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Identification and Preliminary Characterization of a New Chemical Affecting Glucosyltransferase Activities Involved in Plant Cell Wall Biosynthesis

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Molecular Plant 2008年第6期1卷 977-989页

作者： Olga Zabotina Erik Maim Georgia Drakakaki Vincent Bulone Natasha Raikhel Institute for Integrative Genome Biology Center for Plant Cell Biology and Department of Botany and Plant Sciences University of California Riverside CA92521 USA School of Biotechnology Royal Institute of Technology (KTH) AlbaNova University Center Stockholm SE-106 91 Sweden Present address: Department of Biochemistry Biophysics and Molecular Biology Iowa State University Ames IA 50014 USA

Chemical genetics as a part of chemical genomics is a powerful and fast developing approach to dissect biological processes that may be difficult to characterize using conventional genetics because of gene redundancy or lethality and, in the case of polysaccharide biosynthesis, plant flexibility. Polysaccharide synthetic enzymes are located in two main compartments--the Golgi apparatus and plasma membrane-and can be studied in vitro using membrane fractions. Here, we first developed a high-throughput assay that allowed the screening of a library of chemicals with a potential effect on glycosyltransferase activities. Out of the 4800 chemicals screened for their effect on Golgi glucosyltransferases, 66 compounds from the primary screen had an effect on carbohydrate biosynthesis. Ten of these compounds were confirmed to inhibit glucose incorporation after a second screen. One compound exhibiting a strong inhibition effect （ID 6240780 named chemical A） was selected and further studied. It reversibly inhibits the transfer of glucose from UDP-glucose by Golgi membranes, but activates the plasma membrane-bound callose synthase. The inhibition effect is dependent on the chemical structure of the compound, which does not affect endomembrane morphology of the plant cells, but causes changes in cell wall composition. Chemical A represents a novel drug with a great potential for the study of the mechanisms of Golgi and plasma membrane-bound glucosyltransferases.

关键词： cell walls callose synthase chemical screening and identification glycosyltransferase Golgi plasma membrane

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Immunomagnetic removal of cryo-damaged human spermatozoa

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Asian Journal of Andrology 2005年第1期7卷 61-69,页

作者： Uwe Paasch Sonja Grunewald Katja Wuendrich Tbrsten Jope Hans-Jurgen Glander Division of Andrology Department of Dermatology University of Leipzig Leipzig D-04103 Germany

Aim: To estimate the dissipation of mitochondrial transmembrane potential (mTMR,Δψ_m) and activation of sperm caspases (aCP) as signs of apoptosis in human spermatozoa during cryopreservation and to evaluate the efficiency of immunomagnetic cell separation (MACS) of these spermatozoa via annexin V-binding. Methods: The mTMP and aCP in fresh and cryopreserved spermatozoa were detected by fluorescence microscopy and by Western blots. The sperm suspensions were divided into two sperm fractions (with intact and deteriorated membranes) by magnetic cell separation (MiniMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) in dependence on their binding to superparamagnetic annexin V-microbeads (AN-MB). Results: The cryopreservation decreased the portion of spermatozoa with intact mTMP from 80.1% ± 7.2 % to 53.5 % ± 13.1% and increased the spermatozoa with activated pancaspases (aCP) from 21.8 % ± 2.6 % to 47.7 % ± 5.8 % (n = 10; mean ± SEM; P < 0.01). The activation of caspases 1, 3, 8, and 9 in the cryopreserved spermatozoa was confirmed by Western blots (n = 22). MACS reduced significantly the percentage of cryopreserved spermatozoa with dissipated mTMP to 8.1 ± 3.9 (P < 0.01) and also those with aCP to 9.3 % ± 2.2 %. Western blot analyses confirmed the increase of the activated caspase3, 9, and 8 in the AN-MB-positive fraction (P < 0.05) compared with the AN-MB-negative fraction. The MACS separation effect was confirmed by anti-annexin V-antibodies. There was no significant influence of the separation column and the magnetic field on the sperm functions. Conclusion: The cryopreservation impaired the mTMP and enhanced the activation status of caspases in human spermatozoa. The immunomagnetic sperm separation via binding of AN-MB could deplete low quality spermatozoa from cryopreserved semen samples.

