Functional expression of mouse insulin-like growth factor-I with food-grade vector in Lactococcus lactis NZ9000.

AIMS: To functionally express the recombinant mouse insulin-like growth factor-I (rtmIGF-I) in Lactococcus lactis NZ9000 with a food-grade vector. METHODS AND RESULTS: The rtmIGF-I encoding sequence was inserted into secreted food-grade vector pLEB688 and transformed into L. lactis NZ9000. The expression of the recombinant protein rtmIGF-I was confirmed by tricine-SDS-PAGE analysis and Western blot. The concentration of this recombinant protein was 3 mg l(-1) in the medium fraction. Further experiment demonstrated that the recombinant protein was biologically active and promoted NIH3T3 cell proliferation in a concentration-dependent manner. CONCLUSIONS: The rtmIGF-I was expressed in L. lactis and located into the medium fraction. The optimal final concentration which could promote NIH3T3 cell proliferation after incubation was 100 ng ml(-1) . SIGNIFICANCE AND IMPACT OF THE STUDY: The rtmIGF-I was functionally expressed in L. lactis NZ9000 with a food-grade vector. Thus, the recombinant L. lactis NZ9000 could act as a host for the production of rtmIGF-I for further study. The recombinant strain could serve as an IGF-I delivery system.

Homoharringtonine in combination with cytarabine and aclarubicin resulted in high complete remission rate after the first induction therapy in patients with de novo acute myeloid leukemia.

To assess the efficacy and toxicity of HAA regimen (homoharritonine 4 mg/m2/day, days 1-3; cytarabine 150 mg/m2/day, days 1-7; aclarubicin 12 mg/m2/day, days 1-7) as an induction therapy in the treatment of de novo acute myeloid leukemia (AML), 48 patients with newly diagnosed AML, aged 35 (14-57) years, were entered into this clinical study. The median follow-up was 26 months. Eighty-three percent of patients achieved complete remission (CR), and the first single course of induction HAA regimen resulted in CR rate of 79%. The CR rate of 100, 82 and 33% were achieved in patients with favorable, intermediate and unfavorable cytogenetics, respectively. For all patients who achieved CR, the median time from the initiation of the induction therapy to the evaluation of the remission status was 32 days. For all patients, the estimated 3 years overall survival (OS) rate was 53%, whereas for patients with M5, the estimated OS rate at 3 years was 75%. The toxicities associated with HAA regimen were acceptable, and the most common toxicity was infection. This study suggested that HAA regimen might be a well-tolerable, effective induction regimen in young adult patients with AML.

[Noninvasive measurement of gastric emptying rates and gastric motility].

A non-invasive measuring system is developed for the assessment of gastric evacuation and motility by means of epigastric impedance measurement. FIR digital filters realized on personal computer were used for signal filtering and cancellation of respiratory interference. Electrodes of concentric type placed anterior and posterior were proved to be more effective for epigastric impedance measurement.

[The amelioration of electrode techniques for testing cerebral potentials].

This article introduces the research work on the new amelioration of electrode techniques for testing cerebral potentials, and the using methods and their advantages & disadvantages in operating the new cerebral electrode.

Positive and negative hematopoietic cytokines produced by bone marrow endothelial cells.

Recently, cytokines and interleukins such as SCF, GM-CSF, G-CSF, TGF-beta, IL-6, IL-7, IL-8, IL-11 have been reported to be elaborated by endothelial cells. For further study, serum free bone marrow endothelial cell conditioned medium (BMEC-CM) was collected and ultrafiltrated by using a centriprep 10. The concentrated retentate (R-BMEC-CM) contained some substances whose molecular weight was more than 10 000 daltons. The filtrate (F-BMEC-CM) contained some substances whose molecular weight was less than 10 000 daltons. The effects of R-BMEC-CM and F-BMEC-CM on the growth of haematopoietic progenitors and the expression of cytokine and interleukin mRNAs of BMEC were investigated. The results showed that R-BMEC-CM stimulated the growth of CFU-GM, HPP-CFC, BFU-E, CFU-E, and CFU-Meg; while F-BMEC-CM inhibited the growth of these progenitors. Using the method of hybridizing to the Atlas cDNA Array, we were able to detect the presence of mRNAs of cytokines and interleukins in bone marrow endothelial cells. Our finding of the existence of mRNAs of SCF, GM-CSF, IL-6, TGF-beta, IL-1, and IL-11 in these cells was in agreement with the data reported previously. Furthermore, we detected mRNAs of MIP-2, Thymosion-beta4, PDGF, MSP-1, IFN-gamma, IL-13 and inhibin, which are related to haematopoiesis. Among these cytokines and interleukins, SCF, GM-CSF, IL-6, IL-1, and IL-11 are haematopoietic stimulators which may be responsible for the stimulative effects on the growth of haematopoietic progenitors. One of our new findings, the thymosin-beta4, is a small molecular haematopoietic inhibitor. It may be responsible for the inhibitory effect of F-BMEC-CM on haematopoietic progenitors. The presence of mRNAs of BMP, MSP-1, MIP-2, PDGF and IL-13 suggests that bone marrow endothelial cells might elaborate these substances. Their influence on haematopoietic progenitors needs further study.

