Two-dimensional electrophoresis(2-DE) is an important technique in proteomics research.The 2-DE system for proteome of Monascus ruber was established by comparing and analyzing the infection caused by different kinds of mediums,lysis buffer and the condition of rehydration.By cultivating the Monascus ruber with YES for 6 days,extracting total protein by TCA-acetone,lysis buffer with 8 mol/L urea,2 mol/L thiourea,4 % CHAPS,1 % DTT and 2 % Bio-lyte,an ideal 2-DE map with higher resolution and better legibility was obtained,which laid a foundation for the further studies on proteome of Monascus ruber.

ObjectiveTo investigate Schwann cells whether survival and migration after transplanted to central nervous system for a long-term.MethodsThe Schwann cells of rat were expended in vitro, the part of them were labeled with 5'-bromodeoxyuridine (BrdU) and transplanted to rat's middle brain injured by electric needle stimulus, the others were labeled with Hoechst 33342, seeded to PLGA scaffold, and transplanted to rat's transected spinal cord. 8 and 11 months later, rat brain and spinal cord were taken out respectively, examined by BrdU immunohistochemistry and fluorescence microscope.ResultsBrdU positive cells could be seen after 8 months and migrated toward cerebral cortex. Hoechst 33342 positive cells could be identified in scaffold and transected spinal cord after 11 months under fluorescence microscope.ConclusionGrafted Schwann cells can survive in central nervous system for a long-term and migrate toward distance.

OBJECTIVETo establish an efficient and stable method for protein extraction of Streptococcus mutans.

METHODSThe collected bacteria were treated by freeze-thaw and ultrasonic (method 1), ultrasonic (method 2), boiling (method 3), boiling and ultrasonic (method 4), respectively. The index such as state of bacteria broken, concentration of extracted protein and SDS-PAGE of protein were employed to evaluate the effects of above four methods.

RESULTSBeside the method 3, the other three methods could break the bacteria effectively, of which ultrasonic was the key factor. The pattern of SDS-PAGE which treated by method 1, method 2 and method 4 was almost same, but method 1 resulted in the best abundance. There was significantly difference among the four protein concentration extracted by four methods (P < 0.05). All methods exhibited good stability and reproducibility.

CONCLUSIONMethod of freeze-thaw and ultrasonic resulted in an efficient proteins extraction of Streptococcus mutans which demonstrated good stability and reproducibility and easy to handle.

Bacterial Proteins ; Reproducibility of Results ; Streptococcus mutans

OBJECTIVETo investigate the antibacterial activity of decoction of Radix glycyrrhizae against Streptococcus mutans (S. mutans) in vitro.

METHODSThe decoction of Radix glycyrrhizae was prepared by boiling particles of Radix glycyrrhizae, the diameter was 0.2-3.2 mm. In distilled water and filtered, the filtrate was collected for study. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of the decoction against S. mutans were detected using double dilution. The effect of decoction on growth and acidogenic profile of S. mutans were investigated by detecting the Abs of bacteria suspension and the pH value of medium at definite time intervals(0, 3, 7, 12, 23, 40 h) during cultured.

RESULTSThe MIC determined for decoction was 50 mg x mL(-1) and there was no bactericidal effect when concentration of decoction lower than 100 mg x mL(-1). The decoction inhibitted multiplication of bacteria significantly and the effects became stronger with concentration increasing. The decoction also inhibitted S. mutans producing acid and the effect became stronger with concentration increasing. The most efficient inhibition were observed when incubated 12 hours.

CONCLUSIONThe decoction of Radix glycyrrhizae can inhibite the growth and acid-production of S. mutans in vitro.

Anti-Bacterial Agents ; Bacteria ; In Vitro Techniques ; Microbial Sensitivity Tests ; Plant Extracts ; Streptococcus mutans

OBJECTIVETo explore the expansion method of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after ex vivo expansion.

METHODSNK cells were isolated from peripheral blood mononuclear cells (PBMNCs) by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II, and cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were semi-exchanged with fresh media and cytokines every 3 days. Evaluation for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-gamma secretion before and after the culture period.

RESULTSCD3(-) CD56(+) cells concentration increased from (11.2 +/- 5.2)% to (94.2 +/- 3.5)%. In group IL-2 + IL-15 and IL-2 + IL-15 + IL-12 group, cells were expanded 50.5 +/- 4.3 and 52.3 +/- 6.7 - fold, respectively, being significantly higher than that in other three groups [(15.4 +/- 1.1 fold in IL-2 group, 19.9 +/- 3.9 fold in IL-2 + IL-12 group, 6.1 +/- 1.0 fold in control group)] (P<0.01), but no significant difference between each other (P>0.05). The purity of CD3(-) CD56(+) NK cells was over 94% in all groups except the control. The perforin and granzyme B mRNA expressions of expanded NK cells in four experimental groups were significantly higher than those of before expansion (P<0.01) and the expressions in IL-2 + IL-15 and in IL-2 + IL-12 + IL-15 group were significant higher than in other three groups (P<0.01) while no significant difference between each other (P>0.05). IFN-gamma levels in the supernatants of four experiment groups were significantly higher than that in control group (P<0.01) and its levels order was IL-2 + IL-15 + IL-12 group > IL-2 + IL-12 group > IL-2 + IL-15 group > IL-2 group (P<0.01).

