Caveolin‐1 down‐regulation is required for Wnt5a‐Frizzled 2 signalling in Ha‐RasV12‐induced cell transformation

Caveolin‐1 (Cav1) is down‐regulated during MK4 (MDCK cells harbouring inducible Ha‐Ras^V12 gene) transformation by Ha‐Ras^V12. Cav1 overexpression abrogates the Ha‐Ras^V12‐driven transformation of MK4 cells; however, the targeted down‐regulation of Cav1 is not sufficient to mimic this transformation. Cav1‐silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction‐related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I‐CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha‐Ras^V12‐inducing MK4 cells increased exosome‐like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I‐CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I‐CM (MK4+I‐EXs). Wnt5a, a downstream product of Ha‐Ras^V12, was markedly secreted by MK4+I‐CM and MK4+I‐EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha‐Ras^V12‐ and MK4+I‐CM‐induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down‐regulation, either by Ha‐Ras^V12 or targeted shRNA, increased frizzled‐2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I‐EXs in MDCK cells. These data suggest that Cav1‐dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha‐Ras^V12‐Wnt5a‐Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha‐Ras^V12‐driven cell transformation.     

Structure-based design and structure-activity relationships of 1,2,3,4-tetrahydroisoquinoline derivatives as potential PDE4 inhibitors

This paper describes our medicinal chemistry efforts on 7-(cyclopentyloxy)-6-methoxy1,2,3,4-tetrahydroisoquinoline scaffold: design, synthesis and biological evaluation using conformational restriction approach and bioisosteric replacement strategy. Biological data revealed that the majority of the synthesized compounds of this series displayed moderate to potent inhibitory activity against PDE4B and strong inhibition of LPS-induced TNFα release. Among them, compound 19 exhibited the strongest inhibition against PDE4B with an IC₅₀ of 0.88?µM and 21 times more potent selectivity toward PDE4B over PDE4D when compared to rolipram. A primary structure-activity relationship study showed that the attachment of CH₃O group or CF₃O group to the phenyl ring at the para-position was helpful to enhance the inhibitory activity against PDE4B. Moreover, sulfonamide group played a key role in improving the inhibitory activity against PDE4B and subtype selectivity. In addition, the attachment of the additional rigid substituents at the C-3 position of 1,2,3,4-tetrahydroisoquinoline ring was favored to subtype selectivity, which was consistent well with the observed docking simulation.     

Cloning and expression analysis of a water stress-induced gene from Brassica oleracea.

Plant growth and productivity are greatly affected by water stress, such as drought and salinity. Here we report on the cloning and expression analysis of a water stress-induced gene from Brassica oleracea (designated as BoWS, GenBank accession number AY571333) by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoWS consisted of 594 bp and contained a 285 bp open reading frame (ORF) encoding a 95-amino-acid protein. The deduced protein had a calculated molecular mass of 10.53 kDa and an isoelectric point of 6.93. The sequence similarity and comparative analysis showed that BoWS was 84% identical to Arabidopsis thaliana putative water stress-induced protein (GenBank accession number AAM67282). Southern blot analysis indicated that BoWS was a low-copy gene. Northern blot analysis revealed that the expression of BoWS was upregulated by abscisic acid (ABA), mannitol, NaCl, drought, salicylic acid (SA) and hydrogen peroxide (H(2)O(2)). Our results indicate that BoWS is extremely related to the water-deficit stress in B. oleracea.     

用改良的MTT法测定rhG-CSF活性

ＭＴＴ测定法是根据线粒体脱氢酶催化ＭＴＴ形成蓝色甲■的多少来检测活细胞数和功能状态的,但原始方法中存在着一些问题,如敏感性偏低、有机溶剂产生蛋白质沉淀以及产物的溶解度偏低等。为了摸索测定 ｒｈＧ－ＣＳＦ活性的最适条件,我们以 ＮＦＳ－６０细胞为对象,比较了多种溶解缓冲液,并且对细胞数、ＭＴＴ浓度及保留时间、溶解液用量等条件进行了选择。结果表明,ＤＭＦ－２０％ＳＤＳ和 ２０％ＳＤＳ的效果最好,测定时细胞数为每孔 １０００个细胞,所加 ＭＴＴ浓度为 １ｍｇ／ｍｌ,保留时间为 ４ ｈ,溶解液的用量为每孔 １００μｌ。     

Breast cancer found at the time of breast reduction.

