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Sample records for casein kinase ii

  1. Nucleolin (C23), a physiological substrate for casein kinase II

    DEFF Research Database (Denmark)

    Schneider, H R; Issinger, O G

    1988-01-01

    Nucleolin (C23), a 110 kDa phosphoprotein, which is mainly found in the nucleolus has been shown to be a physiological substrate for casein kinase II (CKII). Nucleolin was identified and characterized by immunodetection using an anti-nucleolin antibody. Phosphopeptide patterns from nucleolin...... phosphorylated by purified casein kinase II and of phosphorylated nucleolin which had been isolated from tumor cells grown in the presence of [32P]-o-phosphate, were identical. The partial tryptic digest revealed nine phosphopeptides. Nucleolin isolated from Krebs II mouse ascites cells was phosphorylated...... by purified casein kinase II with about two moles phosphate per one mole of nucleolin....

  2. Enhanced casein kinase II activity in human tumour cell cultures

    DEFF Research Database (Denmark)

    Prowald, K; Fischer, H; Issinger, O G

    1984-01-01

    Casein kinase II (CKII) activity is enhanced as much as 2-3 fold in established and 4-5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells...

  3. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    subunits are highly conserved during evolution. The relationship between CK-2 alpha from humans and plants is still 73%. Similar relationships are reported for the beta-subunit. Chromosomal assignment of CK-2 alpha shows two gene loci, one of which is a pseudogene. They are located on different chromosomes......, no genetic changes are necessarily involved; the observed changes may be entirely due to a signal transduction pathway where CK-2 could be phosphorylated by another kinase(s). CK-2 cDNAs from various organisms have been isolated and characterized. From the deduced amino acid sequence it turns out that CK-2......-subunit affecting: (i) stability, (ii) enzyme specificity and (iii) enzyme activity. The question where CK-2 and its subunits are located throughout the cell cycle has also been addressed, mainly because of the large discrepancies that still exist between results obtained by different investigators. Tissue...

  4. Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

    DEFF Research Database (Denmark)

    Grankowski, N; Boldyreff, B; Issinger, O G

    1991-01-01

    cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most...... of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step...... on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses...

  5. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  6. Functional analysis of Trichoderma reesei CKIIα2, a catalytic subunit of casein kinase II.

    Science.gov (United States)

    Wang, Mingyu; Yang, Hui; Zhang, Meiling; Liu, Kuimei; Wang, Hanbin; Luo, Yi; Fang, Xu

    2015-07-01

    Trichoderma reesei is the most important industrial cellulase-producing filamentous fungus. Although its molecular physiology has been investigated, the signal transduction pathways are not fully understood. In particular, the role of casein kinase II (CKII) is not yet clear. In this work, we carried out functional investigations on a catalytic subunit of CKII, CKIIα2. Comparison of the phenotypic features of T. reesei parent and Δck2α2 strains showed significant changes following ck2α2 disruption. T. reesei Δck2α2 form significantly smaller mycelial pellets in glucose-containing liquid minimum media, have shorter and fewer branch hyphae, produce smaller amounts of chitinases, produce more spores, show more robust growth on glucose-containing agar plates, and consume glucose at a significantly higher rate. Suggestions can be made that CKIIα2 governs chitinase expression, and the disruption of ck2α2 results in lower levels of chitinase production, leading to a weaker cell wall disruption capability, further resulting in weaker hyphal branching, which eventually leads to smaller mycelial pellets in liquid media. Further conclusions can be made that CKIIα2 is involved in repression of sporulation and glucose metabolism, which is consistent with the proposal that CKIIα2 represses global metabolism. These observations make the deletion of ck2α2 a potentially beneficial genetic disruption for T. reesei during industrial applications, as smaller mycelial pellets, more spores and more robust glucose metabolism are all desired traits for industrial fermentation. This work reports novel unique functions of a CKII catalytic subunit and is also the first genetic and physiological investigation on CKII in T. reesei.

  7. Growth-dependent modulation of casein kinase II and its substrate nucleolin in primary human cell cultures and HeLa cells

    DEFF Research Database (Denmark)

    Schneider, H R; Issinger, O G

    1989-01-01

    We have previously provided evidence that casein kinase II (CKII) and its substrate nucleolin increase concomitantly during certain development stages during embryogenesis (Schneider et al., Eur. J. Biochem. 161, 733-738). We now show that during normal growth of primary cell cultures and HeLa...

  8. Casein kinase II induced polymerization of soluble TDP-43 into filaments is inhibited by heat shock proteins.

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    Yari Carlomagno

    Full Text Available BACKGROUND: Trans-activation Response DNA-binding Protein-43 (TDP-43 lesions are observed in Amyotrophic Lateral Sclerosis (ALS, Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP and 25-50% of Alzheimer's Disease (AD cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10-12 nm filaments though wider 18-20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades. RESULTS: The protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization. CONCLUSION

  9. Casein kinase II is required for proper cell division and acts as a negative regulator of centrosome duplication in Caenorhabditis elegans embryos

    Science.gov (United States)

    Medley, Jeffrey C.; Kabara, Megan M.; Stubenvoll, Michael D.; DeMeyer, Lauren E.

    2017-01-01

    ABSTRACT Centrosomes are the primary microtubule-organizing centers that orchestrate microtubule dynamics during the cell cycle. The correct number of centrosomes is pivotal for establishing bipolar mitotic spindles that ensure accurate segregation of chromosomes. Thus, centrioles must duplicate once per cell cycle, one daughter per mother centriole, the process of which requires highly coordinated actions among core factors and modulators. Protein phosphorylation is shown to regulate the stability, localization and activity of centrosome proteins. Here, we report the function of Casein kinase II (CK2) in early Caenorhabditis elegans embryos. The catalytic subunit (KIN-3/CK2α) of CK2 localizes to nuclei, centrosomes and midbodies. Inactivating CK2 leads to cell division defects, including chromosome missegregation, cytokinesis failure and aberrant centrosome behavior. Furthermore, depletion or inhibiting kinase activity of CK2 results in elevated ZYG-1 levels at centrosomes, restoring centrosome duplication and embryonic viability to zyg-1 mutants. Our data suggest that CK2 functions in cell division and negatively regulates centrosome duplication in a kinase-dependent manner. PMID:27881437

  10. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

  11. Drosophila casein kinase I alpha regulates homolog pairing and genome organization by modulating condensin II subunit Cap-H2 levels.

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    Huy Q Nguyen

    Full Text Available The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.

  12. Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

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    Nguyen, Huy Q.; Nye, Jonathan; Buster, Daniel W.; Klebba, Joseph E.; Rogers, Gregory C.; Bosco, Giovanni

    2015-01-01

    The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCFSlimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein. PMID:25723539

  13. Pharmacological and safety evaluation of CIGB-300, a casein kinase 2 inhibitor peptide, administered intralesionally to patients with cervical cancer stage IB2/II

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    Soriano-García JL

    2013-08-01

    Full Text Available CIGB-300 is a pro-apoptotic casein kinase 2 inhibitor peptide with potential anticancer action. An open-label and dose scaling Phase I trial was carried out to investigate the peptide tumor uptake, pharmacokinetics, toxicity, and levels of a CIGB-300 response biomarker in patients with cervical cancer stage IB2/II. Fourteen patients were included; six of them received 35 mg, 6 received 70 mg and the two remaining patients received 245 mg of CIGB-300 prior chemoradiotherapy. CIGB-300 was applied by intratumor injections during 5-consecutive days. For pharmacokinetic and biodistribution studies, the peptide was radiolabeled with 99mTc in the first administration and whole body gammagraphy and plasma testing were done during 48 h. Data showed that the maximum tolerated dose was 70 mg for CIGB-300 in this clinical setting. Furthermore, an allergic-like syndrome was identified as the dose limiting toxicity, which was well-correlated with plasmatic histamine levels. Importantly, the mean tumor uptake was 14.9 mg and 10.4 mg for CIGB-300 doses of 35 and 70 mg, respectively. Also, the kidneys were the main target organ for drug elimination. Finally, treatment with CIGB-300 significantly reduced the B23/nucleophosmin levels in tumor specimens. CIGB-300 meets potentialities to be tested in future trials in a neoadjuvant setting prior to chemoradiotherapy in cervical cancer.

  14. Identification of a BET Family Bromodomain/Casein Kinase II/TAF-Containing Complex as a Regulator of Mitotic Condensin Function

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    Hyun-Soo Kim

    2014-03-01

    Full Text Available Condensin is a central regulator of mitotic genome structure with mutants showing poorly condensed chromosomes and profound segregation defects. Here, we identify NCT, a complex comprising the Nrc1 BET-family tandem bromodomain protein (SPAC631.02, casein kinase II (CKII, and several TAFs, as a regulator of condensin function. We show that NCT and condensin bind similar genomic regions but only briefly colocalize during the periods of chromosome condensation and decondensation. This pattern of NCT binding at the core centromere, the region of maximal condensin enrichment, tracks the abundance of acetylated histone H4, as regulated by the Hat1-Mis16 acetyltransferase complex and recognized by the first Nrc1 bromodomain. Strikingly, mutants in NCT or Hat1-Mis16 restore the formation of segregation-competent chromosomes in cells containing defective condensin. These results are consistent with a model where NCT targets CKII to chromatin in a cell-cycle-directed manner in order to modulate the activity of condensin during chromosome condensation and decondensation.

  15. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

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    Albertella, M.R.; Jones, H.; Thomson, W. [Oxford Univ. (United Kingdom)] [and others

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  16. Ser2 is the autophosphorylation site in the beta subunit from bicistronically expressed human casein kinase-2 and from native rat liver casein kinase-2 beta

    DEFF Research Database (Denmark)

    Boldyreff, B; James, P; Staudenmann, W;

    1993-01-01

    Human casein kinase-2 (CK-2) subunits alpha and beta were bicistronically expressed in bacteria. The recombinant holoenzyme shared all investigated properties with the native CK-2 from mammalian sources (rat liver, Krebs II mouse ascites tumour cells). Contrary to recombinant human CK-2 produced...

  17. Casein kinase-2 structure-function relationship

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A

    1992-01-01

    Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form......, consequently, stimulation of activity. This indicates that residue(s) located in the 171-209 sequence is essential for reconstitution. Also a four residue's N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55......-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme. However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete. There were also intermediate complexes, free alpha-subunit and beta...

  18. Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity

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    Park, J.-W.; Moon, C.H.; Harmache, A.; Wargo, A.R.; Purcell, M.K.; Bremont, M.; Kurath, G.

    2011-01-01

    casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 μm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

  19. Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity.

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    Park, J W; Moon, C H; Harmache, A; Wargo, A R; Purcell, M K; Bremont, M; Kurath, G

    2011-02-01

    casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 μm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.

  20. Expression of casein kinase 2 during mouse embryogenesis

    DEFF Research Database (Denmark)

    Mestres, P; Boldyreff, B; Ebensperger, C;

    1994-01-01

    This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level by immunohisto......This paper deals with the expression and distribution of casein kinase 2 (CK-2) subunits in mouse embryos at different developmental stages. Expression was investigated at the mRNA level of CK-2 alpha- and beta-subunits by in situ hybridization and distribution at the protein level...

  1. Purification and properties of a ribosomal casein kinase from rabbit reticulocytes

    DEFF Research Database (Denmark)

    Issinger, O G

    1977-01-01

    suggested that the casein kinase is a dimer composed of subunits of identical molecular weight. The enzyme utilizes GTP as well as ATP as a phosphoryl donor. It preferentially phosphorylates acidic proteins, in particular the model substrates casein and phosvitin. Casein kinase is cyclic AMP...

  2. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I;

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  3. Subcellular localization of casein kinase I

    DEFF Research Database (Denmark)

    Grankowski, N; Issinger, O G

    1990-01-01

    An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus....

  4. Mayaro virus infection cycle relies on casein kinase 2 activity.

    Science.gov (United States)

    Barroso, Madalena M S; Lima, Carla S; Silva-Neto, Mário A C; Da Poian, Andrea T

    2002-09-06

    Replication of Mayaro virus in Vero cells induces dramatic cytopathic effects and cell death. In this study, we have evaluated the role of casein kinase 2 (CK2) during Mayaro virus infection cycle. We found that CK2 was activated during the initial stages of infection ( approximately 36% after 4h). This activation was further confirmed when the enzyme was partially purified from the cellular lysate either by Mono Q 5/5Hr column or heparin-agarose column. Using this later column, we found that the elution profile of CK2 activity from infected cells was different from that obtained for control cell enzyme, suggesting a structural modification of CK2 after infection. Treatment of infected cells with a cell-permeable inhibitor of CK2, dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB), abolished the cytopathic effect in a dose-dependent manner. Together this set of data demonstrates for the first time that CK2 activity in host cells is required in Mayaro virus infection cycle.

  5. Casein kinase Iepsilon modulates the signaling specificities of dishevelled.

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    Cong, Feng; Schweizer, Liang; Varmus, Harold

    2004-03-01

    Wnt signaling is critical to many aspects of development, and aberrant activation of the Wnt signaling pathway can cause cancer. Dishevelled (Dvl) protein plays a central role in this pathway by transducing the signal from the Wnt receptor complex to the beta-catenin destruction complex. Dvl also plays a pivotal role in the planar cell polarity pathway that involves the c-Jun N-terminal kinase (JNK). How functions of Dvl are regulated in these two distinct pathways is not clear. We show that deleting the C-terminal two-thirds of Dvl, which includes the PDZ and DEP domains and is essential for Dvl-induced JNK activation, rendered the molecule a much more potent activator of the beta-catenin pathway. We also found that casein kinase Iepsilon (CKIepsilon), a previously identified positive regulator of Wnt signaling, stimulated Dvl activity in the Wnt pathway, but dramatically inhibited Dvl activity in the JNK pathway. Consistent with this, overexpression of CKIepsilon in Drosophila melanogaster stimulated Wnt signaling and disrupted planar cell polarity. We also observed a correlation between the localization and the signaling activity of Dvl in the beta-catenin pathway and the JNK pathway. Furthermore, by using RNA interference, we demonstrate that the Drosophila CKIepsilon homologue Double time positively regulates the beta-catenin pathway through Dvl and negatively regulates the Dvl-induced JNK pathway. We suggest that CKIepsilon functions as a molecular switch to direct Dvl from the JNK pathway to the beta-catenin pathway, possibly by altering the conformation of the C terminus of Dvl.

