Combination of arsenic trioxide and BCNU synergistically triggers redox-mediated autophagic cell death in human solid tumors

Arsenic trioxide (As₂O₃) is an effective treatment for relapsed or refractory acute promyelocytic leukemia (APL). After the discovery of As₂O₃ as a promising treatment for APL, several studies investigated the use of As₂O₃ as a single agent in the treatment of solid tumors; however, its therapeutic efficacy is limited. Thus, the systematic study of the combination of As₂O₃ with other clinically used chemotherapeutic drugs to improve its therapeutic efficacy in treating human solid tumors is merited. In this study, we demonstrate for the first time, using isobologram analysis, that As₂O₃ exhibits a synergistic interaction with N,N′-bis(2-chloroethyl)-N-nitrosourea (BCNU). The synergistic augmentation of the cytotoxicity of As₂O₃ with BCNU is in part through the autophagic cell death machinery in human solid tumor cells. As₂O₃ and BCNU in combination produce enhanced cytotoxicity via the depletion of reduced glutathione (GSH) and augmentation of reaction oxygen species (ROS) production. Further analysis indicated that the extension of GSH depletion by this combined regimen occurs through the inhibition of the catalytic activity of glutathione reductase. Blocking ROS production with antioxidants or ROS scavengers effectively inhibits cell death and autophagy formation, indicating that redox-mediated autophagic cell death involves the synergism of As₂O₃ with BCNU. Taken together, this is the first evidence that BCNU could help to extend the therapeutic spectrum of As₂O₃. These findings will be useful in designing future clinical trials of combination chemotherapy with As₂O₃ and BCNU, with the potential for broad use against a variety of solid tumors.     

出芽短梗霉胞外酸性漆酶

通过愈创木酚法平板检测10株出芽短梗霉,发现5株菌能够分泌胞外多酚氧化酶,反应最适pH在2.0左右,均属于酸性多酚氧化酶。菌株NG的酶活最高,达110 U/mL。添加H2O2、EDTA以及过氧化氢酶不显著影响菌株NG胞外酶活,表明NG分泌的多酚氧化酶中不含有锰过氧化物酶（MnP）和不依赖Mn2＋的过氧化物酶（MiP）,属于漆酶（Lac）。     

14-3-3 proteins in neurodegeneration

Among the first reported functions of 14-3-3 proteins was the regulation of tyrosine hydroxylase (TH) activity suggesting a possible involvement of 14-3-3 proteins in Parkinson's disease. Since then the relevance of 14-3-3 proteins in the pathogenesis of chronic as well as acute neurodegenerative diseases, including Alzheimer's disease, polyglutamine diseases, amyotrophic lateral sclerosis and stroke has been recognized. The reported function of 14-3-3 proteins in this context are as diverse as the mechanism involved in neurodegeneration, reaching from basal cellular processes like apoptosis, over involvement in features common to many neurodegenerative diseases, like protein stabilization and aggregation, to very specific processes responsible for the selective vulnerability of cellular populations in single neurodegenerative diseases.Here, we review what is currently known of the function of 14-3-3 proteins in nervous tissue focussing on the properties of 14-3-3 proteins important in neurodegenerative disease pathogenesis.     

Acidic stress promotes a glioma stem cell phenotype

Malignant gliomas are lethal cancers that display cellular hierarchies with cancer stem cells at the apex. Glioma stem cells (GSCs) are not uniformly distributed, but rather located in specialized niches, suggesting that the cancer stem cell phenotype is regulated by the tumor microenvironment. Indeed, recent studies show that hypoxia and its molecular responses regulate cancer stem cell maintenance. We now demonstrate that acidic conditions, independent of restricted oxygen, promote the expression of GSC markers, self-renewal and tumor growth. GSCs exert paracrine effects on tumor growth through elaboration of angiogenic factors, and low pH conditions augment this expression associated with induction of hypoxia inducible factor 2α (HIF2α), a GSC-specific regulator. Induction of HIF2α and other GSC markers by acidic stress can be reverted by elevating pH in vitro, suggesting that raising intratumoral pH may be beneficial for targeting the GSC phenotype. Together, our results suggest that exposure to low pH promotes malignancy through the induction of a cancer stem cell phenotype, and that culturing cancer cells at lower pH reflective of endogenous tumor conditions may better retain the cellular heterogeneity found in tumors.     

