The mitochondrial enzyme glutaminase C (GAC) is highly expressed in a variety of cancer cells, resulting in increased glutamine metabolism and cancer development. Therefore, GAC has become a potential target for anti-tumor drug development. However, current GAC inhibitors shared similar structural characteristics, few new scaffolds were reported. By conducting a prokaryotic Escherichia coli expression system, human GAC protein of high-purity was obtained through lysozyme digestion combined with ultrasound dissociation, and cobalt magnetic beads purification, Moreover, we performed studies to validate interaction between small molecules and GAC protein through thermal shift assay, drug affinity responsive target stability assay, protein crosslinking and GAC enzyme activity detection. Meanwhile, a comprehensive small molecule-protein interaction confirmation and systematic pharmacodynamic study in vitro were carried out on compound C19, which was a reported GAC inhibitor screened from the Enamine database. Results showed that C19 directly bind to GAC protein, disturbed GAC tetramers formation, and inhibited its enzyme catalytic activity. By interfering GAC function, C19 dose-dependently suppressed GAC-mediated glutamine metabolism, reduced glutamate in cancer cells, and thus alleviated A549 and NCI-H1299 non-small cell lung cancer cell growth. Together, C19 was identified as a lead compound, providing a new strategy for the structural design of drugs targeting GAC.

Adenoma, Pleomorphic ; diagnosis ; diagnostic imaging ; surgery ; Adolescent ; Adult ; Aged ; Angiography, Digital Subtraction ; Carcinoma, Adenoid Cystic ; diagnosis ; diagnostic imaging ; surgery ; Chemotherapy, Adjuvant ; Child ; Female ; Follow-Up Studies ; Head and Neck Neoplasms ; diagnosis ; diagnostic imaging ; surgery ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Neurilemmoma ; diagnosis ; diagnostic imaging ; surgery ; Oral Surgical Procedures ; methods ; Postoperative Complications ; Radiotherapy, Adjuvant ; Tomography, X-Ray Computed ; Young Adult

Lipase-mediated biodiesel production becomes increasingly important because of mild reaction conditions, pollution free during the process and easy product separation. Compared with traditional immobilized lipase, whole cell biocatalyst is promising for biodiesel production because it is easy to prepare and has higher enzyme activity recovery. Rhizopus oryzae IFO4697 can be used as the catalyst for biodiesel production. To further study the stability of the whole cell biocatalyst is extremely important for its further application on large scale. This paper focuses on the stability study of Rhizopus oryzae IFO4697 when used for the methanolysis of renewable oils for biodiesel production. The results showed that water content was important for achieving high catalytic activity and good stability of the biocatalyst. The optimum water content was found to be 5%-15%. Both particle size and desiccation methods showed no obvious effect on the stability of the biocatalyst. With GA cross-linking pretreatment, the stability of the biocatalyst could be improved significantly. When Rhizopus oryzae IFO4697 repeatedly used for next batch reaction, direct vacuum filtration was found to be a good way for the maintenance of good stability of the biocatalyst. Under the optimum reaction conditions, the methyl ester yield could keep over 80% during 20 repeated reaction batches.
Biocatalysis ; Bioelectric Energy Sources ; microbiology ; Biofuels ; Cells, Immobilized ; metabolism ; Lipase ; metabolism ; Rhizopus ; metabolism ; Soybean Oil ; metabolism

OBJECTIVETo evaluate the effect of aging on the proliferative and differentiation capacity of human periodontal ligament stem cells (PDLSCs).

METHODSHuman periodontal ligament tissues were obtained from surgically extracted third molars from 6 subjects aged 18-20 years (group A) and 6 subjects aged 45-50 years (group B). The proliferative capacity of PDLSCs isolated from the tissues was examined with MTT assay, and the osteogenic and adipogenic differentiation capacity of the cells were evaluated using alizarin red staining and oil red O staining. SA-βG expression was analyzed to assess the cell senescence. In both groups, PDLSCs were induced for osteogenic differentiation for 7 days, and the differentiation ability of the cells was assessed by examining alkaline phosphatase (ALP) activity and by detecting the expressions of osteocalcin (OCN) and ALP using Western blotting.

