in the CdCl2 group (P <0. 05). Compared with the CdCl2 group, UMERRP with different molecular weights could obviously decreased CdCl2 induced micronuclear rates, chromosome aberration rates, and apoptosis rates (P <0. 05). Of them, BMSC micronuclear rates and chromosome aberration rates decreased most obvious in UMERRP groups with molecular weight below 10 000 (P <0. 05). The apoptosis rate decreased most obviously in UMERRP groups with molecular weight ranging 100 000 and 200 000 (P <0. 05).

CONCLUSIONUMERRP could reduce CdCl2 induced micronuclear rates, chromosome aberration rates, and apoptosis rates.

Apoptosis ; Bone Marrow ; Cadmium Chloride ; toxicity ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Hematopoietic Stem Cells ; Humans ; Mesenchymal Stromal Cells ; Ultrafiltration

ObjectiveTo introduction of perforator flaps,muscle flaps pedicled by descending branch of lateral circumflex femoral artery,method and their clinical application that multiple soft tissue defects of hand are repaire by muliplefoliated tissue flap only branch lateral circumflex femoral artery.MethodsFifteen patients with multiple soft tissue defects of hand were repaired muliplefoliated tissue flap only pedicled bydescending branch lateral circumflex femoral artery.At first,the anterolateral thigh perforator flap was designed and harvested according to the soft tissue defects of hand, then the descending branch lateral circumflex femoral artery was dissected at the same time the segmented perforator flap,fascia lata flap,rectus femoris muscle flap, vastus lateralis muscle flap, vastus intermedius muscle flap and distal spatium intermusculare flap were harvested in need according to distance among soft tissue defects.The muliplefoliated tissue flap was harvested only pedicled by descending branch lateral circumflex femoral artery, at last muscle flaps and fascia lata flaps were covered by skin graft, so the multiple soft tissue defects of hand were repaired in one time.ResultsNo vascular crisis happened. All skin grafts survived well, the contour of all repaired soft tissue defects was good and protective feeling was recovered by skin grafts of all flaps. All cases were got follow-up and the range was from 6 to 20 months(the average was 8.7 months).Wound of donor site healed well, muscle strength of quadriceps and motion of knee were normal. Three cases were excellent,nine cases were well and 3 cases were good, according to upper extremity function evaluation criteria of Chinese Medical Society for the Surgery of the Hand, the rate of good was 80 percent.ConclusionMultiple soft tissue defects of hand can be repaired by muliplefoliated flap only pedicled by descending branch of lateral circumflex femoral artery. Its advantages included reduction of operation time and treatment, good recovery of hand contour and function. It is a good method to repair multiple soft tissue defects of hand.

OBJECTIVE To have a systematic pathomechanism view of three chest impediment-syndromes of Qi Deficiency and Blood Stasis syndrome(QDBS),Qi Stagnation and Blood Stasis syn-drome (QSBS), Cold Obstruction and Qi Stagnation syndrome(COQS) and further investigate the changed metabolome and related pathways for screening potential biomarkers in rat plasma. METHODS According to clinical pathogeny, three kinds of syndrome models were established to simulate the disease of chest impediment. Plasma metabonomics based on UPLC-Q-TOF/MS was applied in this research to detected small molecule metabolites for identifyingthe special potential biomarkers of three chest impediment syndromes, respectively. RESULTS Significant metabolic differences were observed between thecontrol group and three syndrome groups. Furthermore, three syndrome groups were distinguished clearly by pattern recognition method.The particular metabolites contributing most to the classification of three chest impediment syndromes were identified. In the QSBS group, the potential biomarkers could include 2-keto-glutaramic acid, L-methionine, L-homocysteic acid, octadecanamide, stearoylglycine,behenic acid,linoleylcarnitine,lysoPC(14:1(9Z)),indoxyl sulfate and cholic acid.In the COQS group, they could be aminoadipic acid, palmitic amide, oleamide, lysoPC(P-16:0), lysoPC(P-18:0), lysoPC(20:2(11Z,14Z)), 9-HETE and tauroursodeoxycholic acid. Moreover, 4-pyridoxic acid, L-palmi-toylcarnitine, lysoPC(20:0), lysoPC (22:5 (4Z,7Z,10Z,13Z,16Z)), 3- hydroxyhexadecanoic acid and arachidonic acid could be the potential biomarkers for the QDBS group. CONCLUSION Three chest impediment syndromes have their own potential biomarkers.Each special metabolite has its owndifferent metabolic pathway.Both metabolismof cysteine and methionine,and metabolism of alanine,aspartate and glutamate are the main pathways in regulation of metabolic disorders in QSBS syndrome. Lysine biosynthesis and degradation,fatty acid metabolism,and glycerophospholipid metabolism are the main pathways in regulation of metabolic disorders in COQS syndrome.Arachidonic acid metabolism, fatty acid metabolism,fatty acid elongation in mitochondria,and vitamin B6 metabolism are the main pathways in regulation of metabolic disorders in QDBS syndrome.These endogenous substances were indicated as the special potential biomarkers for three chest impediment syndromes and worth studying in depth.

