Generation and selection of immunized Fab phage display library against human B cell lymphoma

The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody （HAMA） response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains （VH） and variable light domains （VL） were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.     

Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.     

Polarization states of diffracted light. Changes accompanying fiber activation.

Measurement of the state of optical polarization of light diffracted from single, skinned and intact fibers of anterior tibialis muscle from Rana pipiens revealed a dependence upon rigor, activation, and sarcomere length (SL) change. Changes in total birefringence, delta nT, and differential field ratio value, rT, were determined. In a relaxed, skinned fiber the total birefringence value, delta nT, decreases as sarcomere length is increased from 2.1 microns to approximately 2.8-3.0 microns. From there it increases significantly to a value of approximately 1.8 x 10(-3) at a sarcomere length of 3.6 microns. The differential field ratio, rT, also shows a biphasic response to increasing sarcomere length, first exhibiting a rapid decrease over shorter SL and leveling out after the SL is beyond 3.0 microns. In comparison, relaxed intact fibers change substantially less upon sarcomere length change, showing little change in birefringence and a small bi-phasic change in rT. Skinned fibers were activated using a solution that has the same ionic strength as the relaxing solution and allows repeatable, and sustained activation. A decrease in both delta nT and rT was observed upon fiber activation. The decrease in delta nT and rT was slightly larger at shorter sarcomere lengths than at longer lengths. Relaxed fibers placed in rigor showed changes in delta nT and rT similar to those observed in activated fibers. These results are consistent with the hypothesis that, after activation, a significant portion of the thick filament cross-bridges rotate towards the actin filament resulting in redistribution of the interfilament mass content. They are also consistent with an average orientation of crossbridges in the overlap region different from that in the nonoverlap region.     

Isolation of pheromone precursor genes of Magnaporthe grisea.

In heterothallic ascomycetes one mating partner serves as the source of female tissue and is fertilized with spermatia from a partner of the opposite mating type. The role of pheromone signaling in mating is thought to involve recognition of cells of the opposite mating type. We have isolated two putative pheromone precursor genes of Magnaporthe grisea. The genes are present in both mating types of the fungus but they are expressed in a mating type-specific manner. The MF1-1 gene, expressed in Mat1-1 strains, is predicted to encode a 26-amino-acid polypeptide that is processed to produce a lipopeptide pheromone. The MF2-1 gene, expressed in Mat1-2 strains, is predicted to encode a precursor polypeptide that is processed by a Kex2-like protease to yield a pheromone with striking similarity to the predicted pheromone sequence of a close relative, Cryphonectria parasitica. Expression of the M. grisea putative pheromone precursor genes was observed under defined nutritional conditions and in field isolates. This suggests that the requirement for complex media for mating and the poor fertility of field isolates may not be due to limitation of pheromone precursor gene expression. Detection of putative pheromone precursor gene mRNA in conidia suggests that pheromones may be important for the fertility of conidia acting as spermatia.     

A bifunctional transposon mini-Tn5gfp-km which can be used to select for promoter fusions and report gene expression levels in Agrobacterium tumefaciens

A mini-Tn5 transposon derivative, mini-Tn5gfp-km, has been constructed which contained a promoter-less artificial operon consisting of two open reading frames, green fluorescent protein (GFP) and neomycin phosphotransferase II (NptII). When this transposon was used to mutagenize Agrobacterium tumefaciens, all the mutants selected in the presence of kanamycin exhibited GFP expression, which could be conveniently monitored by a fluorometer. The transposon appeared to be bifunctional and could provide both selection and reporter functions. Even the mutants showing minimal levels of GFP expression were still resistant to kanamycin. This suggests that this transposon can be used to select for insertions downstream of both weak and strong promoters, as long as the insertions themselves are non-lethal. This system was used to identify A. tumefaciens genes that were upregulated in response to acidic pH. Screening only 20 colonies led to identification of two promoters that were specifically induced by low pH and one promoter that was specifically induced by acetosyringone in a minimal medium of pH 5.5.     

Design and synthesis of 4-phenyl piperidine compounds targeting the mu receptor

Small molecule mu agonists based on the 4-phenyl piperidine scaffold were designed and synthesized to further investigate the therapeutic potential of loperamide analogs. The resulting compounds show excellent agonistic activity towards the human mu receptor with interesting SAR trends within the series.     

人食管癌细胞株PTEN的激光共聚焦扫描显微镜检测

目的对人胚食管上皮永生化细胞株SHEE、SHEEMT、食管癌细胞株EC8712中PTEN表达情况进行定量比较和定位观察.方法采用激光共聚焦扫描显微镜光学切片和荧光探针的双重标记技术对三株细胞中PTEN的表达和分布情况进行检测.结果人食管癌细胞中PTEN主要表达在细胞浆和细胞核,在人胚食管癌上皮永生化细胞株SHEE、SHEEMT主要表达在细胞浆,食管癌细胞EC8712中细胞核表达增多,差异有统计学意义(P<0.01);PTEN在三种细胞株中表达强弱顺序为SHEE>SHEEMT>EC8712,差异有统计学意义(P<0.01).结论PTEN在SHEE、SHEEMT和EC8712分化程度不同的细胞株中均表达,表达和分布位置与分化程度相关.     

Identification of the gene imds-60 in mouse epididymis

Sperm maturation, including the acquisition of motility and the full ability to fertilize oocyte, occurs during its transit through the dynamic environment of the epididymis. However, the roles of many genes involved in the process of sperm maturation still remain to be found. Based on an expressed sequence tag named imds-60, which was first found in uterus but is highly expressed in epididymis, the full-length cDNA sequence of imds-60 with a complete open reading frame was obtained in mouse epididymis by GenBank searching, polymerase chain reaction-based procedures, and 5＇- and 3＇-rapid amplification of cDNA ends. This protein was predicted to have an N-terminal signal peptide and a C-terminal DNase I-like domain with nine transmembrane motifs in the middle part of the protein. Northern blot analysis showed that the mRNA of imds-60 was highly expressed in epididymis but at a rather lower level in uterus, seminal vesicle gland, and stomach. Further study revealed that the mRNA of imds-60 is only expressed in corpus and cauda regions of epididymis, not in caput. It is regulated partially by androgen and peaked in male mice aged from 3 weeks to adult. The imds-60 protein might play an important role in cell communication during sperm maturation.     

Characteristics of the bioreactor landfill system using an anaerobic-aerobic process for nitrogen removal

A sequential upflow anaerobic sludge blanket (UASB) and air-lift loop sludge blanket (ALSB) treatment was introduced into leachate recirculation to remove organic matter and ammonia from leachate in a lab-scale bioreactor landfill. The results showed that the sequential anaerobic-aerobic process might remove above 90% of COD and near to 100% of NH4+ -N from leachate under the optimum organic loading rate (OLR). The total COD removal efficiency was over 98% as the OLR increased to 6.8-7.7 g/l d, but the effluent COD concentration increased to 2.9-4.8 g/l in the UASB reactor, which inhibited the activity of nitrifying bacteria in the subsequent ALSB reactor. The NO3- -N concentration in recycled leachate reached 270 mg/l after treatment by the sequential anaerobic-aerobic process, but the landfill reactor could efficiently denitrify the nitrate. After 56 days operation, the leachate TN and NH4+ -N concentrations decreased to less than 200 mg/l in the bioreactor landfill system. The COD concentration was about 200 mg/l with less than 8 mg/l BOD in recycled leachate at the late stage. In addition, it was found that nitrate in recycled leachate had a negative effect on waste decomposition.