RESULTSHuman PDLSCs were successfully isolated from the 12 teeth and were characterized as MSCs. The PDLSCs derived from donors of different ages were all capable of osteogenic and adipogenic differentiation, but their proliferative and osteogenic differentiation capacity decreased with the donors' age. The cells also exhibited an age- related increase in adipogenic differentiation capacity and SA-βG expression. In both groups, the cells induced in osteogenic medium showed increased OCN expression and ALP activation, and the increments were more obvious in group A.

CONCLUSIONHuman PDLSCs can be isolated from periodontal ligament tissues even from donors of advanced ages, but their proliferative and differentiation capacity decreases and their adipogenic differentiation capacity increases with age.

BACKGROUNDOsteosarcoma (OS) is the most common primary malignant tumor of bone. Mouse models of human OS can invariably provide greater insight into the complex mechanisms that underlie the development and pathogenesis of this aggressive tumor. Bioluminescence technology favored tracing cancer cells in vivo. In this study, an OS model was described and evaluated using human OS cell line, Saos2, labeled with luciferase (Saos2-luc).

METHODSSaos2 cells were infected by lentivirus loading a firefly luciferase gene. Luciferase expression of Saos2-luc cells was characterized both in vitro and in vivo. Specific biologic and oncologic features of Saos2-luc cells were analyzed. The OS was established as orthotopic xenografts in nude mice. Both orthotopic tumors and spontaneous lung metastasis were analyzed.

RESULTSTumorigenesis and spontaneous lung metastasis in nude mice could be monitored in vivo through in vivo imaging system. The enhancement in proliferation, migration and invasion abilities and the attenuation in adhesion ability were observed in Saos2-luc cells compared with Saos2 cells. Furthermore, there were the up-regulation of Osteocalcin, CCR10, CXCR1 and ID1 and the down-regulation of ALP, collagen I, CCR1, CCR3, CXCR3, NID and N-cadherin in Saos2-luc cells compare to Saos2 cells. The rate of spontaneous lung metastasis in Saos2-luc cells was higher than that in Saos2 cells, although without significant difference.

CONCLUSIONSLentivirus transfection may cause alteration of gene expression profiles and further biological functions. This model can be used in the elucidation of molecular mechanisms of tumorigenesis and the screening of new therapeutic agents.

Animals ; Carcinogenesis ; pathology ; Cell Line, Tumor ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Metastasis ; pathology ; Osteosarcoma ; pathology ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Transplantation, Heterologous

A quick HPLC-ESI-MS/MS method was established for simultaneous determination of three chemical compositions in Usnea, including usnic acid, diffractaic acid, and ramalic acid. The separation was performed on a chromatographic column of Agilent ZORBAX SB-C, (4.6 mm x 250 mm, 5 µm), and the mobile phase was methanol (0.05% formic acid)-0.05% formic acid solution (4 mmol ammonium acetate), with an isocratic elution at a flow rate of 0.8 ml · min⁻¹. Multiple reaction monitoring scanning mode (MRM) was performed combined with the ion switching technology in positive and negative ion switching mode to apply for the quantitative determination. The calibration curves for the above three compounds were linear in corresponding injection amount. Their average recoveries were 95.0%-105.1%, with RSDs of 1.1%-5.2%. The method was simple, rapid, accurate with high repeatability, which could provide a reference for overcalling evaluation the quality of Usnea.
Chromatography, High Pressure Liquid ; methods ; Hydroxybenzoates ; analysis ; Spectrometry, Mass, Electrospray Ionization ; methods ; Tandem Mass Spectrometry ; methods ; Usnea ; chemistry

OBJECTIVETo explore the effect of acupoint massage dominant early comprehensive intervention on the prognosis of premature infants with brain injury.

