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    Biotechnology Information curation system mesh medical subject headings mw molecular weight ncbi the national center
    Curation System Mesh Medical Subject Headings Mw Molecular Weight Ncbi The National Center, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Article Title: BBP: Brucella genome annotation with literature mining and curation
    Article Snippet: .. Abbreviations Ajax Asynchronous JavaScript and XML BBP Brucella Bioinformatics Portal CDD The Conserved Domain Database CMR TIGR Comprehensive Microbial Resource COG The Clusters of Orthologous Groups EC Enzyme Commission GMOD Generic Software Components for Model Organism Databases GO Gene Ontology Limix Literature Mining and Curation System MeSH Medical Subject Headings MW Molecular weight NCBI The National Center for Biotechnology Information NIAID The National Institute of Allergy and Infectious Diseases NIH National Institutes of Health NLP Natural Language Processing SOD Superoxide Dismutase PI Isoelectric points TIGR The Institute for Genomic Research .. Authors' contributions ZX: Current webmaster, software programmer, and database administrator.

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    Article Title: BBP: Brucella genome annotation with literature mining and curation
    Article Snippet: .. Abbreviations Ajax Asynchronous JavaScript and XML BBP Brucella Bioinformatics Portal CDD The Conserved Domain Database CMR TIGR Comprehensive Microbial Resource COG The Clusters of Orthologous Groups EC Enzyme Commission GMOD Generic Software Components for Model Organism Databases GO Gene Ontology Limix Literature Mining and Curation System MeSH Medical Subject Headings MW Molecular weight NCBI The National Center for Biotechnology Information NIAID The National Institute of Allergy and Infectious Diseases NIH National Institutes of Health NLP Natural Language Processing SOD Superoxide Dismutase PI Isoelectric points TIGR The Institute for Genomic Research .. Authors' contributions ZX: Current webmaster, software programmer, and database administrator.

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    Biotechnology Information ddbj
    <t>DDBJ</t> record associated with <t>UniProt</t> Q6Q250 showing the related CDS sequence, with coding region outside of the known deposited mRNA sequence
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    Biotechnology Information dysferlin construct
    <t>Mini-dysferlin</t> C72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca 2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC 50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC 50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlin C72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlin C72 from five calcium dose–response curves ( EC 50 of ∼ 250 μM Ca 2+ , 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlin C72 is indicated with black arrows.
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    Biotechnology Information chemical molecules pubchem
    Steroid structure. (A) structure of the C21 steroid progesterone (P, used as an example), with carbon numbering and steroid ring numbering. In the storied graphics in Figures 1B and 2 , the H groups and the relative bonds will be omitted (with the exclusion of the H in 5α-reduced steroids - androstanes and pregnanes). Methyl groups will be indicated by the bonds only without the CH 3 group. (B) structures of C21 pregnene (Δ 4 and Δ 5 , i.e., double bond between C4 and C5 or between C5 and C6, respectively), pregnane (5α-reduced steroid), C19 androstene (Δ 4 , Δ 5 ) and androstane and C18 (A-ring)-aromatic estrogens. Chemical structures were designed with the aid of Sketcher V2.4 (Ihlenfeldt et al., 2009 ), available online at <t>PubChem</t> ( www.ncbi.nlm.nih.gov ; pubchem.ncbi.nlm.nih.gov ) (Kim et al., 2016 ).
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    Biotechnology Information chloroplast 16s rrna gene sequence
    Results of MRPP ordination for bacterial community composition in P. astreoides larvae, based on <t>16S</t> <t>rRNA</t> gene T-RFLP profiles. Ordination plots of T-RFLP profiles, according to the ( a ) developmental stage, ( b ) collection year and ( c ) collection location.
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    DDBJ record associated with UniProt Q6Q250 showing the related CDS sequence, with coding region outside of the known deposited mRNA sequence

    Journal: Journal of Biomedical Semantics

    Article Title: FALDO: a semantic standard for describing the location of nucleotide and protein feature annotation

    doi: 10.1186/s13326-016-0067-z

    Figure Lengend Snippet: DDBJ record associated with UniProt Q6Q250 showing the related CDS sequence, with coding region outside of the known deposited mRNA sequence

    Article Snippet: Discussion and prototyping with representatives from major sequence databases such as UniProt [ ], DDBJ (DNA Data Bank of Japan) [ ] (part of the INSDC partnership with the National Center for Biotechnology Information (NCBI)-GenBank [ ] and European Molecular Biology Laboratory (EMBL)-Bank [ ]), and a number of glycomics databases (BCSDB [ ], GlycomeDB [ ], GLYCOSCIENCES.de [ ], JCGGDB, RINGS [ ] and UniCarbKB [ ]) and assorted open source developers during these meetings led to the development of the Feature Annotation Location Description Ontology (FALDO).