  6. The beta subunit of casein kinase II

    DEFF Research Database (Denmark)

    Boldyreff, B; Piontek, K; Schmidt-Spaniol, I;

    1991-01-01

    cDNAs encoding the beta subunit of pig and mouse CKII were isolated. The porcine cDNA was expressed as a fusion protein in Escherichia coli and used for the production of anti-CKII-beta subunit specific antibodies....

  7. Phosphorylation of acidic ribosomal proteins from rabbit reticulocytes by a ribosome-associated casein kinase

    DEFF Research Database (Denmark)

    Issinger, O G

    1977-01-01

    Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate polyacryl......Two acidic proteins from 80-S ribosomes were isolated and purified to homogeneity. The purified acidic proteins could be phosphorylated by casein kinase using [gamma-32P]ATP and [gamma-32P]GTP as a phosphoryl donor. The proteins became phosphorylated in situ, too. Sodium dodecyl sulfate...

  8. Cloning and sequencing of the casein kinase 2 alpha subunit from Zea mays

    DEFF Research Database (Denmark)

    Dobrowolska, G; Boldyreff, B; Issinger, O G

    1991-01-01

    The nucleotide sequence of the cDNA coding for the alpha subunit of casein kinase 2 of Zea mays has been determined. The cDNA clone contains an open reading frame of 996 nucleotides encoding a polypeptide comprising 332 amino acids. The primary amino acid sequence exhibits 75% identity to the alpha...

  9. Reconstitution of normal and hyperactivated forms of casein kinase-2 by variably mutated beta-subunits

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A;

    1993-01-01

    Twenty-one mutants of the noncatalytic beta-subunit of human casein kinase-2 have been created, expressed in Escherichia coli, and purified to homogeneity. They are either modified at the autophosphorylation site (mutants beta delta 1-4 and beta A 5,6) or bear variable deletions in their C...

  10. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

    Science.gov (United States)

    Luz, Simão; Kongsuphol, Patthara; Mendes, Ana Isabel; Romeiras, Francisco; Sousa, Marisa; Schreiber, Rainer; Matos, Paulo; Jordan, Peter; Mehta, Anil; Amaral, Margarida D; Kunzelmann, Karl; Farinha, Carlos M

    2011-11-01

    Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

  11. Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells

    DEFF Research Database (Denmark)

    Schneider, H R; Reichert, G H; Issinger, O G

    1986-01-01

    , increased phosphorylation of a 110-kDa protein is observed. Treatment of the embryo extracts with heparin, a highly specific inhibitor of CKII activity, results in a drastic reduction of the 110-kDa protein phosphorylation indicating that the protein might be a CKII-specific substrate. Rapidly proliferating...... mouse tumour cells also show an enhanced CKII activity. Here too, a 110-kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP- and cGMP-dependent protein kinases) only show a basal level of enzyme...

  12. Structural Basis for the Potent and Selective Inhibition of Casein Kinase 1 Epsilon

    Energy Technology Data Exchange (ETDEWEB)

    Long, Alexander M. [Amgen Inc., Cambridge, MA (United States); Zhao, Huilin [Amgen Inc., Cambridge, MA (United States); Huang, Xin [Amgen Inc., Cambridge, MA (United States)

    2012-10-29

    Casein kinase 1 epsilon (CK1ε) and its closest homologue CK1δ are key regulators of diverse cellular processes. We report two crystal structures of PF4800567, a potent and selective inhibitor of CK1ε, bound to the kinase domains of human CK1ε and CK1δ as well as one apo CK1ε crystal structure. These structures provide a molecular basis for the strong and specific inhibitor interactions with CK1ε and suggest clues for further development of CK1δ inhibitors.

  13. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-beta-catenin signaling

    NARCIS (Netherlands)

    Cruciat, C.M.; Dolde, C.; de Groot, R.E.; Ohkawara, B.; Reinhard, C.; Korswagen, H.C.; Niehrs, C.

    2013-01-01

    Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-beta-catenin network, where i

  14. Casein kinase 1δ/ε inhibitor PF-5006739 attenuates opioid drug-seeking behavior.

    Science.gov (United States)

    Wager, Travis T; Chandrasekaran, Ramalakshmi Y; Bradley, Jenifer; Rubitski, David; Berke, Helen; Mente, Scot; Butler, Todd; Doran, Angela; Chang, Cheng; Fisher, Katherine; Knafels, John; Liu, Shenping; Ohren, Jeff; Marconi, Michael; DeMarco, George; Sneed, Blossom; Walton, Kevin; Horton, David; Rosado, Amy; Mead, Andy

    2014-12-17

    Casein kinase 1 delta (CK1δ) and casein kinase 1 epsilon (CK1ε) inhibitors are potential therapeutic agents for a range of psychiatric disorders. The feasibility of developing a CNS kinase inhibitor has been limited by an inability to identify safe brain-penetrant compounds with high kinome selectivity. Guided by structure-based drug design, potent and selective CK1δ/ε inhibitors have now been identified that address this gap, through the design and synthesis of novel 4-[4-(4-fluorophenyl)-1-(piperidin-4-yl)-1H-imidazol-5-yl]pyrimidin-2-amine derivatives. PF-5006739 (6) possesses a desirable profile, with low nanomolar in vitro potency for CK1δ/ε (IC50 = 3.9 and 17.0 nM, respectively) and high kinome selectivity. In vivo, 6 demonstrated robust centrally mediated circadian rhythm phase-delaying effects in both nocturnal and diurnal animal models. Further, 6 dose-dependently attenuated opioid drug-seeking behavior in a rodent operant reinstatement model in animals trained to self-administer fentanyl. Collectively, our data supports further development of 6 as a promising candidate to test the hypothesis of CK1δ/ε inhibition in treating multiple indications in the clinic.

  15. Interactions between Casein kinase Iepsilon (CKIepsilon and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.

    Directory of Open Access Journals (Sweden)

    Caroline Lund Dahlberg

    Full Text Available BACKGROUND: Members of the Casein Kinase I (CKI family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. CONCLUSIONS/SIGNIFICANCE: The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.

  16. Casein kinase 1 proteomics reveal prohibitin 2 function in molecular clock.

    Directory of Open Access Journals (Sweden)

    Lorna S Kategaya

    Full Text Available Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite with external cues (e.g., daylight and food. In vertebrates, both casein kinase 1 delta and epsilon (CK1δ and CK1ε regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1δ and CK1ε mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2 as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1ε expression increases PHB2 protein levels and, unexpectedly, knocking down CK1δ decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1δ and CK1ε differentially regulate the expression of PHB2.

  17. Casein kinase G may be the target of spermine during progesterone-induced oocyte maturation

    Institute of Scientific and Technical Information of China (English)

    LIRUNSHENG; JIAKETSO

    1993-01-01

    Casein kinase G (CKG) with more than 2500-fold enrichraent was purified from Bufo bufo gargarizans ovaries. The catalytic activity of the enzyme was found to be associated with its 42 kD subunit, and its 26 kD subunit was found to be the major tsrget for the enzyme autophos phorylation. Each fuU-grown oocyte contained 1.9 units of CKG corresponding to an intracellular concentration of 93 nM. After injecting an amount of 0,38 units of the enzyme into the oocyte, approximately 50% of the progesterone-induoed maturation was inhibited. The inhibitory effect was enhanced in oocytes pretreated with spermine, which was consistent with the results that the enzyme was activated in vitro in the presence of spermine, The MPF-induced oocyte maturation was delayed and even prohibited in the kinase-microinjected oocytes. A 55 kD oocyte protein was identified as an suhstrate of CKG both in vivo and in vitro, and the enhancement of the 55 kD protein phosphory[ation was associated with kinase inhibition on maturation and on protein synthesis in kinase-microinjected oocytes. As the endogenous spermine level decreased in the course of progesteroneinduced oocyte maturation. 55 kD protein was dephosphorylated, Heparin, a specific inhibitor of CKG, potentiated the progesterone-induced oocyte maturation. Altogether the experimental reSults indicated Strongly that CKG may be the physiological target of spermine.

  18. Casein kinase 1 proteomics reveal prohibitin 2 function in molecular clock.

    Science.gov (United States)

    Kategaya, Lorna S; Hilliard, Aisha; Zhang, Louying; Asara, John M; Ptáček, Louis J; Fu, Ying-Hui

    2012-01-01

    Throughout the day, clock proteins synchronize changes in animal physiology (e.g., wakefulness and appetite) with external cues (e.g., daylight and food). In vertebrates, both casein kinase 1 delta and epsilon (CK1δ and CK1ε) regulate these circadian changes by phosphorylating other core clock proteins. In addition, CK1 can regulate circadian-dependent transcription in a non-catalytic manner, however, the mechanism is unknown. Furthermore, the extent of functional redundancy between these closely related kinases is debated. To further advance knowledge about CK1δ and CK1ε mechanisms of action in the biological clock, we first carried out proteomic analysis of both kinases in human cells. Next, we tested interesting candidates in a cell-based circadian readout which resulted in the discovery of PROHIBITIN 2 (PHB2) as a modulator of period length. Decreasing the expression of PHB2 increases circadian-driven transcription, thus revealing PHB2 acts as an inhibitor in the molecular clock. While stable binding of PHB2 to either kinase was not detected, knocking down CK1ε expression increases PHB2 protein levels and, unexpectedly, knocking down CK1δ decreases PHB2 transcript levels. Thus, isolating CK1 protein complexes led to the identification of PHB2 as an inhibitor of circadian transcription. Furthermore, we show that CK1δ and CK1ε differentially regulate the expression of PHB2.

  19. Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O;

    1992-01-01

    Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce...

  20. Ligand-protein interactions of selective casein kinase 1δ inhibitors.

    Science.gov (United States)

    Mente, Scot; Arnold, Eric; Butler, Todd; Chakrapani, Subramanyam; Chandrasekaran, Ramalakshmi; Cherry, Kevin; DiRico, Ken; Doran, Angela; Fisher, Katherine; Galatsis, Paul; Green, Michael; Hayward, Matthew; Humphrey, John; Knafels, John; Li, Jianke; Liu, Shenping; Marconi, Michael; McDonald, Scott; Ohren, Jeff; Paradis, Vanessa; Sneed, Blossom; Walton, Kevin; Wager, Travis

    2013-09-12

    Casein kinase 1δ (CK1δ) and 1ε (CK1ε) are believed to be necessary enzymes for the regulation of circadian rhythms in all mammals. On the basis of our previously published work demonstrating a CK1ε-preferring compound to be an ineffective circadian clock modulator, we have synthesized a series of pyrazole-substitued pyridine inhibitors, selective for the CK1δ isoform. Additionally, using structure-based drug design, we have been able to exploit differences in the hinge region between CK1δ and p38 to find selective inhibitors that have minimal p38 activity. The SAR, brain exposure, and the effect of these inhibitors on mouse circadian rhythms are described. The in vivo evaluation of these inhibitors demonstrates that selective inhibition of CK1δ at sufficient central exposure levels is capable of modulating circadian rhythms.

  1. Circadian and pharmacological regulation of casein kinase I in the hamster suprachiasmatic nucleus

    Indian Academy of Sciences (India)

    Patricia V. Agostino; Santiago A. Plano; Diego A. Golombek

    2008-12-01

    In mammals, the mechanism for the generation of circadian rhythms and entrainment by light–dark (LD) cycles resides in the hypothalamic suprachiasmatic nuclei (SCN), and the principal signal that adjusts this biological clock with environmental timing is the light:dark cycle. Within the SCN, rhythms are generated by a complex of molecular feedback loops that regulate the transcription of clock genes, including per and cry. Posttranslational modification plays an essential role in the regulation of biological rhythms; in particular, clock gene phosphorylation by casein kinase I, both epsilon (CKI) and delta (CKI), regulates key molecular mechanisms in the circadian clock. In this paper, we report for the first time that CKI activity undergoes a significant circadian rhythm in the SCN (peaking at circadian time 12, the start of the subjective night), and its pharmacological inhibition alters photic entrainment of the clock, indicating that CKI may be a key element in this pathway.

  2. Casein Kinase 2 Reverses Tail-Independent Inactivation of Kinesin-1

    Science.gov (United States)

    Xu, Jing; Reddy, Babu J. N.; Anand, Preetha; Shu, Zhanyong; Cermelli, Silvia; Mattson, Michelle K.; Tripathy, Suvranta K.; Hoss, Matthew T.; James, Nikita S.; King, Stephen J.; Huang, Lan; Bardwell, Lee; Gross, Steven P.

    2013-01-01

    Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signaling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (~ 4-fold) without altering kinesin’s single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results provide the first direct evidence of a protein kinase up-regulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin. PMID:22453827

  3. Characterization of the alpha and beta subunits of casein kinase 2 by far-UV CD spectroscopy

    DEFF Research Database (Denmark)

    Issinger, O G; Brockel, C; Boldyreff, B;

    1992-01-01

    Although Chou-Fasman calculations of the secondary structure of recombinant casein kinase 2 subunits alpha and beta suggest they have a similar overall conformation, circular dichroism (CD) studies show that substantial differences in the conformation of the two subunits exist. In addition......, no changes in the far-UV CD spectrum of the alpha subunit are observed in the presence of casein or the synthetic decapeptide substrate RRRDDDSDDD. Furthermore, the alpha-helical structure of the alpha subunit (but not the beta subunit) can be increased in the presence of stoichiometric amounts of heparin...

  4. Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein

    Science.gov (United States)

    Homma, Miwako Kato; Li, Dongxia; Krebs, Edwin G.; Yuasa, Yasuhito; Homma, Yoshimi

    2002-01-01

    Mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in in vitro kinase reaction, indicating association of APC with protein kinase activity. The kinase activity complexed with APC was sensitive to heparin and used GTP as phosphoryl donor, suggesting an involvement of casein kinase 2 (CK2). Both CK2α- and β-subunits were found to associate with APC in immunoprecipitates as well as in pull-down assays, with preferential interaction of APC with tetrameric CK2 holoenzyme. In synchronized cell populations, the association of APC with CK2 was cell cycle dependent, with the highest association in G2/M. Unexpectedly, APC immunoprecipitates containing full-length APC protein inhibited CK2 in vitro, whereas immunoprecipitates of truncated APC had little effect. This was confirmed by using recombinant APC, and the inhibitory region was localized to the C terminus of APC between residues 2086 and 2394. Overexpression of this fragment in SW480 cells suppressed cell proliferation rates as well as tumorigenesis. These results demonstrate a previously uncharacterized functional interaction between the tumor suppressor protein APC and CK2 and suggest that growth-inhibitory effects of APC may be regulated by inhibition of CK2. PMID:11972058

  5. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-β-catenin signaling.