In order to develop potent antidiabetic agents that have inhibitory effect to a-glucosidase, twelve β-acetamido ketone derivatives such as N-{[(substituted-4-oxo-thiochroman-3-yl)phenyl]-methyl}acetamide are designed and synthesized through one-pot Dakin-West reaction. Their chemical structures are confirmed by 1H NMR, 13C NMR, IR and HR-MS. In vitro α-glucosidase inhibition assays of compounds 4a-41 were carried out using glucose oxidase method. The result indicated that most of them possess inhibitory activity in vitro. Compound 4k showed the most potent inhibitory activity with 87.3% inhibition of α-glucosidase at the concentration of 5.39 mmol x L(-1). The structure-activity relationship of these β-acetamido ketone derivatives was discussed preliminarily. Moreover, the molecular docking method was used to study the interaction mode of compound 4k and α-glucosidase. Our results will be helpful for designing of α-glucosidase inhibitors in the future.
Acetamides ; Glycoside Hydrolase Inhibitors ; chemical synthesis ; pharmacology ; Hypoglycemic Agents ; chemical synthesis ; pharmacology ; Molecular Docking Simulation ; Structure-Activity Relationship ; alpha-Glucosidases ; metabolism

OBJECTIVETo determine the ability of adhering and invading to host cells and the related pathologic changes of Leptospira spp. with different virulence.

METHODSA special Fontana silver staining method was developed to observe the ability of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and serogroup Pomona serovar pomona strain 56608, and L.biflexa serogroup Samaranga serovar patoc strain Patoc I to adhere Vero and J774A.1 cells. Ultrastructural lesions of the infected cells were examined by electron microscopy. By using flow cytometry with fluorescein labeling of FITC-Annexin V/PI,apoptosis and necrosis of the Vero and J774A.1 cells induced by the leptospiral strains before and after inactivation with ultraviolet treatment were detected, respectively.

RESULTSThe adhering rates of L.interrogans strains 56601 and 56608 to the Vero cells were 24.2% and 22.9% (P>0.05), while to the J774A.1 cells were 49.0% and 46.9% (P>0.05), respectively. L.biflexa strain Patoc I did not adhere to these two host cells. After the two strains of L.interrogans invaded two different cell lines, the special phagosomes containing the Leptospira were formed and similar cell ultrastructural lesions, such as chromatilysis and chromatin condensation, vacuolar degeneration, mitochondrion swelling and mitochondrial crista disappearance, and endoplasmic reticulum swelling and paramembranous ribosome disappearance, were observed. The apoptosis rates in the Vero cells caused by the L.interrogans strains 56601 and 56608 before and after ultraviolet inactivation were 84.4%, 82.8% and 77.9%, 86.1%, respectively. The L.interrogans strain 56601 mainly induced the terminal apoptosis in Vero cells with the rates of 68.0% and 52.9% before or after inactivation, while the L.interrogans strain 56608 mainly induced the early apoptosis in Vero cells with apoptosis rates of 64.1% and 50.1% before or after inactivation. In the J774A.1 cells, the L.interrogans strain 56608 caused cell necrosis (before and after inactivation) and apoptosis that was dominated by terminal apoptosis.

CONCLUSIONThe established Fontana silver staining method can be used to observe adhesion of L.interrogans.L.interrogans can invade host cells through endocytosis which causes untrastructural lesions. The necrosis or apoptosis induced by L.interrogans are affected by different host cell lines but not the virulence of L.interrogans strains.