METHODSTotally 210 premature infants with brain injury were assigned to the intervention group (112 cases) and the control group (98 cases). All patients received routine therapy (medicinal + routine care instructions). Patients in the intervention group additionally received acupoint massage. Those with abnormal early motion received physical sports treatment. Those with upper limbs dysfunction or with fine movement disorders received occupational therapy. Premature infants' development quotient (DQ) was performed at corrected age of 6 and 12 months by using neuropsychological development examination table for 0 - 6 years old children. The incidence of cerebral palsy was statistically calculated.

RESULTSAt corrected age of 6 months, DQ of gross motor, fine motor, language three functional areas was higher in the intervention group than in the control group with significant difference (P < 0.05). At corrected age of 12 months, DQ of gross motor, fine motor, language, social and adaptive capacities was higher in the intervention group than in the control groupwith significant difference (P < 0.05). The incidence of cerebral palsy was 4.46% (5/112) in the intervention group and 12.24% (12/98) in the control group (P < 0.05).

CONCLUSIONAcupoint massage dominant early comprehensive intervention could obviously improve the intelligence development level and lower the incidence of cerebral palsy in premature infants with brain injury.

Acupuncture Points ; Brain Injuries ; therapy ; Cerebral Palsy ; prevention & control ; Early Medical Intervention ; Female ; Humans ; Infant ; Infant, Newborn ; Infant, Premature ; Male ; Massage ; Prognosis

Aim To investigate the effect of ginsen-oside metabolite compound K ( CK) on migration and invasion of human hepatocellular carcinoma line HepG2, and the possible signaling pathway underlying these processes .Methods HepG2 cells were exposed to ginsenoside CK (0, 10, 20, 40, 80 μmol· L-1 ) for 24 h.The cell viability was examined by MTT as-say, and the ability of migration and invasion was ob-served with the wound healing and transwell assay .The expression of E-cadherin , N-cadherin and other related signal molecules such as p-ERK, ERK, p-Akt, Akt were detected by Western blot .Results The cell via-bility was significantly reduced by ginsenoside CK (20, 40, 80 μmol· L-1) (P<0.01).The ability of cell migration and invasion was significantly inhibited after exposure to ginsenoside CK .After treatment with ginsenoside CK (20, 40, 80 μmol · L-1 ) in HepG2 cells, the expression of E-cadherin markedly in-creased, while N-cadherin expression significantly de-creased.Meanwhile, the expression of p-ERK and p-Akt decreased after treated with ginsenoside CK .Con-clusion Ginsenoside CK inhibits the migration and invasion of human hepatocellular carcinoma line HepG2, which may be through suppression of ERK and Akt signaling .

OBJECTIVETo investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS).

METHODSMutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A β-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.

RESULTSWhen grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The β-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain.

CONCLUSIONYpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Bacterial Outer Membrane Proteins ; secretion ; Bacterial Secretion Systems ; genetics ; Genes, Bacterial ; Plasmids ; Protein Interaction Mapping ; Yersinia pestis ; genetics ; metabolism ; pathogenicity

OBJECTIVETo evaluate clinical outcomes and pronouncing and swallowing functions after supracricoid partial laryngectomy (SCPL).

METHODEight-five cases underwent supracricoid partial laryngectomy from 1990 to 2008 were reviewed.

RESULTSThe 3- and 5-year accumulative survival rates were 90.4% and 81.6%, respectively. 97.6% (83/85) patients were decannulated. 89.4% (76/85) were able to eat normally within 6 weeks after surgery and the swallowing functions in the rest patients were recovered by training in different time after surgery. Fiber laryngoscope showed the reconstructed throats could close well with good swallowing functions six months after the operation.

CONCLUSIONSCPL is a good choice in selected cases with laryngeal cancer, with satisfactory pronouncing and swallowing functions after surgery.

Cricoid Cartilage ; surgery ; Deglutition ; Humans ; Laryngeal Neoplasms ; surgery ; Laryngectomy ; methods ; Pharynx ; Retrospective Studies ; Survival Rate ; Treatment Outcome