An ultramicrochemical test for mycoplasmal contamination of cultured cells.

A sensitive ultramicrochemical enzyme test for mycoplasmal contamination of cultured cells, based on the determination of the activity of adenosine phosphorylase, is described. The test was performed by assaying the enzymatic conversion of [8-14C]adenine and ribose-1-phosphate to [8-14C]adenosine by incubating a plastic leaflet carrying a counted number of cells (1 to 10). These leaflets were isolated from the bottom of the same plastic film dish in which the cells were cultured for experimental or diagnostic purposes, e.g. prenatal diagnosis or inborn errors of metabolism. The present test should be several 1000-fold more sensitive than the originally reported enzymatic method because (a) the adenosine-phosphorylase reaction is measured in the nucleoside forming direction which is by far the most active; and (b) the assay is performed with the cells and not with the culture medium. The latter is of special importance for the detection of those low-grade contamination in which most of the mycoplasma particles are attached to cell membranes.     

An ultramicrochemical test for mycoplasmal contamination of cultured cells

Summary A sensitive ultramicrochemical enzyme test for mycoplasmal contamination of cultured cells, based on the determination of the activity of adenosine phosphorylase, is described. The test was performed by assaying the enzymatic conversion of [8-¹⁴C]adenine and ribose-1-phosphate to [8-¹⁴C]adenosine by incubating a plastic leaflet carrying a counted number of cells (1 to 10). These leaflets were isolated from the bottom of the same plastic film dish in which the cells were cultured for experimental or diagnostic purposes, e.g. prenatal diagnosis or inborn errors of metabolism. The present test should be several 1000-fold more sensitive than the originally reported enzymatic method because (a) the adenosinephosphorylase reaction is measured in the nucleoside forming direction which is by far the most active; and (b) the assay is performed with the cells and not with the culture medium. The latter is of special importance for the detection of those low-grade contaminations in which most of the mycoplasma particles are attached to cell membranes. This investigation was partly supported by FUNGO (The Netherlands), INSERM (France) (A. T. P. 36.76.68), and DGRST (France) (No. 75.50.004).     

Surface-Enhanced Raman Scattering from Individual Au Nanoparticles on Au Films

We investigated the effect of optical thick metal films on the surface-enhanced Raman scattering (SERS) activity of individual Au nanoparticle (NP) monomers and dimers. The film presence is revealed to be positive for the SERS activity of individual NP monomers, while it is not always positive for the electromagnetic enhancement at hot spots for SERS of the dimer, which is explained well by our numerical simulations. The polarized SERS signals from the NP dimer are elucidated well in terms of the plasmon hybridization of the dimer. SERS contributions both from individual NP surfaces and the junction between the NP and its supporting substrate were discussed as well.     

Conservation tillage methods affect soil water use and spring maize yield in a semi-humid drought-prone area of China

In the arid areas of Northwest China, water has become a key limiting factor for agricultural development, so conservation tillage management has become an important measure to improve water use efficiency and crop yield. Our experiment, conducted from 2015 to 2018, explored the effects of conventional flat planting with no mulch (NP), with straw mulch (SM), and with plastic mulch (PM); and also ridge and furrow planting without mulch (RF) and ridge-furrow planting with plastic film and straw mulch (RFPFM) on soil nutrients, soil water storage, water use efficiency, and crop yield when compared with traditional no mulch and no fertilization cultivation (CK). Four years of experiments showed that compared to CK, SM and RFPFM had significantly increased soil surface nutrient content and increased organic matter content, and the water depletion during the maize growth period of the NP, SM, PM, RF, and RFPFM treatments increased by 9.7%, 0.6%, 8.6%, 4.4%, and 14.2%, respectively. For four years, the average spring maize yields of the NP, SM, PM, RF, and RFPFM treatments increased by 36%, 87%, 115%, 40%, and 119%, compared to those of CK. The PM and RFPFM treatments' water use efficiencies were significantly different from those of the other treatments and the basic decreasing trend of water use efficiency was RFPFM > PM > SM > RF > NP > CK. In summary, we showed that because ridge-furrow planting with plastic film mulch improved both soil nutrients and crop yields, it is a preferred cultivation method for agricultural water utilization in arid areas.     