关键词： spermatozoa cryopreservation immunomagnetic separation plasma membrane annexin V apoptosis caspases mitochondria

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Cloning and expression of SLC10A4,a putative organic anion transport protein

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World Journal of Gastroenterology 2006年第42期12卷 6797-6805页

作者： Patrick L Splinter Konstantinos N Lazaridis Paul A Dawson Nicholas F LaRusso The Center for Basic Research in Digestive Disease Division of Gastroenterology and Hepatology Mayo Medical School Clinic and Foundation Rochester Minnesota 55905 United States Department of Internal Medicine-Gastroenter-ology Wake Forest University School of Medicine Winston-Salem North Carolina 27157 United States

AIM: To determine if novel bile acid transporters may be expressed in human tissues. METHODS: SLC10A1 (NTCP) was used as a probe to search the NCBI database for homology to previously uncharacterized ESTs. The homology search identified an EST (termed SLC10A4) that shares sequence identity with SLC10A1 and SLC10A2 (ASBT). We performed Northern blot analysis and RT-PCR to determine the tissue distribution of SLC10A4. SLC10A4 was cloned in frame with an epitope tag and overexpressed in CHO cells to determine cellular localization and functional analysis of bile acid uptake. RESULTS: Northern analysis revealed that SLC10A4 mRNA is ubiquitously expressed in human tissues with the highest levels of mRNA expression in brain, placenta, and liver. In SLC10A4-transfected CHO cells, immunoblotting analysis and immunofluorescence staining demonstrated a 49-kDa protein that is expressed at the plasma membrane and intracellular compartments. Functional analysis of SLC10A4 showed no significant taurocholate uptake in the presence of sodium when compared to untransfected CHO cells. CONCLUSION: To date, we have shown that this protein has no capacity to transport taurocholate relative to SLC10A1; however, given its ubiquitous tissue distribution, it may play a more active role in transporting other endogenous organic anions.

关键词： SLC10A4 Bile acid transport Biliaryepithelium Taurocholate plasma membrane

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Kinetics of the H＋-ATPase from Dry and 5-Hours- Imbibed Maize Embryos in Its Native, Solubilized, and Reconstituted Forms

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Molecular Plant 2011年第3期4卷 505-515页

作者： Sobeida Sanchez-Nieto Consuelo Enriquez-Arredondo Fernando Guzmbn-Chavez Rolando Hern&ndez-Munoz Jorge Ramirez Marina Gavilanes-Ruiz Departamento de Bioquimica Facultad de Quimica Conjunto E. Universidad Nacional Autonoma de Mexico Cd Universitaria 04360 Mexico DF Mexico Instituto de Fisiologfa Celular Universidad Nacional Autonoma de Mexico Cd Universitaria 04360 Mexico DF Mexico

membranes undergo recovery upon rehydration in seed germination. Previous work has described that the plasma membrane H＋-ATPase from maize embryos adopts two different forms at 0 and 5 h of imbibition. We investigated how the kinetics of these two forms could be affected by alterations in the plasma membrane （PM）. In comparison to the O-h, PMs from the 5-h imbibed embryos showed changes in glycerophospholipid composition, decrease in leakage, and increase in fluidity. Kinetics of the PM H^-ATPase from 0 and 5-h imbibed embryos showed negative cooperativity, With the removal of the membrane environment, the activity of the enzymes shifted to a more complex kinetics, displaying two enzyme components. Lipid reconstitution produced one component with positive cooperativity. In all cases, enzymes from 0 and 5-h imbibed embryos presented similar kinetics with some quantitative differences. These results indicate that the two enzyme forms have the potential ability to respond to changes in the membrane enyiror^rpent, but the fact that they do not show differences in the native membranes at 0 or 5 h implies that modifications in the membrane are not drastic enough to alter their kinetics, or that they are able to preserve their boundary lipids or associated proteins and thus retain the same kinetic behavior.

关键词： ATPase activity H^-ATPase maize germination plasma membrane H+-ATPase plasma membrane Zea mays.

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Protective Effects of Glycinebetaine on Brassica chinensis Under Salt Stress

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Acta Botanica Sinica 2001年第8期43卷 809-814页

作者：许雯孙梅好朱亚芳苏维埃中国科学院上海植物生理研究所上海200032

Brassica chinensis L. were foliarly applied with glycinebetaine (GB), as this species is unable to synthesis GB and sensitive to osmotic stress such as salt. The exogenous GB was easily absorbed and transported by the leaf of B. chinensis . Its application (0-20 mmol/L) enhanced the plant tolerance to salt stress. The treatment of 15 mmol/L GB significantly decreased the Na + accumulation in leaf and root under NaCl stress. This difference in accumulating Na + and K + is caused by higher selectivity of root absorption. Furthermore, GB increased H +_ATPase activity of root plasma membrane evidently. This result strongly suggested that in root the decreased Na + accumulation was caused by the GB accumulation that enhanced the extrusion of Na + from the cell in some way through plasma membrane transporter, e.g. Na +/H + antiport driven by H +_ATPase. The GB application was also found to stabilize the plasma membrane, to decrease the loss of chlorophyll, and to stimulate the osmosis induced proline response under salt stress.

