OBJECTIVEFunctionally, erythropoietin (EPO) can promote the proliferation and growth of erythroid progenitor cells, and it is widely used in the treatment of anemia in chronic diseases caused by tumor and inflammation. However, it is unclear whether EPO has any effect on tumor cell iron metabolism and tumor cell proliferation. The purpose of this study was to explore the effects of recombinant human EPO (rhEPO) on the expression of transferrin receptor (TfR, CD(71) antigen) of leukemic cell K562 and its relation to cell cycle.

METHODSIn vitro culture of K562 cell was performed with additions of various concentrations of rhEPO and Fe. Treatments were terminated at 24 h and 72 h, respectively. Then each group of cells was incubated with FITC-IgG antibody to CD(71) or PI, a kind of DNA dye. And TfR expression and DNA synthesis status were analyzed by flow-cytometry.

RESULTS(1) The expression of TfR by K562 cells increased significantly when incubated for 72 h with different concentrations of rhEPO. The measurement values of 5 U/ml, 10 U/ml and 20 U/ml groups were 12.2 +/- 1.40, 10.7 +/- 0.99 and 11.1 +/- 0.90, respectively. They were markedly increased when compared with that of control group (6.27 +/- 0.11, P < 0.05). (2) When incubated with rhEPO (5 u/ml) alone or combined with FeCl(3) (100 micro mol/L), the percentages of cells in S phase were 51.1% and 59.6%, respectively. They significantly increased when compared with that of control group (42.9%, P < 0.05).

CONCLUSIONSIron is very important for the proliferation of both normal cells and leukemic cells. It is essential to the activity of ribonucleotide reductase (RR). The authors hypothesized that rhEPO would increase the expression of TfR and intracellular iron content of leukemic cells, which would enhance the DNA synthesis and cell proliferation. Therefore, the clinical application of rhEPO to promote erythropoiesis of cancer patients should be cautious.

Cell Cycle ; drug effects ; Erythropoietin ; pharmacology ; Flow Cytometry ; Humans ; K562 Cells ; Receptors, Transferrin ; metabolism ; Recombinant Proteins

OBJECTIVETo investigate the effects of recombinant human testis sperm binding protein (TSBP) on human sperm motility parameters in vitro.

METHODSSperm specimens obtained from 22 healthy fertile men were prepared by the Percoll gradient-centrifugation technique. The sperm suspension was incubated with recombinant His6-TSBP at the concentration of 0.01 mg/ml or 0.1 mg/ml at 37 degrees C for 1 or 3 hours in vitro. The combination of the recombinant protein and sperm membrane was determined by Western blot, and the sperm motility parameters were analyzed by computer-aided sperm analysis (CASA). The same procedure was performed for 12 asthenospermia patients.

RESULTSIn the 22 healthy volunteers, the percentage of forward motile sperm was increased after incubated with 0.1 mg/ml recombinant protein for 1 h (P < 0.05), both forward motile sperm percentage and motility were increased after incubated with recombinant protein at the same concentration for 3 h (P < 0.05), but no effect was observed after incubation with 0.01 mg/ml recombinant protein. In the 12 asthenospermia patients, the forward motile sperm percentage was increased after incubated with 0.1 mg/ml recombinant protein for 3 h (P < 0.05), but no statistically significant difference was observed in sperm motility.

CONCLUSIONRecombinant His6-TSBP at the concentration of 0.1 mg/ml can increase sperm motility in healthy fertile men and the forward motile sperm percentage in both healthy fertile men and asthenospermia patients in vitro.

Adult ; Humans ; Infertility, Male ; metabolism ; Male ; Recombinant Proteins ; pharmacology ; Seminal Plasma Proteins ; pharmacology ; Sperm Motility ; drug effects

Fungi immunoregulatory proteins family is effective in immunological regulation and anti-tumor. We used Pichia pastoris expression system for recombinant expression of Lz-8, an immunomodulatory protein isolated from fruiting body of Ganoderma lucidum. The Gs115 (mut+) strains of P. pastoris was used as host cells. PCR and sequencing of DNA showed that Lz-8 cDNA was successfully integrated into the P. pastoris genome. Electrophoresis (SDS-PAGE), matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and immunological techniques were used to identify recombinant Lz-8 (rLz-8). Lz-8 expressed in Escherichia coli, the Pichia system requires further optimization to obtain more active fungi immunomodulatory protein. Lz-8 was expressed in P. pastoris successfully, and polyacrylamide gel electrophoresis in the presence of SDS-PAGE gave a single band with an apparent Mr=14,000 D. MALDI-TOF-MS also showed that molecular weight of rLz-8 was 12,722 D. Aggregation was observed from sheep red blood cells in the presence of purified rLz-8 within the concentration range of 12.5-50 microg/mL. However, no aggregation was seen at concentration greater than 50 microg/mL for any type of human red blood cell. The dose at 0.5 mg/kg of rLz-8 induced macrophage cytophagocytesis, and set interferon as control at 0.5 mg/kg. These results suggested that active and stable rLz-8 was obtained in P. pastoris expression system.
Fungal Proteins ; biosynthesis ; genetics ; immunology ; Immunocompetence ; immunology ; Macrophages ; immunology ; Phagocytosis ; drug effects ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