    Techniques: Sequencing

    Mini-dysferlin C72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca 2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC 50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC 50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlin C72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlin C72 from five calcium dose–response curves ( EC 50 of ∼ 250 μM Ca 2+ , 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlin C72 is indicated with black arrows.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Mini-dysferlin C72 formation requires ∼200 μM extracellular calcium, broadly correlating with the extracellular calcium concentration required for calcium-dependent membrane repair of injured muscle cells. (A) Development of a flow cytometry membrane repair assay reveals 100–200 μM as the activating concentration of extracellular Ca 2+ required for calcium-dependent membrane repair pathways in cultured human muscle cells. (B) Treatment of primary human muscle cells with the calpain inhibitor calpeptin shows dose-dependent inhibition of cell survival, with an IC 50 of 11.8 ± 5.8 μM (a representative dose–response curve is shown; the calculated IC 50 is derived from four independent dose–response curves performed on different days, one with singlet samples at each dose, three in duplicate). C) Representative Western blot of a dose–response curve showing increasing formation of cleaved mini-dysferlin C72 with increasing concentrations of extracellular calcium. (D) Pooled densitometric quantification of levels of cleaved mini-dysferlin C72 from five calcium dose–response curves ( EC 50 of ∼ 250 μM Ca 2+ , 95% confidence interval). (E, F) In vitro digestion of dysferlin-exon 40a with 0.2 A.U. of purified calpain-1 (E) and calpain-2 (F). Mini-dysferlin C72 is indicated with black arrows.

    Article Snippet: Constructs Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Concentration Assay, Flow Cytometry, Cytometry, Cell Culture, Inhibition, Derivative Assay, Western Blot, In Vitro, Purification

    Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Dysferlin is cleaved in multiple cells types independent of MG53. (A, B) Injury-activated formation of mini-dysferlin C72 is calcium dependent and blocked by calpeptin and occurs in multiple cell lineages. (A) Cells were cultured to confluence and damaged by scraping in the presence or absence of Ca 2+ or the presence of Ca 2+ plus the calpain inhibitor calpeptin (Calp). Cell pellets were lysed in RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. One PVDF membrane was probed with Hamlet-1, which detects the dysferlin C-terminus and mini-dysferlin C72 (black arrowhead). The duplicate PVDF membrane was probed with Romeo, detecting the dysferlin N-terminus and corresponding cleaved N-terminal fragment (gray arrowhead). Membranes were reprobed with anti-MG53 or anti-GAPDH to show equal loading. (B) Mouse astrocytes and human umbilical vein endothelial cells do not express MG53, and thus formation of mini-dysferlin C72 occurs independently of MG53.

    Article Snippet: Constructs Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Cell Culture, SDS Page

    The calpain cleavage site in dysferlin is predicted to reside in exon 40a. (A) The apparent molecular weight of mini-dysferlin C72 (72 kDa) predicts that cleavage of dysferlin occurs between exons 40 and 41, between C2DE and C2E. (B) Exon 40a bears a consensus site for calpain cleavage (GPS-CCD, ccd.biocuckoo.org; Liu et al. , 2011 ). (C) Alignment of exon 40a between dysferlin paralogues reveals only moderate preservation of amino acid sequence. However, exon 40a sequences in all species possess a putative calpain cleavage site, in each case with maximum likelihood of cleavage LAPTNTA–SPPSSPH.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: The calpain cleavage site in dysferlin is predicted to reside in exon 40a. (A) The apparent molecular weight of mini-dysferlin C72 (72 kDa) predicts that cleavage of dysferlin occurs between exons 40 and 41, between C2DE and C2E. (B) Exon 40a bears a consensus site for calpain cleavage (GPS-CCD, ccd.biocuckoo.org; Liu et al. , 2011 ). (C) Alignment of exon 40a between dysferlin paralogues reveals only moderate preservation of amino acid sequence. However, exon 40a sequences in all species possess a putative calpain cleavage site, in each case with maximum likelihood of cleavage LAPTNTA–SPPSSPH.

