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    Enzyme substrates for visualizing acidic organelles
    1.
    发明申请
    Enzyme substrates for visualizing acidic organelles 审中-公开
    用于显示酸性细胞器的酶底物

    公开(公告)号：US20130252268A1

    公开(公告)日：2013-09-26

    申请号：US13986535

    申请日：2013-05-14

    申请人： John J. Naleway ,  Daniel J. Coleman

    发明人： John J. Naleway ,  Daniel J. Coleman

    IPC分类号： C12Q1/44 ,  C12Q1/34

    CPC分类号： C12Q1/44 ,  C12Q1/34 ,  G01N33/5076 ,  G01N33/531 ,  G01N33/573 ,  G01N33/582

    摘要： The present invention relates to the visualization of acidic organelles based upon organelle enzyme activity. The organelle substrates of the invention are specific for enzyme activity of the organelle and label these organelles, such as lysosomes, rendering them visible and easily observed. Substrates of the present invention include substrates that produce a fluorescent signal. The fluorogenic acidic organelle enzyme substrates of this invention are designed to provide high fluorescence at low pH values and are derivatized to permit membrane permeation through both outer and organelle membranes of intact cells and can be used for staining cells at very low concentrations. They can be used for monitoring enzyme activity in cells at very low concentrations and are not toxic to living cells or tissues.

    摘要翻译： 本发明涉及基于细胞器活性的酸性细胞器的可视化。 本发明的细胞器底物对于细胞器的酶活性是特异性的并且标记这些细胞器，例如溶酶体，使得它们可见并容易观察。 本发明的基板包括产生荧光信号的基板。 本发明的荧光酸性细胞器酶底物被设计为在低pH值下提供高荧光，并且被衍生化以允许通过完整细胞的外部和细胞器膜的膜渗透，并且可以用于以非常低的浓度染色细胞。 它们可用于以非常低的浓度监测细胞中的酶活性，对活细胞或组织无毒性。

    Modified luciola cruciata luciferase gene and protein
    2.
    发明申请
    Modified luciola cruciata luciferase gene and protein 有权
    修饰的luciola cruciata萤光素酶基因和蛋白质

    公开(公告)号：US20120028257A1

    公开(公告)日：2012-02-02

    申请号：US12928337

    申请日：2010-12-09

    申请人： Ying Jiang ,  Daniel J. Coleman ,  John J. Naleway ,  Gabriele M. Cook

    发明人： Ying Jiang ,  Daniel J. Coleman ,  John J. Naleway ,  Gabriele M. Cook

    IPC分类号： C12Q1/66 ,  C12N15/12 ,  C12Q1/68

    CPC分类号： C12Y113/12007 ,  C12N9/0069 ,  C12Q1/008 ,  C12Q1/66

    摘要： A codon optimized and stabilized luciferase gene based upon the sequence of the natural luciferase gene isolated from Luciola cruciata (Japanese firefly) and a novel recombinant DNA characterized by incorporating this new gene coding for a novel luciferase into a vector DNA for improved activities in mammalian cells, are disclosed. This new luciferase exhibits long-wavelength light emission, as well as improved thermostability and higher expression levels in mammalian cell systems, compared to native luciferase. Assays using this new enzyme for measuring various biological metabolic functions are described.

    摘要翻译： 基于从Luciola cruciata（日本萤火虫）分离的天然荧光素酶基因的序列的密码子优化和稳定的荧光素酶基因和新型重组DNA，其特征在于将编码新的荧光素酶的该新基因并入载体DNA中以改善哺乳动物细胞的活性 ，被披露。 与天然荧光素酶相比，这种新的荧光素酶表现出长波长发光，以及哺乳动物细胞系统中改善的热稳定性和更高的表达水平。 描述了使用这种新酶测量各种生物代谢功能的测定法。

    MODIFIED LUCIOLA CRUCIATA LUCIFERASE PROTEIN
    3.
    发明申请
    MODIFIED LUCIOLA CRUCIATA LUCIFERASE PROTEIN 有权
    改良的LUCIOLA CRUCIATA LUCIFERASE蛋白

