重组人VEGF165的表达纯化及单抗的初步筛选

血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)是抑制肿瘤生长和转移的重要靶点。为获得抗VEGF单抗细胞株,构建了rhVEGF₁₆₅工程菌,并利用复合自动诱导获得高效表达。经纯化获得高纯度rhVEGF₁₆₅蛋白,经检测具有促人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)增殖活性,其EC₅₀为2.4ng/ml。免疫小鼠,获得了3株能稳定分泌抗VEGF单抗的杂交瘤细胞株,为开发VEGF治疗性单抗提供了重要基础。     

CD8+ T cells and IFN-γ mediate the time-dependent accumulation of infected red blood cells in deep organs during experimental cerebral malaria

Background

Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood.

Methods and Findings

Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8⁺ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4⁺ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs.

Conclusions

CD8⁺ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues.     

OAS1蛋白单克隆抗体的制备及其表征

目的:用OAS1蛋白免疫小鼠,获得OAS1特异性单克隆抗体,为OAS1的含量测定提供基础。方法:通过全基因合成的方法获得目的基因序列,转化大肠杆菌BL21细胞诱导His-OAS1及OAS1蛋白表达,纯化后用作抗原免疫小鼠,取脾融合,筛选稳定分泌抗体的阳性细胞株,制备并纯化单抗,通过SDS-PAGE,ELISA,Western blot等方法进行检测。结果:体外高效表达OAS1蛋白,并成功制备特异性单克隆抗体,效价在5×10^-11mol/L以上,亲和常数为3.37×10⁸ L·mol^-1。结论:获得高亲和力OAS1单克隆抗体,为其含量的检测奠定了基础。     

csd alleles in the red dwarf honey bee (Apis florea,Hymenoptera: Apidae) show exceptionally high nucleotide diversity

Abstract The single locus complementary sex determination (sl‐csd) gene is the primary gene determining the gender of honey bees (Apis spp.). While the csd gene has been well studied in the Western honey bee (Apis mellifera), and comparable data exist in both the Eastern honey bee (Apis cerana) and the giant honey bee (Apis dorsata), no studies have been conducted in the red dwarf honey bee, Apis florea. In this study we cloned the genomic region 3 of the A. florea csd gene from 60 workers, and identified 12 csd alleles. Analysis showed that similar to A. mellifera, region 3 of the csd gene contains a RS domain at the N terminal, a proline‐rich domain at the C terminal, and a hypervariable region in the middle. However, the A. florea csd gene possessed a much higher level of nucleotide diversity, compared to A. mellifera, A. cerana and Apis dorsata. We also show that similar to the other three Apis species, in A. florea, nonsynonymous mutations in the csd gene are selectively favored in young alleles.     

Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT signalings

Purpose

To explore the effects of Icaritin on chronic myeloid leukemia (CML) cells and underlying mechanisms.

Method

CML cells were incubated with various concentration of Icaritin for 48 hours, the cell proliferation was analyzed by MTT and the apoptosis was assessed with Annexin V and Hoechst 33258 staining. Cell hemoglobinization was determined. Western blotting was used to evaluate the expressions of MAPK/ERK/JNK signal pathway and Jak-2/Phorpho-Stat3/Phorsph-Akt network-related protein. NOD-SCID nude mice were applied to demonstrate the anti-leukemia effect of Icaritin in vivo.

Results

Icaritin potently inhibited proliferation of K562 cells (IC50 was 8 µM) and primary CML cells (IC50 was 13.4 µM for CML-CP and 18 µM for CML-BC), induced CML cells apoptosis and promoted the erythroid differentiation of K562 cells with time-dependent manner. Furthermore, Icaritin was able to suppress the growth of primary CD34+ leukemia cells (CML) and Imatinib-resistant cells, and to induce apoptosis. In mouse leukemia model, Icaritin could prolong lifespan of NOD-SCID nude mice inoculated with K562 cells as effective as Imatinib without suppression of bone marrow. Icaritin could up-regulate phospho-JNK or phospho-C-Jun and down-regulate phospho-ERK, phospho-P-38, Jak-2, phospho-Stat3 and phospho-Akt expression with dose- or time-dependent manner. Icaritin had no influence both on c-Abl and phospho-c-Abl protein expression and mRNA levels of Bcr/Abl.