    Article Snippet: Constructs Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Molecular Weight, Preserving, Sequencing

    Exon 40a–containing dysferlin is ubiquitously expressed, and mini-dysferlin C72 can be generated in multiple tissues. (A) Exon 40a is widely expressed in human tissues (∼40–60% transcripts), with lower relative levels in skeletal muscle, heart, and brain (∼10–15% transcripts). Dysferlin alternately spliced exons 5a, 17, and 40a were PCR amplified from a human tissue cDNA panel (Clontech) using primers flanking each of the exons. PCR amplification was performed for 30, 35, and 40 cycles to derive a simple standard curve and control for saturation. Ctrl; plasmid control. (B) Endogenous dysferlin from multiple tissues is cleaved by calpains in vitro, releasing mini-dysferlin C72 . Mouse tissues were sectioned and lysed in RIPA, and endogenous dysferlin was immunoprecipitated with Romeo and protein G–Sepharose. Dysferlin-bound Sepharose beads were incubated with 0.2 active unit (A.U.) of purified recombinant calpain-1 at 30°C for 10 s in the presence of 2 mM CaCl 2 . Dysferlin was detected by Western analysis with the C-terminal antibody Hamlet-1. Mini-dysferlin C72­ is indicated with a black arrow. (C) An anti–exon 40a antibody (α-40a) is specific to exon 40a-containing dysferlin in transfected HEK293 cells. Membranes were probed with anti–exon 40a and then reprobed with Hamlet-1 to reveal total dysferlin expression. GAPDH indicates even loading. (D) Anti–exon 40a antibody recognizes full-length dysferlin-exon 40a and cleaved mini-dysferlin C72 but not the N-terminal counterfragment. Dysferlin was immunopurified from transfected HEK293 cells and subject to in vitro calpain cleavage. R1 (Romeo) reveals the N-terminal counterfragment, H1 reveals mini-dysferlin C72 , and α-40a shows reactivity to full-length dysferlin and mini-dysferlin C72 . (E) Dysferlin exon 40a is expressed at similar levels in human muscle and heart. Total dysferlin was immunoprecipitated with Hamlet-1 from three control human muscles (1–3, ages 5, 18, and 37 yr, respectively, from young adults subject to testing for malignant hypothermia and shown to be normal) and two human hearts (1 and 2, donor hearts from young adults). Dysferlin-exon 40a was identified by Western blot with pAb α-40a. Membranes were reprobed with Romeo to reveal total immunoprecipitated dysferlin.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Exon 40a–containing dysferlin is ubiquitously expressed, and mini-dysferlin C72 can be generated in multiple tissues. (A) Exon 40a is widely expressed in human tissues (∼40–60% transcripts), with lower relative levels in skeletal muscle, heart, and brain (∼10–15% transcripts). Dysferlin alternately spliced exons 5a, 17, and 40a were PCR amplified from a human tissue cDNA panel (Clontech) using primers flanking each of the exons. PCR amplification was performed for 30, 35, and 40 cycles to derive a simple standard curve and control for saturation. Ctrl; plasmid control. (B) Endogenous dysferlin from multiple tissues is cleaved by calpains in vitro, releasing mini-dysferlin C72 . Mouse tissues were sectioned and lysed in RIPA, and endogenous dysferlin was immunoprecipitated with Romeo and protein G–Sepharose. Dysferlin-bound Sepharose beads were incubated with 0.2 active unit (A.U.) of purified recombinant calpain-1 at 30°C for 10 s in the presence of 2 mM CaCl 2 . Dysferlin was detected by Western analysis with the C-terminal antibody Hamlet-1. Mini-dysferlin C72­ is indicated with a black arrow. (C) An anti–exon 40a antibody (α-40a) is specific to exon 40a-containing dysferlin in transfected HEK293 cells. Membranes were probed with anti–exon 40a and then reprobed with Hamlet-1 to reveal total dysferlin expression. GAPDH indicates even loading. (D) Anti–exon 40a antibody recognizes full-length dysferlin-exon 40a and cleaved mini-dysferlin C72 but not the N-terminal counterfragment. Dysferlin was immunopurified from transfected HEK293 cells and subject to in vitro calpain cleavage. R1 (Romeo) reveals the N-terminal counterfragment, H1 reveals mini-dysferlin C72 , and α-40a shows reactivity to full-length dysferlin and mini-dysferlin C72 . (E) Dysferlin exon 40a is expressed at similar levels in human muscle and heart. Total dysferlin was immunoprecipitated with Hamlet-1 from three control human muscles (1–3, ages 5, 18, and 37 yr, respectively, from young adults subject to testing for malignant hypothermia and shown to be normal) and two human hearts (1 and 2, donor hearts from young adults). Dysferlin-exon 40a was identified by Western blot with pAb α-40a. Membranes were reprobed with Romeo to reveal total immunoprecipitated dysferlin.

