2', 3', 5'-Tri-O-acetyl-N6-(3-hydroxylaniline)adenosine (WS070117) is a derivative compound of natural product cordycepin. It has significant lipids regulating activity and low toxicity which has been proved by in vitro and in vivo experiments. In this study, 1H NMR-based metabolomics was used to investigate the dose-related effects of WS070117 on hyperlipidemia of high-fat-fed hamsters. The hyperlipidemic hamsters were administrated with six different doses of WS070117, including 3, 12, 50, 100, 200 and 400 mg x kg(-1) x d(-1). 1H NMR spectra of hamster serum were visually and statistically analyzed using two multivariate analyses: principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). As a result, WS070117-treated groups showed dose-related regulation of metabolites associated with lipid metabolism, choline metabolism and glucose metabolism. The dose of 3 mg x kg(-1) x d(-1) of WS070117 only exhibited a little lipids regulating activity. However, the doses of 12 and 50 mg x kg(-1) x d(-1) of WS070117 both regulated the contents of metabolites to reverse significantly toward normal levels. When the dose of WS070117 reached 100 mg x kg(-1) x d(-1), it was more effective than positive control drugs. The work suggested that NMR-based metabolomics might be a valuable approach to evaluate dose-related effects of lipids regulating compounds.
Adenosine ; analogs & derivatives ; pharmacology ; Animals ; Cricetinae ; Hyperlipidemias ; metabolism ; Least-Squares Analysis ; Lipid Metabolism ; drug effects ; Magnetic Resonance Spectroscopy ; Metabolomics ; Multivariate Analysis ; Principal Component Analysis

OBJECTIVETo investigate the characteristics of gene mutations in unexplained infantile epileptic encephalopathy (EE).

METHODSA total of 47 infants with unexplained infantile EE were enrolled, and next-generation sequencing was used to analyze gene mutations in these infants and their parents.

RESULTSOf all 47 infants, 23 were found to have gene mutations, among whom 13 had de novo mutations and 10 had heterozygous mutations inherited from their father or mother. Among the 23 infants with gene mutations, 17 were found to have the gene mutations related to EE (among whom 14 had ion channel gene mutations), 2 had the gene mutations related to congenital inherited metabolic diseases, 2 had the gene mutations related to brain structural abnormality, and 2 had the gene mutations related to mental retardation.

CONCLUSIONSUnexplained infantile EE may have gene mutations, mainly ion channel gene mutations.

OBJECTIVETo investigate the allocation and management of large medical equipment (LME) in Xuzhou, Jiangsu Province, China, in order to make the best use of LME to meet the medical needs of local people.

METHODSThe research collected data from all hospitals that have LME in Xuzhou using questionnaire; 38 (97.4%) hospitals returned the questionnaire.

RESULTSIn Xuzhou, there are a total of 71 pieces of LME, each serving 126 600 people in an area of 163 km(2). Sixty-two percent of them are allocated in urban areas, with Gini coefficient at 0.52, indicating imbalance and biased allocation of LME.

CONCLUSIONThe allocation of LME in Xuzhou is out of control and unfairly allocated.

China ; Equipment and Supplies ; Hospitals ; Medical Laboratory Science ; Surveys and Questionnaires

OBJECTIVETo explore whether the inhibitory effect of Genipin on uncoupling protein-2 (UCP-2) in mitochondria is involved in energy metabolism of androgen-independent PC3 prostate cancer cells.

METHODSPC3 prostate cancer cells were cultured and treated with Genipin at the concentrations of 40, 80, and 160 μmol/L for 48 hours. Then the proliferation of the cells was detected by MTT assay, the expression of UCP-2 mRNA determined by RT-PCR, and the content of intracellular pyruvic acid (PA) and the activity of succinate dehydrogenase (SDH) in the mitochondria measured by visible spectrophotometry.

RESULTSWith the increased concentration of Genipin, the proliferative activity of the PC-3 cells, the expression level of UCP-2 mRNA, the content of intracellular PA and the activity of SDH in the cells were all decreased, namely, with the enhanced inhibitory effect of Genipin on UCP-2, a trend of reduction was observed in the proliferation of the cells, intracellular PA content, and SDH activity in the mitochondria.

CONCLUSIONGenipin is involved in the energy metabolism of androgen-independent PC3 prostate cancer cells by reducing the content of intracellular PA and the activity of SDH in the mitochondria, which may be associated with its inhibitory effect on UCP-2.

