Addressing the stability of C-capped dipeptide efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa

Synthetic optimization of a biologically labile class of dipeptides that function as efflux pump inhibitors to potentiate the antibacterial agent levofloxacin in Pseudomonas aeruginosa has led to the discovery of a related series of compounds that are completely stable in a variety of biological matrices. Other than the stability profile, the in vitro profile of the new series is essentially identical to that observed with the original one. A prototypical compound from the new series demonstrates potentiation in an in vivo model of infection.     

Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein Promotes Protective Immune Responses in Mice

The Cap protein of porcine circovirus type 2 (PCV2) that serves as a major host-protective immunogen was used to develop recombinant vaccines for control of PCV2-associated diseases. Growing research data have demonstrated the high effectiveness of flagellin as an adjuvant for humoral and cellular immune responses. Here, a recombinant protein was designed by fusing a modified version of bacterial flagellin to PCV2 Cap protein and expressed in a baculovirus system. When administered without adjuvant to BALB/c mice, the flagellin-Cap fusion protein elicited stronger PCV2-specific IgG antibody response, higher neutralizing antibody levels, milder histopathological changes and lower viremia, as well as higher secretion of cytokines such as TNF-α and IFN-γ that conferred better protection against virus challenge than those in the recombinant Cap alone-inoculated mice. These results suggest that the recombinant Cap protein when fused to flagellin could elicit better humoral and cellular immune responses against PCV2 infection in a mouse model, thereby acting as an attractive candidate vaccine for control of the PCV2-associated diseases.     

大豆苗期茎秆相关性状对荫蔽的响应

通过设置自然光照和遮荫环境2个处理,对51份大豆种质资源的苗期茎秆相关性状进行研究。结果表明:大豆茎秆相关性状在自然光照和遮荫环境下的变异系数分别为8.40%~78.06%和11.27%~84.68%,除第1、2节节间长度外,其他各性状在荫蔽环境下的变异系数均高于在自然光照下的变异系数。苗期荫蔽胁迫导致大豆株高、最低分枝高度、各节间长度极显著增加(P0.01),而茎粗、分枝数、主茎节数极显著降低。对2种光照环境下的株高指数进行分析发现,株高指数IL以及I2、I4和I6在2种光照环境下差异达到显著水平(P0.05)。相关分析表明,在荫蔽环境下,除第1节外,大豆株高与各节间长度呈显著或极显著正相关,与主茎节数相关性不显著。通径分析表明,自然光照下,第10(0.752)、11(0.736)节对株高的正向作用最大,而在荫蔽环境下,第7(0.752)、10(0.732)节对株高的正向效应较大。在筛选的材料中,有6份材料的株高在2种光照环境下差异不显著,可为套作大豆品种选育奠定基础。     

CD3-epsilon overexpressed in prothymocytes acts as an oncogene.

BACKGROUND: Upon engagement of the T cell receptor for antigen, its associated CD3 proteins recruit signal transduction molecules, which in turn regulate T lymphocyte proliferation, apoptosis, and thymocyte development. Because some signal transducing molecules recruited by CD3-epsilon, i.e., p56lck and p59fyn, are oncogenic and since we previously found that overexpression of CD3-epsilon transgenes causes a block in T lymphocyte and NK cell development, we tested the hypothesis that aberrant CD3-epsilon signaling leads both to abnormal T lymphocyte death and lymphomagenesis. MATERIALS AND METHODS: Ten independently derived transgenic mouse lines were generated with four different genomic CD3-epsilon constructs. Mice either homozygous or hemizygous for each transgene were analyzed for an arrest in T lymphocyte development and for the occurrence of T cell lymphomas. RESULTS: Aggressive clonal T cell lymphomas developed at very high frequencies in seven mouse lines with intermediate levels of copies of CD3-epsilon derived transgenes. However, these lymphomas were not found when high copy numbers of CD3-epsilon transgenes caused a complete block in early thymic development or when a transgene was used in which the exons coding for the CD3-epsilon protein were deleted. Analyses of a series of double mutant mice, tgCD3-epsilon x RAG-2null, indicated that lymphomagenesis was initiated in lineage-committed prothymocytes, i.e., before rearrangement of the T cell receptor genes. In addition, the transgene coding for the CD3-epsilon cytoplasmic domain and its transmembrane region induced a T cell differentiation signal in premalignant tgCD3-epsilon x RAG-2null mice. CONCLUSION: The nonenzymatic CD3-epsilon protein acted as a potent oncogene when overexpressed early in T lymphocyte development. Lymphomagenesis was dependent on signal transduction events initiated by the cytoplasmic domain of CD3-epsilon.     