    Article Snippet: Constructs Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Generated, Polymerase Chain Reaction, Amplification, Plasmid Preparation, In Vitro, Immunoprecipitation, Incubation, Purification, Recombinant, Western Blot, Transfection, Expressing

    Dysferlin exon 40a and calpain recruit to sites of membrane injury. Cultured MO3.13 secondary oligodendrocytes (row 1) and primary human myotubes (row 2) were shot with 4-μm silica beads using a Bio-Rad Helios Gene Gun, fixed at 10 s postinjury in cold 3% paraformaldehyde, and then permeabilized and immunolabeled (see Materials and Methods ). Romeo was applied for 2 h before Hamlet-1 to bias the detection of the N-terminal dysferlin epitope. Dysferlin was detectable only at sites of membrane injury with Hamlet-1 (rows 1 and 2). Staining with an antibody raised to dysferlin exon 40a revealed exon 40a–containing dysferlin recruits to sites of injury within 10 s (row 3). Calpain-2 was detectable at sites of membrane injury at 2 s (T2, row 4) and 10 s postdamage (T10, row 5). Large-injury sites often showed a void of negative labeling for calpain-2 (T10, row 6), suggesting that calpain might be extracted or escape from large injuries. Scale bar, 5 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Dysferlin exon 40a and calpain recruit to sites of membrane injury. Cultured MO3.13 secondary oligodendrocytes (row 1) and primary human myotubes (row 2) were shot with 4-μm silica beads using a Bio-Rad Helios Gene Gun, fixed at 10 s postinjury in cold 3% paraformaldehyde, and then permeabilized and immunolabeled (see Materials and Methods ). Romeo was applied for 2 h before Hamlet-1 to bias the detection of the N-terminal dysferlin epitope. Dysferlin was detectable only at sites of membrane injury with Hamlet-1 (rows 1 and 2). Staining with an antibody raised to dysferlin exon 40a revealed exon 40a–containing dysferlin recruits to sites of injury within 10 s (row 3). Calpain-2 was detectable at sites of membrane injury at 2 s (T2, row 4) and 10 s postdamage (T10, row 5). Large-injury sites often showed a void of negative labeling for calpain-2 (T10, row 6), suggesting that calpain might be extracted or escape from large injuries. Scale bar, 5 μm.

    Article Snippet: Constructs Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Cell Culture, Immunolabeling, Staining, Labeling