[The effects of a novel aminosteroid on WEHI-3B leukemia cells].

OBJECTIVE: To evaluate the effect of a novel aminosteroid(KH) on WEHI-3B cells. METHODS: The effects and mechanism of KH on proliferation, differentiation and apoptosis of WEHI-3B cells in vitro were studied by semi-solid colony culture, MTT assay, morphologic examination, NBT reduction test, NSE assay, DNA fragmentation in gel electrophoresis and RT-PCR of c-myc oncogene. RESULTS: The growth of leukemic colony was inhibited by KH(10(-8)-10(-4) mol.L-1) after treatment of KH for 7 days. The percentages of NBT and NSE positive cells were increased from 40.38% to 71.17% and 48.25% to 79.25%, respectively as well as the typical ladders of DNA fragments in gel electrophoresis were also observed after WEHI-3B cells were treated with KH(10(-8)-10(-4) mol.L-1) for 5 days. The c-myc mRNA expression of WEHI-3B cells was decreased by 58.7% after WEHI-3B cells were treated with KH(10(-8) mol.L-1) for 5 days. CONCLUSION: KH might suppress the proliferation of WEHI-3B cells, induce the differentiation of WEHI-3B cells into macrophage-like cells and facilitate the cell apoptosis. There is relationship between the above effects and c-myc mRNA expression of WEHI-3B cells.

[The anti-leukemia effect of Sophora flavescens and its mechanism].

By using CFU-GM/CFU-L colony assay, NBT/MTT reductant test and DNA fragmentation analysis, we studied the effects of Sophora flavescens (SF) on CFU-GM proliferative ratio in human normal bone marrow/umbilical cord blood and on proliferation, differentiation and apoptosis in human acute myelogenous leukemia HL-60 cells. The results showed that 5, 10, 15, 20 micrograms.microliter-1 of SF significantly inhibited proliferation and induced apoptosis in the HL-60 cells in a dose-dependent fashion. Fifteen micrograms.microliter-1 of SF also induced differentiation in HL-60 cells. Furthermore, cytotoxic activity of SF(5-15 micrograms.microliter-1) was not apparent on human normal hematopoietic progenitors(CFU-GM). The results indicate that an appropriate concentration of SF has a selective antileukemic effect. Thus, these are important impetuses for further research of SF as an anticancer agent.

[Effect of curcumin on the proliferation of murine CFU-GM and WEHI-3B cells].

OBJECTIVE: To evaluate the effect of curcumin on the proliferation of hematopoietic progenitor cells or leukemic stem cells. METHODS: Mouse bone marrow cells (colony forming unit-granulocyte and macrophage, CFU-GM) and WEHI-3B cells were observed using the colony assays. RESULTS: The curcumin significantly inhibited the proliferation of both CFU-GM and WEHI-3B cells in a dose-dependent manner ranging from 10(-8) to 10(-4) mol.L-1; their IC50S were 1.036 x 10(-5) mol.L-1 and 1.220 x 10(-6) mol.L-1 of curcumin respectively. CONCLUSION: The proliferation of murine CFU-GM and WEHI-3B cells can be suppressed by curcumin. The inhibitory effect of curcumin on the proliferation of WEHI-3B cells is stronger than that of CFU-GM.

[Effects of serum-free murine bone marrow endothelial cell conditioned medium on the growth of CFU-E and BFU-E].

Serum-free murine bone marrow endothelial cell conditioned medium(mBMEC-cm) was collected. The mBMEC-cm was ultrafiltered in Centriprep. The concentrated retentive substance of mBMEC-cm (MW > 10,000) and filtrate of mBMEC-cm(MW < 10,000) were obtained. The effects of mBMEC-cm on the growth of CFU-E and BFU-E were investigated. The results showed: 1. The retentive substance of mBMEC-cm significantly enhanced the growth of CFU-E and BFU-E. The effects of mBMEC-cm(2%-6%, V/V) on the growth of CFU-E and BFU-E were dose-dependent. 2. The filtrate of mBMEC-cm markedly inhibited the growth of CFU-E and BFU-E. The effects of the filtrate of mBMEC-cm on the growth of CFU-E and BFU-E were also dose-dependent. The mechanism of regulation will be studied further.

[Effect of a novel aminosteroid on animal model of murine myelomonocytic leukemia].

To evaluate the effect of a novel aminosteroid on murine myelomonocytic leukemia, BALB/c mice were treated by the novel aminosteroid for 7 days after receiving 1 x 10(6) WEHI-3B(myelomonocytic leukemia) cells intraperitoneally. The counts and classification of white blood cells obtained from tail blood were determined on the day before transplanting WEHI-3B cells and 7, 14, 21, 28 days after transplantation. Undifferentiated cell percentage of bone marrow and spleen weight were checked 30 days after transplantation. The results demonstrated that the above indexes of mice treated with the novel aminosteroid were decreased compared with those treated with normal saline(NS). Decrease of the indexes showed a dose-dependent relationship ranging from 5-20 mg.kg-1.d-1. It is suggested that leukemia mice could be effectively treated by the novel aminosteroid and the efficacy maybe paralleled with the dosage of the novel aminosteroid.

