Expression and purification of an FGF9 fusion protein in E. coli,and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration

Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity. Both purified forms of rhFGF9, with and without fusion tags, significantly stimulated the proliferation of NIH3T3 cells. The FGF9 subfamily, including FGF9, FGF16, and FGF20, in addition to rhFGF16, rhFGF9, and rhFGF20, were shown to stimulate the proliferation and migration of HuH7 human hepatocellular carcinoma (HCC) cells. Mechanistic studies revealed that the stimulation of HuH7 cell proliferation and migration with rhFGF9 and rhFGF20 were associated with the activation of the extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) pathways and matrix metalloproteinase-26 (MMP26). Inhibition of the ERK and NF-κB pathways blocked cell migration, and NF-κB was demonstrated to be regulated by ERK. Therefore, the present study demonstrates a simple method for the preparation of biologically active rhFGF9 protein. Furthermore, the results indicate that exogenous rhFGF9- and rhFGF20-activated ERK/NF-κB signal transduction pathways play important roles in the regulation of HCC cell proliferation and migration, and this discovery helps to find the potential for new solutions of the treatment of liver cancer.

     

Quantitative detection of BCR–ABL fusion gene and its application in monitoring chronic myeloid leukemia treatment

The BCR–ABL fusion gene in chromosome translocation, t (9; 22), and its product, p210BCR/ABL oncogenic tyrosine kinase, is the underlying molecular mechanism that leads to the development of CML. Quantitative detection of BCR–ABL fusion gene has become a reliable approach to diagnose and monitor CML. The aim of this study was to evaluate a Roche t (9; 22) kit in CML diagnosis, monitoring treatment responses, and identification of relapse. Using BCR–ABL fusion gene-expressing K562 cells, a series of standard samples were prepared and used to establish a curve for the calculation of BCR–ABL fusion gene expression in patient samples. Our results indicate that PCR detection system with aforementioned kit has good reproducibility. In addition, the relative concentration of BCR–ABL measured by PCR was in agreement with the patient’s response to the Imatinib treatment and bone marrow morphology remission. Furthermore, we found that the relative concentration of BCR–ABL fusion gene increased 1–3 months before CML relapse was clinically and cytogenetically diagnosed, suggesting that the PCR-based BCR–ABL fusion gene detection with t (9; 22) kit is able to diagnose the recurrence of CML at least 1 month earlier than the classic cytogenetic analysis. In conclusion, detection of BCR–ABL fusion gene expression in CML using Roche t (9; 22) kit has great clinical value in the primary diagnosis, monitoring treatment responses, and identification of relapse in CML patients.     

Enhanced chilling tolerance in Zoysia matrella by pre-treatment with salicylic acid, calcium chloride, hydrogen peroxide or 6-benzylaminopurine

Following leaf application of salicylic acid (SA), calcium chloride, hydrogen peroxide and 6-benzylaminopurine (BA), Manila grass (Zoysia matrella) plants were exposed to day/night temperature of 7/2 °C for 120 h in a growth chamber. The lower content of malondialdehyde (MDA) and H₂O₂ and higher activities of ascorbate peroxidase (APX), guaiacol peroxidase (POD) and catalase (CAT) during exposure to low temperature in pre-treated plants in comparison with control plants demonstrated that these compounds improved the chilling tolerance of Manila grass.     

冀东地区西伯利亚白刺开发利用价值研究

西伯利亚白刺是华北沿海地区仅有的一个白剌种,在冀东滨海地区有一定的野生群落分布。研究结果表明,西伯利亚白刺具有极强的耐盐能力,0—50cm土体平均全盐含量最高达到1．97％,极端最高值达到3．99％。其果汁含有非常丰富的维生素C,高达444．07mg／100g,仅次于酸枣,排在各类蔬菜、水果的第二位,是开发功能饮料的上好原料。果实成熟后的嫩茎叶含粗蛋白28．80％,粗纤维8．86％,粗脂肪2．24％,是优质的饲料。种子脂肪含量17．2％,其中油酸含量25．02％,亚油酸含量69．02％,是生产优质保健食用油的好原料。所以,西伯利亚白刺是开发利用重盐碱地和沿海滩涂的优良植物,极具开发价值。     

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.     