    Cleavage of dysferlin to form mini-dysferlin C72 is conferred by exon 40a. (A) Untransfected HEK293 cells, as well as HEK293 transfected with dysferlin expression constructs with (+40a) or without exon 40a, were subjected to scrape injury 24 h posttransfection in the presence or absence of calcium. Only dysferlin expression constructs bearing exon 40a demonstrate injury-activated, calcium-dependent formation of the C-terminal mini-dysferlin C72 fragment (lane 6, Hamlet-1 and anti-Myc, black arrows). The N-terminal counterfragment can be detected with Romeo-1 (lane 6, gray arrow). Membranes were reprobed for loading controls GAPDH and β-tubulin. (B) Ubiquitous calpains specifically cleave exon 40a–containing dysferlin. MEFs were transfected by electroporation with dysferlin expression constructs with or without exon 40a and harvested 24 h posttransfection via scrape injury in the presence of calcium. Injury-activated formation of mini-dysferlin C72 requires exon 40a and is observed in wild-type MEFs (WT) but not in MEFs from CAPNS1 -knockout mice (−/−) deficient for calpain-1 and -2. Retroviral rescue of CAPNS1 in knockout (−/−R) MEFs restores calpain expression (see CAPN2 immunoblot) to levels exceeding that in WT cells and increases injury-induced dysferlin cleavage. Mini-dysferlin C72 is indicated with asterisks. (C) Dysferlin bearing exon 40a is specifically cleaved by either calpain-1 or -2 in vitro, forming mini-dysferlin C72 . Enhanced GFP–dysferlin FLAG was immunoprecipitated with anti-dysferlin (Romeo) and protein G–Sepharose (see Materials and Methods ). Sepharose beads were incubated with the indicated dilutions of purified recombinant calpain-1 or -2 at 30°C for 3 min in the presence of 2 mM CaCl 2 . Digested samples were analyzed by SDS–PAGE and Western blot. Dysferlin bearing exon 40a is specifically cleaved by both calpain-1 and -2 to form mini-dysferlin C72 (black arrowhead), whereas dysferlin without exon 40a remains uncleaved.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Cleavage of dysferlin to form mini-dysferlin C72 is conferred by exon 40a. (A) Untransfected HEK293 cells, as well as HEK293 transfected with dysferlin expression constructs with (+40a) or without exon 40a, were subjected to scrape injury 24 h posttransfection in the presence or absence of calcium. Only dysferlin expression constructs bearing exon 40a demonstrate injury-activated, calcium-dependent formation of the C-terminal mini-dysferlin C72 fragment (lane 6, Hamlet-1 and anti-Myc, black arrows). The N-terminal counterfragment can be detected with Romeo-1 (lane 6, gray arrow). Membranes were reprobed for loading controls GAPDH and β-tubulin. (B) Ubiquitous calpains specifically cleave exon 40a–containing dysferlin. MEFs were transfected by electroporation with dysferlin expression constructs with or without exon 40a and harvested 24 h posttransfection via scrape injury in the presence of calcium. Injury-activated formation of mini-dysferlin C72 requires exon 40a and is observed in wild-type MEFs (WT) but not in MEFs from CAPNS1 -knockout mice (−/−) deficient for calpain-1 and -2. Retroviral rescue of CAPNS1 in knockout (−/−R) MEFs restores calpain expression (see CAPN2 immunoblot) to levels exceeding that in WT cells and increases injury-induced dysferlin cleavage. Mini-dysferlin C72 is indicated with asterisks. (C) Dysferlin bearing exon 40a is specifically cleaved by either calpain-1 or -2 in vitro, forming mini-dysferlin C72 . Enhanced GFP–dysferlin FLAG was immunoprecipitated with anti-dysferlin (Romeo) and protein G–Sepharose (see Materials and Methods ). Sepharose beads were incubated with the indicated dilutions of purified recombinant calpain-1 or -2 at 30°C for 3 min in the presence of 2 mM CaCl 2 . Digested samples were analyzed by SDS–PAGE and Western blot. Dysferlin bearing exon 40a is specifically cleaved by both calpain-1 and -2 to form mini-dysferlin C72 (black arrowhead), whereas dysferlin without exon 40a remains uncleaved.

    Article Snippet: Constructs Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Transfection, Expressing, Construct, Electroporation, Knock-Out, Mouse Assay, In Vitro, Immunoprecipitation, Incubation, Purification, Recombinant, SDS Page, Western Blot

