Purification and characterization of two extracellular polyhydroxyalkanoate depolymerases from Pseudomonas mendocina

Two polyhydroxyalkanoate depolymerases, PHAase I and PHAase II, were purified to homogeneity from the culture supernatant of an effective PHA-degrading bacterium, Pseudomonas mendocina DS04-T. The molecular masses of PHAase I and PHAase II were determined by SDS-PAGE as 59.4 and 33.8 kDa, respectively. Their optimum pH values were 8.5 and 8, respectively. Enzymatic activity was optimal at 50 °C. Both purified enzymes could degrade PHB, PHBV, and P(3HB-co-4HB). Addition of Na⁺ and K⁺ slightly increased the rate of PHAase II. EDTA significantly inhibited PHAase II but not PHAase I. Mercaptoethanol and H₂O₂ also inhibited the activities of both enzymes.     

中国家蚕抗菌肽人工合成类CMⅣ基因在甜菜夜蛾虫体中的表达

将人工合成的中国家蚕抗菌肽类CMⅣ基因与抗菌肽信号肽基因连接 ,经EcoRⅠ、HindⅢ双酶切后 ,克隆于pFASTBacⅠ的EcoRⅠ、HindⅢ酶切位点之间 ,得到重组转座载体pFASTBac ABP ,经测序证明阳性克隆正确。将重组转座载体转化HD1 0Bac大肠杆菌 ,得到重组Bacmid ABP。将重组Bacmid转染sf2 1细胞及感染甜菜夜蛾 (Laphygmaexigua)幼虫 ,在培养细胞上清及虫体血淋巴中均测到抗菌活性。经Northernblotting证明感染甜菜夜蛾幼虫中有类CMⅣmRNA的存在。且表达产物在酸性电泳中电泳行为与天然抗菌肽CMⅣ组分相似。为进一步利用昆虫细胞及虫体生产抗菌肽药物打下了基础。     

Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-β

Background

Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-β could target to nerve cells and improve nerve regeneration.

Methods

Laminin-binding assay and sustained release assay of NGF-β fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested.

Findings

LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages.

Conclusion

Fused with NtA, NGF-β could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries.     

Simulated Microgravity Induces the Proliferative Inhibition and Morphological Changes in Porcine Granulosa Cells

Astronauts are always faced with serious health problems during prolonged spaceflights. Previous studies have shown that weightlessness significantly affects the physiological function of female astronauts, including a change in reproductive hormones and ovarian cells, such as granulosa and theca cells. However, the effects of microgravity on these cells have not been well characterized, especially in granulosa cells. This study aimed to investigate the effects of simulated microgravity (SMG) on the proliferation and morphology of porcine granulosa cells (pGCs). pGC proliferation from the SMG group was inhibited, demonstrated by the reduced O.D. value and cell density in the WST-1 assay and cell number counting. SMG-induced pGCs exhibited an increased ratio of cells in the G0/G1 phase and a decreased ratio of cells in the S and G2/M phase. Western blot analysis indicated a down-regulation of cyclin D1, cyclin-dependent kinase 4 (cdk4), and cyclin-dependent kinase 6 (cdk6), leading to the prevention of the G1-S transition and inducing the arrest phase. pGCs under the SMG condition showed an increase in nuclear area. This caused a reduction in nuclear shape value in pGCs under the SMG condition. SMG-induced pGCs exhibited different morphologies, including fibroblast-like shape, rhomboid shape, and pebble-like shape. These results revealed that SMG inhibited proliferation and induced morphological changes in pGCs.     

ALPK2 acts as tumor promotor in development of bladder cancer through targeting DEPDC1A

Bladder cancer is one of the most common malignant tumors in the urinary system. The development and improvement of treatment efficiency require the deepening of the understanding of its molecular mechanism. This study investigated the role of ALPK2, which is rarely studied in malignant tumors, in the development of bladder cancer. Our results showed the upregulation of ALPK2 in bladder cancer, and data mining of TCGA database showed the association between ALPK2 and pathological parameters of patients with bladder cancer. In vitro and in vivo experiments demonstrated that knockdown of ALPK2 could inhibit bladder cancer development through regulating cell proliferation, cell apoptosis, and cell migration. Additionally, DEPDC1A is identified as a potential downstream of ALPK2 with direct interaction, whose overexpression/downregulation can inhibit/promote the malignant behavioral of bladder cancer cells. Moreover, the overexpression of DEPDC1A can rescue the inhibitory effects of ALPK2 knockdown on bladder cancer. In conclusion, ALPK2 exerts a cancer-promoting role in the development of bladder cancer by regulating DEPDC1A, which may become a promising target to improve the treatment strategy of bladder cancer.Subject terms: Cancer models, Bladder cancer     

Intratumoral heterogeneity determines discordant results of diagnostic tests for human epidermal growth factor receptor (HER) 2 in gastric cancer specimens

Accurate assessment of human epidermal growth factor receptor (HER) 2 is essential for efficient selection of patients who may benefit from therapies targeting this surface receptor (e.g., trastuzumab). Intratumoral heterogeneity of HER2 expression may potentially contribute to inaccurate assessment of HER2 status. To clarify intratumoral heterogeneity of HER2 expression and its potential clinical impact on assessment of HER2 status, we analyzed 148 endoscopic biopsy specimens and 117 excisional tumor specimens collected from 148 patients with primary gastric cancer. Specifically, we assessed HER2 protein overexpression and gene amplification using, respectively, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). There were 28 IHC-positive cases and 25 FISH-positive cases among these 148 patients. Heterogeneous HER2 protein expression was demonstrated in 23 of 29 (79.3%) IHC-positive cases, while gene expression heterogeneity was found in 11 of 25 (44.0%) FISH-positive cases. Intratumoral heterogeneity was the main reason of discordant results between IHC and FISH or between endoscopic biopsy and excisional tumor specimens. The clinical significance and impact of intratumoral HER2 expression heterogeneity on treatment outcome in gastric cancer require further studies.     

Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine

Two multimode Hg(II) sensors, L‐MethBQA and L‐CysBQA, were obtained by fusing methionine or S‐methyl cysteine, into a bis‐quinolyl amine‐based chiral podand scaffold. Quinolyl groups serve as the fluorophore and possess nitrogen lone pairs capable of chelating metal ions. On exposure to Hg²⁺ or Zn²⁺, these sensors show signal enhancement in fluorescence. However, Cu²⁺ quenches their fluorescence in 30:70 acetontrile/water. L‐CysBQA complexes with Hg²⁺, producing an exciton‐coupled circular dichroism spectrum with the opposite sign to the one that is produced by Cu²⁺ or Zn²⁺ complexation. L‐CysBQA binds Hg²⁺ more strongly than Zn²⁺ and is shown to differentiate Hg²⁺ from other metal ions, such as Zn²⁺, Cu²⁺, Ni²⁺, and Pb²⁺, exceptionally well. The synergistic use of relatively soft sulfur, quinoline‐based chiral ligands and chiroptically enhanced fluorescence detection results in high sensitivity and selectivity for Hg²⁺. Chirality, 2011. © 2011 Wiley‐Liss, Inc.