    Science.gov (United States)

    Cruciat, Cristina-Maria; Dolde, Christine; de Groot, Reinoud E A; Ohkawara, Bisei; Reinhard, Carmen; Korswagen, Hendrik C; Niehrs, Christof

    2013-03-22

    Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-β-catenin network, where it acts as a regulatory subunit of CK1ε: In a Wnt-dependent manner, DDX3 binds CK1ε and directly stimulates its kinase activity, and promotes phosphorylation of the scaffold protein dishevelled. DDX3 is required for Wnt-β-catenin signaling in mammalian cells and during Xenopus and Caenorhabditis elegans development. The results also suggest that the kinase-stimulatory function extends to other DDX and CK1 members, opening fresh perspectives for one of the longest-studied protein kinase families.

  6. Functional analysis of Casein Kinase 1 in a minimal circadian system.

    Directory of Open Access Journals (Sweden)

    Gerben van Ooijen

    Full Text Available The Earth's rotation has driven the evolution of cellular circadian clocks to facilitate anticipation of the solar cycle. Some evidence for timekeeping mechanism conserved from early unicellular life through to modern organisms was recently identified, but the components of this oscillator are currently unknown. Although very few clock components appear to be shared across higher species, Casein Kinase 1 (CK1 is known to affect timekeeping across metazoans and fungi, but has not previously been implicated in the circadian clock in the plant kingdom. We now show that modulation of CK1 function lengthens circadian rhythms in Ostreococcustauri, a unicellular marine algal species at the base of the green lineage, separated from humans by ~1.5 billion years of evolution. CK1 contributes to timekeeping in a phase-dependent manner, indicating clock-mediated gating of CK1 activity. Label-free proteomic analyses upon overexpression as well as inhibition revealed CK1-responsive phosphorylation events on a set of target proteins, including highly conserved potentially clock-relevant cellular regulator proteins. These results have major implications for our understanding of cellular timekeeping and can inform future studies in any circadian organism.

  7. Circadian metabolic regulation through crosstalk between casein kinase 1δ and transcriptional coactivator PGC-1α.

    Science.gov (United States)

    Li, Siming; Chen, Xiao-Wei; Yu, Lei; Saltiel, Alan R; Lin, Jiandie D

    2011-12-01

    Circadian clock coordinates behavior and physiology in mammals in response to light and feeding cycles. Disruption of normal clock function is associated with increased risk for cardiovascular and metabolic diseases, underscoring the emerging concept that temporal regulation of tissue metabolism is a fundamental aspect of energy homeostasis. We have previously demonstrated that transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), coordinates circadian metabolic rhythms through simultaneous regulation of metabolic and clock gene expression. In this study, we found that PGC-1α physically interacts with, and is phosphorylated by, casein kinase 1δ (CK1δ), a core component of the circadian pacemaker. CK1δ represses the transcriptional function of PGC-1α in cultured hepatocytes, resulting in decreased gluconeogenic gene expression and glucose secretion. At the molecular level, CK1δ phosphorylation of PGC-1α within its arginine/serine-rich domain enhances its degradation through the proteasome system. Together, these results elucidate a novel mechanism through which circadian pacemaker transduces timing signals to the metabolic regulatory network that controls hepatic energy metabolism.

  8. Regulation of Ceramide Synthase by Casein Kinase 2-dependent Phosphorylation in Saccharomyces cerevisiae*

    Science.gov (United States)

    Fresques, Tara; Niles, Brad; Aronova, Sofia; Mogri, Huzefa; Rakhshandehroo, Taha; Powers, Ted

    2015-01-01

    Complex sphingolipids are important components of eukaryotic cell membranes and, together with their biosynthetic precursors, including sphingoid long chain bases and ceramides, have important signaling functions crucial for cell growth and survival. Ceramides are produced at the endoplasmic reticulum (ER) membrane by a multicomponent enzyme complex termed ceramide synthase (CerS). In budding yeast, this complex is composed of two catalytic subunits, Lac1 and Lag1, as well as an essential regulatory subunit, Lip1. Proper formation of ceramides by CerS has been shown previously to require the Cka2 subunit of casein kinase 2 (CK2), a ubiquitous enzyme with multiple cellular functions, but the precise mechanism involved has remained unidentified. Here we present evidence that Lac1 and Lag1 are direct targets for CK2 and that phosphorylation at conserved positions within the C-terminal cytoplasmic domain of each protein is required for optimal CerS activity. Our data suggest that phosphorylation of Lac1 and Lag1 is important for proper localization and distribution of CerS within the ER membrane and that phosphorylation of these sites is functionally linked to the COP I-dependent C-terminal dilysine ER retrieval pathway. Together, our data identify CK2 as an important regulator of sphingolipid metabolism, and additionally, because both ceramides and CK2 have been implicated in the regulation of cancer, our findings may lead to an enhanced understanding of their relationship in health and disease. PMID:25429105

  9. Regulation of ceramide synthase by casein kinase 2-dependent phosphorylation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Fresques, Tara; Niles, Brad; Aronova, Sofia; Mogri, Huzefa; Rakhshandehroo, Taha; Powers, Ted

    2015-01-16

    Complex sphingolipids are important components of eukaryotic cell membranes and, together with their biosynthetic precursors, including sphingoid long chain bases and ceramides, have important signaling functions crucial for cell growth and survival. Ceramides are produced at the endoplasmic reticulum (ER) membrane by a multicomponent enzyme complex termed ceramide synthase (CerS). In budding yeast, this complex is composed of two catalytic subunits, Lac1 and Lag1, as well as an essential regulatory subunit, Lip1. Proper formation of ceramides by CerS has been shown previously to require the Cka2 subunit of casein kinase 2 (CK2), a ubiquitous enzyme with multiple cellular functions, but the precise mechanism involved has remained unidentified. Here we present evidence that Lac1 and Lag1 are direct targets for CK2 and that phosphorylation at conserved positions within the C-terminal cytoplasmic domain of each protein is required for optimal CerS activity. Our data suggest that phosphorylation of Lac1 and Lag1 is important for proper localization and distribution of CerS within the ER membrane and that phosphorylation of these sites is functionally linked to the COP I-dependent C-terminal dilysine ER retrieval pathway. Together, our data identify CK2 as an important regulator of sphingolipid metabolism, and additionally, because both ceramides and CK2 have been implicated in the regulation of cancer, our findings may lead to an enhanced understanding of their relationship in health and disease.

  10. Regulation of Ikaros function by casein kinase 2 and protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    Amy; K; Erbe; Aleksandar; Savic; Sinisa; Dovat

    2011-01-01

    The Ikaros gene encodes a zinc finger,DNA-binding protein that regulates gene transcription and chromatin remodeling.Ikaros is a master regulator of hematopoiesis and an established tumor suppressor.Moderate alteration of Ikaros activity (e.g.haploinsufficiency) appears to be sufficient to promote malignant transformation in human hematopoietic cells.This raises questions about the mechanisms that normally regulate Ikaros function and the potential of these mechanisms to contribute to the development of leukemia.The focus of this review is the regulation of Ikaros function by phosphorylation/dephosphorylation.Site-specific phosphorylation of Ikaros by casein kinase 2 (CK2) controls Ikaros DNA-binding ability and subcellular localization.As a consequence,the ability of Ikaros to regulate cell cycle progression,chromatin remodeling,target gene expression,and thymocyte differentiation are controlled by CK2.In addition,hyperphosphorylation of Ikaros by CK2 leads to decreased Ikaros levels due to ubiquitinmediated degradation.Dephosphorylation of Ikaros by protein phosphatase 1 (PP1) acts in opposition to CK2 to increase Ikaros stability and restore Ikaros DNA binding ability and pericentromeric localization.Thus,the CK2 and PP1 pathways act in concert to regulate Ikaros activity in hematopoiesis and as a tumor suppressor.This highlights the importance of these signal transduction pathways as potential mediators of leukemogenesis via their role in regulating the activities of Ikaros.

  11. Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

    Institute of Scientific and Technical Information of China (English)

    彭寨玉; 余新炳; 吴忠道; 徐劲; 吴德; 李孜

    2004-01-01

    Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.

  12. The autophosphorylation and p34cdc2 phosphorylation sites of casein kinase-2 beta-subunit are not essential for reconstituting the fully-active heterotetrameric holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;

    1993-01-01

    Two mutants of human casein kinase-2 beta-subunit with short deletions at either their amino (delta 1-4) or carboxy (delta 209-215) terminal side have been created that have lost the capability to undergo autophosphorylation and p34cdc2 mediated phosphorylation, respectively. Both mutants give rise...

  13. Phosphorylation of the C proteins in heterogeneous ribonucleoprotein (hnRNP) particles in HeLa cells: Characterization of in vivo phosphorylation, comparison with in vitro phosphorylation using casein kinase II, and preliminary studies on the effects of phosphorylation on particle structure

    Energy Technology Data Exchange (ETDEWEB)

    Kleiman, N.J.

    1989-01-01

    Newly formed pre-messenger RNA associates with protein to form heterogeneous ribonucleoprotein (hnRNP) particles. In HeLa cells, hnRNP particles contain six core proteins. Two proteins, termed C{sub 1} and C{sub 2}, are phosphorylated in vitro by casein kinase 11 (CKII). C{sub 1} protein became {sup 32}P-labeled after HeLa cells were incubated with ({sup 32}P)-orthophosphate in vivo (ibid). Because phosphorylation is a ubiquitous regulatory mechanism, C protein phosphorylation was studied in greater detail. C protein phosphorylation in hnRNP particles was investigated in HeLa cells incubated with ({sup 32}P)-orthophosphate in vivo. Immunoblotting in pH 3.5-10 isoelectric focusing (IEF) gels indicated that C proteins focus only at pH 5.0. In pH 4.5-5.5 IEF gels, individually purified C, and 2 proteins resolve into the same four closely spaced, {sup 32}P-labeled bands. A fifth, unlabeled, more basic species was detached when hnRNP particles were purified without NaF. All {sup 32}P-labeled species contained identical amounts of {sup 32}P per unit protein suggesting that charge heterogeneity is not due to differential phosphorylation. Attempts to detect bound carbohydrate were unsuccessful. {sup 32}P-labeled phosphate was readily removed by potato acid phosphatase. E. coli alkaline phosphatase and snake venom phosphodiesterase were ineffective. {sup 32}P-label was found exclusively in phosphoserine. One-dimensional peptide mapping with chymotrypsin and S. aureus protease detected two phosphorylated peptides. C protein phosphorylation was also investigated in vitro. Incubation of hnRNP particles with rabbit liver CKII and {sup 32}P-ATP followed by IEF in pH 4.5-5.5 gels indicated that all four C protein species were {sup 32}P-labeled. {sup 32}P-label was found exclusively in phosphoserine.

  14. The association of lysozyme with casein

    NARCIS (Netherlands)

    Roos, de A.L.; Walstra, P.; Geurts, T.J.

    1998-01-01

    The association of hen eggs’ lysozyme with caseins was studied by using three casein substrates: (I) solutions of the various caseins, (II) artificially made casein micelles of various compositions and (III) caseins adsorbed onto soya-oil emulsion droplets. In solution, lysozyme associated most stro

  15. Casein kinase 2α regulates multidrug resistance-associated protein 1 function via phosphorylation of Thr249.

    Science.gov (United States)

    Stolarczyk, Elzbieta I; Reiling, Cassandra J; Pickin, Kerry A; Coppage, Ryan; Knecht, Marc R; Paumi, Christian M

    2012-09-01

    We have shown previously that the function of Ycf1p, yeast ortholog of multidrug resistance-associated protein 1 (MRP1), is regulated by yeast casein kinase 2α (Cka1p) via phosphorylation at Ser251. In this study, we explored whether casein kinase 2α (CK2α), the human homolog of Cka1p, regulates MRP1 by phosphorylation at the semiconserved site Thr249. Knockdown of CK2α in MCF7-derived cells expressing MRP1 [MRP1 CK2α(-)] resulted in increased doxorubicin sensitivity. MRP1-dependent transport of leukotriene C(4) and estradiol-17β-d-glucuronide into vesicles derived from MRP1 CK2α(-) cells was decreased compared with MRP1 vesicles. Moreover, mutation of Thr249 to alanine (MRP1-T249A) also resulted in decreased MRP1-dependent transport, whereas a phosphomimicking mutation (MRP1-T249E) led to dramatic increase in MRP1-dependent transport. Studies in tissue culture confirmed these findings, showing increased intracellular doxorubicin accumulation in MRP1 CK2α(-) and MRP1-T249A cells compared with MRP1 cells. Inhibition of CK2 kinase by 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole resulted in increased doxorubicin accumulation in MRP1 cells, but not in MRP1 CK2α(-), MRP1-T249A, or MRP1-T249E cells, suggesting that CK2α regulates MRP1 function via phosphorylation of Thr249. Indeed, CK2α and MRP1 interact physically, and recombinant CK2 phosphorylates MRP1-derived peptide in vitro in a Thr249-dependent manner, whereas knockdown of CK2α results in decreased phosphorylation at MRP1-Thr249. The role of CK2 in regulating MRP1 was confirmed in other cancer cell lines where CK2 inhibition decreased MRP1-mediated efflux of doxorubicin and increased doxorubicin cytotoxicity. This study supports a model in which CK2α potentiates MRP1 function via direct phosphorylation of Thr249.

  16. Activated CaMKII Couples GluN2B and Casein Kinase 2 to Control Synaptic NMDA Receptors

    Directory of Open Access Journals (Sweden)

    Antonio Sanz-Clemente

    2013-03-01

    Full Text Available Synaptic activity triggers a profound reorganization of the molecular composition of excitatory synapses. For example, NMDA receptors are removed from synapses in an activity- and calcium-dependent manner, via casein kinase 2 (CK2 phosphorylation of the PDZ ligand of the GluN2B subunit (S1480. However, how synaptic activity drives this process remains unclear because CK2 is a constitutively active kinase, which is not directly regulated by calcium. We show here that activated CaMKII couples GluN2B and CK2 to form a trimolecular complex and increases CK2-mediated phosphorylation of GluN2B S1480. In addition, a GluN2B mutant, which contains an insert to mimic the GluN2A sequence and cannot bind to CaMKII, displays reduced S1480 phosphorylation and increased surface expression. We find that although disrupting GluN2B/CaMKII binding reduces synapse number, it increases synaptic-GluN2B content. Therefore, the GluN2B/CaMKII association controls synapse density and PSD composition in an activity-dependent manner, including recruitment of CK2 for the removal of GluN2B from synapses.