    公开(公告)号：US20120034634A1

    公开(公告)日：2012-02-09

    申请号：US12800830

    申请日：2010-05-24

    申请人： Daniel J. Coleman ,  John J. Naleway ,  Gabriele M. Cook

    发明人： Daniel J. Coleman ,  John J. Naleway ,  Gabriele M. Cook

    IPC分类号： C12N9/02 ,  C07H21/04 ,  C12Q1/66

    CPC分类号： C12N15/85 ,  C07K14/43577 ,  C12N9/0069 ,  C12N2800/107

    摘要： A codon optimized and stabilized luciferase gene based upon the sequence of the natural luciferase gene isolated from Luciola cruciata (Japanese firefly) and a novel recombinant DNA characterized by incorporating this new gene coding for a novel luciferase into a vector DNA for improved activities in mammalian cells, are disclosed. This new luciferase exhibits long-wavelength light emission, as well as improved thermostability and higher expression levels in mammalian cell systems, compared to native luciferase.

    摘要翻译： 基于从Luciola cruciata（日本萤火虫）分离的天然荧光素酶基因的序列的密码子优化和稳定的荧光素酶基因和新型重组DNA，其特征在于将编码新的荧光素酶的该新基因并入载体DNA中以改善哺乳动物细胞的活性 ，被披露。 与天然荧光素酶相比，这种新的荧光素酶表现出长波长发光，以及哺乳动物细胞系统中改善的热稳定性和更高的表达水平。

    Fluorescent haloalkyl derivatives of reporter molecules well retained in cells
    5.
    发明授权
    Fluorescent haloalkyl derivatives of reporter molecules well retained in cells 失效
    报道分子的荧光卤代烷基衍生物保留在细胞中

    公开(公告)号：US5362628A

    公开(公告)日：1994-11-08

    申请号：US26633

    申请日：1993-03-05

    申请人： Richard P. Haugland ,  Yu-Zhong Zhang ,  Ram Sabnis ,  Nels A. Olson ,  John J. Naleway ,  Rosaria P. Haugland

    发明人： Richard P. Haugland ,  Yu-Zhong Zhang ,  Ram Sabnis ,  Nels A. Olson ,  John J. Naleway ,  Rosaria P. Haugland

    IPC分类号： C12Q1/04 ,  C12Q1/34 ,  G01N33/535 ,  C12Q1/00 ,  A61K37/02 ,  C07H15/24 ,  G01N33/40

    CPC分类号： C12Q1/04 ,  C12Q1/34 ,  G01N33/535 ,  C12Q2334/00 ,  C12Q2334/20 ,  C12Q2334/40 ,  C12Q2337/20 ,  G01N2333/924 ,  Y10S530/802

    摘要： The subject invention provides a method for analyzing the metabolic activity in cells by improving the retention of a detectable reporter molecule only in intact cells where a particular enzyme is present. In particular, improved retention results from a two part process involving conjugation of haloalkyl-substituted derivatives of a reporter molecule with intracellular cysteine-containing peptides while unblocking the reporter molecule. The method for analyzing metabolic activity of cells involves the use of a substrate having the formXR-REPORTER-BLOCKwherein -BLOCK is a group selected to be removable by action of a specific analyte, to give REPORTER spectral properties different from those of the substrate,-REPORTER- is a molecule that, when no longer bound to BLOCK by a BLOCK-REPORTER bond, has spectral properities different from those of the substrate, andXR-- is a haloalkyl moiety that can covalently react with an intracellular thiol (Z--S--H) to form a thioether conjugate (Z--S--R--).After the substrate enters the cells, the analyte removes BLOCK to make REPORTER detectable by the change in spectral properties, and the haloalkyl XR reacts with the intracellular thiol to form the thioether conjugate inside the cells, which is well-retained in the cells.