In this study, peaT1 gene was subcloned into the Pichia pastoris expression vector pPIC9K, which contained both the methanol-inducible promoter and the transcription terminator of the AOX1 gene, resulting the plasmid pPIC9K-peaT1. The recombinant plasmid was linearized by Sal I or Bgl II and transformed into P. pastoris GS115 by electroporation method. Recombinant strain was screened by Minimal Dextrose Medium and further confirmed by PCR. The gene was in frame integrated into the Pichia genome through homologous recombination, resulting the recombinant strain. Regulated by the alpha-Factor, promoter of AOX1 gene and termination signal of yeast genomic, the recombinant protein was expressed and secreted into the supernatant. The SDS-PAGE analysis indicated that the apparent molecular weight of target protein was about 35 kD. Bioassay results showed that the inhibition rate of the expressed protein against TMV was 30.37%. The supernatant was collected and then purified by anion exchange chromatography. This protein can promote seedling growth of wheat obviously.
Alternaria ; genetics ; Fungal Proteins ; biosynthesis ; genetics ; pharmacology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Triticum ; drug effects ; growth & development

OBJECTIVETo investigate the penetrating ability of fusion protein PEP-1-EGFP with human umbilical vein endothelial cells.

METHODSTwo prokaryotic expression plasmids pET15b-EGFP and pET15b-PEP-1-EGFP were constructed and transformed into E. coli BL21 (DE3) to express EGFP and fusion protein PEP-1-EGFP, respectively. The expressed EGFP and PEP-1-EGFP were purified with Ni(2+) -resin affinity chromatography, and their capabilities of transduction into human umbilical vein endothelial cells were evaluated. The time- and dose-dependent transduction of the fusion protein PEP-1-EGFP and its stability in the human umbilical vein endothelial cells were observed. The toxicity of the fusion protein PEP-1-EGFP was detected by MTT method.

RESULTSEGFP failed to be transduced into human umbilical vein endothelial cells, whereas PEP-1-EGFP fusion protein was transduced into cells shortly in 5 minutes. Its transduction was time- and dose-dependent and the fluorescence in the cells were detected even 27 hours later. No cytotoxicity of the fusion protein PEP-1-EGFP to human umbilical vein endothelial cells was detected even when the dose reached up to 200 micromol/L.

CONCLUSIONPEP-1-EGFP fusion protein can efficiently transduce the target protein into human umbilical vein endothelial cells, which provides a basis for future researches on the transduction of antioxidant enzymes mediated by the cell-penetrating peptide, PEP-1, in ischemia-reperfusion injury therapy.

Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Green Fluorescent Proteins ; metabolism ; Humans ; Peptides ; metabolism ; Protein Transport ; Recombinant Fusion Proteins ; metabolism ; toxicity ; Umbilical Veins ; cytology

We used ribosome engineering technology, with which antibiotic-resistant strains are resulted from mutations on microbial ribosome, to screen a high butanol-producing Clostridium strain. A novel mutant strain S3 with high butanol production and tolerance was obtained from the original Clostridium acetobutylicum L7 with the presence of mutagen of streptomycin. Butanol of 12.48 g/L and ethanol of 1.70 g/L were achieved in S3, 11.2% and 50%, respectively higher than the parent strain. The conversion rate of glucose to butanol increased from 0.19 to 0.22, and fermentation time was 9 h shorter. This caused an increase in butanol productivity by 30.5%, reaching 0.24 g/(Lh). The mutant butanol tolerance was increased from 12 g/L to 14 g/L, the viscosity of fermentation broth was dramatically decreased to 4 mPa/s, 60% lower than the parent strain. In addition, the genetic stability of mutant strain S3 was also favorable. These results demonstrate that ribosome engineering technology may be a promising process for developing high butanol-producing strains.
Butanols ; metabolism ; Clostridium acetobutylicum ; drug effects ; genetics ; metabolism ; Fermentation ; Genetic Engineering ; Mutation ; Recombinant Proteins ; biosynthesis ; genetics ; Ribosomes ; genetics ; Streptomycin ; pharmacology