[Study on the optimum experimental conditions for the steady growth of human K-562 cell line].

The results of the assays of growth state, semisolid colony culture, cytomorphological and chromosome analysis for K-562 cell line showed that the cells had the similar biological characteristics with the cells which were originally established. It suggests that the growth state of K-562 cells under the experimental conditions in our laboratory are steady.

[The effects of cytokines produced by bone marrow endothelial cells on the regulation of hematopoiesis].

In this study, the effects of bone marrow endothelial cell conditioned medium (BMEC-CM) on hematopoietic progenitors and the expression of cytokines mRNA of BMEC were investigated. BMEC-CM was ultrafiltrated in Centriprep-10, an ultrafiltering concentrator. The retentate (MW>10 kD) and filtrate (MW<10 kD) were obtained. The effects of different components of BMEC-CM on the murine hematopoietic progenitors were examined. The results showed that the retentate of BMEC-CM (MW>10 kD) significantly promoted the growth of CFU-GM, CFU-E, BFU-E, CFU-Meg and HPP-CFC, while the growth of CFU-GM, CFU-E, BFU-E, CFU-Meg and HPP-CFC were markedly inhibited by the filtrate of BMEC-CM (MW<10 kD). The effects of BMEC-CM were dose-dependent. The presence of mRNAs for GM-CSF, TGF-beta, bone morphogenetic protein 2A, basic fibroblast growth factor receptor, endothelin-2, macrophage-stimulating 1 (MSP-1), thymosin-beta10, connective tissue growth factor, macrophage inflammatory protein 2 alpha (MIP-2alpha), IL-6, IL-11, IL-13 and IFN-gamma mRNA were successfully detected by Atlas Arrays.

Effect of Cordyceps sinensis on erythropoiesis in mouse bone marrow.

The effect of Cordyceps sinensis crystal (CS-Cr) on stimulating proliferation of erythroid progenitor cells (CFU-E and BFU-E) in LACA mouse marrow in vivo and in vitro by methyl cellulose gel culture system is reported. The numbers of CFU-E and BFU-E were increased after 5 consecutive daily treatment with 100, 150 and 200 mg/kg of CS-Cr with a peak at 150 mg/kg. Higher doses (> 150 mg/kg) of CS-Cr resulted in a reduction of the peak of CFU-E and BFU-E and then, the numbers returned to the control level with increased doses. The cytosine arabinoside (Ara-C) suicide test showed significant increases in the percentage of CFU-E and BFU-E in S-phase after CS-Cr treatment. Pretreatment of mice with CS-Cr could protect CFU-E and BFU-E against the cytotoxic agent--harringtonine. Addition of CS-Cr to culture system also promoted the generation of CFU-E and BFU-E at concentrations of 150-200 micrograms/ml in vitro. With a liquid culture technique, a stimulatory action of CS-Cr on fibroblast colony-forming units (CFU-F) proliferation was seen in vivo and in vitro.

Dissecting the hematopoietic microenvironment. IX. Further characterization of murine bone marrow stromal cells.

We have previously shown the adherent nontransformed, nonimmortalized murine bone marrow stromal cell (BMSC) population to consist of phagocytic macrophage and endothelial-like cells and nonphagocytic fibroblasts. Both colonial and near confluent growth of each cell type was obtained following magnetic bead separation, subsequent passaging, and sustained culture with fetal bovine serum and cytokines. Monoclonal antibody staining of antigenic determinants was used to characterize the phenotype of the stromal cell population in primary platings of murine colony-forming unit fibroblast and long-term bone marrow cultures. The antibodies MECA-99, MECA-32, and MJ7-18, raised against murine vascular endothelial antigenic determinants, and von Willebrand's factor all stained selectively for the rounded endothelial-like cells. Endothelial-like cells as well as macrophages expressed the myeloid surface antigens F4/80, 7/4, and Mac-1 under our culture conditions. The cytoskeleton of the stromal fibroblasts in culture was shown to express smooth muscle-specific actin isoforms, as evidenced by positive staining of stress fibers for alpha smooth muscle-1, CGA-7 (alpha/gamma isoforms), and HHF-35 (recognizes all muscle-specific actins). Under culture conditions, stromal fibroblasts were also found to be positive for a polyclonal smooth muscle myosin. It was found that these fibroblasts stained for collagens type I, III, and IV in our cultures. Although collagen type IV is considered a by-product of endothelial cells, endothelial-like cells in our cultures did not stain for any of the collagen types. We propose a classification listing for murine BMSCs as macrophages, endothelial-like cells, and fibroblasts that display smooth muscle-like characteristics in culture.