关键词： glycinebetaine Brassica chinensis proline Na + accumulation salt stress plasma membrane H +_ATPase

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Dissecting Multiple Arabidopsis CC-NBS-LRR Proteins Structure and Localization

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Journal of Biosciences and Medicines 2024年第7期12卷 87-99页

作者： Jianzhong Huang Xiuying Guan Xiaoju Zhong Peng Jia Hongbin Zhang Kai Chen Zhuojun Li Guangyu Chen Chuitang Zeng Key Research Laboratory of Chronic Disease Fuzhou Medical University Fuzhou China Technology Innovation Center of Chronic Disease Research in Fuzhou City Fuzhou Science and Technology Bureau Fuzhou China

NBS-LRR (nucleotide binding sites and leucine rich repeat) protein plays a crucial role as sentries and as defense activators in plants. The structure and function of NBS-LRR proteins are closely related. Previous articles have announced that the activated ZAR1 (HopZ-Activated Resistance 1) forms a pentamer in the plasma membrane, which is a calcium permeable channel that can trigger plant immune signaling and cell death. However, the structure of galore NBS-LRRs in Arabidopsis is not yet clear. The functional sites of distinct NBS-LRR in cells may vary. In addition, identifying pathogens and activating defense regions may occur in different subcellular compartments. Therefore, dissecting the specific structure and positioning of NBS-LRRs is an indispensable step in understanding their functions. In this article, we exploit AlphaFold to predict the structure of some designed NBS-LRRs, and utilize Agroinfiltration transient expression system, combined with biochemical fractionation, to dissect the localization of these NBS-LRR receptors from Arabidopsis. Structural data indicates that the identified NBS-LRRs share analogous conformation. membrane fractionation assay demonstrates these NBS-LRRs are mainly associated with the membrane. These data show that the Ca2+-permeable channel activity may be evolutionarily conserved in NBS-LRR of Arabidopsis, and this study provides some reference clues for analyzing the structure and localization patterns of other plant immune receptors.

关键词： Arabidopsis Calcium Permeation Channel Pentamer plasma membrane

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Optimization the Cell Wall Degrading Enzymes and Technique for Isolation of Protoplasts in Potato

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Journal of Pharmacy and Pharmacology 2016年第4期4卷 179-182页

作者： Le Minh Phuong Hana Vodickova Brigitazamecnikova Jaromir Lachman Czech University of Life Science in Prague Department of Chemistry Kamycka 961H 29 Suchdol Praha 6 Czech Republic Czech University of Life Science in Prague Department of Chemistry Kamycka 961/129 Suchdol Praha 6 Czech Repubfic

plasma membrane of plant cells is surrounded by cellulose wall and adjacent cells are joined together by a thick pectin rich matrix. Separation of plant cells and removal of the cell wall experimentally, by either a mechanical or an enzymatic process, results in the production ofprotoplast. Protoplasts are useful tools to study the uptake and transport ofmacromolecules and production of somatic hybrids. Protoplasts can be obtained from all types of actively growing young and healthy tissues. The most convenient and widely used source of plant protoplasts is leaf. Juvenile seedling tissues, cotyledons are other alternative tissues most frequently used for protoplasts isolation. All the environmental and genotypic factors, which affect the cell wall thickenings and compactness indirectly, influence the number of protoplasts recovered. Protoplasts are isolated by two methods, mechanical and enzymatic. The enzyme mixture solution of celluiose/macerozyme is used to digest the cell wall. The critical factors affecting the obtaning ofprotoplasts are the kinds of cell wall degrading enzymes, the physiological state of plant leaves, the type of osmotic stabilizers and the composition of reaction solution. With the improvement of technique and enzyme combination rate, the yield of collected protoplasts will be increased higher.

关键词： plasma membrane protoplast enzyme cell wall potato plant.

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The effects of Phosphorylation on K-Ras4B protein’s distribution

The effects of Phosphorylation on K-Ras4B protein’s distrib...