    Calpain cleaves otoferlin and myoferlin in addition to dysferlin. (A) Calpain rapidly cleaves immunoprecipitated ferlin proteins in vitro. Dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag were immunoprecipitated with anti-myc and protein G–Sepharose (see Materials and Methods ). Dysferlin-bound Sepharose beads were incubated with purified 0.2 A.U. of recombinant calpain-1 at 30°C for 2 or 10 s in the presence of 2 mM CaCl 2. Proteolysis was rapidly inhibited by reconstitution of the reaction in SDS lysis buffer and heating to 94°C. Digested samples were analyzed by SDS–PAGE and Western blot. Top, C-terminal fragments detected with anti-myc (dysferlin) or anti-Flag (myoferlin and otoferlin). Bottom, N-terminal fragments detected by N-terminal (Romeo-dysferlin) or internal antibodies (7D2, myoferlin; C12, otoferlin). (B) Dysferlin and otoferlin display damage-dependent cleavage, whereas myoferlin cleavage appears to be constitutive. HEK293 cells were transfected with dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag and lysed in calcium-free RIPA (lane 1), RIPA containing 900 μM calcium (permissive for calpain cleavage), or damaged by scraping in the presence of calcium. Scraped cell pellets were lysed in calcium-free RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. Dysferlin was detected with anti-Myc; otoferlin and myoferlin were detected with anti-Flag. (C) Diagram of the predicted calpain cleavage sites within dysferlin, otoferlin, and myoferlin (schematic produced using DOG 2.0; Ren et al. , 2009 ). Molecular weight calculation of the cleaved C-terminal modules was used to elucidate the most likely calpain cleavage site ( ccd.biocuckoo.org ). In each case, the C-terminal fragments released by calpain cleavage represent transmembrane-anchored, dual-C2-domain modules.

    Journal: Molecular Biology of the Cell

    Article Title: Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    doi: 10.1091/mbc.E14-04-0947

    Figure Lengend Snippet: Calpain cleaves otoferlin and myoferlin in addition to dysferlin. (A) Calpain rapidly cleaves immunoprecipitated ferlin proteins in vitro. Dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag were immunoprecipitated with anti-myc and protein G–Sepharose (see Materials and Methods ). Dysferlin-bound Sepharose beads were incubated with purified 0.2 A.U. of recombinant calpain-1 at 30°C for 2 or 10 s in the presence of 2 mM CaCl 2. Proteolysis was rapidly inhibited by reconstitution of the reaction in SDS lysis buffer and heating to 94°C. Digested samples were analyzed by SDS–PAGE and Western blot. Top, C-terminal fragments detected with anti-myc (dysferlin) or anti-Flag (myoferlin and otoferlin). Bottom, N-terminal fragments detected by N-terminal (Romeo-dysferlin) or internal antibodies (7D2, myoferlin; C12, otoferlin). (B) Dysferlin and otoferlin display damage-dependent cleavage, whereas myoferlin cleavage appears to be constitutive. HEK293 cells were transfected with dysferlin MycHis , otoferlin MycFlag , and myoferlin MycFlag and lysed in calcium-free RIPA (lane 1), RIPA containing 900 μM calcium (permissive for calpain cleavage), or damaged by scraping in the presence of calcium. Scraped cell pellets were lysed in calcium-free RIPA, and 10 μg of protein was separated by SDS–PAGE and transferred onto PVDF membrane. Dysferlin was detected with anti-Myc; otoferlin and myoferlin were detected with anti-Flag. (C) Diagram of the predicted calpain cleavage sites within dysferlin, otoferlin, and myoferlin (schematic produced using DOG 2.0; Ren et al. , 2009 ). Molecular weight calculation of the cleaved C-terminal modules was used to elucidate the most likely calpain cleavage site ( ccd.biocuckoo.org ). In each case, the C-terminal fragments released by calpain cleavage represent transmembrane-anchored, dual-C2-domain modules.

    Article Snippet: Constructs Our dysferlin construct (EGFP-FL-DYSF pcDNA3.1, National Center for Biotechnology Information [NCBI] reference sequence NP_003485.1) was a generous gift from Kate Bushby (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subcloned into pIRES2 EGFP (OriGene).