  17. Ricinine: a pyridone alkaloid from Ricinus communis that activates the Wnt signaling pathway through casein kinase 1α.

    Science.gov (United States)

    Ohishi, Kensuke; Toume, Kazufumi; Arai, Midori A; Sadhu, Samir K; Ahmed, Firoj; Mizoguchi, Takamasa; Itoh, Motoyuki; Ishibashi, Masami

    2014-09-01

    Wnt signaling plays important roles in proliferation, differentiation, development of cells, and various diseases. Activity-guided fractionation of the MeOH extract of the Ricinus communis stem led to the isolation of four compounds (1-4). The TCF/β-catenin transcription activities of 1 and 3 were 2.2 and 2.5 fold higher at 20 and 30μM, respectively. Cells treated with ricinine (1) had higher β-catenin and lower of p-β-catenin (ser 33, 37, 45, Thr 41) protein levels, whereas glycogen synthase kinase 3β (GSK3β) and casein kinase 1α (CK1α) protein levels remained unchanged. Cells treated with pyrvinium, an activator of CK1α, had lower β-catenin levels. However, the combined treatment of pyrvinium and 1 led to higher β-catenin levels than those in cells treated with pyrvinium alone, which suggested that 1 inhibited CK1α activity. Furthermore, 1 increased β-catenin protein levels in zebrafish embryos. These results indicated that 1 activated the Wnt signaling pathway by inhibiting CK1α.

  18. Casein Kinase 1δ Is an APC/CCdh1 Substrate that Regulates Cerebellar Granule Cell Neurogenesis

    Directory of Open Access Journals (Sweden)

    Clara Penas

    2015-04-01

    Full Text Available Although casein kinase 1δ (CK1δ is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/CCdh1 ubiquitin ligase, and conditional deletion of the APC/CCdh1 activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/CCdh1 also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/CCdh1 controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target.

  19. FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes

    Science.gov (United States)

    Kuga, Takahisa; Sasaki, Mitsuho; Mikami, Toshinari; Miake, Yasuo; Adachi, Jun; Shimizu, Maiko; Saito, Youhei; Koura, Minako; Takeda, Yasunori; Matsuda, Junichiro; Tomonaga, Takeshi; Nakayama, Yuji

    2016-01-01

    FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts. PMID:27222304

  20. Identification of a secreted casein kinase 1 in Leishmania donovani: effect of protein over expression on parasite growth and virulence.

    Directory of Open Access Journals (Sweden)

    Mary Dan-Goor

    Full Text Available Casein kinase 1 (CK1 plays an important role in eukaryotic signaling pathways, and their substrates include key regulatory proteins involved in cell differentiation, proliferation and chromosome segregation. The Leishmania genome encodes six potential CK1 isoforms, of which five have orthologs in other trypanosomatidae. Leishmania donovani CK1 isoform 4 (Ldck1.4, orthologous to LmjF27.1780 is unique to Leishmania and contains a putative secretion signal peptide. The full-length gene and three shorter constructs were cloned and expressed in E. coli as His-tag proteins. Only the full-length 62.3 kDa protein showed protein kinase activity indicating that the N-terminal and C-terminal domains are essential for protein activity. LdCK1.4-FLAG was stably over expressed in L. donovani, and shown by immunofluorescence to be localized primarily in the cytosol. Western blotting using anti-FLAG and anti-CK1.4 antibodies showed that this CK1 isoform is expressed and secreted by promastigotes. Over expression of LdCK1.4 had a significant effect on promastigote growth in culture with these parasites growing to higher cell densities than the control parasites (wild-type or Ld:luciferase, P<0.001. Analysis by flow cytometry showed a higher percentage, ∼4-5-fold, of virulent metacyclic promastigotes on day 3 among the LdCK1.4 parasites. Finally, parasites over expressing LdCK1.4 gave significantly higher infections of mouse peritoneal macrophages compared to wild-type parasites, 28.6% versus 6.3%, respectively (p = 0.0005. These results suggest that LdCK1.4 plays an important role in parasite survival and virulence. Further studies are needed to validate CK1.4 as a therapeutic target in Leishmania.

  1. Casein kinase Ⅱ interacts with prion protein in vitro and forms complex with na-tive prion protein in vivo

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein to pathologic isoform. Casein kinase Ⅱ (CK2) is a ubiquitously expressed and evolutiouarily conserved pleiotropic protein kinase that is essential for viability. To explore the possible molecular interaction between CK2 and prion protein (PrP), the full-length sequences of human CK2α and CK2β complementary DNA were amplified with reverse transcription-polymerase chain reaction using the total messenger RNA from cell line SH-SY5Y as the template; then, the fusion proteins histidine-CK2α and glutathione S-transferase-histidine-CK2β were expressed in Escherichia coll. The interaction between CK2 and PrP was evaluated with co-immunoprecipi-tation and pull-down assays. The results demonstrated that recombinant PrP bound specifically with CK2α, but not with CK2β. The native CK2 and PrP in hamster brains interacted with each other, forming protein complexes. Three different glycosylated forms of PrP (diglycosylated, monoglycosylated and unglycosylated PrP) from normal brains interacted with the CK2α subunit, though the unglycosylated PrP seemed to have a stronger binding ability with CK2α subunit. The domain responsible for interacting with CK2α was located at the C-terminal segment of PrP (residues 91-231). This study proposed reliable experimental data for the molecular interaction between PrP and CK2α (both in recombinant and native categories), scientific clues for further assessing the potential biological significance of the PrP-CK2 interaction, and the possible role of CK2 in the pathogenesis of prion diseases.

  2. Casein kinase 1 alpha regulates chromosome congression and separation during mouse oocyte meiotic maturation and early embryo development.

    Science.gov (United States)

    Wang, Lu; Lu, Angeleem; Zhou, Hong-Xia; Sun, Ran; Zhao, Jie; Zhou, Cheng-Jie; Shen, Jiang-Peng; Wu, Sha-Na; Liang, Cheng-Guang

    2013-01-01

    Casein kinase I alpha (CK1α) is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1) extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP), an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.

  3. Casein kinase 1 alpha regulates chromosome congression and separation during mouse oocyte meiotic maturation and early embryo development.

    Directory of Open Access Journals (Sweden)

    Lu Wang

    Full Text Available Casein kinase I alpha (CK1α is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1 extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP, an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.

  4. Cyclooxygenase 2-dependent and independent activation of Akt through casein kinase 2α contributes to human bladder cancer cell survival

    Directory of Open Access Journals (Sweden)

    Fujimoto Kiyohide

    2011-05-01

    Full Text Available Abstract Background Survival rate for patients presenting muscle invasive bladder cancer is very low, and useful therapeutic target has not been identified yet. In the present study, new COX2 downstream signals involved in urothelial carcinoma cell survival were investigated in vitro and in vivo. Methods COX2 gene was silenced by siRNA transfection. Orthotopic implantation animal model and transurethral instillation of siRNA with atelocollagen was constructed to examine the effects of COX2 knockdown in vivo. Cell cycle was examined by flowcytoketry. Surgical specimens derived from patients with urinary bladder cancer (all were initially diagnosed cases were used for immunohistochemical analysis of the indicated protein expression in urothelial carcinoma cells. Results Treatment with the COX2 inhibitor or knockdown of COX2 reduced expression of casein kinase (CK 2 α, a phophorylated Akt and urokinase type plasminogen activator (uPA, resulting in p27 induction, cell cycle arrest at G1 phase and cell growth suppression in human urothelial carcinoma cell lines expressing COX2. Silencing of CK2α exhibited the similar effects. Even in UMUC3 cells lacking the COX2 gene, COX2 inhibition also inhibited cell growth through down-regulation of the CK2α-Akt/uPA axis. The mouse orthotropic bladder cancer model demonstrated that the COX2 inhibitor, meloxicam significantly reduced CK2α, phosphorylated Akt and uPA expression, whereas induced p27 by which growth and invasiveness of bladder cancer cells were strongly inhibited. Immunohistochemically, high expression of COX2, CK2α and phosphorylated form of Akt was found in high-grade, invasive carcinomas as well as carcinoma in situ, but not in low-grade and noninvasive phenotypes. Conclusions COX2-dependent and independent activation of CK2α-Akt/uPA signal is mainly involved in urothelial carcinoma cell survival, moreover, not only COX2 but also CK2α could be direct targets of COX2 inhibitors.

  5. CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O

    2002-01-01

    An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

  6. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Directory of Open Access Journals (Sweden)

    Sanne H. Olesen

    2015-01-01

    Full Text Available The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein–protein interaction (PPI inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2 did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM, while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  7. Stability of the human Hsp90-p50Cdc37 chaperone complex against nucleotides and Hsp90 inhibitors, and the influence of phosphorylation by casein kinase 2.

    Science.gov (United States)

    Olesen, Sanne H; Ingles, Donna J; Zhu, Jin-Yi; Martin, Mathew P; Betzi, Stephane; Georg, Gunda I; Tash, Joseph S; Schönbrunn, Ernst

    2015-01-19

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands.

  8. Stability of the Human Hsp90-p50Cdc37 Chaperone Complex against Nucleotides and Hsp90 Inhibitors, and the Influence of Phosphorylation by Casein Kinase 2

    Science.gov (United States)

    Olesen, Sanne H.; Ingles, Donna J.; Zhu, Jin-Yi; Martin, Mathew P.; Betzi, Stephane; Georg, Gunda I.; Tash, Joseph S.; Schönbrunn, Ernst

    2015-01-01

    The molecular chaperone Hsp90 is regulated by co-chaperones such as p50Cdc37, which recruits a wide selection of client protein kinases. Targeted disruption of the Hsp90-p50Cdc37 complex by protein-protein interaction (PPI) inhibitors has emerged as an alternative strategy to treat diseases characterized by aberrant Hsp90 activity. Using isothermal microcalorimetry, ELISA and GST-pull down assays we evaluated reported Hsp90 inhibitors and nucleotides for their ability to inhibit formation of the human Hsp90β-p50Cdc37 complex, reconstituted in-vitro from full-length proteins. Hsp90 inhibitors, including the proposed PPI inhibitors gedunin and H2-gamendazole, did not affect the interaction of Hsp90 with p50Cdc37 in vitro. Phosphorylation of Hsp90 and p50Cdc37 by casein kinase 2 (CK2) did not alter the thermodynamic signature of complex formation. However, the phosphorylated complex was vulnerable to disruption by ADP (IC50 = 32 µM), while ATP, AMPPNP and Hsp90 inhibitors remained largely ineffective. The differential inhibitory activity of ADP suggests that phosphorylation by CK2 primes the complex for dissociation in response to a drop in ATP/ADP levels. The approach applied herein provides robust assays for a comprehensive biochemical evaluation of potential effectors of the Hsp90-p50Cdc37 complex, such as phosphorylation by a kinase or the interaction with small molecule ligands. PMID:25608045

  9. Structural dynamics of Casein Kinase I (CKI) from malarial parasite Plasmodium falciparum (Isolate 3D7): Insights from theoretical modelling and molecular simulations.

    Science.gov (United States)

    Dehury, Budheswar; Behera, Santosh Kumar; Mahapatra, Namita

    2017-01-01

    The protein kinases (PKs), belonging to serine/threonine kinase (STKs), are important drug targets for a wide spectrum of diseases in human. Among protein kinases, the Casein Kinases (CKs) are vastly expanded in various organisms, where, the malarial parasite Plasmodium falciparum possesses a single member i.e., PfCKI, which can phosphorylate various proteins in parasite extracts in vitro condition. But, the structure-function relationship of PfCKI and dynamics of ATP binding is yet to be understood. Henceforth, an attempt was made to study the dynamics, stability, and ATP binding mechanisms of PfCKI through computational modelling, docking, molecular dynamics (MD) simulations, and MM/PBSA binding free energy estimation. Bi-lobed catalytic domain of PfCKI shares a high degree of secondary structure topology with CKI domains of rice, human, and mouse indicating co-evolution of these kinases. Molecular docking study revealed that ATP binds to the active site where the glycine-rich ATP-binding motif (G16-X-G18-X-X-G21) along with few conserved residues plays a crucial role maintaining stability of the complex. Structural superposition of PfCKI with close structural homologs depicted that the location and length of important loops are different, indicating the dynamic properties of these loops among CKIs, which is consistent with principal component analysis (PCA). PCA displayed that the overall global motion of ATP-bound form is comparatively higher than that of apo form. The present study provides insights into the structural features of PfCKI, which could contribute towards further understanding of related protein structures, dynamics of catalysis and phosphorylation mechanism in these important STKs from malarial parasite in near future.

  10. Characterization of casein kinase II in human colonic carcinomas after heterotransplantation into nude mice

    DEFF Research Database (Denmark)

    Seitz, G; Münstermann, U; Schneider, H R

    1989-01-01

    they were derived. Immunoblots using a polyclonal CKII-specific antibody showed that the increase of activity was due to a higher expression of the enzyme. Immunohistochemical studies on cross sections of nude mouse tumours showed that most of the CKII molecules were located at the peripheral part...

  11. Casein kinase II is elevated in solid human tumours and rapidly proliferating non-neoplastic tissue

    DEFF Research Database (Denmark)

    Münstermann, U; Fritz, G; Seitz, G

    1990-01-01

    extracts from solid tumours followed by immunostaining with an anti-CKII polyclonal antibody, (b) immunohistochemical staining of cells from tissue sections and (c) by activity measurements using the CKII-specific synthetic peptide (RRRDDDSDDD). The maximum observed activity in the colorectal carcinomas...

  12. The Rho kinases I and II regulate different aspects of myosin II activity

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Multhaupt, Hinke A B; Couchman, John R

    2005-01-01

    persistent ROCK II and guanine triphosphate-bound RhoA. In contrast, the microfilament cytoskeleton was enhanced by ROCK II down-regulation. Phagocytic uptake of fibronectin-coated beads was strongly down-regulated in ROCK II-depleted cells but not those lacking ROCK I. These effects originated in part from......The homologous mammalian rho kinases (ROCK I and II) are assumed to be functionally redundant, based largely on kinase construct overexpression. As downstream effectors of Rho GTPases, their major substrates are myosin light chain and myosin phosphatase. Both kinases are implicated in microfilament...... bundle assembly and smooth muscle contractility. Here, analysis of fibroblast adhesion to fibronectin revealed that although ROCK II was more abundant, its activity was always lower than ROCK I. Specific reduction of ROCK I by siRNA resulted in loss of stress fibers and focal adhesions, despite...