Conclusion

Icaritin from Chinese herb medicine may be a potential anti-CML agent with low adverse effect. The mechanism of anti-leukemia for Icaritin is involved in the regulation of Bcr/Abl downstream signaling. Icaritin may be useful for an alternative therapeutic choice of Imatinib-resistant forms of CML.     

Effects of dietary zinc levels on the activities of enzymes, weights of organs, and the concentrations of zinc and copper in growing rats

Zinc (Zn) is an essential nutrient that is required in humans and animals for many physiological functions, including immune and antioxidant function, growth, and reproduction. The present study was performed to investigate the effects of three Zn levels, including Zn adequate (35.94 mg/kg, as a control), Zn deficiency (3.15 mg/kg), and Zn overload (347.50 mg/kg) in growing male rats for 6 wk. This allowed for evaluation of the effects that these Zn levels might have on body weight, organ weight, enzymes activities, and tissues concentrations of Zn and Cu. The results showed that Zn deficiency has negative effects on growth, organ weight, and biological parameters such as alkaline phosphatase (ALP) and Cu−Zn superoxide dismutase (Cu−Zn SOD) activities, whereas Zn overload played an effective role in promoting growth, improving the developments of organs and enhancing immune system. Hepatic metallothionein (MT) concentration showed an identical increase tendency in rats fed both Zn-deficient and Zn-overload diets. The actual mechanism of reduction of Cu concentration of jejunum in rats fed a Zn-overload diet might involve the modulation or inhibition of a Cu transporter protein by Zn and not by the induction of MT.     

Protein flexibility prediction by an all-atom mean-field statistical theory

We extended a mean-field model to proteins with all atomic detail. The all-atom mean-field model was used to calculate the dynamic and thermodynamic properties of a three-helix bundle fragment of Staphylococcal protein A (Protein Data Bank [PDB] ID 1BDD) and alpha-spectrin SH3 domain protein (PDB ID 1SHG). We show that a model with all-atomic detail provides a significantly more accurate prediction of flexibility of residues in proteins than does a coarse-grained residue-level model. The accuracy of flexibility prediction is further confirmed by application of the method to 18 additional proteins with the largest size of 224 residues.     

Proteomic analysis of early‐response to mechanical stress in neonatal rat mandibular condylar chondrocytes

The objectives of this study were to investigate the early response to mechanical stress in neonatal rat mandibular chondrocytes by proteomic analysis. To evaluate its molecular mechanism, chondrocytes were isolated and cultured in vitro, then loaded mechanical stress by four‐point bending system on different patterns. Morphological observation, flow cytometric analysis, and MTT assays indicated that 4,000 µstrain loading for 60 min was an appropriate mechanical stimulus for the following proteome analysis, which produced a transient but obvious inhibitory effect on the cell cycle. Therefore, we took a proteomic approach to identify significantly differential expression proteins in chondrocytes under this mechanical stress. Using 2‐DE and MALDI‐TOF, we identified seven differentially expressed proteins including the MAPK pathway inhibitor RKIP, cytoskeleton proteins, actin and vimentin, and other selected proteins. Some differentially expressed proteins were validated by both Western blot analysis and fluorescent staining of cytoskeleton at different loading times. The vimentin and RKIP responsive expression were also proven in vivo in oral orthopedic treatment rats, which was in line with the result in vitro. The histological changes in cartilage also showed the inhibition effect. Furthermore, the expressional level of phosphorylated ERK was increased, which demonstrates the changes in MAPK activity. Taken together, these data indicate that mechanical stress resulted in vimentin expression changes first and then led to the subsequent changes in actin expression, MAPK pathway regulated by RKIP and heat shock protein GRP75. All those changes contributed to the cytoskeleton remolding and cell cycle inhibition, finally led to condylar remodeling. J. Cell. Physiol. 223:610–622, 2010. © 2010 Wiley‐Liss, Inc.     

The effect of elevated temperature on spawning of introduced pumpkinseed Lepomis gibbosus in Europe

In an experimental comparison of pumpkinseed Lepomis gibbosus reproduction under ambient and climate change water temperature regimes, spawning occurred earlier in the season, which is likely to lead to greater young-of-the-year survival with concomitant implications in the U.K. under warmer climatic conditions.