SPARC在子宫内膜癌组织中的表达及意义

目的：探讨富含半胱氨酸的酸性分泌型蛋白（secreted protien,acidic and rich in cysteine,SPARC）在子宫内膜癌组织中的表达及其临床意义。方法：应用组织芯片技术研究SPARC在正常子宫内膜、增生的子宫内膜和子宫内膜癌组织中的表达情况。结果：SPARC在正常子宫内膜、增生的子宫内膜和子宫内膜癌组织中的阳性表达率分别为96.55%、76.79%、59.50%,差异具有统计学意义（P〈0.05）。在子宫内膜癌中,SPARC的表达强度与手术-病理分期、组织学分级、肌层浸润深度和淋巴结转移相关,差异具有统计学意义（P〈0.05）。结论：SPARC表达的缺失与子宫内膜癌的发生、发展密切相关,检测SPARC可为子宫内膜癌的早期诊断、进一步治疗及预后判断提供一定的理论依据。     

Oligodendrocyte progenitor cells proliferate and survive in an immature state following treatment with an axolemma-enriched fraction

The ability of an AEF (axolemma-enriched fraction) to influence the proliferation, survival and differentiation of OPC (oligodendrocyte progenitor cells) was evaluated. Following addition of AEF to cultured OPC, the AEF associated with the outer surface of OPC so that subsequent metabolic events were likely mediated by direct AEF-OPC contact. Addition of AEF to the cultured OPC resulted in a dose- and time-dependent increase in proliferation that was partially dependent on Akt (protein kinase B) and MAPK (mitogen-activated protein kinase) activation. The major mitogen in an AEF-SE (soluble 2.0 M NaCl extract of the AEF) was identified as aFGF (acidic fibroblast growth factor) and accounted for 50% of the mitogenicity. The remaining 50% of the mitogenicity had properties consistent with bFGF (basic fibroblast growth factor) but was not unequivocally identified. Under conditions that limit the survival of OPC in culture, AEF treatment prolonged the survival of the OPC. Antigenic and morphological examination of the AEF-treated OPC indicated that the AEF treatment helped the OPC survive in a more immature state. The potential downstream metabolic pathways potentially activated in OPC by AEF and the consequences of these activated pathways are discussed. The results of these studies are consistent with the view that direct contact of axons with OPC stimulates their proliferation and survival while preventing their differentiation.     

Temporal dynamics of epidermal responses of guppies Poecilia reticulata to a sublethal range of waterborne zinc concentrations

This study assessed the histological changes in the epidermis of guppies Poecilia reticulata induced by waterborne zinc (Zn). Laboratory‐reared P. reticulata fry were maintained individually in separate vessels containing artificial water (8 µg l^?1 Zn) to which 0, 15, 30, 60 or 120 µg l^?1 Zn was added. Their epidermal response to Zn was monitored regularly over 4 weeks. Compared with controls, mucus was rapidly released and mucous cell numbers decreased at all concentrations. Thereafter mucous release, epidermal thickness, numbers and size of mucous cells fluctuated at a rate that varied with Zn concentration, but fluctuations declined after day 18. Results clearly highlight the dynamic nature of the epidermal response to sublethal concentrations of waterborne Zn. In general, low concentrations of Zn induced a rapid response with reduced numbers and size of mucous cells and shift in mucin composition, and a subsequent thickening of the epidermis. Epidermal thickness and mucous cell area fluctuated over time but were normal after a month of exposure to low Zn concentrations. The number of mucous cells, however, remained low. Virtually all mucous cells from fish maintained in 15 and 60 µg l^?1 Zn contained acidic mucins throughout the month, whereas fish maintained at 30 µg l^?1 Zn responded by production of neutral mucins during the first 12 days followed by a mixture of neutral and acidic mucins. At 120 µg l^?1 Zn, the most dramatic effects were the gradual but sustained decrease in numbers and area of mucous cells, and the shift to acidic mucins in these cells. Thus, as concentration of Zn increased, the epidermal responses indicated a disturbed host response (dramatic decline in mucous cell numbers, with mixed composition of mucins), which may have been less effective in preventing Zn uptake across the epithelium.     