  13. 酪蛋白激酶2结合并磷酸化ataxin-3%Casein kinase 2 interacts with and phosphorylates ataxin-3

    Institute of Scientific and Technical Information of China (English)

    陶瑞松; 费尔康; 应征; 王洪枫; 王光辉

    2008-01-01

    目的 马查多-约瑟夫病/脊髓小脑共济失调3型,是由MJDI基因产物ataxin-3的C-末端的多聚谷氨酰胺发生重复扩展突变而引起的一种常染色体显性遗传的神经退行性疾病,目前它的发病机制还不清楚.很多研究表明磷酸化修饰作用在很多神经退行性疾病的发病过程中起到重要作用,然而已知可以磷酸化ataxin-3的激酶仍然很少.本研究的目的是探讨酪蛋白激酶2(Casein kinase 2,CK2)对于ataxin-3的磷酸化作用.方法 通过GST pulldown和免疫共沉淀技术鉴定ataxin-3和CK2的相互作用.通过体外磷酸化技术检测CK2对ataxin-3的磷酸化.结果 (1)正常和扩展突变型ataxin-3在体外与CK2α、β亚单位均发生相互作用;(2)在293细胞中正常和扩展突变型ataxin-3只和CK2β亚单位相互作用,而与α亚单位没有结合;(3)正常和扩展突变型ataxin-3都可以被CK2磷酸化.结论 Ataxin-3是蛋白激酶CK2的底物.%Objective Machado-Joseph disease(MJD)/Spinocerebellar ataxia type 3(SCA3)is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJDI gene product,ataxin-3.The precise mechanism of the MJD/SCA3 pathogenesis remains unclear.A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases.However,few kinases are known to phosphorylate ataxin-3.The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 (CK2).Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase (GST) pull-down assay and co-immunoprecipition assay.The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays.Results (1) Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro.(2) In 293 cells,both wild type and expanded ataxin-3 interacted with CK2β,but not CK2α.(3) CK2 phosphorylated wild type and

  14. Immunohistochemical and Proteomic Evaluation of Nuclear Ubiquitous Casein and Cyclin-Dependent Kinases Substrate in Invasive Ductal Carcinoma of the Breast

    Directory of Open Access Journals (Sweden)

    Piotr Ziółkowski

    2009-01-01

    Full Text Available Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS is 27 kDa chromosomal protein of unknown function. Its amino acid composition as well as structure of its DNA binding domain resembles that of high-mobility group A, HMGA proteins. HMGA proteins are associated with various malignancies. Since changes in expression of HMGA are considered as marker of tumor progression, it is possible that similar changes in expression of NUCKS could be useful tool in diagnosis and prognosis of breast cancer. For identification and analysis of NUCKS we used proteomic and histochemical methods. Analysis of patient-matched samples of normal and breast cancer by mass spectrometry revealed elevated levels of NUCKS in protein extracts from ductal breast cancers. We elicited specific antibodies against NUCKS and used them for immunohistochemistry in invasive ductal carcinoma of breast. We found high expression of NUCKS in 84.3% of cancer cells. We suggest that such overexpression of NUCKS can play significant role in breast cancer biology.

  15. Identification of a novel crosstalk between casein kinase 2α and NPM-ALK in ALK-positive anaplastic large cell lymphoma.

    Science.gov (United States)

    Armanious, Hanan; Gelebart, Pascal; Anand, Mona; Lai, Raymond

    2013-02-01

    It was previously reported that β-catenin contributes to the tumorigenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL), and the oncogenic effects of β-catenin in these tumors are promoted by NPM-ALK, an abnormal fusion protein characteristic of ALK(+)ALCL. In this study, we hypothesized that NPM-ALK promotes the oncogenic activity of β-catenin via its functional interactions with the Wnt canonical pathway (WCP). To test this hypothesis, we examined if NPM-ALK modulates the gene expression of various members in the WCP. Using a Wnt pathway-specific oligonucleotide array and Western blots, we found that the expression of casein kinase 2α (CK2α) was substantially downregulated in ALK(+)ALCL cells in response to siRNA knockdown of NPM-ALK. CK2α is biologically important in ALK(+)ALCL, as its inhibition using 4,5,6,7-tetrabromobenzotriazole or siRNA resulted in a significant decrease in cell growth and a substantial decrease in the β-catenin protein level. Furthermore, CK2α co-immunoprecipitated with NPM-ALK and regulated its level of serine phosphorylation, a feature previously shown to correlate with the oncogenic potential of this fusion protein. To conclude, this study has revealed a novel crosstalk between NPM-ALK and CK2α, and our data supports the model that these two molecules work synergistically to promote the tumorigenicity of these lymphomas.

  16. Immunohistochemical study of nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 in invasive breast carcinoma of no special type.

    Science.gov (United States)

    Symonowicz, Krzysztof; Duś-Szachniewicz, Kamila; Woźniak, Marta; Murawski, Marek; Kołodziej, Paweł; Osiecka, Beata; Jurczyszyn, Kamil; Ziółkowski, Piotr

    2014-10-01

    The aim of the present study was to investigate the immunohistochemical expression of nuclear ubiquitous casein and cyclin-dependent kinases substrate 1 (NUCKS1) in invasive breast carcinoma of no special type, in association with clinicopathological characteristics, including the tumor grade, frequency of lymph node involvement and distant metastasis. In addition, associations between NUCKS1 and other tumor subtype markers, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), Ki-67 and cytokeratin 5/6 (CK 5/6), were investigated. NUCKS1 expression was shown to be associated with the formation of distant metastases and lymph node involvement. Furthermore, an association between the presence of NUCKS1 and histological grading was observed. The results confirmed that the expression of NUCKS1 in low grade invasive breast carcinoma of no special type was significantly less common compared with cases of high grade carcinoma. With regard to the additional tumor subtype markers, NUCKS1 expression was demonstrated to be significantly associated with Ki-67 and CK 5/6; however, no association was identified with ER, PR and HER2. Therefore, NUCKS1 may be a novel prognostic marker in the histopathological evaluation of invasive breast carcinoma of no special type.

  17. Suppression of casein kinase 2 sensitizes tumor cells to antitumor TRAIL therapy by regulating the phosphorylation and localization of p65 in prostate cancer.

    Science.gov (United States)

    Gang, Xiaokun; Wang, Yao; Wang, Yingdi; Zhao, Yu; Ding, Liya; Zhao, Jingwen; Sun, Lin; Wang, Guixia

    2015-09-01

    In the United States, prostate cancer (PCa) is the most commonly diagnosed cancer in males. For PCa at the late hormone-refractory stage, substantial improvement in treatment strategies is critically needed. TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, but both intrinsic and acquired resistance to TRAIL poses a huge problem in establishing clinically effective TRAIL therapies. In the present study, we examined the role played by casein kinase 2 (CK2) in the TRAIL‑induced nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB) pathway in a PCa cell line. Downregulation of CK2 combined with a sub-dose of TRAIL suppressed p65 phosphorylation at serine 536. The combination treatment of TRAIL and the CK2 inhibitor decreased p65 nuclear translocation. Under the treatment of a sub-dose of TRAIL, downregulation of CK2, using both genetic and pharmacological approaches, decreased the transcriptional activity of NF-κB and the expression of NF-κB downstream anti-apoptosis genes. Therefore, we provided novel molecular mechanistic insight reporting that CK2 regulates the sensitivity of PCa cells to the antitumor effect of TRAIL. This is important for understanding how the TRAIL pathway is disrupted in PCa and may help to develop an effective combinatorial therapy for PCa.

  18. The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling.

    Science.gov (United States)

    Schwarz-Romond, Thomas; Asbrand, Christian; Bakkers, Jeroen; Kühl, Michael; Schaeffer, Hans-Joerg; Huelsken, Jörg; Behrens, Jürgen; Hammerschmidt, Matthias; Birchmeier, Walter

    2002-08-15

    Wnt signals control decisive steps in development and can induce the formation of tumors. Canonical Wnt signals control the formation of the embryonic axis, and are mediated by stabilization and interaction of beta-catenin with Lef/Tcf transcription factors. An alternative branch of the Wnt pathway uses JNK to establish planar cell polarity in Drosophila and gastrulation movements in vertebrates. We describe here the vertebrate protein Diversin that interacts with two components of the canonical Wnt pathway, Casein kinase Iepsilon (CKIepsilon) and Axin/Conductin. Diversin recruits CKIepsilon to the beta-catenin degradation complex that consists of Axin/Conductin and GSK3beta and allows efficient phosphorylation of beta-catenin, thereby inhibiting beta-catenin/Tcf signals. Morpholino-based gene ablation in zebrafish shows that Diversin is crucial for axis formation, which depends on beta-catenin signaling. Diversin is also involved in JNK activation and gastrulation movements in zebrafish. Diversin is distantly related to Diego of Drosophila, which functions only in the pathway that controls planar cell polarity. Our data show that Diversin is an essential component of the Wnt-signaling pathway and acts as a molecular switch, which suppresses Wnt signals mediated by the canonical beta-catenin pathway and stimulates signaling via JNK.

  19. LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis1[OPEN

    Science.gov (United States)

    Min, Ling; Hu, Qin; Li, Yaoyao; Xu, Jiao; Ma, Yizan; Zhu, Longfu; Yang, Xiyan; Zhang, Xianlong

    2015-01-01

    Somatic embryogenesis (SE) is an efficient tool for the propagation of plant species and also, a useful model for studying the regulatory networks in embryo development. However, the regulatory networks underlying the transition from nonembryogenic callus to somatic embryos during SE remain poorly understood. Here, we describe an upland cotton (Gossypium hirsutum) CASEIN KINASE I gene, GhCKI, which is a unique key regulatory factor that strongly affects SE. Overexpressing GhCKI halted the formation of embryoids and plant regeneration because of a block in the transition from nonembryogenic callus to somatic embryos. In contrast, defective GhCKI in plants facilitated SE. To better understand the mechanism by which GhCKI regulates SE, the regulatory network was analyzed. A direct upstream negative regulator protein, cotton LEAFY COTYLEDON1, was identified to be targeted to a cis-element, CTTTTC, in the promoter of GhCKI. Moreover, GhCKI interacted with and phosphorylated cotton CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF transcription factor15 by coordinately regulating the expression of cotton PHYTOCHROME INTERACTING FACTOR4, finally disrupting auxin homeostasis, which led to increased cell proliferation and aborted somatic embryo formation in GhCKI-overexpressing somatic cells. Our results show a complex process of SE that is negatively regulated by GhCKI through a complex regulatory network. PMID:26491146

  20. LEAFY COTYLEDON1-CASEIN KINASE I-TCP15-PHYTOCHROME INTERACTING FACTOR4 Network Regulates Somatic Embryogenesis by Regulating Auxin Homeostasis.

    Science.gov (United States)

    Min, Ling; Hu, Qin; Li, Yaoyao; Xu, Jiao; Ma, Yizan; Zhu, Longfu; Yang, Xiyan; Zhang, Xianlong

    2015-12-01

    Somatic embryogenesis (SE) is an efficient tool for the propagation of plant species and also, a useful model for studying the regulatory networks in embryo development. However, the regulatory networks underlying the transition from nonembryogenic callus to somatic embryos during SE remain poorly understood. Here, we describe an upland cotton (Gossypium hirsutum) CASEIN KINASE I gene, GhCKI, which is a unique key regulatory factor that strongly affects SE. Overexpressing GhCKI halted the formation of embryoids and plant regeneration because of a block in the transition from nonembryogenic callus to somatic embryos. In contrast, defective GhCKI in plants facilitated SE. To better understand the mechanism by which GhCKI regulates SE, the regulatory network was analyzed. A direct upstream negative regulator protein, cotton LEAFY COTYLEDON1, was identified to be targeted to a cis-element, CTTTTC, in the promoter of GhCKI. Moreover, GhCKI interacted with and phosphorylated cotton CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF transcription factor15 by coordinately regulating the expression of cotton PHYTOCHROME INTERACTING FACTOR4, finally disrupting auxin homeostasis, which led to increased cell proliferation and aborted somatic embryo formation in GhCKI-overexpressing somatic cells. Our results show a complex process of SE that is negatively regulated by GhCKI through a complex regulatory network.

  1. Drosophila mbm is a nucleolar myc and casein kinase 2 target required for ribosome biogenesis and cell growth of central brain neuroblasts.

    Science.gov (United States)

    Hovhanyan, Anna; Herter, Eva K; Pfannstiel, Jens; Gallant, Peter; Raabe, Thomas

    2014-05-01

    Proper cell growth is a prerequisite for maintaining repeated cell divisions. Cells need to translate information about intracellular nutrient availability and growth cues from energy-sensing organs into growth-promoting processes, such as sufficient supply with ribosomes for protein synthesis. Mutations in the mushroom body miniature (mbm) gene impair proliferation of neural progenitor cells (neuroblasts) in the central brain of Drosophila melanogaster. Yet the molecular function of Mbm has so far been unknown. Here we show that mbm does not affect the molecular machinery controlling asymmetric cell division of neuroblasts but instead decreases their cell size. Mbm is a nucleolar protein required for small ribosomal subunit biogenesis in neuroblasts. Accordingly, levels of protein synthesis are reduced in mbm neuroblasts. Mbm expression is transcriptionally regulated by Myc, which, among other functions, relays information from nutrient-dependent signaling pathways to ribosomal gene expression. At the posttranslational level, Mbm becomes phosphorylated by casein kinase 2 (CK2), which has an impact on localization of the protein. We conclude that Mbm is a new part of the Myc target network involved in ribosome biogenesis, which, together with CK2-mediated signals, enables neuroblasts to synthesize sufficient amounts of proteins required for proper cell growth.

  2. Casein Kinase 2 Is a Novel Regulator of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Trafficking.

    Science.gov (United States)

    Chan, Ting; Cheung, Florence Shin Gee; Zheng, Jian; Lu, Xiaoxi; Zhu, Ling; Grewal, Thomas; Murray, Michael; Zhou, Fanfan

    2016-01-04

    Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated.

