Listeria monocytogenes Mutants Carrying Newcastle Disease Virus F Gene Fused to its actA and plcB： In vitro Expression and Immunogenicity in Chickens

Recombinant Listeria monocytogenes mutants carrying Newcastle disease virus (NDV) fusionprotein gene F were constructed by homologous recombination.NDV F or its truncated fragment Fa wasused as the model heterologous gene to be integrated into actA or plcB downstream of their signal sequences.Correct orientation of the inserted genes was verified by polymerase chain reaction amplification of F or Fa.The inserted F and Fa were expressed in the two recombinants Lm-AactA-F and Lm-AplcB-Fa as shown bysodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.Both recombinantsexhibited reduced virulence to embryonated eggs and mice by about 1.5-2.5 logs as compared with theparent wild strain 10403S. They were also less invasive than strain 10403S (P<0.05). Chickens receiving therecombinant strains orally or intraperitoneally were partially protected from virulent NDV challenge possiblydue to enhancement of non-specific immunity because the antibody titers against the homologous virus strainor the recombinant truncated fusion protein were marginal. Further research is needed in other animal modelsto see if the low antibody response results from insufficient expression of the heterologous genes as a resultof failure of L. monocytogenes or its recombinants to persist or replicate in chickens.     

Effects of divercin V41 combined to NaCl content, phenol (liquid smoke) concentration and pH on Listeria monocytogenes ScottA growth in BHI broth by an experimental design approach

AIMS: To investigate the main effects and interactions of different factors : divercin V41 (0-4 ng ml(-1)), NaCl content (0.5-5.5% w v(-1)), phenol (liquid smoke) concentration (0-8 ppm), and pH (5.5-7.5) on Listeria monocytogenes ScottA growth. METHODS AND RESULTS: Experiments were carried out in BHI broth using a central composite design. Divercin V41 (div41), NaCl content and pH were found to be the most influential factors whereas phenol concentration in liquid smoke had no effect on L. monocytogenes ScottA growth in our experimental domain. The combined effects of div41, NaCl content and pH decreased L. monocytogenes ScottA maximum specific growth rate (mu(max)) from 0.34 to 0.01 h(-1) and led to a significant increase in lag time (t(lag)) from 5.5 to 25 h. CONCLUSION: In this study, NaCl, pH and phenol conditions were similar to those currently observed in smoked salmon production. This shows that L. monocytogenes ScottA growth could be efficiently delayed by the use of div41 in addition to the usual technological hurdles. SIGNIFICANCE AND IMPACT OF THE STUDY: In conclusion, the technological hurdles of cold smoked salmon production could be further optimized and combined with the use of div41 or the div41 producer strain to improve the food safety of the product.     

cAMP对转化细胞中几种基因表达及CREB DNA结合活性的影响

 从癌基因、抑癌基因及转录因子 CREB(c AMP反应序列结合蛋白 )对 CRE DNA序列结合活性的相关性 ,对 db- c AMP处理的小鼠 C3H10 T1 /2转化细胞增殖抑制作用进行了研究 .实验结果表明 ,转化细胞中 PKA(蛋白激酶 A)活性显著低于正常细胞 ,而 PKC(蛋白激酶 C)活性则显著高于正常细胞 .斑点印迹和 Northern印迹分析显示转化细胞中 c- myc和 Ca M(钙调素 )基因表达明显高于正常细胞 ,而 p53基因和 Rb基因表达则明显低于正常细胞 ,这些差别与 C3H10 T1/ 2 转化细胞增殖失控有关 .转化细胞经 db- c AMP(1 mmol/L)处理后 ,细胞增殖受到明显抑制 ,db- c AMP处理0 .5h后 ,转化细胞中 PKA活性便明显增强 ,PKC活性则被显著抑制 ,处理 2 h后 ,c- myc和 Ca M基因表达下降 ,而 p53和 Rb基因表达则增强 ,这些变化与 c AMP抑制 C3H10 T1/ 2 转化细胞增殖有密切联系 .凝胶阻滞电泳分析显示 db- c AMP(1 mmol/L )处理短时间内 ,CREB对 CRE DNA序列无结合活性 ,1 2 h后开始出现较弱的结合活性 ,2 4 h后才明显加强 ,表明在 db- c AMP处理的早期 ,调控区中含有 CRE序列的基因不参与 db- c AMP对细胞增殖抑制的调节 ,即与 CREB磷酸化及其相应的 DNA结合活性无相关性 .     

S100A6 (calcyclin) deficiency induces senescence‐like changes in cell cycle,morphology and functional characteristics of mouse NIH 3T3 fibroblasts

S100A6 (calcyclin) is a calcium binding protein with two EF‐hand structures expressed mostly in fibroblasts and epithelial cells. We have established a NIH 3T3 fibroblast cell line stably transfected with siRNA against S100A6 to examine the effect of S100A6 deficiency on non‐transformed cell physiology. We found that NIH 3T3 fibroblasts with decreased level of S100A6 manifested altered cell morphology and proliferated at a much slower pace than the control cells. Cell cycle analysis showed that a large population of these cells lost the ability to respond to serum and persisted in the G0/G1 phase. Furthermore, fibroblasts with diminished S100A6 level exhibited morphological changes and biochemical features of cellular senescence as revealed by β‐galactosidase and gelatinase assays. Also, S100A6 deficiency induced changes in the actin cytoskeleton and had a profound impact on cell adhesion and migration. Thus, we have shown that the S100A6 protein is involved in multiple aspects of fibroblast physiology and that its presence ensures normal fibroblast proliferation and function. J. Cell. Biochem. 109: 576–584, 2010. © 2009 Wiley‐Liss, Inc.     