Inorganic carbon limitation and mixotrophic growth in Chlamydomonas from an acidic mining lake

Plankton communities in acidic mining lakes (pH 2.5-3.3) are species-poor because they face extreme environmental conditions, e.g. 150mg l(-1) Fe2+ +Fe3+. We investigated the growth characteristics of the dominant pigmented species, the flagellate Chlamydomonas acidophila, in semi-continuous culture experiments under in situ conditions. The following hypotheses were tested: (1) Low inorganic carbon (IC) concentrations in the epilimnion (e.g. 0.3 mg l(-1)) arising from the low pH limit phototrophic growth (H-1); (2) the additional use of dissolved organic carbon (mixotrophy) leads to higher growth rates under IC-limitation (H-2), and (3) phagotrophy is not relevant (H-3). H-1 was supported as the culture experiments, in situ PAR and IC concentrations indicated that IC potentially limited phototrophic growth in the mixed surface layers. H-2 was also supported: mixotrophic growth always exceeded pure phototrophic growth even when photosynthesis was saturated. Dark growth in filtered lake water illuminated prior to inoculation provided evidence that Chlamydomonas was able to use the natural DOC. The alga did not grow on bacteria, thus confirming H-3. Chlamydomonas exhibited a remarkable resistance to starvation in the dark. The compensation light intensity (ca. 20 micromol photons m(-2) s(-1)) and the maximum phototrophic growth (1.50 d(-1)) fell within the range of algae from non-acidic waters. Overall, Chlamydomonas, a typical r-strategist in circum-neutral systems, showed characteristics of a K-strategist in the stable, acidic lake environment in achieving moderate growth rates and minimizing metabolic losses.     

抗人胎盘酸性铁蛋白单链抗体的构建、表达与鉴定

目的:制备人胎盘酸性铁蛋白(PAF),构建、表达并鉴定抗人PAF单链可变区抗体片段(scFv)。方法:设计以SfiⅠ、NotⅠ为酶切位点、以(Gly4Ser)3为linker的2对引物,从抗人PAF单克隆抗体可变区基因的克隆载体中扩增VH和VL基因,用重叠延伸PCR在VH和VL基因间引入连接短肽,构建VH-linker-VL的scFv基因。经SfiⅠ、NotⅠ酶切后克隆到原核分泌型表达载体pUC19/119上,转化大肠杆菌TG1和筛选后测序验证,IPTG诱导表达,SDS-PAGE鉴定其相对分子质量(Mr)。以人PAF为抗原,scFv为一抗,抗His单克隆抗体为二抗,间接ELISA鉴定其抗体活性。结果:重叠延伸PCR扩增产物经凝胶电泳可见约700bp的条带,DNA序列分析证明2株抗体具有完整的scFv序列,并含有c-myc和His。IPTG诱导阳性菌表达产物经SDS-PAGE鉴定有Mr约27000的显示条带,符合scFv与表达标签融合蛋白的理论值;间接ELISA证明表达产物可与PAF结合。结论:成功构建并表达了抗人PAF单链抗体,为进一步建立检测PAF的间接ELISA奠定了基础。     

Evolutionary and biochemical differences between human and monkey acidic mammalian chitinases

Acidic mammalian chitinase (AMCase), an enzyme implicated in the pathology of asthma, is capable of chitin cleavage at a low pH optimum. The corresponding gene (CHIA) can be found in genome databases of a variety of mammals, but the enzyme properties of only the human and mouse proteins were extensively studied. We wanted to compare enzymes of closely related species, such as humans and macaques. In our attempt to study macaque AMCase, we searched for CHIA-like genes in human and macaque genomes. We found that both genomes contain several additional CHIA-like sequences. In humans, CHIA-L1 (hCHIA-L1) is an apparent pseudogene and has the highest homology to CHIA. To determine which of the two genes is functional in monkeys, we assessed their tissue expression levels. In our experiments, CHIA-L1 expression was not detected in human stomach tissue, while CHIA was expressed at high levels. However, in the cynomolgus macaque stomach tissue, the expression pattern of these two genes was reversed: CHIA-L1 was expressed at high levels and CHIA was undetectable. We hypothesized that in macaques CHIA-L1 (mCHIA-L1), and not CHIA, is a gene encoding an acidic chitinase, and cloned it, using the sequence of human CHIA-L1 as a guide for the primer design. We named the new enzyme MACase (Macaca Acidic Chitinase) to emphasize its differences from AMCase. MACase shares a similar tissue expression pattern and pH optimum with human AMCase, but is 50 times more active in our enzymatic activity assay. DNA sequence of the mCHIA-L1 has higher percentage identity to the human pseudogene hCHIA-L1 (91.7%) than to hCHIA (84%). Our results suggest alternate evolutionary paths for human and monkey acidic chitinases.