Hypoxia facilitates the survival of nucleus pulposus cells in serum deprivation by down-regulating excessive autophagy through restricting ROS generation

Nucleus pulposus (NP) cells reside in a hypoxic environment in vivo, while the mechanisms of how NP cells maintain survival under hypoxia are not clear. Autophagy is an important physiological response to hypoxia and implicated in the survival regulation in most types of cells. This study was designed to investigate the role of autophagy in the survival of NP cells under hypoxia. We found that appropriate autophagy activity was beneficial to the survival of NP cells in serum deprivation, while excessive autophagy led to death of the NP cells. Hypoxia facilitated the survival of NP cells in serum deprivation by down-regulating excessive autophagy. Hypoxia down-regulated the autophagy activity of NP cells through restricting the production of reactive oxygen species (ROS) and inactivating the AMPK/mTOR signaling pathway, and possibly through a pathway involving HIF-1α. We believed that understanding the autophagy response of NP cells to hypoxia and its role in cell survival had important clinical significance in the prevention and treatment of degenerative discogenic diseases.     

DNA and nuclear protein characteristics in non-neoplastic and neoplastic endometrial tissue

Feulgen-DNA and nuclear protein (NP) measurements were performed on non-neoplastic and neoplastic endometrium. Non-neoplastic endometrial cells were exclusively characterized by euploid nuclear DNA content. The NP content may vary significantly in relation to the proliferative stage as reflected by a 2-3-fold increase NP/DNA ratio in growing as compared to growth arrested cells. Endometrial adenocarcinomas could be subdivided into euploid and aneuploid types. The euploid tumors were found to exhibit DNA and NP characteristics comparable with those of normal tissue. In contrast, aneuploid tumors showed DNA and NP characteristics indicating increased proliferative activity as well as a pronounced disorder between the DNA and protein cycle.     

Nuclear protein content and Ki-67 immunoreactivity in nonneoplastic and neoplastic thyroid cells.

The nuclear DNA content in thyroid tumor cells has been shown to be closely related to the malignant potential of the neoplasm. Besides DNA, nuclear protein (NP) constitutes the major mass of the nucleus. The NP content may vary significantly in relation to the proliferative stage in growing as compared to growth-arrested cells. The increase in NP content associated with the transition from G0 to G1 occurs before the onset of DNA synthesis and may be used to assess growth activity. The nuclear DNA and NP contents were analyzed in 90 nonneoplastic lesions and 75 benign and 62 malignant thyroid tumors. All nonneoplastic specimens were euploid, and 1 of 90 was growth activated. In the group of benign tumors, 59 were euploid, and 16 were aneuploid. Among these there were 5 (9%) of 59 and 6 (38%) of 16 growth-activated specimens, respectively. In the group of malignant tumors 57 of 62 were classified as euploid, and in this group 12 (21%) showed growth activity according to the NP content. Five of 62 were aneuploid, and 3 (60%) of these 5 tumors were growth activated. Evaluation of the growth activity by means of monoclonal antibody Ki-67 was performed on a subgroup of 32 thyroid specimens, both nonneoplastic and neoplastic lesions. Ki-67 immunoreactivity was observed in 0-1.1% cells of 6 nonneoplastic lesions, in 0-3.1% cells of 14 benign cells and in 0.2-3.9% cells of 12 malignant thyroid tumors. Growth activity, as reflected by the NP/DNA ratio and Ki-67 immunoreactivity, appears to be low both in nonneoplastic thyroid lesions and thyroid tumors.     

太阳能消毒对温室土壤环境效应及防治黄瓜根结线虫病效果

采用室内测定与大田试验相结合的方法,研究了太阳能不同消毒方式对温室土壤环境的效应及对温室黄瓜根结线虫病的控制效果.结果表明,垄沟式覆盖地膜处理对温室土壤温度、土壤酶的活性、微生物数量的影响最明显,处理16d,棚室10、20、30、40、50cm深土壤的最高温度依次是59.1、57.7、56.6、48.9、47.6℃,平均每天超过55、50、45℃持续时间分别为7.5、8.5h和16h;土壤温度的升高,有利于提高对根结线虫的杀灭效果.0～20cm土壤脲酶、蔗糖酶、碱性磷酸酶、过氧化氢酶分别降低43.3%、18.7%、20.1%和13.1%;土壤真菌、细菌、放线菌数量分别降低96.0%、84.8%、53.9%.垄沟式未覆膜处理对土壤环境的影响次之,平面式未覆膜的影响最小,酶活性降低及土壤微生物数量下降对土壤的活性有一定的负效应.垄沟式覆膜太阳能消毒对温室黄瓜根结线虫控制效果最显著,持效期最长,能有效杀灭温室0～50cm土壤内根结线虫;处理后第1年和第2年对温室黄瓜根结线虫控制效果均达到100%,第3年防效96.7%,第5年仍达72.8%.垄沟式未覆膜控制效果次之;平面式未覆膜控制效果最差,持效期最短.     