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第十届全国化学生物学学术会议

作者： Si-Yu Zhang Fang-Yi Li Yong-Xiang Chen Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology （Ministry of Education） Department of Chemistry Tsinghua University

K-Ras4 B is one of the most urgent targets for cancer therapy.[1] Its biological activity depends on its localization to the plasma membrane（PM）, which is mainly mediated by a polybasic domain and a S-farnesylated C-terminus. Besides, previous studies have shown that conditional phosphorylation at Ser181 of K-Ras4 B might relate to K-Ras4 B ’ s cellular distribution and signaling activity.[2] However, there are discordant conclusions toward the roles that phosphorylation plays. Herein, We developed an efficient strategy for producing full-length K-Ras4 B protein bearing farnesyl, phosphate, and methyl modifications. With this system, we observed that phosphorylation at Ser181 weakens the membrane binding affinity of K-Ras4 B in dependent manner on membrane fluidity, but does not fully inhibit its binding *** the other hand, phosphorylation does not affect the interaction between K-Ras4 B and its cytosolic shuttle protein PDE δ. After delivered into cells, K-Ras4 B containing nonhydrolyzable phosphoserine mimetic displayed a decreasing PM distribution compared with the nonphosphorylable one. Thus, phosphorylation might facilitate the dissociation of K-Ras4 B from the PM. In addition, we found that phosphorylation slightly change the thermotropic properties of K-Ras4 B-incorporated membrane.[3]

关键词： K-Ras4B protein synthesis phosphorylation plasma membrane

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Rab5 dependent membrane endocytosis is required for Semaphorin3A induced axon growth cone collapse

Rab5 dependent membrane endocytosis is required for Semaphor...

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中国神经科学学会第九届全国学术会议暨第五次会员代表大会

作者： Kong-Yan WU~1,Ai-Li SHENG~1,Wen-Wen LIU~2,An-Qi GENG~1,Xing-Yu JIANG~2,Xiao-Bing YUAN~1, Zhen-Ge LUO~(1,*) 1 Institute of Neuroscience,State Key Laboratory of Neuroscience,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200031,China 2 CAS Key Lab for Biological Effects of Nanomaterials and Nanosafety,National Center for NanoScience and Technology,Beijing 100190,China

＜正＞Guidance cues are critical for axon navigation and ultimately for the wiring of the neuro-circuits during nervous system *** cones of growing axons can sense both attractive and repulsive cues in the environment and make a corresponding ***3A,a negative guidance cues,can collapse growth cones of outgrowing axons,however,the intracellular molecular mechanisms are not well *** we show that the small GTPase Rab5 is involved in growth one collapse in response to ***5 activity assay shows that Rab5 is transiently activated after treatment with *** cone collapse is abolished by down-regulation of Rab5 by specific siRNA in response to *** advantage of live cell FRET assay,we demonstrate that semaphorin3A can activate Rab5 and promote the retrograde trafficking of Rab5 positive endosomes in growth ***5 is required for endocytosis of the growth cone plasma membrane during growth cone collapse. Over-expression of Rab5 leads to axon growth cones collapse in vitro and impedes projecting axons growth in vivo. These findings indicate that in response to negative guidance molecules semaphorin3A,axon growth cones driving endocytosis of the plasma membrane associated with the activated function of Rab5 resulted in growth cone collapse.

关键词： growth cone Semaphorin3A Rab5 endocytosis plasma membrane FRET microfluidic culture system electroporation

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Antitumor Activity of Recombinant Antimicrobial Peptid Penaeidin-2 against Kidney Cancer Cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2014年第4期34卷 529-534页

作者：孟明翔宁建芳于京佑陈丹丹孟小林徐进平张杰 Department of Urology Renmin Hospital of Wuhan University State Key Laboratory of Virology College of Life Sciences Wuhan University

Penaeidin-2（Pen-2） is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins show similar activities to the native Pen-2 protein. Previous researches have shown that some antimicrobial peptides（AMPs） exhibit cytotoxic activity against cancer cells. To date, there have been no studies on the antitumor effects of Pen-2. This study evaluated the potential of recombinant pen-2（rPen-2） in the selective killing of kidney cancer cell lines ACHN and A498, and its action mechanism. MTT assays found the maximal growth inhibition of HK-2, ACHN and A498 cells treated with 100 μg/mL rPen-2 at 48 h was 13.2%, 62.4%, and 70.4%, respectively. DNA-specific fluorescent dye staining showed a high percentage of apoptosis on cancer cells. Flow cytometry revealed that the apoptosis rate of HK-2, ACHN and A498 cells was 15.2%, 55.2%, and 61.5% at 48 h respectively, suggesting that rPen-2 induced higher apoptosis rate in cancer cells than in HK-2 cells. Laser confocal scanning microscopy demonstrated that the plasma membrane was the key site where rPen-2 interacted with and destroyed tumor cells. Scanning electron microscopy showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2. These results suggest that rPen-2 is a novel potential therapeutic agent that may be useful in treating kidney cancers.

关键词： recombinant Penaeidin-2 kidney cancer cell plasma membrane apoptosis lysis

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