  3. The plastid casein kinase 2 phosphorylates Rubisco activase at the Thr-78 site but is not essential for regulation of Rubisco activation state

    Directory of Open Access Journals (Sweden)

    Sang Yeol eKim

    2016-03-01

    Full Text Available Rubisco activase (RCA is essential for the activation of Rubisco, the carboxylating enzyme of photosynthesis. In Arabidopsis, RCA is composed of a large RCAα and small RCAβ isoform that are formed by alternative splicing of a single gene (At2g39730. The activity of Rubisco is controlled in response to changes in irradiance by regulation of RCA activity, which is known to involve a redox-sensitive disulfide bond located in the carboxy-terminal extension of the RCAα subunit. Additionally, phosphorylation of RCA threonine-78 (Thr-78 has been reported to occur in the dark suggesting that phosphorylation may also be associated with dark-inactivation of RCA and deactivation of Rubisco. In the present study, we developed site-specific antibodies to monitor phosphorylation of RCA at the Thr-78 site and used non-reducing SDS-PAGE to monitor the redox status of the RCAα subunit. By immunoblotting, phosphorylation of both RCA isoforms occurred at low light and in the dark and feeding peroxide or DTT to leaf segments indicated that redox status of the chloroplast stroma was a critical factor controlling RCA phosphorylation. Use of a knockout mutant identified the plastid-targeted casein kinase 2 (cpCK2α as the major protein kinase involved in RCA phosphorylation. Studies with recombinant cpCK2α and synthetic peptide substrates identified acidic residues at the -1, +2 and +3 positions surrounding Thr-78 as strong positive recognition elements. The cpck2 knockout mutant had strongly reduced phosphorylation at the Thr-78 site but was similar to wild type plants in terms of induction kinetics of photosynthesis following transfer from darkness or low light to high light, suggesting that if phosphorylation of RCA Thr-78 plays a direct role it would be redundant to redox regulation for control of Rubisco activation state under normal conditions.

  4. A mechanism for regulation of chloroplast LHC II kinase by plastoquinol and thioredoxin.

    Science.gov (United States)

    Puthiyaveetil, Sujith

    2011-06-23

    State transitions are acclimatory responses to changes in light quality in photosynthesis. They involve the redistribution of absorbed excitation energy between photosystems I and II. In plants and green algae, this redistribution is produced by reversible phosphorylation of the chloroplast light harvesting complex II (LHC II). The LHC II kinase is activated by reduced plastoquinone (PQ) in photosystem II-specific low light. In high light, when PQ is also reduced, LHC II kinase becomes inactivated by thioredoxin. Based on newly identified amino acid sequence features of LHC II kinase and other considerations, a mechanism is suggested for its redox regulation.

  5. Casein kinase 2 down-regulation and activation by polybasic peptides are mediated by acidic residues in the 55-64 region of the beta-subunit. A study with calmodulin as phosphorylatable substrate

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Issinger, O G;

    1994-01-01

    The noncatalytic beta-subunit is responsible for the latency of casein kinase 2 (CK2) activity toward calmodulin. Twenty-one mutants of the beta-subunit bearing either deletions or Ala substitutions for charged residues in the 5-6, 55-70, and 171-178 sequences have been assayed for their ability...... insensitive to 42 nM polylysine, which conversely promotes a more than 10-fold increase of calmodulin phosphorylation with wild-type beta.(ABSTRACT TRUNCATED AT 250 WORDS)...

  6. Safety and preliminary efficacy data of a novel Casein Kinase 2 (CK2 peptide inhibitor administered intralesionally at four dose levels in patients with cervical malignancies

    Directory of Open Access Journals (Sweden)

    Alonso Daniel F

    2009-05-01

    Full Text Available Abstract Background Cervical cancer is now considered the second leading cause of death among women worldwide, and its incidence has reached alarming levels, especially in developing countries. Similarly, high grade squamous intraepithelial lesion (HSIL, the precursor stage for cervical cancer, represents a growing health problem among younger women as the HSIL management regimes that have been developed are not fully effective. From the etiological point of view, the presence of Human Papillomavirus (HPV has been demonstrated to play a crucial role for developing cervical malignancies, and viral DNA has been detected in 99.7% of cervical tumors at the later stages. CIGB-300 is a novel cyclic synthetic peptide that induces apoptosis in malignant cells and elicits antitumor activity in cancer animal models. CIGB-300 impairs the Casein Kinase (CK2 phosphorylation, by targeting the substrate's phosphoaceptor domain. Based on the perspectives of CIGB-300 to treat cancer, this "first-in-human" study investigated its safety and tolerability in patients with cervical malignancies. Methods Thirty-one women with colposcopically and histologically diagnosed microinvasive or pre-invasive cervical cancer were enrolled in a dose escalating study. CIGB-300 was administered sequentially at 14, 70, 245 and 490 mg by intralesional injections during 5 consecutive days to groups of 7 – 10 patients. Toxicity was monitored daily until fifteen days after the end of treatment, when patients underwent conization. Digital colposcopy, histology, and HPV status were also evaluated. Results No maximum-tolerated dose or dose-limiting toxicity was achieved. The most frequent local events were pain, bleeding, hematoma and erythema at the injection site. The systemic adverse events were rash, facial edema, itching, hot flashes, and localized cramps. 75% of the patients experienced a significant lesion reduction at colposcopy and 19% exhibited full histological regression

  7. Transient Receptor Potential Melastatin 7 Cation Channel Kinase: New Player in Angiotensin II-Induced Hypertension.

    Science.gov (United States)

    Antunes, Tayze T; Callera, Glaucia E; He, Ying; Yogi, Alvaro; Ryazanov, Alexey G; Ryazanova, Lillia V; Zhai, Alexander; Stewart, Duncan J; Shrier, Alvin; Touyz, Rhian M

    2016-04-01

    Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein comprising a magnesium (Mg(2+))/cation channel and a kinase domain. We previously demonstrated that vasoactive agents regulate vascular TRPM7. Whether TRPM7 plays a role in the pathophysiology of hypertension and associated cardiovascular dysfunction is unknown. We studied TRPM7 kinase-deficient mice (TRPM7Δkinase; heterozygous for TRPM7 kinase) and wild-type (WT) mice infused with angiotensin II (Ang II; 400 ng/kg per minute, 4 weeks). TRPM7 kinase expression was lower in heart and aorta from TRPM7Δkinase versus WT mice, effects that were further reduced by Ang II infusion. Plasma Mg(2+) was lower in TRPM7Δkinase versus WT mice in basal and stimulated conditions. Ang II increased blood pressure in both strains with exaggerated responses in TRPM7Δkinase versus WT groups (Phypertension is exaggerated, cardiac remodeling and left ventricular dysfunction are amplified, and endothelial function is impaired. These processes are associated with hypomagnesemia, blunted TRPM7 kinase expression/signaling, endothelial nitric oxide synthase downregulation, and proinflammatory vascular responses. Our findings identify TRPM7 kinase as a novel player in Ang II-induced hypertension and associated vascular and target organ damage.

  8. Phosphorylation of as1-casein is regulated by different genes

    NARCIS (Netherlands)

    Bijl, E.; Valenberg, van H.J.F.; Huppertz, T.; Hooijdonk, van A.C.M.; Bovenhuis, H.

    2014-01-01

    Casein phosphorylation is a posttranslational modification catalyzed by kinase enzymes that attach phosphate groups to specific AA in the protein sequence. This modification is one of the key factors responsible for the stabilization of calcium phosphate nanoclusters in casein micelles and for the i

  9. Bovine β-casein

    NARCIS (Netherlands)

    Atamer, Zeynep; Post, Antonie E.; Schubert, Thomas; Holder, Aline; Boom, Remko Marcel; Hinrichs, Jörg

    2017-01-01

    In recent years there has been an increasing interest in pure casein fractions, particularly β-casein due to its physiochemical properties as well as its bio- and techno-functional properties. A range of methods has been developed for the fractionation of casein into its individual proteins. The

  10. Calmodulin kinase II inhibition protects against structural heart disease.

    Science.gov (United States)

    Zhang, Rong; Khoo, Michelle S C; Wu, Yuejin; Yang, Yingbo; Grueter, Chad E; Ni, Gemin; Price, Edward E; Thiel, William; Guatimosim, Silvia; Song, Long-Sheng; Madu, Ernest C; Shah, Anisha N; Vishnivetskaya, Tatiana A; Atkinson, James B; Gurevich, Vsevolod V; Salama, Guy; Lederer, W J; Colbran, Roger J; Anderson, Mark E

    2005-04-01

    Beta-adrenergic receptor (betaAR) stimulation increases cytosolic Ca(2+) to physiologically augment cardiac contraction, whereas excessive betaAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a recently identified downstream element of the betaAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in betaAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive betaAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and betaAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to betaAR signaling.

  11. The effect of polylysine on casein-kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O;

    1992-01-01

    alpha subunit. The concentration of polylysine required for half-maximal stimulation is comparable to CK2 concentration and increases by increasing CK2 concentration, suggesting that polylysine primarily interacts with the enzyme, rather than with the peptide substrate.......The mechanism by which polybasic peptides stimulate the activity of casein kinase 2 (CK2) has been studied by comparing the effect of polylysine on the phosphorylation of a variety of protein and peptide substrates by the native CK2 holoenzyme and by its recombinant catalytic alpha subunit, either...... phosphorylated by either CK2 holoenzyme or the recombinant alpha subunit with 5.8-fold and 2.8-fold stimulation by polylysine, respectively. The recombinant beta subunit of CK2 is itself a good exogenous substrate for the enzyme, its phosphorylation, however, is inhibited rather than enhanced by polylysine...

  12. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    Science.gov (United States)

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  13. The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II

    DEFF Research Database (Denmark)

    Elmlund, Hans; Baraznenok, Vera; Lindahl, Martin

    2006-01-01

    CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8......-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II....

  14. Effect of oxidative stress on Rho kinase II and smooth muscle contraction in rat stomach.

    Science.gov (United States)

    Al-Shboul, Othman; Mustafa, Ayman

    2015-06-01

    Recent studies have shown that both Rho kinase signaling and oxidative stress are involved in the pathogenesis of a number of human diseases, such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the gastrointestinal (GI) smooth muscle Rho kinase pathway. The aim of the current study was to investigate the effect of oxidative stress on Rho kinase II and muscle contraction in rat stomach. The peroxynitrite donor 3-morpholinosydnonimine (SIN-1), hydrogen peroxide (H2O2), and peroxynitrite were used to induce oxidative stress. Rho kinase II expression and ACh-induced activity were measured in control and oxidant-treated cells via specifically designed enzyme-linked immunosorbent assay (ELISA) and activity assay kits, respectively. Single smooth muscle cell contraction was measured via scanning micrometry in the presence or absence of the Rho kinase blocker, Y-27632 dihydrochloride. All oxidant agents significantly increased ACh-induced Rho kinase II activity without affecting its expression level. Most important, oxidative stress induced by all three agents augmented ACh-stimulated muscle cell contraction, which was significantly inhibited by Y-27632. In conclusion, oxidative stress activates Rho kinase II and enhances contraction in rat gastric muscle, suggesting an important role in GI motility disorders associated with oxidative stress.

  15. Phosphorylation of the mRNA cap binding protein and eIF-4A by different protein kinases

    Energy Technology Data Exchange (ETDEWEB)

    Hagedorn, C.H.

    1987-05-01

    These studies were done to determine the identity of a protein kinase that phosphorylates the mRNA cap binding protein (CBP). Two chromatographic steps (dye and ligand and ion exchange HPLC) produced a 500x purification of an enzyme activity in rabbit reticulocytes that phosphorylated CBP at serine residues. Isoelectric focusing analysis of kinase treated CBP demonstrated 5 isoelectric species of which the 2 most anodic species were phosphorylated (contained /sup 32/P). This kinase activity phosphorylated CBP when it was isolated or in the eIF-4F complex. Purified protein kinase C, cAMP or cGMP dependent protein kinase, casein kinase I or II, myosin light chain kinase or insulin receptor kinase did not significantly phosphorylate isolated CBP or CBP in the eIF-4F complex. However, cAMP and cGMP dependent protein kinases and casein kinase II phosphorylated eIF-4A but did not phosphorylate the 46 kDa component of eIF-4F. cAMP dependent protein kinase phosphorylated a approx. 220 kDa protein doublet in eIF-4F preparations. These studies indicate that CBP kinase activity probably represents a previously unidentified protein kinase. In addition, eIF-4A appears to be phosphorylated by several protein kinases whereas the 46 kDa component of the eIF-4F complex was not.

  16. Protein kinase C-beta II (PKC-betaII) expression in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise; Lindebjerg, Jan; Lahn, Michael;

    2009-01-01

    PURPOSE: Current development of targeted agents for the treatment of colorectal cancer include the clinical evaluation of kinase inhibitors, such as enzastaurin, a serine/threonine kinase inhibitor designed to suppress signaling through Protein Kinase C (PKC) and AKT pathways. Little is known abo...

  17. Natural variation in casein composition of milk

    NARCIS (Netherlands)

    Bijl, E.

    2014-01-01

    Bovine milk contains 3-4 % protein and almost 80% of the milk protein fraction consist of four caseins; αs1-casein, β-casein, αs2-casein and κ-casein. Most of the caseins in milk are assembled in casein micelles, which consist of several thousands of indi

  18. Slit and Netrin-1 guide cranial motor axon pathfinding via Rho-kinase, myosin light chain kinase and myosin II

    Directory of Open Access Journals (Sweden)

    Drescher Uwe

    2010-06-01

    Full Text Available Abstract Background In the developing hindbrain, cranial motor axon guidance depends on diffusible repellent factors produced by the floor plate. Our previous studies have suggested that candidate molecules for mediating this effect are Slits, Netrin-1 and Semaphorin3A (Sema3A. It is unknown to what extent these factors contribute to floor plate-derived chemorepulsion of motor axons, and the downstream signalling pathways are largely unclear. Results In this study, we have used a combination of in vitro and in vivo approaches to identify the components of floor plate chemorepulsion and their downstream signalling pathways. Using in vitro motor axon deflection assays, we demonstrate that Slits and Netrin-1, but not Sema3A, contribute to floor plate repulsion. We also find that the axon pathways of dorsally projecting branchiomotor neurons are disrupted in Netrin-1 mutant mice and in chick embryos expressing dominant-negative Unc5a receptors, indicating an in vivo role for Netrin-1. We further demonstrate that Slit and Netrin-1 signalling are mediated by Rho-kinase (ROCK and myosin light chain kinase (MLCK, which regulate myosin II activity, controlling actin retrograde flow in the growth cone. We show that MLCK, ROCK and myosin II are required for Slit and Netrin-1-mediated growth cone collapse of cranial motor axons. Inhibition of these molecules in explant cultures, or genetic manipulation of RhoA or myosin II function in vivo causes characteristic cranial motor axon pathfinding errors, including the inability to exit the midline, and loss of turning towards exit points. Conclusions Our findings suggest that both Slits and Netrin-1 contribute to floor plate-derived chemorepulsion of cranial motor axons. They further indicate that RhoA/ROCK, MLCK and myosin II are components of Slit and Netrin-1 signalling pathways, and suggest that these pathways are of key importance in cranial motor axon navigation.