CONCLUSIONHigh purity NK cells isolated by negative selection using miniMACS can be efficiently expanded with IL-2 + IL-15, and their biological functions were enhanced.

Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Granzymes ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukin-12 ; pharmacology ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukins ; pharmacology ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; metabolism ; Perforin ; metabolism

Objective To study the characteristics of different papillary renal cell carcinoma (PRCC)subtypes and their prognosis after nephrectomy.Methods Clinical data of 14 PRCC patients(7 males,7 females)with ages ranging from 20-77 in our institute from 2005 to 2011 were retrospectively reviewed.There were 5 tumors in the left kidney and 9 tumors in the right kidney.The average maximum tumor diameter was 3.8(1.6-7.8)cm.Patients presented with gross hematuria(n =3),flank pain(n =3),palpable abdominal mass(n =1)or asymptomatic(n =7).The TNM stages were 8 T1aN0M0,2 T1bN0M0,1 T1aN0M1,1 T2aN0M0,1 T3aN0M0 and 1 T3aN1 M0.Six patients were treated with radical nephrectomy,8 cases were treated with partial nephrectomy.Results There were 6 type Ⅰ and 8 type Ⅱ PRCCs cases.In pathology,type Ⅰ PRCC showed papillae covered by small cells with scanty basophilic cytoplasm,and arranged in a single layer on the papillary basement membrane with low nuclear grade.Type Ⅱ PRCC was composed of cells with higher nuclear grade,abundant eosinophilic cytoplasm,and pseudostratified nuclei on papillary cores.There were 12 well-differentiated cases,2 moderate-differentiated cases and no poorly differentiated case.Follow-up was carried out from 12to 80 months.During the follow-up,1 patient with type Ⅰ PRCC developed multiple lung metastases 26 month after surgery and deteriorated into hepatic and bone metastases at 34 month after surgery.We offered the patient with targeted therapy and the patient was still alive.There was 1 type Ⅱ PRCC patient died with multiple metastases at 42 month after surgery.Others were still alive without local recurrence and metastasis.Conclusions PRCC is not a common subtype of renal cell carcinoma in China.Early stage PRCC patient would achieve good prognosis after treated with nephrectomy.Targeted therapy is a good treatment option for metastatic papillary renal cell carcinoma patients.

The authors describe here the procedures for using the gelatin half-embedding method to obtain thin spinal cord slices with attached dorsal roots and performing visually guided whole-cell patch-clamp recording of postsynaptic currents evoked by primary afferent fibers in rat spinal dorsal horn. A segment of spinal cord with attached dorsal roots was prepared and half-embedded in an agar block with 20% (w/v) gelatin. Thin spinal cord slices with attached dorsal roots were obtained with a vibratome and whole-cell patch-clamp configuration was established under the infrared observation. At the holding potential of -70 mV, spontaneous excitatory postsynaptic currents (EPSCs) and dorsal root stimulation-evoked EPSCs were recorded as inward currents. According to the conduction velocity of afferent fibers and stimulus threshold, evoked EPSCs that are mediated by A-like or C-like fibers were distinguished. At the holding potential of 0 mV, spontaneous inhibitory postsynaptic currents (IPSCs) and dorsal root stimulation-evoked IPSCs were recorded as outward currents. Using 5 micromol/L strychnine or 20 micromol/L bicuculline, GABAergic or glycinergic evoked IPSCs could be isolated. Using visual patch-clamp method synaptic transmission can be accurately assessed by measuring postsynaptic currents of the dorsal horn neurons. More importantly, with the aid of infrared observation, the incidence of failure to establish a clamp configuration can be greatly reduced and it becomes easier to make recordings from the neurons in deep dorsal horn laminae. Thus, the present research approach an effective approach to study the modulation of primary afferent synaptic transmission.
Animals ; Electrophysiology ; Evoked Potentials ; physiology ; Excitatory Postsynaptic Potentials ; physiology ; Female ; Male ; Neurons, Afferent ; physiology ; Patch-Clamp Techniques ; methods ; Posterior Horn Cells ; physiology ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; cytology ; physiology ; Synaptic Transmission ; physiology

BACKGROUNDRetinopathy of prematurity (ROP) has become one of the leading causes of visual loss in children. Vascular endothelial growth factor A (VEGF-A) is the principal stimulator of angiogenesis. VEGF was differentially spliced from exon 8 to exons 8a and 8b to form two families: the pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family. Previous research has shown variable effeteness of bevacizumab in inhibiting retinal neovascularization in ROP. This study aimed to investigate whether the effectiveness of this inhibition depends on the relative ratio of the two VEGF isoforms.