Separation of the transforming growth factor from cells of murine sarcoma induced by polymeric film

Use was made of the cell line PS-103 induced by subcutaneous implantation of plastic film to CBA mice. The conditioned medium from PS-103 was shown to stimulate anchorage independence of PS-103 itself and several pseudonormal cells: NRK and NIH/3T3. The peak of the colony-stimulating activity after gel filtration was displayed by fractions with a molecular weight of 15 000, which were acid- and heat-resistant. All those characteristics correlated with the traits of the transforming growth factors (TGF). The significance of the TGF for carcinogenesis induced by plastic films is discussed.     

p-Nonylphenol acts as a promoter in the BALB/3T3 cell transformation

p-Nonylphenol (NP) has attracted attention as an estrogenic contaminant, and the environmental pollution by NP has been found to be extensive. NP is classified as a phenolic antioxidant based on the chemical activity and structure. Some phenolic antioxidants are known to induce and/or enhance carcinogenesis. We examined the effects of NP on the two-stage transformation of BALB/3T3 cells, a model of two-stage carcinogenesis. The treatment by NP in the promotion phase markedly enhanced the transformation of the cells pre-treated with a subthreshold dose of a carcinogen, 3-methylcholanthrene (MCA), but not that of non-pretreated cells. The promoting activity of NP was approximately one hundredth of that of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, in the cell transformation. The treatment by NP in the initiation phase did not induce cell transformation with and without post-treatment by TPA. These results indicate that NP acts as a pure promoter of cell transformation implying that it may cause the enhancement of carcinogenesis in vivo. The enhancement by NP of MCA-initiated transformation was suggested not to be mediated by estrogen receptors in BALB/3T3 cells because 17 beta-estradiol did not promote cell transformation in our experiments, and it has been reported that BALB/3T3 cells do not express estrogen receptors at a detectable level.     

Envelope glycoproteins of hantavirus can mediate cell-cell fusion independently

Hantaviruses (HTVs) are enveloped viruses and can induce low PH-dependent cell fusion. In this report we molecularly cloned viral glycoproteins (GPs) cDNA and nucleocapsid (NP) cDNA of two strains of Hantaan virus and one strain of Seoul virus and expressed in Vero E6 cells under control of a CMV promoter. The examinations of viral gene expressions were carried out by IFA and immune-precipitation. After treatment with low PH (PH 5.8) medium the syncytium were observed in the cells transfected with the GPs clones while in the cells transfected with the NP clones we did not find this phenomenon. Furthermore cotransfection of the NP and GPs did not enhance fusion activity. Treatment with anti-GP monoclonal antibodies could inhibit fusion activity whereas the antibodies against NP could not. These results indicated that GPs can mediate cell-cell fusion independently.     

Biodegradation of a polyvinyl alcohol-starch blend plastic film

Attempts were made to elucidate the degradation mechanism of a polyvinyl alcohol (PVA)-starch blend plastic. A part of the starch fraction of this plastic was dissolved into an aqueous phase in a control test. Treatment with a PVA-degrading bacterium or enzyme gave a maximal weight loss of approximately 70% and film breakage occurred. Since this plastic contains 40% PVA, it is apparent that not only the PVA fraction but also a considerable portion of the starch fraction was lost from the film by treatment with the PVA-degrading enzyme. As the PVA-degrading bacterium and enzyme used here showed no starch-degrading activity, loss of the starch fraction seems to depend on its dissolution with degradation of the PVA fraction. These experimental results indicated that the degradation of the PVA fraction is an important requisite for complete degradation or decomposition of this plastic film.     