  19. Fibronectin matrix assembly requires distinct contributions from Rho kinases I and -II

    DEFF Research Database (Denmark)

    Yoneda, Atsuko; Ushakov, Dmitriy; Multhaupt, Hinke A B;

    2006-01-01

    Extracellular matrix is integral to tissue architecture and regulates many aspects of cell behavior. Fibronectin matrix assembly involves the actin cytoskeleton and the small GTPase RhoA, but downstream signaling is not understood. Here, down-regulation of either rho kinase isoform (ROCK I or -II...

  20. Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II.

    Science.gov (United States)

    Cingolani, Francesca; Casasampere, Mireia; Sanllehí, Pol; Casas, Josefina; Bujons, Jordi; Fabrias, Gemma

    2014-08-01

    Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (Ki = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites.

  1. Using bacteria to determine protein kinase specificity and predict target substrates.

    Directory of Open Access Journals (Sweden)

    Michael F Chou

    Full Text Available The identification of protein kinase targets remains a significant bottleneck for our understanding of signal transduction in normal and diseased cellular states. Kinases recognize their substrates in part through sequence motifs on substrate proteins, which, to date, have most effectively been elucidated using combinatorial peptide library approaches. Here, we present and demonstrate the ProPeL method for easy and accurate discovery of kinase specificity motifs through the use of native bacterial proteomes that serve as in vivo libraries for thousands of simultaneous phosphorylation reactions. Using recombinant kinases expressed in E. coli followed by mass spectrometry, the approach accurately recapitulated the well-established motif preferences of human basophilic (Protein Kinase A and acidophilic (Casein Kinase II kinases. These motifs, derived for PKA and CK II using only bacterial sequence data, were then further validated by utilizing them in conjunction with the scan-x software program to computationally predict known human phosphorylation sites with high confidence.

  2. Electrochemiluminescence resonance energy transfer between graphene quantum dots and graphene oxide for sensitive protein kinase activity and inhibitor sensing

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Ru-Ping; Qiu, Wei-Bin; Zhao, Hui-Fang; Xiang, Cai-Yun; Qiu, Jian-Ding, E-mail: jdqiu@ncu.edu.cn

    2016-01-21

    Herein, a novel electrochemiluminescence resonance energy transfer (ECL-RET) biosensor using graphene quantum dots (GQDs) as donor and graphene oxide (GO) as acceptor for monitoring the activity of protein kinase was presented for the first time. Anti-phosphoserine antibody conjugated graphene oxide (Ab-GO) nonocomposite could be captured onto the phosphorylated peptide/GQDs modified electrode surface through antibody–antigen interaction in the presence of casein kinase II (CK2) and adenosine 5′-triphosphate (ATP), resulting in ECL from the GQDs quenching by closely contacting GO. This ECL quenching degree was positively correlated with CK2 activity. Therefore, on the basis of ECL-RET between GQDs and GO, the activity of protein kinase can be detected sensitively. This biosensor can also be used for quantitative analysis CK2 activity in serum samples and qualitative screening kinase inhibition, indicating the potential application of the developed method in biochemical fundamental research and clinical diagnosis. - Highlights: • We reported a novel ECL-RET biosensor for sensitive analysis of casein kinase II activity. • The successful ECL-RET between GQDs and GO could be established. • GQDs was employed for casein kinase II activity monitoring and inhibition assay. • Highly sensitive detection of CK2 activity and inhibition was achieved.

  3. Mitogen-activated protein kinase-activated protein kinase 2 in angiotensin II-induced inflammation and hypertension: regulation of oxidative stress.

    Science.gov (United States)

    Ebrahimian, Talin; Li, Melissa Wei; Lemarié, Catherine A; Simeone, Stefania M C; Pagano, Patrick J; Gaestel, Matthias; Paradis, Pierre; Wassmann, Sven; Schiffrin, Ernesto L

    2011-02-01

    Vascular oxidative stress and inflammation play an important role in angiotensin II-induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase-activated protein kinase 2 (MK2), a downstream target of p38 mitogen-activated protein kinase, is involved in angiotensin II-induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II-induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II-induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II-induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II-induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II-induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II-induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation

  4. Roles of an unconventional protein kinase and myosin II in amoeba osmotic shock responses.

    Science.gov (United States)

    Betapudi, Venkaiah; Egelhoff, Thomas T

    2009-12-01

    The contractile vacuole (CV) is a dynamic organelle that enables Dictyostelium amoeba and other protist to maintain osmotic homeostasis by expelling excess water. In the present study, we have uncovered a mechanism that coordinates the mechanics of the CV with myosin II, regulated by VwkA, an unconventional protein kinase that is conserved in an array of protozoa. Green fluorescent protein (GFP)-VwkA fusion proteins localize persistently to the CV during both filling and expulsion phases of water. In vwkA null cells, the established CV marker dajumin still localizes to the CV, but these structures are large, spherical and severely impaired for discharge. Furthermore, myosin II cortical localization and assembly are abnormal in vwkA null cells. Parallel analysis of wild-type cells treated with myosin II inhibitors or of myosin II null cells also results in enlarged CVs with impaired dynamics. We suggest that the myosin II cortical cytoskeleton, regulated by VwkA, serves a critical conserved role in the periodic contractions of the CV, as part of the osmotic protective mechanism of protozoa.

  5. Creatine kinase activity in patients with diabetes mellitus type I and type II.

    Science.gov (United States)

    Jevrić-Causević, Adlija; Malenica, Maja; Dujić, Tanja

    2006-08-01

    Diabetes mellitus can be looked upon as an array of diseases, all of which exhibit common symptoms. While pathogenesis of IDDM (insulin dependant diabetes mellitus) is well understood, the same is not true for diabetes mellitus type II. In the latter case, relative contribution of the two factors (insulin resistance or decreased insulin secretion) varies individually, being highly increased in peripheral tissues and strictly dependant on insulin for glucose uptake. Moreover, in patients with diabetes mellitus type II, disbalance at the level of regulation of glucose metabolism as well as lipid metabolism has been noted in skeletal muscles. It is normal to assume that in this type of diabetes, these changes are reflected at the level of total activity of enzyme creatine kinase. This experimental work was performed on a group of 80 regular patients of Sarajevo General Hospital. Forty of those patients were classified as patients with diabetes type I and forty as patients with diabetes type II. Each group of patients was carefully chosen and constituted of equal number of males and females. The same was applied for adequate controls. Concentration of glucose was determined for each patient with GOD method, while activity of creatine kinase was determined with CK-NAC activated kit. Statistical analysis of the results was performed with SPSS software for Windows. Obtained results point out highly expressed differences in enzyme activity between two populations examined. Changes in enzyme activity are more expressed in patients with diabetes type II. Positive correlation between concentration of glucose and serum activity of the enzyme is seen in both categories of diabetic patients which is not the case for the patients in control group. At the same time, correlation between age and type of diabetes does exist . This is not followed at the level of enzyme activity or concentration of glucose.

  6. Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors.

    Science.gov (United States)

    Canning, Peter; Ruan, Qui; Schwerd, Tobias; Hrdinka, Matous; Maki, Jenny L; Saleh, Danish; Suebsuwong, Chalada; Ray, Soumya; Brennan, Paul E; Cuny, Gregory D; Uhlig, Holm H; Gyrd-Hansen, Mads; Degterev, Alexei; Bullock, Alex N

    2015-09-17

    RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers.

  7. Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus.

    Science.gov (United States)

    Kłopocka, W; Redowicz, M J

    2004-10-01

    The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebae's endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin-myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion.

  8. Casein nomenclature, structure and association

    Science.gov (United States)

    This chapter of the Encyclopedia of Dairy Science deals with the recent developments in the nomenclature, classification, structures, and associations of the major milk proteins: the caseins. Identification of the caseins continues to be based upon their primary structures. Significant findings re...

  9. Ablation of phosphoinositide-3-kinase class II alpha suppresses hepatoma cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Ng, Stanley K.L. [Singapore Immunology Network A-STAR (Singapore); Neo, Soek-Ying, E-mail: neo_soek_ying@sics.a-star.edu.sg [Singapore Immunology Network A-STAR (Singapore); Yap, Yann-Wan [Singapore Immunology Network A-STAR (Singapore); Karuturi, R. Krishna Murthy; Loh, Evelyn S.L. [Genome Institute of Singapore A-STAR (Singapore); Liau, Kui-Hin [Department of General Surgery, Tan Tock Seng Hospital (Singapore); Ren, Ee-Chee, E-mail: ren_ee_chee@immunol.a-star.edu.sg [Singapore Immunology Network A-STAR (Singapore); Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore (Singapore)

    2009-09-18

    Cancer such as hepatocellular carcinoma (HCC) is characterized by complex perturbations in multiple signaling pathways, including the phosphoinositide-3-kinase (PI3K/AKT) pathways. Herein we investigated the role of PI3K catalytic isoforms, particularly class II isoforms in HCC proliferation. Among the siRNAs tested against the eight known catalytic PI3K isoforms, specific ablation of class II PI3K alpha (PIK3C2{alpha}) was the most effective in impairing cell growth and this was accompanied by concomitant decrease in PIK3C2{alpha} mRNA and protein levels. Colony formation ability of cells deficient for PIK3C2{alpha} was markedly reduced and growth arrest was associated with increased caspase 3 levels. A small but significant difference in gene dosage and expression levels was detected between tumor and non-tumor tissues in a cohort of 19 HCC patients. Taken together, these data suggest for the first time that in addition to class I PI3Ks in cancer, class II PIK3C2{alpha} can modulate HCC cell growth.

  10. α-Calcium calmodulin kinase II modulates the temporal structure of hippocampal bursting patterns.

    Directory of Open Access Journals (Sweden)

    Jeiwon Cho

    Full Text Available The alpha calcium calmodulin kinase II (α-CaMKII is known to play a key role in CA1/CA3 synaptic plasticity, hippocampal place cell stability and spatial learning. Additionally, there is evidence from hippocampal electrophysiological slice studies that this kinase has a role in regulating ion channels that control neuronal excitability. Here, we report in vivo single unit studies, with α-CaMKII mutant mice, in which threonine 305 was replaced with an aspartate (α-CaMKII(T305D mutants, that indicate that this kinase modulates spike patterns in hippocampal pyramidal neurons. Previous studies showed that α-CaMKII(T305D mutants have abnormalities in both hippocampal LTP and hippocampal-dependent learning. We found that besides decreased place cell stability, which could be caused by their LTP impairments, the hippocampal CA1 spike patterns of α-CaMKII(T305D mutants were profoundly abnormal. Although overall firing rate, and overall burst frequency were not significantly altered in these mutants, inter-burst intervals, mean number of intra-burst spikes, ratio of intra-burst spikes to total spikes, and mean intra-burst intervals were significantly altered. In particular, the intra burst intervals of place cells in α-CaMKII(T305D mutants showed higher variability than controls. These results provide in vivo evidence that besides its well-known function in synaptic plasticity, α-CaMKII, and in particular its inhibitory phosphorylation at threonine 305, also have a role in shaping the temporal structure of hippocampal burst patterns. These results suggest that some of the molecular processes involved in acquiring information may also shape the patterns used to encode this information.

  11. Bcl10 is phosphorylated on Ser138 by Ca2+/calmodulin-dependent protein kinase II.

    Science.gov (United States)

    Ishiguro, Kazuhiro; Ando, Takafumi; Goto, Hidemi; Xavier, Ramnik

    2007-03-01

    Ordered assembly of scaffold proteins Carma1-Bcl10-Malt1 determines NF-kappaB activation following T cell receptor (TCR) engagement. Carma1-Bcl10 interaction and the signaling pathway are controlled by Carma1 phosphorylation, which are induced by PKCtheta and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition to Carma1 phosphorylation, previous studies have demonstrated that Bcl10 is phosphorylated in the C-terminal Ser/Thr rich region following TCR engagement. However the kinases that phosphorylate Bcl10 are incompletely understood. Here we show that CaMKII phosphorylates Bcl10 on Ser138. Furthermore, a CaMKII inhibitor, KN93, and CaMKII siRNA substantially reduce Bcl10 phosphorylation induced by phorbol myristate acetate/ionomycin. S138A mutation prolongs Bcl10-induced NF-kappaB activation, suggesting that Bcl10 phosphorylation is involved in attenuation of NF-kappaB activation. These findings suggest that CaMKII modulates NF-kappaB activation via phosphorylating Bcl10 as well as Carma1.

  12. [The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis].

    Science.gov (United States)

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Omel'ianchuk, L V

    2008-09-01

    The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.

  13. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    Science.gov (United States)

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  14. Chymosin-induced hydrolysis of caseins: Influence of degree of phosphorylation of alpha-s1-casein and genetic variants of beta-casein

    NARCIS (Netherlands)

    Bijl, E.; Valenberg, van H.J.F.; Sikkes, S.; Jumelet, S.; Sala, G.; Olieman, K.; Hooijdonk, van A.C.M.; Huppertz, T.

    2014-01-01

    The objective of this study was to investigate the impact of natural variations in aS1-casein and b-casein composition of milk on chymosin-induced hydrolysis of these caseins in milk gels and in sodium caseinate solutions. At 50% casein degradation, 15% more of aS1-casein with eight phosphate groups

  15. Hyperexcitability and changes in activities of Ca2+/calmodulin-dependent kinase II and mitogen-activated protein kinase in the hippocampus of rats exposed to 1-bromopropane.

