Cryopreservation of in vitro-grown shoot-tips of <Emphasis Type=Italic>Rabdosia rubescens</Emphasis> by encapsulation-dehydration and evaluation of their genetic stability

Shoot-tips of Rabdosia rubescens, excised from in vitro-grown proliferating shoots that were cold-hardened at 5°C for 3 weeks, were encapsulated in alginate beads. Subsequently, these were precultured in a mixture of 0.4 M sucrose and 2 M glycerol for 1 h and then desiccated with silica-gel to about 21% water content prior to freezing in liquid nitrogen. After thawing, about 85% of cryopreserved shoot-tips grew into true-to-type shoots and with enhanced rooting capacity. Eight single-bud sibling lines were used to assess genetic stability of these encapsulated shoot-tips. When the relative DNA content was measured by flow cytometry (FCM), no changes were observed between controls and cryopreserved shoots. Using a sequence-related amplified polymorphism (SRAP) assay, it was observed that seven out of eight cryopreserved lines showed identical banding patterns; while the eighth line displayed an absent band, amounting to a low variance rate of 0.01%. These findings suggested that it was necessary to monitor the genetic stability of recovered cryopreserved R. rubescens shoots.     

Effects of a moderate-intensity static magnetic field and adriamycin on K562 cells

The aim of this study was to investigate whether a moderate‐intensity static magnetic field (SMF) can enhance the killing effect of adriamycin (ADM) on K562 cells, and to explore the effects of SMF combined with ADM on K562 cells. We analyzed the metabolic activity of cells, cell cycle distribution, DNA damage, change in cell ultrastructure, and P‐glycoprotein (P‐gp) expression after K562 cells were exposed continuously to a uniform 8.8 mT SMF for 12 h, with or without ADM. Our results showed that the SMF combined with ADM (25 ng/ml) significantly inhibited the metabolic activity of K562 cells (P < 0.05), while neither ADM nor the SMF alone affected the metabolic activity of these cells. Cell ultrastructure was altered in the SMF + ADM group. For example, cell membrane was depressed, some protuberances were observable, and vacuoles in the cytoplasm became larger. Cells were arrested at the G2/M phase and DNA damage increased after cells were treated with the SMF plus ADM. ADM also induced the P‐gp expression. In contrast, in the SMF group and SMF + ADM group, the P‐gp expression was decreased compared with the ADM group. Taken together, our results showed that the 8.8 mT SMF enhanced the cytotoxity potency of ADM on K562 cells, and the decrease in P‐gp expression may be one reason underlying this effect. Bioelectromagnetics 32:191–199, 2011. © 2010 Wiley‐Liss, Inc.     

ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.     

Increased Expression of SLC2A9 Decreases Urate Excretion From the Kidney

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.     

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.     

SLC5A9/SGLT4, a new Na+-dependent glucose transporter, is an essential transporter for mannose, 1,5-anhydro-D-glucitol, and fructose

We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) > D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).     

The N- and C-terminal mutations in tryptophan permease Tat2 confer cell growth in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> under high-pressure and low-temperature conditions

Tryptophan uptake appears to be the limiting factor in growth of tryptophan auxotrophic Saccharomyces cerevisiae strains under the conditions of high hydrostatic pressure and low temperature. When the cells are subjected to a pressure of 25 MPa, tryptophan permease Tat2 is degraded in a manner dependent on ubiquitination by Rsp5. One of the high-pressure growth-conferring genes, HPG2, was shown to be allelic to TAT2. The HPG2-1 (Tat2^E27F) mutation site is located within the ExKS motif in the N-terminus, and the HPG2-2 (Tat2^D563N) and HPG2-3 (Tat2^E570K) mutation sites are located at the KQEIAE sequence in the C-terminus. The HPG2 mutations enhance the stability of Tat2 during high-pressure or low-temperature incubation, leading to cell growth under these stressful conditions. These results suggest that the cytoplasmic tails are involved in Rsp5-mediated ubiquitination of Tat2 under high-pressure or low-temperature conditions.Communicated by K. Horikoshi     

大麻素抑制大鼠三叉神经节神经元ATP激活电流

本文在培养的大鼠三叉神经节(trigeminal ganglion,TG)神经元上采用全细胞膜片钳技术,探讨大麻素对大鼠TG神经元ATP激活电流(ATP-activated currents,IATP)的影响.结果显示(1)胞外给予ATP,大部分受检细胞(67/75,89.33%)可记录到一个内向电流,且具有剂量依赖性.该电流可被P2X嘌呤受体特异性拮抗剂PPADS所阻断.(2)预加WIN55212-2[大麻素受体1(cannabinoid receptor 1,CB1受体)激动剂]可对IATP产生抑制作用,此作用呈剂量依赖性,并可被CB1受体特异性拮抗剂AM281阻断.预加不同浓度的WIN55212-2(1x10-13、1x10-12、1x10-11、1x10-10、1x10-9和1x10-8mol/L)对IATP(1x10-4mol/L ATP)的抑制作用分别为(8.14±3.14)%、(20.11±2.72)%、(46.62±3.51)%、(72.16±5.64)%、(80.21±2.80)%和(80.59±3.55)%.(3)预加WIN55212-2后IATP的浓度-反应曲线明显下移;最大反应浓度时的IATP幅值减小了(58.02±4.21)%,而阈值基本不变;预加WIN55212.2前后曲线的EC50值非常接近(1.15x10-4mol/L vs 1.27x10-4 mol/L).(4)预加forskolin[腺苷酸环化酶(adenylyl cyclase,AC)激动剂]或8-Br-cAMP可以逆转WIN55212-2对IATP的抑制作用.以上结果表明,大麻素可能作用于CB1受体,通过抑制AC-cAMP-PKA途径发挥对IATP的抑制作用.     

金川多肋骨牦牛体外触摸鉴别法的解剖学验证及应用

目的通过屠宰和解剖学试验,验证金川多肋骨牦牛体外触摸鉴别方法的准确性,为科研和生产应用建立简捷实用、准确可靠的方法。方法对试验牦牛进行触摸鉴别,根据鉴定结果,依照肋骨对数将牦牛分为A、B两组(A组具15对肋骨,B组具14对肋骨),从A、B两组中随机抽取25%的个体进行屠宰和解剖实验。应用触摸鉴别法在中心产区和分布区对15对肋骨牦牛的比例进行普查。结果触摸鉴别准确率为96.15%;19头肋骨数为15对的牦牛中,胸椎为15,腰椎数为5的个体有18头,约占95%。而腰椎数为4的个体仅1头,占5.26%;26头屠宰牦牛中,真肋+弓肋+浮肋组型为8+7+0个体的比例为68.42%;8+6+1个体比例为31.58%。结论触摸鉴别法简捷、准确率高,可以在生产和科研中推广应用;15对肋骨牦牛中,大多数个体的胸椎多出1个,而腰椎数正常,且60%以上个体肋骨组型为8+7+0,无浮肋;中心产区,多肋牦牛比例为52.08%,分布区为30.21%。