TGFβ regulates Galectin-3 expression through canonical Smad3 signaling pathway in nucleus pulposus cells: implications in intervertebral disc degeneration

Galectin-3 is highly expressed in notochordal nucleus pulposus (NP) and thought to play important physiological roles; however, regulation of its expression remains largely unexplored. The aim of the study was to investigate if TGFβ regulates Galectin-3 expression in NP cells. TGFβ treatment resulted in decreased Galectin-3 expression. Bioinformatic analysis using JASPAR and MatInspector databases cross-referenced with published ChIP-Seq data showed nine locations of highly probable Smad3 binding in the LGALS3 proximal promoter. In NP cells, TGFβ treatment resulted in decreased activity of reporters harboring several 5′ deletions of the proximal Galectin-3 promoter. While transfection of NP cells with constitutively active (CA)-ALK5 resulted in decreased promoter activity, DN-ALK5 blocked the suppressive effect of TGFβ on the promoter. The suppressive effect of Smad3 on the Galectin-3 promoter was confirmed using gain- and loss-of-function studies. Transfection with DN-Smad3 or Smad7 blocked TGFβ mediated suppression of promoter activity. We also measured Galectin-3 promoter activity in Smad3 null and wild type cells. Noteworthy, promoter activity was suppressed by TGFβ only in wild type cells. Likewise, stable silencing of Smad3 in NP cells using sh-Smad3 significantly blocked TGFβ-dependent decrease in Galectin-3 expression. Treatment of human NP cells isolated from tissues with different grades of degeneration showed that Galectin-3 expression was responsive to TGF-β-mediated suppression. Importantly, Galectin-3 synergized effects of TNF-α on inflammatory gene expression by NP cells. Together these studies suggest that TGFβ, through Smad3 controls Galectin-3 expression in NP cells and may have implications in the intervertebral disc degeneration.     

Counting total cell numbers microscopically on stained films

Two main sources of error in the conventional stained film cell count method, i.e. errors from imperfect spreading of the suspension and uneven distribution of cells in the film, have been eliminated by counting all the cells in the film. Staining solution and sodium alginate were added to the suspension before spreading over the slide. With a microsyringe, 20-μl suspension was spread in two bands of ca 1.7 mm × 60 mm each. Then total cells could be counted easily in the x 100 magnification field by moving the slide in one direction. Results were satisfactory. Coefficients of variation were 2.6 and 1.1% in two separate yeast determinations each with five replicates. For counting higher magnifications, a suitable image analyser for automating the method is believed worthy of study.     

Protective effect of antioxidants against para-nonylphenol-induced inhibition of cell growth in Saccharomyces cerevisiae

The cell growth-modulating activity of an endocrine disruptor, p-nonylphenol (NP), was estimated using the yeast Saccharomyces cerevisiae as a simple model of eukaryotic cells. NP caused a dose-dependent suppressive effect on cell growth of S. cerevisiae at 10, 25 and 50 microM. The NP-induced cell growth inhibition was restored when concomitantly lipophilic antioxidants such as alpha-tocopherol and beta-carotene were supplied, but not the hydrophilic antioxidants ascorbic acid or (-)epigallocatechin gallate (EGCG). The cellular oxygen consumption of S. cerevisiae was also inhibited in a dose-dependent fashion by the extracellular addition of NP, and pretreatment with alpha-tocopherol and beta-carotene suppressed NP-induced inhibition of cellular oxygen consumption, but ascorbic acid and EGCG were not effective. Furthermore, NP caused a marked generation of radical oxygen species (ROS) in S. cerevisiae, which was suppressed by treatment with alpha-tocopherol and beta-carotene, but not with ascorbic acid and EGCG. However, NP did not show a significant inhibitory effect on cell growth and survival of mitochondria-deficient petite mutant cells and they showed a relatively weak ROS-generating activity compared with parent yeast cells. These results suggest that NP-induced inhibition of cell growth and oxygen consumption in S. cerevisiae might be possibly associated with ROS generation in yeast mitochondria. The significance of this finding is discussed from the viewpoint of NP-induced oxidative stress against eukaryotic cells.     

生物降解地膜应用与地膜残留污染防控

地膜是农业生产中重要的生产资料之一,过去30多年,地膜用量和农作物覆膜面积一直稳定增长,2014年地膜用量达到了144万t,覆膜面积超过1 800万hm~2,该技术对保障我国农产品安全供给作出了重大贡献。与此同时,地膜残留污染问题越来越严重,一些农田的地膜残留量超过250 kg/hm~2,在我国西北的局部区域,地膜残留已经对农田土壤、作物生长发育、农事作业等造成严重影响和危害。生物降解地膜成为替代普通PE地膜、解决地膜残留污染的一种有效措施和手段,目前生物降解地膜正处于产品研发和评价试验的关键阶段,部分生物降解地膜产品在马铃薯、花生、烟草生产上显示出良好的效果,但总体上还面临巨大挑战,主要包括需要进一步提高地膜产品质量,提高地膜产品破裂和降解可控性,改善地膜的增温保墒能力,实现满足农作物对地膜覆盖功能的要求;此外,需要降低生物降解地膜综合成本,促进生物降解地膜规模化应用。总体上,随着技术进步和农业生产环境的变化,生物降解地膜应用将具有良好的前景。     

Bromodomain-containing protein 4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity in NP cells

An imbalance between matrix synthesis and degradation is the hallmark of intervertebral disc degeneration while inflammatory cytokines contribute to the imbalance. Bromodomain and extra-terminal domain (BET) family is associated with the pathogenesis of inflammation, and inhibition of BRD4, a vital member of BET family, plays an anti-inflammatory role in many diseases. However, it remains elusive whether BRD4 plays a similar role in nucleus pulposus (NP) cells and participates in the pathogenesis of intervertebral disc degeneration. The present study aims to observe whether BRD4 inhibition regulates matrix metabolism by controlling autophagy and NLRP3 inflammasome activity. Besides, the relationship was investigated among nuclear factor κB (NF-κB) signaling, autophagy and NLRP3 inflammasome in NP cells. Here, real-time polymerase chain reaction, western blot analysis and adenoviral GFP-LC3 vector transduction in vitro were used, and it was revealed that BRD4 inhibition alleviated the matrix degradation and increased autophagy in the presence or absence of tumor necrosis factor α. Moreover, p65 knockdown or treatment with JQ1 and Bay11-7082 demonstrated that BRD4 inhibition attenuated NLRP3 inflammasome activity through NF-κB signaling, while autophagy inhibition by bafilomycin A1 promoted matrix degradation and NLRP3 inflammasome activity in NP cells. In addition, analysis of BRD4 messenger RNA expression in human NP tissues further verified the destructive function of BRD4. Simply, BRD4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity through NF-κB signaling in NP cells.     

Effects of Boron Supplementation on Peripartum Dairy Cows’ Health

In the present study, toxicity of commercial zinc oxide nanoparticles (ZnO NPs) was studied on the bacterium Pseudomonas sp., human promyelocytic leukemia (HL-60) cells, and peripheral blood mononuclear cells (PBMC). The toxicity was assessed by measuring growth, cell viability, and protein expression in bacterial cell. The bacterial growth and viability decreased with increasing concentrations of ZnO NP. Three major proteins, ribosomal protein L1 and L9 along with alkyl hydroperoxides reductase, were upregulated by 1.5-, 1.7-, and 2.0-fold, respectively, after ZnO NP exposure. The results indicated oxidative stress as the leading cause of toxic effect in bacteria. In HL-60 cells, cytotoxic and genotoxic effects along with antioxidant enzyme activity and reactive oxygen species (ROS) generation were studied upon ZnO NP treatment. ZnO NP exhibited dose-dependent increase in cell death after 24-h exposure. The DNA-damaging potential of ZnO NP in HL-60 cells was maximum at 0.05 mg/L concentration. Comet assay showed 70–80% increase in tail DNA at 0.025 to 0.05 mg/L ZnO NP concentration. A significant increase of 1.6-, 1.4-, and 2.0-fold in ROS level was observed after 12 h. Genotoxic potential of ZnO NPs was also demonstrated in PBMC through DNA fragmentation. Thus, ZnO NP, besides being an essential element having antibacterial activity, also showed toxicity towards human cells (HL-60 and PBMC).     

Endogenous expression of matriptase in neural progenitor cells promotes cell migration and neuron differentiation

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.