To study the activities of interleukin(IL)-2 and granulocyte-macrophage colony-stimulating factor(GM-CSF)(hlL-2linGM-CSF). Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia colf (Ecoli) BL21 (DE3) in inclusion body (IB) form. Afler IB was extracted and clarified. it was denatured and purified by afinity chromato graphy. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hlL-2/mGM-CSF in E coli yielded approximately 20 mg protein/L culture and the purity was about 90%．The specific activities of 1L-2 and GM-CSF were 54xl0. IU/mg and 7.1xl0. IU/mg. respectively. Conclusion This research provides important information about the anti. tumor activity of hiL-2/mGM-CSF r/vivo. thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.

0biective To establish an experimental model of early stage avascular necrosis of the femoral head(ANFH)caused by corticosteroid in adult rabbits and to observe the pathological changes with various imaging techniques． Methotis ANFH was induced by fl combination of hypersensifivity vasculitis caused by injection of horse serum and subsequent administration of a high dose of corticosteroid．The pathological changes were detected with digital radiography(DR), computed tomography(CT), magnetic resonance imaging(MRI), ink artery infusion angiography, hematoxylin-eosin staining, and immunohistochemistry．Results The imageological and pathological changes corresponded to the clinical characteristics of early stage ANFH．DR showed bilaterally increased bone density．an unclear epiphyseai line, and blurred texture of cancellous bone．CT showed spot-like low-density imaging of cancellous bone thinner cortical bone, osteoporosis, and an unclear epiphyseal Iine．MRI showed bone marrow edem a and spot-like high signals in T2-weighted imaging in cancellous bone．Ink artery infusion angiography showed fewer obstructed blood vessels in the femoral head．I1E staining of pathological sections showed fewer trabeculae and thin bone, an increased proportion of empty osteocyte lacunae, decreased hematopoiesis, thrombosis, and fat eell hypertrophy． tmmunohistochem istry showed attenuated expression of vascular endothelial growth factor in osteoblasts and chondrocytes, and on the inner membrane of blood vessels． Conclusion Experimental rabbit mode1 of early stage ANFH caused by corticosteroid can be successfully established and provide the foundation for developing effective me山ods to treat early stage ANFH

We have developed a cDNA microarray containing 10250 ESTs to monitor the transcriptional strategy of conidial germination. A total of 1561 genes that had their expression levels specially altered in the process were obtained and hierarchically clustered with respect to their expression profiles. By functional analysis, we provided a global view of an important biological system related to conidial germination, including characterization of the pattern of gene expression at sequential developmental phases, and changes of gene expression profiles corresponding to morphological transitions. We matched the EST sequences to GO terms in the Saccharomyces Genome Database (SGD). A number of homologues of Saccharomyces cerevisiae genes related to signalling pathways and some important cellular processes were found to be involved in T. rubrum germination. These genes and signalling pathways may play roles in distinct steps, such as activating conidial germination, maintenance of isotropic growth, establishment of cell polarity and morphological transitions.

Dermatophytes are the primary causative agent of dermatophytoses, a disease that affects billions of individuals worldwide. Trichophyton rubrum is the most common of the superficial fungi. Although T. rubrum is a recognized pathogen for humans, little is known about how its transcriptional pattern is related to development of the fungus and establishment of disease. It is therefore necessary to identify genes whose expression is relevant to growth, metabolism and virulence of T. rubrum.

Different methods were used to prepare HLA tetramers and the yields of each method were compared. Our results indicate that preliminary refolding of the heavy chain (Hc) and light chain (β2m) yields more monomer than the typical conventional method with urea-solubilized Hc and β2m. We then used the corresponding tetramers to detect cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL). Increasing data suggest that the adoptive transfer of CMV-specific CTL constitutes an effective strategy against CMV infections. We designed a method that efficiently induces CMV-specific CTL to a higher frequency in vitro than is currently achieved. This method increased the percentage of CMV-specific CTL from below 1% to 20% of PBL, accounting for more than 40% of CD8+ T cells. Successful HLA tetramer preparation provides the basis for the subsequent detection of CMVspecific CTL in clinical applications.