    Techniques: Immunoprecipitation, In Vitro, Incubation, Purification, Recombinant, Lysis, SDS Page, Western Blot, Transfection, Produced, Molecular Weight

    Steroid structure. (A) structure of the C21 steroid progesterone (P, used as an example), with carbon numbering and steroid ring numbering. In the storied graphics in Figures 1B and 2 , the H groups and the relative bonds will be omitted (with the exclusion of the H in 5α-reduced steroids - androstanes and pregnanes). Methyl groups will be indicated by the bonds only without the CH 3 group. (B) structures of C21 pregnene (Δ 4 and Δ 5 , i.e., double bond between C4 and C5 or between C5 and C6, respectively), pregnane (5α-reduced steroid), C19 androstene (Δ 4 , Δ 5 ) and androstane and C18 (A-ring)-aromatic estrogens. Chemical structures were designed with the aid of Sketcher V2.4 (Ihlenfeldt et al., 2009 ), available online at PubChem ( www.ncbi.nlm.nih.gov ; pubchem.ncbi.nlm.nih.gov ) (Kim et al., 2016 ).

    Journal: Frontiers in Pharmacology

    Article Title: Intracrine Regulation of Estrogen and Other Sex Steroid Levels in Endometrium and Non-gynecological Tissues; Pathology, Physiology, and Drug Discovery

    doi: 10.3389/fphar.2018.00940

    Figure Lengend Snippet: Steroid structure. (A) structure of the C21 steroid progesterone (P, used as an example), with carbon numbering and steroid ring numbering. In the storied graphics in Figures 1B and 2 , the H groups and the relative bonds will be omitted (with the exclusion of the H in 5α-reduced steroids - androstanes and pregnanes). Methyl groups will be indicated by the bonds only without the CH 3 group. (B) structures of C21 pregnene (Δ 4 and Δ 5 , i.e., double bond between C4 and C5 or between C5 and C6, respectively), pregnane (5α-reduced steroid), C19 androstene (Δ 4 , Δ 5 ) and androstane and C18 (A-ring)-aromatic estrogens. Chemical structures were designed with the aid of Sketcher V2.4 (Ihlenfeldt et al., 2009 ), available online at PubChem ( www.ncbi.nlm.nih.gov ; pubchem.ncbi.nlm.nih.gov ) (Kim et al., 2016 ).

    Article Snippet: We also would like to thank the management and curator of the online databases we made use of, namely: the database of chemical molecules PubChem ( www.ncbi.nlm.nih.gov ; pubchem.ncbi.nlm.nih.gov ) maintained by the National Center for Biotechnology Information (NCBI; National Library of Medicine/United States National Institutes of Health - NIH); Chemical Abstracts Service (CAS), maintained by the American Chemical Society ( www.cas.org ); Human Metabolome Data Base (HMDB, www.hmdb.ca ), funded and maintained by Genome Canada; Chemical Book ( www.chemicalbook.com ), funded by industrial partners; Chemical Entities of Biological Interest (ChEBI; www.ebi.ac.uk/chebi/init.do ), curated by the European Bioinformatics Institute of the European Molecular Biology Laboratory (EMBL); drug and drug target database Drugbank ( www.drugbank.ca/drugs ), University of Alberta and The Metabolomics Innovation Centre; GeneCards ( www.genecards.org ), developed and maintained by the Crown Human Genome Center at the Weizmann Institute of Science; Online Mendelian Inheritance in Man® (OMIM®, https://omim.org/ ), McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD); Mouse Genome Informatics (MGI; www.informatics.jax.org ), Mouse Genome Database at the Mouse Genome Informatics website, The Jackson Laboratory, Bar Harbor, Maine.

    Techniques:

    Results of MRPP ordination for bacterial community composition in P. astreoides larvae, based on 16S rRNA gene T-RFLP profiles. Ordination plots of T-RFLP profiles, according to the ( a ) developmental stage, ( b ) collection year and ( c ) collection location.

    Journal: The ISME Journal

    Article Title: Diversity and dynamics of bacterial communities in early life stages of the Caribbean coral Porites astreoides

    doi: 10.1038/ismej.2011.144

    Figure Lengend Snippet: Results of MRPP ordination for bacterial community composition in P. astreoides larvae, based on 16S rRNA gene T-RFLP profiles. Ordination plots of T-RFLP profiles, according to the ( a ) developmental stage, ( b ) collection year and ( c ) collection location.

    Article Snippet: Though > 96 products were cloned and sequenced from each stage, many of the sequenced clones matched most closely to eukaryotic 18S rRNA or chloroplast 16S rRNA gene sequence (GenBank/NCBI(National Center for Biotechnology Information)).

    Techniques: