Four strains of thermophilic cellulolytic anaeobic bacteria were isolated from fresh feces, heat compost, cellulolytic mixed culture with a method based on adherence of cellulolytic bacteria to cellulose. The cells of isolates were straight or slightly curved rods that were 0.4 micron-0.6 micron x 3 microns-15 microns, Gram negative, strictly anaerobic, sulfate reduction negative, spore-forming bacteria. Most of the cells had oval terminal spores, while subterminal spores, middle spores, two or more spores also could be observed and spore formation could occurred in any position. The isolates degraded cellulose filter paper, cellulose powder Whatman CF II, microcrystalline cellulose, cellulose powder MN300 and unpretreated maize stem core, sugarcane residue and rice straw. The pH and temperature ranges for growth on cellulose were 6.2-8.9 and 45 degrees C-65 degrees C respectively with the optima, 7.0-7.5 and 55 degrees C-60 degrees C, respectively. The major fermentation products from cellulose were acetic acid, ethanol, CO2, H2. The isolates could ferment cellobiose, glucose, fructose, maltose, and sorbital. The phylogenetic analysis based on 16S rDNA suggested strain EVA1 was the closest relative of Clostridium thermocellum with 99.8% sequence similarity.

The objective of this study was to investigate micromechanical properties and electromotility of cochlear outer hair cells in vivo. Extracochlear electrically stimulation with sinusoid alternating current was delivered to cochlea of guinea pigs. Sound pressure level was recorded from ear canal by microphone and the electrically evoked otoacoustic emissions (EEOE) and their distortion products (DPEEOE) were analyzed with FFT spectrum analyzer. The EEOE in 3 kHz to 33 kHz were recorded from 15 guinea-pigs in 18 guinea-pigs, the transfer function was more smooth in 8 kHz to 31 kHz. The DP EEOE were very clear when F1 = 6 kHz, F2 = 7.2 kHz, and the current intensity higher than 100 microA. While the intensity increased to 300 microA, two distortion products, F2-F1 and 2(2F1-F2)-F1, could be seen in addition to 2F1-F2. The input-output function showed that EEOE and DP EEOE I/O function were linear when lower electric intensity stimulation was given, but they displayed compress non-linear features when higher intensity current was delivered. The authors conclude that EEOE and DP EEOE are expression of electrophonic hearing characteriged by wide frequency bandpass appearance, wide dynamic range and non-linear features, so they are good tools for studying the mechanical properties of outer hair cells and the integrate function of Corti's organ.

To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) and multidrug resistance gene (MDR1) increase resistance to 1,3-Bis(2-Chloroethy1)-1-Nitrosourea (BCNU) and P-glycoprotein effluxed drugs, the present authors obtained a full length cDNA fragment encoding MGMT from liver tissue of a patient with cholelithiasis by RT-PCR. A bicistronic retroviral vector G1Na-MGMT-IRES-MDR1 cDNA was constructed and transfected the packaging cell lines GP + E86 and PA317 by electric performation method, using the medium containing VCR and BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, cord blood CD34+ cells were enriched with a high-gradient magnetic cell sorting system (MACS), and then transfected repeatedly with supernatant of retrovirus containing human MGMT and MDR1cDNA under stimulation of hemapoietic growth factors. PCR, RT-PCR, Southern blot, Northern blot, Western blot, FACS and MTT assay were used to evaluate the transfer and expression of the double genes in cord blood CD34+ cells. The cDNA encoding MGMT was verified by DNA sequencing and the bicistronic retroviral vector was confirmed by restriction endonuclease analysis. The purity of cord blood CD34+ cells was approximately 92% and recover rate was 75%, the highest titer of recombinant amphotropic retrovirus in the supernatant was up to 5.8 x 10(5) cfu/ml. The efficiency of gene transduction was 18% and 20% tested by colony formation and PCR, respectively. No helper virus was found by both nested PCR and rescue assay. The results showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The MTT analysis showed a 4.5 to 7.8-fold increase of resistance of transducted cells to BCNU and P-glycoprotein effluxed drug as compared with the nontransduced cells. This study provided a foundation for ameliorating combination chemotherapy toxicity in tumor clinical trial.