Animals ; Apoptosis ; physiology ; Cell Adhesion ; Cells, Cultured ; Cercopithecus aethiops ; Endocytosis ; Humans ; Leptospira interrogans ; classification ; pathogenicity ; Macrophages ; microbiology ; ultrastructure ; Serotyping ; Vero Cells ; Virulence

The alpha-amylase (EC 3.2.1.1) from the Gram-positive Alicyclobacillus acidocaldarius was one kind of thermoacidophilic enzyme, with optimal temperature and pH of 75 degrees C and 3, respectively. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3901bp long, comprising one open reading frame encoding a polypeptide of 1301 amino acids. The calculated molecular weight of the alpha-amylase AMY was about 140kD. The gene amy was expressed in E. coli BL21 (DE3) and Pichia pastoris respectively, and both of the cloned proteins had bioactivity. The activity of amylase expressed in P. pastoris was further testified by amylase activity staining. The alpha-amylase expressed in P. pastoris had been purified and characterized. The apparent molecular weight of that was about 160kD according to SDS-PAGE. The optimum of pH for the enzyme was pH 3.2 as the native enzyme was; but the optimum of temperature was 65 degrees C and a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes in 70 degrees C. So the enzyme expressed by P. pastoris was also thermoacidophilic. Moreover some sequence was cloned by PCR, which ranged from + 1174 bp to + 3288 bp in the gene amy, encoding 705 amino acids with the calculated molecular weight of 79kD. The truncated gene amy' was expressed in E. coli BL21 (DE3) induced by 1 mmol/L IPTG, and the expressed enzyme also retained alpha-amylase activity.
Bacillus ; enzymology ; genetics ; Bacterial Proteins ; genetics ; isolation & purification ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Pichia ; genetics ; metabolism ; alpha-Amylases ; genetics ; isolation & purification ; metabolism

AIMTo identify "Shegan" [Belamcanda chinensis (L.) DC.] and relative medicinal plants of Iris including Iris tectorum Maxim., I. dichotoma Pall., I. germanica L. and I. japonica Thunb. by ribulose 1,5-bisphosphate carboxylase Large Gene (rbcL) sequence analysis.

METHODSGeneral DNA was isolated from the fresh leaves of Belamcanda chinensis and 4 Iris spp. by CTAB. A pair of primers was designed to amplify the rbcL gene and PCR Preps DNA kit was used to purify the PCR products. The rbcL sequences were determined by ABI (Applied Biosystems Inco.) Prism 310 Genetic Analyzer.

RESULTSA fragment of about 750 bp of rbcL gene from Belamcanda chinensis and 4 Iris spp. were amplified and sequenced. The rbcL sequences of Iris tectorum, I. dichotoma Pall. and I. japonica were reported for the first time. The rbcL sequences of 5 species of Iridaceae were aligned and analyzed using Clustal (Version 8.0) and MEGA (Version 2.0.) programs. The nucleotide number of difference is from 1.000 to 20.000. The tranversions is from 0.000 to 9.000 and the transitions is from 0.000 to 14.000. Phylogenetic tree based on rbcL partial sequence data indicated that the eleven samples of 5 species clustered separately.

CONCLUSIONThe sequence variation of rbcL can be used to identify Belamcanda chinensis and 4 species of relative medicinal plants of Iris. The molecular phylogenetic tree accords with the classical taxonomy.

Base Sequence ; Chloroplasts ; genetics ; DNA, Plant ; analysis ; Genes, Plant ; Iridaceae ; classification ; genetics ; Iris Plant ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Plants, Medicinal ; classification ; genetics ; Ribulose-Bisphosphate Carboxylase ; classification ; genetics ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVETo investigate the frequency of JAK2 V617F mutation in 145 myeloproliferative disorders (MPDs) patients, analyze the correlation between JAK2 V617F mutation and clinical features.

METHODSThe JAK2 V617F mutation was detected by direct DNA sequencing of PCR product and allele-specific PCR respectively. The expression of JAK2, phospho-JAK2 and phospho-STAT5 proteins was determined by Western blot. The clinical data of MPDs patients with or without JAK2 V617F mutation was collected and analyzed for evaluating the clinical significance of JAK2 V617F mutation.