Cell Line, Tumor ; drug effects ; Energy Metabolism ; Humans ; Ion Channels ; metabolism ; Iridoids ; pharmacology ; Male ; Mitochondria ; metabolism ; Mitochondrial Proteins ; metabolism ; Prostatic Neoplasms ; metabolism ; Pyruvic Acid ; metabolism ; RNA, Messenger ; Succinate Dehydrogenase ; metabolism ; Uncoupling Protein 2

The interaction among collagen, von Willebrand factor (vWF) and glycoprotein Ib axis is the first step in hemostasis and thrombosis, especially under high shear condition. To develop a new remedy of anti-thrombosis, mRNA from endothelial cells was extracted, and reverse transcription PCR was adopted to amplify DNA of interest. After sequencing, recombinant expression vector was constructed. The amplified DNA fragment of vWF domain A3 was inserted into expression vector with 6 x his taq, pET20b(+), the recombinant was transformed into E coli (strain DE3) and induced by IPTG. Recombinant vWF-A3 was designated as a recombinant fragment comprising residues 918 - 1114 of mature vWF subunit. It was purified through Ni-NTA resin column and refolded in Tris buffer containing GSH and GSSG. The results showed that rvWF-A3 was expressed successfully in E coli (strain DE3), accounting for 46% of total bacterial protein with its purity of over 95%. It was identified that rvWF-A3 is capable to bind collagen and inhibit the wild vWF binding to collagen by competition. It is concluded that rvWF-A3 fragment might be an effective antithrombotic agent for preventing arterial thrombosis.
Cloning, Molecular ; Collagen ; metabolism ; Escherichia coli ; genetics ; Humans ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; von Willebrand Factor ; chemistry ; genetics ; metabolism

This study was aimed to clone the gene coding mouse CXC chemokine receptor 4 (CXCR4), to construct the recombinant lentiviral vector carrying enhanced green fluorescence protein (EGFP) and to explore its expression in eukaryotic cells (293FT cells). The full length CXCR4 gene was cloned by RT-PCR using bone marrow cells from C57BL/6 mouse as template and inserted into PCR-Blunt vector. CXCR4 fragment was generated by digestion with restriction endonuclease and subcloned into a lentiviral vector to generate recombinant lentiviral vector LV-IRES-EGFP-CXCR4, which was co-transfected into 293FT cells together with envelope plasmid and packaging plasmid by lipofectamine 2000. Viruses were gathered and concentrated using ultracentrifuge, and then transfected into 293FT cells. Expression of EGFP was detected by fluorescent microscopy and flow cytometry (FCM). And the expression of CXCR4 protein was detected by Western blot. The results demonstrated that mouse CXCR4 gene was cloned and the lentiviral vector was successfully constructed. The lentiviral particles were correctly packaged, and the virus titers were above 10(8) TU/ml in the supernatant after concentration. Expression of EGFP was detected by fluorescent microscopy in the transfected 293FT cells, and the transfection efficacy > 95% was determined by FCM. Expression of CXCR4 protein detected by FCM and Western blot was significantly higher than that in control group. It is concluded that the CXCR4 gene along with the gene coding EGFP is successfully inserted into a lentiviral vector to construct a recombinant lentiviral vector, which can be expressed in eukaryotic cells.
Animals ; Cell Line ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred C57BL ; Plasmids ; Receptors, CXCR4 ; genetics ; Transfection

Child ; Cord Blood Stem Cell Transplantation ; methods ; Histocompatibility Testing ; standards ; Humans ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; therapy ; Transplantation, Homologous ; methods ; Treatment Outcome

OBJECTIVETo observe the incidence of heparin-induced thrombocytopenia (HIT) in patients received unfractionated heparin (UFH) treatment, and explore the feasibility of monitoring HIT by platelet counts, as well as the significance of HIT-antibody test in HIT diagnosis.

METHODS145 patients received UFH treatment in Vascular Surgery Department were studied. Before and after the UFH treatment, platelet counts, HIT-antibody ELISA test and heparin-induced platelet aggregation (HIPA) were tested.

RESULTSAmong the 145 patients, thrombocytopenia occurred in 40 (27.6%) cases, HIT-antibody ELISA test positive in 59 (40.7%) cases, HIPA test positive in 26 (17.9%) cases. The HIT was diagnosed in 24 (16.5%) cases, and heparin-induced thrombocytopenia and thrombosis (HITTS) occurred in 5 (3.4% in all cases, and 20.8% in HIT patients). In HIT patients, 15 patients (62.5%) were thrombocytopenia, HIT-antibody positive and HIPA test positive. Platelet counts in all of the 24 patients recovered to normal or level before UFH treatment in 3-6 days after heparin withdrawal therapy.

CONCLUSIONHIT can be early diagnosed by monitoring platelet counts, HIT-antibody ELISA test and HIPA test. Withdrawal of heparin therapy in time and use of alternative anticoagulant, HITTS rate might be expected to decline further.