A visible cutaneous scar develops from the excess formation of immature collagen in response to an inflammatory reaction. This study examined the role of epidermal growth factor (EGF) in the formation of cutaneous scars. Twenty Crl:CD-1 (ICR) mice were used and 2 full-thickness skin wounds were made on the dorsum of each mouse. One of the wounds was treated with recombinant human EGF by local application and the other was treated with saline for control until complete healing was achieved. The EGF-treated group's wounds healed faster than the control group's. The width of the scar was smaller by 30% and the area was smaller by 26% in the EGF-treated group. Inflammatory cell numbers were significantly lower in the EGF-treated group. The expression of transforming growth factor (TGF)-beta1 in the EGF-treated group was increased. It was observed that the amount of collagen in the EGF-treated group was larger than the control group. In the EGF-treated group, the visible external scars were less noticeable than that in the control group. These results suggest that EGF can reduce cutaneous scars by suppressing inflammatory reactions, decreasing expression of TGF-beta1, and mediating the formation of collagen.
Animals ; Cicatrix/pathology/*prevention & control ; Collagen/metabolism ; Epidermal Growth Factor/*pharmacology ; Humans ; Inflammation/metabolism ; Mice ; Recombinant Proteins/*pharmacology ; Skin/drug effects/metabolism/pathology ; Wound Healing/*drug effects

We developed the recombinant green fluorescent protein gene yeast cell to screen estrogenic compounds based on two episomal vectors. In the expression vector the expression of human estrogen receptor alpha(hERalpha) was driven by 3-glyceraldehydephosphate dehydrogenase (GPD) promoter; in the reporter vector the expression of the yeast enhanced green fluorescent protein (yEGFP) gene was under the control of the estrogen response element (ERE). The vectors were transformed into yeast cell (W303-1A) to construct GFP recombinant yeast cell. Incubation of the yeast cell with various concentrations of the estrogenic compounds led to expression of the reporter gene product GFP in a dose dependent manner. Compared to other yeast bioassays, the yeast cell for environmental estrogen bioassay based on yEGFP reporter gene did not need cell wall disruption or the addition of a substrate or reagent. This yEGFP assay was performed completely in 96 well plates. So this test system can be used as a rapid and high throughput system for screening estrogenic chemical products, which has the characteristics of the sensitivity, reproducibility and cheapness.
Biological Assay ; methods ; Estrogen Receptor alpha ; genetics ; metabolism ; Estrogens ; analysis ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Proteins ; genetics ; metabolism ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism

Rat's osteoblasts cultured in vitro were stimulated by recombinant bovine basic fibroblast growth factor(rb-bFGF). After 1-2 days, the osteoblast grew long protuberance, the numbers of osteoblasts were greater than the control groups', the vitality of osteoblasts was better than that of control groups. After 1 hour, the expression of c-fos in osteoblast increased when compared with that in the control group. After the osteoblasts having been stimulated for 1-2 days, the expression of c-fos increased more conspicuously. These results show that bFGF can boost the osteoblast to grow and proliferate and can enhance the expression of c-fos gene. The increase of c-fos gene's expression may be an important step in the signal transformation process of all kinds of stimulation boosting osteoblast to proliferate.
Animals ; Cattle ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fibroblast Growth Factor 2 ; pharmacology ; Gene Expression Regulation ; drug effects ; Genes, fos ; drug effects ; Osteoblasts ; cytology ; drug effects ; metabolism ; Proto-Oncogene Proteins c-fos ; metabolism ; Rats ; Rats, Wistar ; Recombinant Proteins ; pharmacology

In order to produce relatively large amounts of recombinant human intestinal trefoil factor and assess its biological activity. The expression plasmid pPIC9-hITF containing AOX1 promotor and the sequences of secreting signal peptides was transformed into the yeast cells. Then through selection, positive transformants were cultivated in fermentation basal salts medium in a 5L fermenter to obtain large amount product with low cost. The secreted peptides were then purified by a combination of ionic exchange chromatography and molecular sieve. To verify the product, electrospray mass spectrometry analyses was used to determine the structure of rhITF and Western Blotting was performed to test the immunological activity. Furthermore, the biological activity of the peptide was examined by experiments from cell to tissue. The nucleotide sequence of rhITF was the same as expected. With a 5-L fermenter, 253mg of hITF was isolated at the purity of 96% from 3.5 L of yeast fermentation broth. The expression level for recombinant human ITF in this yeast system was 73.33mg/L. In our study, we provided a way to gain a production among milligram to gram of recombinant human ITF by the use of a yeast expression system. As human ITF are difficult to purify in any significant amount from tissue extraction, the way described may become a valuable tool in obtaining pure peptide for further studies of trefoil peptide function.
Cell Movement ; drug effects ; Epithelial Cells ; cytology ; drug effects ; Fermentation ; Humans ; Intestine, Small ; cytology ; Peptides ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Trefoil Factor-2