    摘要翻译： 本发明提供了通过仅在存在特定酶的完整细胞中改善可检测报道分子的保留来分析细胞中的代谢活性的方法。 特别地，涉及将报道分子的卤代烷基取代的衍生物与含细胞内半胱氨酸的肽结合的两部分方法的改进的保留结果，同时解除报道分子的阻断。 用于分析细胞代谢活性的方法涉及使用具有XR-REPORTER-BLOCK形式的底物，其中-BLOCK是通过特定分析物的作用被选择为可去除的基团，以产生与底物的不同的REPORTER光谱性质 -REPORTER-是一个分子，当不再通过BLOCK-REPORTER键与BLOCK结合时，具有与底物不同的光谱性质，XR-是可以与胞内硫醇（ZSH）共价反应的卤代烷基部分， 以形成硫醚缀合物（ZSR-）。 在底物进入细胞后，分析物除去BLOCK以通过光谱性质的变化使REPORTER可检测到，并且卤代烷基XR与细胞内硫醇反应以在细胞内形成硫醚缀合物，其在细胞中良好保留。

    Modified Luciola cruciata luciferase gene and protein
    6.
    发明授权
    Modified Luciola cruciata luciferase gene and protein 有权
    修饰的Luciola cruciata萤光素酶基因和蛋白质

    公开(公告)号：US07723502B2

    公开(公告)日：2010-05-25

    申请号：US12287561

    申请日：2008-10-10

    申请人： Daniel J. Coleman ,  John J. Naleway ,  Gabriele M. Cook

    发明人： Daniel J. Coleman ,  John J. Naleway ,  Gabriele M. Cook

    IPC分类号： C07H21/02 ,  C07H21/04 ,  C12P21/06 ,  C12P21/04 ,  C12N15/00

    CPC分类号： C12N15/85 ,  C07K14/43577 ,  C12N9/0069 ,  C12N2800/107

    摘要： A codon optimized and stabilized luciferase gene based upon the sequence of the natural luciferase gene isolated from Luciola cruciata (Japanese firefly) and a novel recombinant DNA characterized by incorporating this new gene coding for a novel luciferase into a vector DNA for improved activities in mammalian cells, are disclosed. This new luciferase exhibits long-wavelength light emission, as well as improved thermostability and higher expression levels in mammalian cell systems, compared to native luciferase.

    摘要翻译： 基于从Luciola cruciata（日本萤火虫）分离的天然荧光素酶基因的序列的密码子优化和稳定的荧光素酶基因和新型重组DNA，其特征在于将编码新的荧光素酶的该新基因并入载体DNA中以改善哺乳动物细胞的活性 ，被披露。 与天然荧光素酶相比，这种新的荧光素酶表现出长波长发光，以及哺乳动物细胞系统中改善的热稳定性和更高的表达水平。

    Long wavelength lipophilic fluorogenic glycosidase substrates
    8.
    发明授权
    Long wavelength lipophilic fluorogenic glycosidase substrates 失效
    长波长磷酸荧光蛋白酶底物