The importance of eggs as a source of speciWc antibodies is well recognized. Egg yolk contains 8-20 mg immunoglobulins (IgY) per milliliter. However, the major problem in separating IgY is to remove the high concentrations of lipids in egg yolk. We Wrst used water dilution method to get the supernatant containing IgY, then puriWed the antibody by caprylic acid–ammonium sulfate method, and obtained speciWc antibody with satisfactory purity and activity. By comparison of these several methods, each has its advantages, one can be chosen to purify IgY according to practical need. The puriWed IgY produced by the immunized chickens can stain the human peripheral blood mononuclear cell eVectively when labeled with Xuorescent FITC.

Interleukin-2 (IL-2) can stimulate T cell proliferation and diVerentiation when binding to its receptor on T cells. It produces a marked eVect by enhancing the cytotoxicity of CD8+ T cells and natural killer cells. Granulocyte-macrophage colony stimulating factor (GM-CSF) is associated with many cells proliferation, such as dendritic cells, macrophages. Here, we report the construction, expression and puriWcation of a bifunctional protein, hIL-2/GM-CSF, which may facilitate interaction between T cells and the antigen presentation cells and improve the eYciency of antigen presentation. We found that the use of chemicals and temperature shift is a peculiar system for induction of the Escherichia coli transformed with an IPTG-regulated hIL-2/GM-CSF expression vector in this research. After renaturation, anion exchange chromatography, metal aYnity chromatography, and strict endotoxin-free cation exchange chromatography, the fusion protein devoid of endotoxin showed high purity. Cell proliferation experiments proved that this bifunctional protein retains both hIL-2 and GM-CSF biological activities. These results will facilitate the numerous subsequent studies on this bifunctional molecule.

Trichophyton rubrum (T. rubrum) is the most common of the superficial fungi. In an effort to better understand the genetic and biochemical makeup of T. rubrum, we generated cDNA libraries from 3 growth stages and used these to isolate 4002 unique expressed sequence tags (ESTs). Sequence comparisons with the Genbank database allowed 1226 of the ESTs to be assigned putative functions or matched with homologs from other organisms. Of the remaining ESTs, 989 were only weakly similar to known sequences and 1787 had no identifiable functions, suggesting that they represent novel genes. We further analyzed the presence of several important genes involved in the growth, metabolism, signal transduction, pathogenesis and drug resistance in T. rubrum. This information was used to newly elucidate important metabolic pathways in T. rubrum. Taken together, our results should form the molecular basis for continued research on the physiological processes and pathogenic mechanisms of T. rubrum, and may lead to a better understanding of fungal drug resistance and identification of new drug targets.

Four human vascular endothelial growth factor receptor Flt-1 cDNA fragments containing extracellular domain loops 2, 1-2, 2-3 and 1-3 respectively were amplified from human placental cDNA library by PCR and used for screening ligand binding domains by yeast two-hybrid system. The result showed that, not only loop 1-3, but also the smaller fragment loop 2-3 could bind to hVEGF165. Recombinant expression plasmids pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1(2-3) were constructed and transformed to Pichia. pastoris host strain GS115, cultured in flasks, and expressed under the induction of 1% methanol. The expressed product existed in supernatant in the form of soluble molecules and contained more than 60% of total protein after being induced for 4d. After being purified by CM-Sepharose FF and Sephacryl S-100 chromatography, its purity reached above 90%. Biological assay in vitro showed that the binding capacity of expressed soluble Flt-1(2-3) to hVEGF165 and its inhibiting effect on the proliferation of human umbilical veins endothelial cells (HUVEC) stimulated with hVEGF165 were close to those of sFlt-1(1-3). Animal test showed that sFlt-1(2-3) could inhibit the formation of regenerate blood vessels stimulated with hVEGF165 significantly.