The present experiments were designed to study the effects of glucose, EDTA, glutamine on the in vitro development of single blastomeres from 2-cell embryos in mouse, and the efficiency of cryopreservation of blastocysts from single blastomers with different vitrification. Single blastomeres derived from female ICR x male BDF1 2-cell embryos were cultured in mKRB with or without glucose, EDTA and glutamine, respectively. The expanded blastocyst rates were significantly different between in mKRB with glucose and without glucose (34% vs 65%); The blastomeres were cultured in mKRB with EDTA and glutamine but glucose, the expanded blastocyst rate (90%) was significantly higher than other groups. The blastocysts derived from single blastomeres were vitrified in liquid nitrogen after equilibration in GFS40 for 0.5-2 min, the survival rate 24%-51%. The blastocysts were pretreated in mPBS with 10% glycerol for 5 min, followed by exposure to GFS40 at 25 degrees C for 0.5 min, then vitrified in liquid nitrogen(two-step method), the survival rate was 61%. However, the survival rates increased to 64% and 70% when the blastocysts were vitrified(one-step method) ater equilibration in EFS40 at 25 degrees C for 0.5-1 min.

To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and multidrug resistance gene (MDR1) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed. The vector was transduced into the packaging cell lines GP + E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDR1cDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4- to 7.2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontransduced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.

The cDNA of human stem cell factor(hSCF) containing signal sequence was cloned into the transfer vector pVL941 of AcNPV to construct a recombinant transfer vector pVL941-SCF. Sf9 cells were cotransfected with wild type viral DNA and pVL914-SCF to produce the recombinant virus AcNPV-SCF by homologous recombination in cell. Southern-hybridization analysis suggested that the recombinant viral DNA contained hSCF cDNA fragment. The Sf9 cells infected with the recombinant baculovirus AcNPV-SCF expressed biologically active rhSCF which was secreted into the cell culture. The synergistic activities of SCF in conjunction with human interleukin-3(hIL-3) was measured by MTT colorimetric method and TF-1 cell line. The expression level of Sf9 cells reached its highest at about 1970 units/ml in the 3rd day after the infection with AcNPV-SCF. Three SCF bands with molecular masses of 18 x 10(3), 20 x 10(3) and 22 x 10(3) were detected by immunoblotting.

Primordial germ cells (PGC) were isolated from 8.5, 10.5, 12.5 days post coitum (dpc) embryos of F1 (Balb/c x ICR), C57BL/6J, 129/svJ, 129/sv-ter mice, and cultured on mitotically inactive MEF or STO feeder layer cells with addition of leukemia inhibitory factor, stem cell factor and basic fibroblast growth factor in cultures. PGCs formed densely packed and AKP positive colonies with pluripotential marker gene (oct-4) expression resembling undifferentiated ES cells in morphology and growth pattern. Five EG cell lines derived from PGCs were established: EG1(8.5 dpc, F1), EG2 and EG3 (8.5 dpc, C57BL/6J), EG4 (10.5 dpc, 129/svJ), EG5 (10.5 dpc, 129/sv-ter). No long term culture was obtained from 12.5 dpc PGCs of 29 embryos. All five EG cell lines cultured on feeder layer cells or in LIF containing medium still remain undifferentiated state at 15 th passage. Under appropriate conditions, EG cells formed embryoid bodies in suspension culture and multiple types of differentiated cells in monolayer culture. When these EG cells were injected in nude mice, they formed teratocacinomas containing differentiated cells such as cartilage, neural tissue and epithelium. These results show that EG1-5 cell lines derived from 8.5, 10.5 dpc embryos are pluripotential.

To explore the relationship between the proliferation, differentiation of rat hepatic stem like cell line WB-F344 and cytokines in vitro.

To dissociate viable isolated Deiters' cells from guinea pig cochlea and perform morphological measurements under inverted microscope; To study electrophysiological properties of Deiters' cells.

Our purpose is to determine whether murine submandibular gland produce hematopoietic growth factors or not and which hematopoietic growth factor are produced by murine submandibular gland.

To assess the effect of Astragalus membranaceus injection (AMI) on megakaryocyte hematopoiesis in anemic mice and explore its mechanism.

To observe the effects of recombinant human thrombopoietin (rhTPO) and rhTPO in combination with recombinant human interleukin-11(rhIL-11) on the megakaryocyte (MK) colony growth and maturation in patients with chronic idiopathic thrombocytopenic purpura (CITP) in vitro.

To investigate the feasibility of using decalcified bone matrix loaded with mesenchymal stem cells (MSCs) derived from adult human bone marrow as scaffolds for bone tissue engineering.

The efficiency of rituximab (Mabthera) is related to CD20 expression density on cell membrane. It is not yet to be solved how to heighten expression level of CD20 on multiple myeloma (MM) cell membrane and to increase the efficacy of Mabthera to MM. This study was designed to observe whether thalidomide could promote the effect of Mabthera on suppressing myeloma cells in vitro and its possible mechanism.