    公开(公告)号：US5242805A

    公开(公告)日：1993-09-07

    申请号：US749255

    申请日：1991-08-23

    申请人： John J. Naleway ,  Yu-zhong Zhang ,  Richard P. Haugland

    发明人： John J. Naleway ,  Yu-zhong Zhang ,  Richard P. Haugland

    IPC分类号： C07H17/00 ,  C12Q1/34

    CPC分类号： C12Q1/34 ,  C07H17/00 ,  C12Q2334/00 ,  G01N2333/924

    摘要： The claimed invention relates to a substrate for evaluating glycosidic enzymes comprising a resorufin derivative of the general formula: ##STR1## wherein Gly is a carbohydrate bonded to resorufin by a glycosidic linkage; where at least one of substituents R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.8, and R.sub.9 is a lipophilic residue of the formula --L(CH.sub.2).sub.n CH.sub.3, where n is greater than 3 and less than 22, and where L is a methylene --CH.sub.2 --, an amide --NHCO--, a sulfonamide --NHSO.sub.2 --, a carboxamide --CONH--, a carboxylate ester --COO--, a urethane --NHCOO--, a urea --NHCONH--, or a thiourea --NHCSNH--; andwhere the remainder of substituents R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.8, and R.sub.9, which may be the same or different, are hydrogen, halogen, or other lipophilic residues, which may be the same or different, containing from about 1 to about 22 carbon atoms of the formula --L'(CH.sub.2).sub.m CH.sub.3, where m is less than 22, and where L' is a methylene --CH.sub.2 --, an amide --NHCO--, a sulfonamide --NHSO.sub.2 --, a carboxamide --CONH--, a carboxylate ester --COO--, a urethane --NHCOO--, a urea --NHCONH--, or a thiourea --NHCSNH--.A preferred embodiment of the invention is a non-fluorescent substrate specifically hydrolyzable by a glycosidase inside a cell to yield, after greater than about 2 minutes, an orange to red fluorescent detection product which is retained inside a viable cell more than about 2 hours at greater than about 15.degree. C. and which is non-toxic to the cell. The substrates are used for evaluating a glycosidic enzyme in living plant or animal cells whether the enzyme is present endogenously; present as a result of manipulation of the cell's genome, or added to the cell exogenously, such as by covalently binding the enzyme to a protein to form an enzyme-protein complex that enters the cell.

    摘要翻译： 所要求保护的发明涉及用于评估糖苷酶的底物，其包含通式如下的再生烯衍生物：其中Gly是通过糖苷键键合至残留的碳水化合物; 其中取代基R 1，R 2，R 4，R 6，R 8和R 9中的至少一个是式-L（CH 2）n CH 3的亲脂性残基，其中n大于3且小于22，并且其中L是亚甲基 - CH 2 - ，酰胺-NHCO-，磺酰胺-NHSO 2 - ，酰胺-CONH-，羧酸酯-COO-，氨基甲酸酯-NHCOO-，脲-NHCONH-或硫脲-NHCSNH-; 并且其中可以相同或不同的取代基R 1，R 2，R 4，R 6，R 8和R 9的其余部分是氢，卤素或可以相同或不同的其它亲脂性残基，其含有约1至 约22个碳原子的式-L'（CH 2）m CH 3，其中m小于22，其中L'是亚甲基-CH 2 - ，酰胺-NHCO-，磺酰胺-NHSO 2 - ，甲酰胺-CONH- ，羧酸酯-COO-，氨基甲酸酯-NHCOO-，脲-NHCONH-或硫脲-NHCSNH-。 本发明的优选实施方案是特异性可由细胞内的糖苷酶水解的非荧光底物，大于约2分钟后，产生橙色至红色荧光检测产物，其在活细胞内保留超过约2小时 大于约15℃，对细胞无毒性。 底物用于评价活体植物或动物细胞中的糖苷酶，无论酶是否内源性存在; 作为细胞基因组的操作或外源性加入到细胞中的结果，例如通过将酶与蛋白质共价结合以形成进入细胞的酶 - 蛋白质复合物。

    Enzyme substrates for visualizing acidic organelles
    10.
    发明授权
    Enzyme substrates for visualizing acidic organelles 有权

    公开(公告)号：US08460862B2

    公开(公告)日：2013-06-11

    申请号：US12381560

    申请日：2009-03-11

    申请人： Daniel J. Coleman ,  John J. Naleway

    发明人： Daniel J. Coleman ,  John J. Naleway

    IPC分类号： C12Q1/00

    CPC分类号： C12Q1/44 ,  C12Q1/34 ,  G01N33/5076 ,  G01N33/531 ,  G01N33/573 ,  G01N33/582

    摘要： The present invention relates to the visualization of acidic organelles based upon organelle enzyme activity. The organelle substrates of the invention are specific for enzyme activity of the organelle and label these organelles, such as lysosomes, rendering them visible and easily observed. Substrates of the present invention include substrates that produce a fluorescent signal. The fluorogenic acidic organelle enzyme substrates of this invention are designed to provide high fluorescence at low pH values and are derivatized to permit membrane permeation through both outer and organelle membranes of intact cells and can be used for staining cells at very low concentrations. They can be used for monitoring enzyme activity in cells at very low concentrations and are not toxic to living cells or tissues.