Adult ; Aged ; Anticoagulants ; adverse effects ; Enzyme-Linked Immunosorbent Assay ; Female ; Heparin ; adverse effects ; Humans ; Male ; Middle Aged ; Platelet Aggregation ; Platelet Count ; Thrombocytopenia ; chemically induced ; diagnosis

OBJECTIVE Tissue plasminogen activator (tPA) is the only approved pharmaco-logical therapy for acute brain ischaemia;however,a major limitation of tPA is the haemorrhagic trans-formation that follows tPA treatment. Here, we determined whether nicotinamide mononucleotide (NMN), a key intermediate of nicotinamide adenine dinucleotide biosynthesis, affects tPA-induced haemorrhagic transformation. METHODS Middle cerebral artery occlusion (MCAO) was achieved in CD1 mice by introducing a filament to the left MCA for 5 h.When the filament was removed for reperfusion, tPA was infused via the tail vein.A single dose of NMN was injected i.p.(300 mg·kg-1).Mice were killed at 24 h post ischaemia, and their brains were evaluated for brain infarction, oedema, haemoglobin content, apoptosis, neuroinflammation, blood-brain barrier (BBB) permeability, the expression of tight junction proteins(TJPs)and the activity/expression of MMPs. RESULTS In the mice infused with tPA at 5 h post ischaemia, there were significant increases in mortality, brain infarction, brain oedema, brain haemoglobin level, neural apoptosis, Iba-1 staining (microglia activation) and myeloperoxidase staining (neutrophil infiltration). All these tPA-induced alterations were significantly prevented by NMN administration. Mechanistically, the delayed tPA treatment increased BBB permeability by down-regulating TJPs, including claudin-1, occludin and zonula occludens-1,and enhancing the activities and protein expression of MMP9 and MMP2. Similarly, NMN administration partly blocked these tPA-induced molecular changes. CONCLUSION Our results demonstrate that NMN ameliorates tPA-induced haemorrhagic transformation in brain ischaemia by maintaining the integrity of the BBB.

OBJECTIVETo investigate lipoprotein lipase (LPL) and serum thymidine kinase (TK) levels in chronic lymphocytic leukemia (CLL) and their correlations with other prognostic factors.

METHODSLPL expression level in peripheral blood samples of 58 CLL patients was detected by semi-quantitative reverse transcription PCR (RT-PCR). Serum TK1 level in 39 CLL patients was detected by enhanced chemiluminescence (ECL) and TK monoclonal antibody (Anti-TK mAb). IgVH mutation status was detected by multiplex PCR and sequencing of purified PCR products. The expression of ZAP-70 protein and CD38 were determined by flow cytometry . Panel probes and fluorescence in situ hybridization (FISH) were used to detect cytogenetic aberrations.

RESULTSThe median LPL expression level in CLL was 0.26 (0 -6.29), while undetectable in normal controls. LPL expression level was significantly correlated with IgVH mutation status, Binet stages, CD38 and cytogenetic aberrations. Patients with unmutated IgVH genes had higher LPL than those with IgVH mutations (P = 0.010). Patients in Binet stage B and C had higher LPL than those in stage A (P = 0.011). LPL level was higher in patients with CD38 > or = 30% (P = 0.001). Higher LPL level was found in patients with unfavorable cytogenetic aberrations (deletion in 17p13 or 11q22) than those with favorable cytogenetics (deletion in 13q as the sole abnormality) (P = 0.002). LPL level was not significantly correlated with sex, age, and ZAP-70 protein (P > 0.05). The level of TK1 was higher in CLL patients than that in normal control (P < 0.05). Patients with higher level of absolute lymphocyte count (ALC), lactate dehydrogenase (LDH), unmutated IgVH genes and ZAP-70 had higher levels of TK1 than those with lower level of ALC, LDH, mutated IgVH genes and ZAP-70 (P = 0.018, P = 0.018, P = 0.030 and P = 0.038, respectively). TK1 level had no correlation with sex, age, Binet stages, CD38, and cytogenetic aberrations (P > 0.05).

CONCLUSIONSLPL expression and serum TK1 levels significantly correlate with other CLL prognostic factors and could be predictive markers for IgVH mutation status. LPL and serum TK1 might be applied to the assessment of prognosis in CLL patients.

ADP-ribosyl Cyclase 1 ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; enzymology ; metabolism ; Lipoprotein Lipase ; blood ; Male ; Middle Aged ; Mutation ; Thymidine Kinase ; blood ; ZAP-70 Protein-Tyrosine Kinase ; metabolism