RESULTS1) The frequency of JAK2 V617F mutation for PV, IMF, ET was 92%, 58%, 50% respectively. Compared with conventional DNA sequencing (PV 84%, IMF 44%, ET 39%, respectively), allele-specific PCR exhibited a higher sensitivity in JAK2 V617F mutation detection. 2) The expression levels of phospho-JAK2 and phospho-STAT5 in peripheral blood mononuclear cells (PBMNCs) were upregulated significantly in JAK2 V617F-positive patients than in JAK2 V617F negative patients. 3) Compared with the patients with no JAK2 V617F mutation, the JAK2 V 617F-positive patients' features were as follows: older age of onset, higher mean leukocyte counts, lower platelet counts and smaller spleen volume. Frequency of thrombosis events in PT, ET, IMF was 17%, 32%, 16% respectively for JAK2 V617F positive group, and 0% (PV), 16% (ET), 5% (IMF) for JAK2 V617F negative group.

CONCLUSIONSMPDs patients display higher frequency of JAK2 V617F mutation. JAK2 V617F mutation positive patients predispose to a thrombosis tendency.

Female ; Humans ; Janus Kinase 2 ; genetics ; metabolism ; Male ; Mutation ; Myeloproliferative Disorders ; genetics ; metabolism ; Phosphorylation ; STAT5 Transcription Factor ; metabolism ; Sequence Analysis, DNA

Objective： This stduy was designed to investigate the molecular mechanism of cell proliferation inhibited by tea polyphenols in nasopharyngeal carcinoma cell line CNE-LMP1. Method： Light microscope, MTT assay, flow cytometry, Western blot,and reporter gene analysis were applied to investigate the effects of tea polyphenols and(-)-epigallocatechin-3-gallate (EGCG) on the cell cycle and related molecular mechanisms. Result： After treatment of CNE-LMP1 cells with tea polyphenols(200 μ g/ml), the number of proliferating cells was obviously decreased as determined by light microscopy, and the survival rate of cells decreased from 100％ to 38.1％ as determined by MTT assay. With the increase of tea polyphenols concentrations, the number of cells in S-phase was obviously decreased from 22.20％ to 13.16％ (200 μ g/ml), and the number of cells in Phase G1 were elevated from 68.50％ to 74.08％ . It suggested that tea polyphenols could arrest the cell cycle at G1/S checkpoint. The transcription and the expression of cyclin D1 were obviously declined 4-5 fold. Furthermore, after treatment with tea polyphenols, transactivities of NFκ B and AP-1 was obviously down-regulated in CNE-LMP1 cells for 5-6 and 7-8 fold, respectively. Conclusion： Tea polyphenols could arrest cell cycle and inhibit cells proliferation of nasopharyngeal carcinoma, and down-regulation of the gene transcription and expression of cyclin D1 might also be involved in these events.

Objective To analyze the expressions of helper T cells (Th) and regulatory T cells (Treg) in peripheral blood of children with dust mite allergic asthma and their clinical significance.Methods Thirty asthmatic children with dust mite positive were included in the study as asthmatic group.Thirty healthy subjects,age and sex matched,were included as healthy control group.Peripheral blood mononuclear cells (PBMC) were isolated by densitygradient centrifugation over Ficoll-Hypaque cushions.The expressions of interferon-γ (IFN-γ),interleukin-4 (IL-4) and Foxp3 in CD4 + T cells with or without dust mite stimulation were assessed by intracellular staining and flow cytometry (FCM).The anti-inducible costimulatory molecule (ICOS),anti-CD2s or anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies were added to PBMC and co-cultured for 3 days after dust mite stimulation.The expressions of Thl (CD4 + IFN-γ+ T cells),Th2 (CD4 + IL-4 + T cells) and Treg(CD4 + CD25 + Foxp3 + T cells) were analyzed.The data were analyzed by using statistical software.Results Compared with the healthy control group,in dust mite positive asthma group,the percentage of Thl cells was remarkably increased (P ＜ 0.05),while that of Treg cells was significantly decreased (P ＜ 0.05),and that of Th2 cells were not significantly different (P ＞ 0.05).Compared with nonstimulated PBMC of asthma group,the percentages of Thl and Treg had no significant variation (all P ＞ 0.05),while that of Th2 cells was notably increased (P ＜ 0.01).In the present of anti-ICOS or anti-CD2s antibodies,no significant variation were found about the percentages of Th1 and Treg (all P ＞ 0.05),while expressions of Th2 cells were significantly decreased (all P ＜0.0001).However,in the present of anti-CTLA-4 antibodies,the percentages of Th1,Th2 and Treg had no significant difference (all P ＞ 0.05).Conclusions There is a constructive,allergen independent Th1 activation and Treg deficiency as well as allergen inductivity,ICOS-and CD2s-dependent Th2 activation in the peripheral blood of children with dust mite allergic asthma.