In a recent study involving 27,500 women who had breast reduction surgery in Ontario, Canada, 17 women who were diagnosed as having breast cancer at the time of their breast reduction surgery were identified. The aims of this study were to (1) describe a population-based series of patients who had breast cancer diagnosed at the time of breast reduction, (2) describe the treatment of these cancers, and (3) compare their survival rate with survival in patients in the general population who had breast cancer. Information about these women, their treatment, and outcome was extracted from hospital records, pathology reports, and reports from regional cancer centers. The chance of finding an invasive breast cancer at the time of breast reduction was 0.06 percent, which is lower than what has been reported previously. Sixty-seven percent of these women were treated with total mastectomy. In the remaining 33 percent, who were treated with partial mastectomy, the entire tumor was removed at the time of breast reduction. Fifty percent of the women were treated with radiation, and 25 percent were treated with chemotherapy or hormonal therapy. Compared with women in the general population of Ontario who have breast cancer, women whose breast cancer is discovered during breast reduction surgery are more likely to be treated with complete mastectomy and less likely to be treated with radiotherapy or chemotherapy. Seventy-one percent of the breast reduction group were axillary node-negative at diagnosis, compared with 58 percent in the general population of women with breast cancer. Survival from breast cancer in women diagnosed at the time of breast reduction (88 percent, 5-year survival) was better than survival from breast cancer in the general population (77 percent). These findings suggest that cancers found in women at the time of breast reduction are less advanced, possibly because they are diagnosed at an earlier stage.     

Sodium (±)‐5‐bromo‐2‐(α‐hydroxypentyl) benzoate ameliorates pressure overload‐induced cardiac hypertrophy and dysfunction through inhibiting autophagy

Sodium (±)‐5‐bromo‐2‐(a‐hydroxypentyl) benzoate (generic name: brozopine, BZP) has been reported to protect against stroke‐induced brain injury and was approved for Phase II clinical trials for treatment of stroke‐related brain damage by the China Food and Drug Administration (CFDA). However, the role of BZP in cardiac diseases, especially in pressure overload‐induced cardiac hypertrophy and heart failure, remains to be investigated. In the present study, angiotensin II stimulation and transverse aortic constriction were employed to induce cardiomyocyte hypertrophy in vitro and in vivo, respectively, prior to the assessment of myocardial cell autophagy. We observed that BZP administration ameliorated cardiomyocyte hypertrophy and excessive autophagic activity. Further results indicated that AMP‐activated protein kinase (AMPK)‐mediated activation of the mammalian target of rapamycin (mTOR) pathway likely played a role in regulation of autophagy by BZP after Ang II stimulation. The activation of AMPK with metformin reversed the BZP‐induced suppression of autophagy. Finally, for the first time, we demonstrated that BZP could protect the heart from pressure overload‐induced hypertrophy and dysfunction, and this effect is associated with its inhibition of maladaptive cardiomyocyte autophagy through the AMPK‐mTOR signalling pathway. These findings indicated that BZP may serve as a promising compound for treatment of pressure overload‐induced cardiac remodelling and heart failure.     

Microsatellite DNA markers for population-genetic studies of blue mackerel (Scomber australasicus) and cross-specific amplification in S. japonicus

Blue mackerel (Scomber australasicus) is targeted by large-scale purse-seiners in the western North Pacific, and its stock structure is still contentious. Herein, we described 10 polymorphic microsatellite loci for blue mackerel. The number of alleles among 32 individuals surveyed ranged from five to 27 (average of 16.2 alleles per locus). Departures from Hardy-Weinberg expectation were observed at two loci. Cross-specific amplification in the congener, S. japonicus, was successful, except for one locus, revealed to be diagnostic for these congeners. These microsatellite loci will be useful tools to address queries in population genetic structure, fishery management unit and taxonomic species status in the genus Scomber.