Hydrogen Sulfide Alleviates Aluminum Toxicity in Germinating Wheat Seedlings

Protective role of hydrogen sulfide (H2S) on seed germination and seedling growth was studied in wheat (Triticum) seeds subjected to aluminum (Al3+) stress. We show that germination and seedling growth of wheat is inhibited by high concentrations of AICI3. At 30 mmol/L AICI3 germination is reduced by about 50% and seedling growth is more dramatically inhibited by this treatment. Pre-incubation of wheat seeds in the H2S donor NaHS alleviates AICI3-induced stress in a dose-dependant manner at an optimal concentration of 0.3 mmol/L. We verified that the role of NaHS in alleviating Al3+ stress could be attributed to H2S/HS- by showing that the level of endogenous H2S increased following NaHS treatment. Furthermore, other sodium salts containing sulfur were ineffective in alleviating Al3+ stress. NaHS pretreatment significantly increased the activities of amylases and esterases and sustained much lower levels of MDA and H2O2 in germinating seeds under Al3+ stress. Moreover, NaHS pretreatment increased the activities of guaiacol peroxidase, ascorbate peroxidase, superoxide dismutase and catalase and decreased that of lipoxygenase. NaHS pretreatment also decreased the uptake of Al3+ in AICI3-treated seed. Taken together these results suggest that H2S could increase antioxidant capability in wheat seeds leading to the alleviation of Al3+ stress.     

Synthetic bivalent CD4-mimetic miniproteins show enhanced anti-HIV activity over the monovalent miniprotein

HIV-1 envelope glycoprotein gp120 is displayed as a trimeric complex on the surface of virion and infected T-cells, making it a typical multivalent target. This paper describes the design and synthesis of bivalent CD4-mimetic miniproteins to target the conserved CD4-binding pockets in the trimeric gp120. Using miniprotein CD4M9 as the model inhibitor, we created bivalent inhibitors in which two CD4M9 moieties were tethered by a spacer of varied length and evaluated their anti-HIV activity using a cell culture assay. The synthetic bivalent miniproteins showed 5-21-fold enhancement in anti-HIV activity over the monovalent miniprotein. The activity enhancement is dependent on the length of the spacer. The study suggests that targeting the oligomeric gp120 complex by novel multivalent ligands offers a valuable strategy for developing highly specific and effective HIV entry inhibitors.     

Functional interaction of monoubiquitinated FANCD2 and BRCA2/FANCD1 in chromatin

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least 11 complementation groups (A, B, C, D1, D2, E, F, G, I, J, and L), and eight FA genes have been cloned. The FANCD1 gene is identical to the breast cancer susceptibility gene, BRCA2. The FA proteins cooperate in a common pathway, but the function of BRCA2/FANCD1 in this pathway remains unknown. Here we show that monoubiquitination of FANCD2, which is activated by DNA damage, is required for targeting of FANCD2 to chromatin, where it interacts with BRCA2. FANCD2-Ub then promotes BRCA2 loading into a chromatin complex. FANCD2(-/-) cells are deficient in the assembly of DNA damage-inducible BRCA2 foci and in chromatin loading of BRCA2. Functional complementation with the FANCD2 cDNA restores BRCA2 foci and its chromatin loading following DNA damage. BRCA2(-/-) cells expressing a carboxy-terminal truncated BRCA2 protein form IR-inducible BRCA2 and FANCD2 foci, but these foci fail to colocalize. Functional complementation of these cells with wild-type BRCA2 restores the interaction of BRCA2 and FANCD2. The C terminus of BRCA2 is therefore required for the functional interaction of BRCA2 and FANCD2 in chromatin. Taken together, our results demonstrate that monoubiquitination of FANCD2, which is regulated by the FA pathway, promotes BRCA2 loading into chromatin complexes. These complexes appear to be required for normal homology-directed DNA repair.     

Inter-rater agreement on chromosome 5 breakage in FISH-based mutagen sensitivity assays (MSAs)

In chromosome breakage assays, validated, universal criteria for selection of cells and classification of chromosome aberrations may enhance their utility for cancer susceptibility screening. To standardize a fluorescence in situ hybridization (FISH) modification of the mutagen sensitivity assay (MSA), scoring criteria were evaluated by web-based validation. Two hundred digital FISH images were assigned random identification numbers. With this set of images, criteria for inclusion of cells and measurement of the frequency of abnormal cells were evaluated by eight observers, all of whom had five or more years of experience. Observers included doctoral and MS/BS level cytogeneticists, and were drawn from a randomized pool of 54 volunteers. Questions addressed were: (1) how uniformly were criteria applied to analysis of a standard digital FISH image set and (2) did concordance vary with educational level? These data suggest inter-rater agreement within a factor of 2 for average breakage frequency, but revealed greater variability in cell selection. These results aid in estimating the components of assay variance due to definitions, technical parameters and biological variables.