METHODSIntravitreal bevacizumab injection (IVB) was performed in the oxygen-induced-retinopathy (OIR) mice on postnatal day 12 (P12) (intravitreal phosphate buffered saline (PBS) injection as control). The Evans blue perfused retina were used to test the retinal neovascularization-leakage (NVL) area and non-perfusion (NP) area.

RESULTSThe retinal NVL and NP area in the IVB group were significantly smaller than the intravitreal PBS injection group (IVP group). On P17, the protein level of total VEGF isoforms was significantly inhibited compared to IVP group (P < 0.05) while VEGF(165)b isoform was slight reduced (P > 0.05). The switch from pro-angiogenic isoforms to anti-angiogenic isoforms after IVB could be found. The relative protein expression of VEGF(165)b isoform was significantly higher in IVB group than in IVP group (P < 0.05) on P17 which was correlated with the reduced ischemia-induced angiogenesis in OIR mice after IVB.

CONCLUSIONSThe anti-angiogenic effectiveness might depend on the relative high expression of VEGF(165)b after intravitreal bevacizumab injection. Anti-angiogenic therapy is a more effective therapy for ROP.

Angiogenesis Inhibitors ; administration & dosage ; Animals ; Animals, Newborn ; Antibodies, Monoclonal, Humanized ; administration & dosage ; Bevacizumab ; Disease Models, Animal ; Intravitreal Injections ; Mice ; Mice, Inbred C57BL ; Protein Isoforms ; analysis ; Retinal Neovascularization ; prevention & control ; Retinopathy of Prematurity ; drug therapy ; Vascular Endothelial Growth Factor A ; analysis

OBJECTIVETo investigate the recovery of rat transected spinal cord injury after implantation of Schwann cells combined with poly (lactide-co-glycolide) (PLGA).

METHODSSchwann cells were expanded, co-cultured with PLGA for 9 days in vitro, and then analyzed with scanning electron microscope (SEM). Rat spinal cord at the level of T(9) was transected. Schwann cells labeled with BrdU and PLGA scaffold were implanted to injury site. After 1, 3, 6 months, BrdU/MBP immunohistochemistry double staining, semi-thin sections stained thionin and ultra-thin section were performed to investigate myelin renew. BBB open field locomotion, motor evoked potential (MEP), compound muscle action potential (CMAP) and somatosensory evoked potential (SEP) were recorded.

RESULTSSchwann cells grew well on PLGA under SEM. BrdU/MBP double positive cells would been seen, remyelination was thin and formed by Schwann cells at 6 months later under electron microscope (EM). BBB behavioral tests revealed no significant difference in recovery comparing with experiment group and control group. The results of MEP, CMAP and SEP showed no significant improvement in the conduction of spinal cord.

CONCLUSIONSThere are the compatibility between Schwann cells and PLGA. Although remyelination was found in morphology, function conduction of spinal cord failed to be established.

Animals ; Cells, Cultured ; Disease Models, Animal ; Evoked Potentials, Motor ; Female ; Immunohistochemistry ; Lactic Acid ; chemistry ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Nerve Regeneration ; Polyglycolic Acid ; chemistry ; Prostheses and Implants ; Rats ; Rats, Wistar ; Schwann Cells ; chemistry ; transplantation ; ultrastructure ; Spinal Cord Injuries ; physiopathology ; surgery ; Tissue Engineering ; methods

Diosgenin can inhibit the growth of A375 and K562 cell lines and induce their apoptosis with an effect on pro-apoptotic members of Bcl-2 family. To study the SAR of diosgenin derivatives, and to improve the anti-tumor activity of diosgenin, a series of novel diosgenin derivatives were designed and synthesized. Their anti-tumor activities in vitro were evaluated. The results revealed that most of the new derivatives had potent effects against K562, A375 and A549 (three tumor cell lines) in vitro, and had no or less effect against H293 and L02 (two normal cell lines). Particularly, some compounds (e.g. 1, 6-8) showed excellent activities on K562 with IC50 values ranging from 1.96 to 4.35 micromol x L(-1).
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Diosgenin ; analogs & derivatives ; chemical synthesis ; chemistry ; pharmacology ; Drug Design ; Humans