The I1-imidazoline receptor in PC12 pheochromocytoma cells activates protein kinases C, extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK).

We sought to further elucidate signal transduction pathways for the I1-imidazoline receptor in PC12 cells by testing involvement of protein kinase C (PKC) isoforms (betaII, epsilon, zeta), and the mitogen-activated protein kinases (MAPK) ERK and JNK. Stimulation of I1-imidazoline receptor with moxonidine increased enzymatic activity of the classical betaII isoform in membranes by about 75% and redistributed the atypical isoform into membranes (40% increase in membrane-bound activity), but the novel isoform of PKC was unaffected. Moxonidine and clonidine also increased by greater than two-fold the proportion of ERK-1 and ERK-2 in the phosphorylated active form. In addition, JNK enzymatic activity was increased by exposure to moxonidine. Activation of ERK and JNK followed similar time courses with peaks at 90 min. The action of moxonidine on ERK activation was blocked by the I1-receptor antagonist efaroxan and by D609, an inhibitor of phosphatidylcholine-selective phospholipase C (PC-PLC), previously implicated as the initial event in I1-receptor signaling. Inhibition or depletion of PKC blocked activation of ERK by moxonidine. Two-day treatment of PC12 cells with the I1/alpha2-agonist clonidine increased cell number by up to 50% in a dose related manner. These data suggest that ERK and JNK, along with PKC, are signaling components of the I1-receptor pathway, and that this receptor may play a role in cell growth.     

T‐cell epitope‐dependent immune response in inbred (C57BL/6J,SJL/J,and C3H/HeN) and transgenic P301S and Tg2576 mice

Alzheimer's disease is characterized by two pathological hallmarks, the intracellular deposition of hyperphosphorylated Tau protein and the extracellular deposition of Aβ_1–40/42, both being targets for immunotherapy. This study evaluates the immunogenic properties of three AD‐specific B‐cell epitopes (Tau_229–237[pT231/pS235], pyroGluAβ_3–8, and Aβ_{37/38–42/43}) linked to five foreign T‐cell epitopes (MVFP, TT, TBC Ag85B, PvT19, and PvT53) by immunizing inbred C57BL/6J (H‐2^b), SJL/J (H‐2^s2), and C3H/HeN (H‐2^k) mice. Two promising candidates with respect to MHC II restriction were selected, and two transgenic mouse models of AD, P301S (H‐2^b/k) and Tg2576 (H‐2^b/s) animals, were immunized with one B‐cell epitope in combination with two T‐cell epitopes. Responders displayed an enhanced immune response compared with wild‐type animals, which supports the vaccine design and the vaccination strategy. The immune response was also characterized by specific IgG subtype titers, which revealed a strong polarization toward the humoral pathway for immunization of phospho‐Tau, whereas for both Aβ vaccines, a mixed cellular/humoral pathway response was observed. Despite the diversity and unpredictability of the immunogenicity of the peptide vaccines, all three peptide vaccine formulations appear to be promising constructs for future evaluation of their therapeutic properties. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser⁴⁶ in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser⁴⁶, and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.     

The effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on cell growth, cell cycle, induction of DNA fragmentation, and activity of caspase 3 in murine leukemia L1210 cells and fibroblast NIH-3T3 cells

Quinazolines are multitarget agents, which have broad spectrum of biological activity, and some of them are now in cancer clinical testing. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects on cancer cell lines HeLa and B16. Quinazoline, at micromolar concentrations, induced morphological changes and necrosis of B16 cells, and at nanomolar concentrations it produced changes of F-actin cytoskeleton. It did not cause changes in the cell cycle, did not induce apoptotic cell death in B16 cells, did not have a mutagenic effect, and did not even behave as a typical intercalating agent. Little significant reduction of tumor volume in intramuscular transplanted B16 cells was observed. The aim of the present study was to examine the cytotoxic effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on murine leukemia L1210 cells and fibroblast NIH-3T3 cells. Induction of cell morphology and cell cycle changes, induction of apoptosis and caspase 3 activity were studied. Quinazoline acted cytotoxically on both cell lines. The sensitivity of leukemia L1210 cells to the quinazoline was higher than that of fibroblast NIH-3T3. The IC(100) was 12 microM for L1210 cells and 24 microM for NIH-3T3 cells. No effect of quinazoline on the cell cycle profile of L1210 and NIH-3T3 was detected, however, quinazoline induced an increase of the sub-G(0) cell fraction, apoptotic DNA fragmentation, and apoptotic morphological changes at a concentration of 12 microM. This quinazoline concentration induced caspase 3 activity. Our results demonstrated that induction of apoptotic cell death via activation of caspase 3 contributed to the cytotoxic effects of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline in murine leukemia L1210 cells.