MicroRNA-329 upregulation impairs the HMGB2/β-catenin pathway and regulates cell biological behaviors in melanoma

Melanoma is responsible for the majority of deaths caused by skin cancer. Antitumor activity of microRNA-329 (miR-329) has been seen in several human cancers. In this study, we identify whether miR-329 serves as a candidate regulator in melanoma. Melanoma-related differentially expressed genes were screened with its potential molecular mechanism predicted. Melanoma tissues and pigmented nevus tissues were collected, where the levels of miR-329 and high-mobility group box 2 (HMGB2) were determined. To characterize the regulatory role of miR-329 on HMGB2 and the β-catenin pathway in melanoma cell activities, miR-329 mimics, miR-329 inhibitors, and siRNA-HMGB2 were transfected into melanoma cells. Cell viability, migration, invasion, cell cycle, and apoptosis were assessed. miR-329 was predicted to influence melanoma by targeting HMGB2 via the β-catenin pathway. High level of HMGB2 and low miR-329 expression were observed in melanoma tissues. HMGB2 was targeted and negatively regulated by miR-329. In melanoma cells transfected with miR-329 mimics or siRNA-HMGB2, cell proliferation, migration, and invasion were impeded, yet cell cycle arrest and apoptosis were promoted, corresponding to decreased levels of β-catenin, cyclin D1, and vimentin and increased levels of GSK3β and E-cadherin. Collectively, our results show that miR-329 can suppress the melanoma progression by downregulating HMGB2 via the β-catenin pathway.     

Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B

The molecular machinery required for autophagy is highly conserved in all eukaryotes as seen by the high degree of conservation of proteins involved in the formation of the autophagosome membranes. Recently, both yeast Apg8p and its rat homologue Map1lc3 were identified as essential constituents of autophagosome membrane as a processed form. In addition, both the yeast and human proteins exist in two modified forms produced by a series of post-translational modifications including a critical C-terminal cleavage after a conserved Gly residue, and the smaller processed form is associated with the autophagosome membranes. Herein, we report the identification and characterization of three human orthologs of the rat Map1LC3, named MAP1LC3A, MAP1LC3B, and MAP1LC3C. We show that the three isoforms of human MAP1LC3 exhibit distinct expression patterns in different human tissues. Importantly, we found that the three isoforms of MAP1LC3 differ in their post-translation modifications. Although MAP1LC3A and MAP1LC3C are produced by the proteolytic cleavage after the conserved C-terminal Gly residue, like their rat counterpart, MAP1LC3B does not undergo C-terminal cleavage and exists in a single modified form. The essential site for the distinct post-translation modification of MAP1LC3B is Lys-122 rather than the conserved Gly-120. Subcellular localization by cell fractionation and immunofluorescence revealed that three human isoforms are associated with membranes involved in the autophagic pathway. These results revealed different regulation of the three human isoforms of MAP1LC3 and implicate that the three isoforms may have different physiological functions.     

Transactivation of the epidermal growth factor receptor is involved in 12-O-tetradecanoylphorbol-13-acetate-induced signal transduction

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion is still not well understood even though it is thought to be related to the protein kinase C/mitogen-activated protein kinase/AP-1 pathway. Recently, TPA was also found to induce epidermal growth factor receptor (EGFR) activity. Here, we investigated whether the EGFR is a necessary component for TPA-induced signal transduction associated with tumor promotion. We demonstrated that potent inhibitors of the EGFR, PD153035 and AG1478, blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs), AP-1 activity, and cell transformation. Egfr gene deficiency blocked TPA-induced ERK activity and AP-1 binding activity. The blocking of the ectodomain of the EGFR by a monoclonal antibody depressed TPA-induced ERK activity and AP-1 DNA binding activity. The use of a neutralizing antibody for heparin-binding EGF, one of the ligands of EGFR, blocked TPA-induced phosphorylation of ERKs. BB-94, a potent inhibitor of matrix metalloproteinases, which are activators of ectodomain shedding of EGFR ligands, also blocked TPA-induced ERK activity, AP-1 DNA binding, and cell transformation but had no effect on EGF-induced signal transduction. Anti-EGFR, anti-heparin-binding EGF, and BB-94 each blocked TPA-induced EGFR phosphorylation, but only anti-EGFR could block EGF-induced EGFR phosphorylation. Based on these results, we conclude that the EGFR is required for mediating TPA-induced signal transduction. EGFR transactivation induced by TPA is a mechanism by which the EGFR mediates TPA-induced tumor promotion-related signal transduction.     

Placental alkaline phosphatase, insulin, and adenine nucleotides or adenosine synergistically promote long-term survival of serum-starved mouse embryo and human fetus fibroblasts

Earlier we showed that in serum-starved fibroblasts placental alkaline phosphatase (PALP) can exert growth factor-like effects. Here we report that in mouse embryo (NIH 3T3) and human fetus (HTB-157) fibroblasts, PALP (200 nM) alone provided full protection against serum starvation-induced cell death for 5 days. After 12 days, substantial effects of PALP on cell survival required the copresence of insulin (500 nM) and ATP or adenosine (100 microM). In serum-starved NIH 3T3 cells, PALP induced activating phosphorylation of p42/p44 mitogen-activated protein (MAP) kinases; insulin, but not ATP, had small additional effects. PALP also stimulated the expression of various cyclins; ATP both prolonged and enhanced PALP-induced expression of cyclins A and E. Finally, ATP/adenosine enhanced activation of Akt kinase by insulin. The results suggest that PALP may be a regulator of growth and remodeling of fetal tissues during the second and third trimester of pregnancy when it is expressed.