    Science.gov (United States)

    Fueta, Yukiko; Fukunaga, Kohji; Ishidao, Toru; Hori, Hajime

    2002-12-20

    Chronic inhalation of 1-bromopropane (1-BP), a substitute of ozone-depleting chlorofluorocarbons, has been suspected of having central neurotoxicity (Clinical Neurology and Neurosurgery 101 (1999) 199; Journal of Occupational Health 44 (2002) 1) for humans. In animal experiments, 1-BP inhalation (1500 ppm) caused hyperexcitability in the CA1 and the dentate gyrus (DG) [Journal of Occupational Health 42 (2000) 149, Journal of Occupational Health 44 (2002) 156]. We studied whether the hyperexcitability is associated with changes of Ca2+/calmodulin-dependent kinase II (CaMKII), mitogen-activated protein kinase (MAPK), and protein kinase C (PKC). Male Wistar rats were exposed to 1-BP for 6 hours in a day in an exposure chamber with a concentration of 700 ppm for 8 weeks. After the inhalation, paired-pulse ratios of field excitatory postsynaptic potentials and population spikes (PSs) were analyzed in the CA1 and DG of hippocampal slices. Control rats were then given fresh air in the inhalation chamber. Semiquantitative immunoblotting analyses of protein kinases using antibodies against active and conventional protein kinases were done using the whole hippocampus. A paired-pulse ratio of PS was increased at the 5 ms interpulse interval in the CA1 and at the 10-20 ms interpulse intervals in the DG. The amount of active MAPK and total amount of CaMKIIalpha and beta were significantly increased by 28, 29, and 46% compared to control, respectively, without any change in PKC activity. In contrast, the amount of active CaMKIIbeta was decreased to 78%. These results suggest that modifications of intracellular signaling cascades are associated with hyperexcitability that occurred in the hippocampal formation of rats exposed to the chronic inhalation of 1-BP.

  16. Mechanical stress triggers cardiomyocyte autophagy through angiotensin II type 1 receptor-mediated p38MAP kinase independently of angiotensin II.

    Directory of Open Access Journals (Sweden)

    Li Lin

    Full Text Available Angiotensin II (Ang II type 1 (AT1 receptor is known to mediate a variety of physiological actions of Ang II including autophagy. However, the role of AT1 receptor in cardiomyocyte autophagy triggered by mechanical stress still remains elusive. The aim of this study was therefore to examine whether and how AT1 receptor participates in cardiomyocyte autophagy induced by mechanical stresses. A 48-hour mechanical stretch and a 4-week transverse aorta constriction (TAC were imposed to cultured cardiomyocytes of neonatal rats and adult male C57B/L6 mice, respectively, to induce cardiomyocyte hypertrophy prior to the assessment of cardiomyocyte autophagy using LC3b-II. Losartan, an AT1 receptor blocker, but not PD123319, the AT2 inhibitor, was found to significantly reduce mechanical stretch-induced LC3b-II upregulation. Moreover, inhibition of p38MAP kinase attenuated not only mechanical stretch-induced cardiomyocyte hypertrophy but also autophagy. To the contrary, inhibition of ERK and JNK suppressed cardiac hypertrophy but not autophagy. Intriguingly, mechanical stretch-induced autophagy was significantly inhibited by Losartan in the absence of Ang II. Taken together, our results indicate that mechanical stress triggers cardiomyocyte autophagy through AT1 receptor-mediated activation of p38MAP kinase independently of Ang II.

  17. Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

    Directory of Open Access Journals (Sweden)

    Stabel Silvia

    2002-04-01

    Full Text Available Abstract Background The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro. Results We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B, alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues. Conclusion The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

  18. Ca2+/calmodulin-dependent kinase II contributes to inhibitor of nuclear factor-kappa B kinase complex activation in Helicobacter pylori infection.

    Science.gov (United States)

    Maubach, Gunter; Sokolova, Olga; Wolfien, Markus; Rothkötter, Hermann-Josef; Naumann, Michael

    2013-09-15

    Helicobacter pylori, a class I carcinogen, induces a proinflammatory response by activating the transcription factor nuclear factor-kappa B (NF-κB) in gastric epithelial cells. This inflammatory condition could lead to chronic gastritis, which is epidemiologically and biologically linked to the development of gastric cancer. So far, there exists no clear knowledge on how H. pylori induces the NF-κB-mediated inflammatory response. In our study, we investigated the role of Ca(2+) /calmodulin-dependent kinase II (CAMKII), calmodulin, protein kinases C (PKCs) and the CARMA3-Bcl10-MALT1 (CBM) complex in conjunction with H. pylori-induced activation of NF-κB via the inhibitor of nuclear factor-kappa B kinase (IKK) complex. We use specific inhibitors and/or RNA interference to assess the contribution of these components. Our results show that CAMKII and calmodulin contribute to IKK complex activation and thus to the induction of NF-κB in response to H. pylori infection, but not in response to TNF-α. Thus, our findings are specific for H. pylori infected cells. Neither the PKCs α, δ, θ, nor the CBM complex itself is involved in the activation of NF-κB by H. pylori. The contribution of CAMKII and calmodulin, but not PKCs/CBM to the induction of an inflammatory response by H. pylori infection augment the understanding of the molecular mechanism involved and provide potential new disease markers for the diagnosis of gastric inflammatory diseases including gastric cancer.

  19. Increased calcium/calmodulin-dependent protein kinase II activity by morphine-sensitization in rat hippocampus.

    Science.gov (United States)

    Kadivar, Mehdi; Farahmandfar, Maryam; Ranjbar, Faezeh Esmaeli; Zarrindast, Mohammad-Reza

    2014-07-01

    Repeated exposure to drugs of abuse, such as morphine, elicits a progressive enhancement of drug-induced behavioral responses, a phenomenon termed behavioral sensitization. These changes in behavior may reflect long-lasting changes in some of the important molecules involved in memory processing such as calcium/calmodulin-dependent protein kinase II (CaMKII). In the present study, we investigated the effect of morphine sensitization on mRNA expression of α and β isoforms and activity of CaMKII in the hippocampus of male rats. Animals were treated for 3 days with saline or morphine (20mg/kg) and following a washout period of 5 days, a challenge dose of morphine (5mg/kg) were administered. The results indicate that morphine administration in pre-treated animals produces behavioral sensitization, as determined by significant increase in locomotion and oral stereotypy behavior. In addition, repeated morphine treatment increased mRNA expression of both α and β isoforms of CaMKII in the hippocampus. The present study also showed that induction of morphine sensitization significantly increased both Ca2+/calmodulin-independent and Ca2+/calmodulin-dependent activities of CaMK II in the rat hippocampus. However, acute administration of morphine (5mg/kg) did not alter either α and β CaMKII mRNA expression or CaMKII activity in the hippocampus. The stimulation effects of morphine sensitization on mRNA expression and activity of CaMKII were completely abolished by administration of naloxone, 30min prior to s.c. injections of morphine (20mg/kg/day×3 days). Our data demonstrated that induction of morphine sensitization could effectively modulate the activity and the mRNA expression of CaMKII in the hippocampus and this effect of morphine was exerted by the activation of opioid receptors.

  20. Role of EGFR transactivation in angiotensin II signaling to extracellular regulated kinase in preglomerular smooth muscle cells.

    Science.gov (United States)

    Andresen, Bradley T; Linnoila, Jenny J; Jackson, Edwin K; Romero, Guillermo G

    2003-03-01

    Angiotensin (Ang) II promotes the phosphorylation of extracellular regulated kinase (ERK); however, the mechanisms leading to Ang II-induced ERK phosphorylation are debated. The currently accepted theory involves transactivation of epidermal growth factor receptor (EGFR). We have shown that generation of phosphatidic acid (PA) is required for the recruitment of Raf to membranes and the activation of ERK by multiple agonists, including Ang II. In the present report, we confirm that phospholipase D-dependent generation of PA is required for Ang II-mediated phosphorylation of ERK in Wistar-Kyoto and spontaneously hypertensive rat preglomerular smooth muscle cells (PGSMCs). However, EGF stimulation does not activate phospholipase D or generate PA. These observations indicate that EGF recruits Raf to membranes via a mechanism that does not involve PA, and thus, Ang II-mediated phosphorylation of ERK is partially independent of EGFR-mediated signaling cascades. We hypothesized that phosphoinositide-3-kinase (PI3K) can also act to recruit Raf to membranes; therefore, inhibition of PI3K should inhibit EGF signaling to ERK. Wortmannin, a PI3K inhibitor, inhibited EGF-mediated phosphorylation of ERK (IC50, approximately 14 nmol/L). To examine the role of the EGFR in Ang II-mediated phosphorylation of ERK we utilized 100 nmol/L wortmannin to inhibit EGFR signaling to ERK and T19N RhoA to block Ang II-mediated ERK phosphorylation. Wortmannin treatment inhibited EGF-mediated but not Ang II-mediated phosphorylation of ERK. Furthermore, T19N RhoA inhibited Ang II-mediated ERK phosphorylation, whereas T19N RhoA had significantly less effect on EGF-mediated ERK phosphorylation. We conclude that transactivation of the EGFR is not primarily responsible for Ang II-mediated activation of ERK in PGSMCs.

  1. Syndecan-2 regulates melanin synthesis via protein kinase C βII-mediated tyrosinase activation.

    Science.gov (United States)

    Jung, Hyejung; Chung, Heesung; Chang, Sung Eun; Choi, Sora; Han, Inn-Oc; Kang, Duk-Hee; Oh, Eok-Soo

    2014-05-01

    Syndecan-2, a transmembrane heparan sulfate proteoglycan that is highly expressed in melanoma cells, regulates melanoma cell functions (e.g. migration). Since melanoma is a malignant tumor of melanocytes, which largely function to synthesize melanin, we investigated the possible involvement of syndecan-2 in melanogenesis. Syndecan-2 expression was increased in human skin melanoma tissues compared with normal skin. In both mouse and human melanoma cells, siRNA-mediated knockdown of syndecan-2 was associated with reduced melanin synthesis, whereas overexpression of syndecan-2 increased melanin synthesis. Similar effects were also detected in human primary epidermal melanocytes. Syndecan-2 expression did not affect the expression of tyrosinase, a key enzyme in melanin synthesis, but instead enhanced the enzymatic activity of tyrosinase by increasing the membrane and melanosome localization of its regulator, protein kinaseII. Furthermore, UVB caused increased syndecan-2 expression, and this up-regulation of syndecan-2 was required for UVB-induced melanin synthesis. Taken together, these data suggest that syndecan-2 regulates melanin synthesis and could be a potential therapeutic target for treating melanin-associated diseases.

  2. Cyclin-dependent kinase 9 links RNA polymerase II transcription to processing of ribosomal RNA.

    Science.gov (United States)

    Burger, Kaspar; Mühl, Bastian; Rohrmoser, Michaela; Coordes, Britta; Heidemann, Martin; Kellner, Markus; Gruber-Eber, Anita; Heissmeyer, Vigo; Strässer, Katja; Eick, Dirk

    2013-07-19

    Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3' extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3' processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3' rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing.

  3. Oxidized calmodulin kinase II regulates conduction following myocardial infarction: a computational analysis.

    Directory of Open Access Journals (Sweden)

    Matthew D Christensen

    2009-12-01

    Full Text Available Calmodulin kinase II (CaMKII mediates critical signaling pathways responsible for divergent functions in the heart including calcium cycling, hypertrophy and apoptosis. Dysfunction in the CaMKII signaling pathway occurs in heart disease and is associated with increased susceptibility to life-threatening arrhythmia. Furthermore, CaMKII inhibition prevents cardiac arrhythmia and improves heart function following myocardial infarction. Recently, a novel mechanism for oxidative CaMKII activation was discovered in the heart. Here, we provide the first report of CaMKII oxidation state in a well-validated, large-animal model of heart disease. Specifically, we observe increased levels of oxidized CaMKII in the infarct border zone (BZ. These unexpected new data identify an alternative activation pathway for CaMKII in common cardiovascular disease. To study the role of oxidation-dependent CaMKII activation in creating a pro-arrhythmia substrate following myocardial infarction, we developed a new mathematical model of CaMKII activity including both oxidative and autophosphorylation activation pathways. Computer simulations using a multicellular mathematical model of the cardiac fiber demonstrate that enhanced CaMKII activity in the infarct BZ, due primarily to increased oxidation, is associated with reduced conduction velocity, increased effective refractory period, and increased susceptibility to formation of conduction block at the BZ margin, a prerequisite for reentry. Furthermore, our model predicts that CaMKII inhibition improves conduction and reduces refractoriness in the BZ, thereby reducing vulnerability to conduction block and reentry. These results identify a novel oxidation-dependent pathway for CaMKII activation in the infarct BZ that may be an effective therapeutic target for improving conduction and reducing heterogeneity in the infarcted heart.

  4. Function of cGMP-dependent protein kinase II in volume load-induced diuresis.

    Science.gov (United States)

    Schramm, Andrea; Schinner, Elisabeth; Huettner, Johannes P; Kees, Frieder; Tauber, Philipp; Hofmann, Franz; Schlossmann, Jens

    2014-10-01

    Atrial natriuretic peptide (ANP)/cGMPs cause diuresis and natriuresis. Their downstream effectors beyond cGMP remain unclear. To elucidate a probable function of cGMP-dependent protein kinase II (cGKII), we investigated renal parameters in different conditions (basal, salt diets, starving, water load) using a genetically modified mouse model (cGKII-KO), but did not detect any striking differences between WT and cGKII-KO. Thus, cGKII is proposed to play only a marginal role in the adjustment of renal concentration ability to varying salt loads without water restriction or starving conditions. When WT mice were subjected to a volume load (performed by application of a 10-mM glucose solution (3% of BW) via feeding needle), they exhibited a potent diuresis. In contrast, urine volume was decreased significantly in cGKII-KO. We showed that AQP2 plasma membrane (PM) abundance was reduced for about 50% in WT upon volume load, therefore, this might be a main cause for the enhanced diuresis. In contrast, cGKII-KO mice almost completely failed to decrease AQP2-PM distribution. This significant difference between both genotypes is not induced by an altered p-Ser256-AQP2 phosphorylation, as phosphorylation at this site decreases similarly in WT and KO. Furthermore, sodium excretion was lowered in cGKII-KO mice during volume load. In summary, cGKII is only involved to a minor extent in the regulation of basal renal concentration ability. By contrast, cGKII-KO mice are not able to handle an acute volume load. Our results suggest that membrane insertion of AQP2 is inhibited by cGMP/cGKII.

  5. Type II kinase inhibitors show an unexpected inhibition mode against Parkinson's disease-linked LRRK2 mutant G2019S.

    Science.gov (United States)

    Liu, Min; Bender, Samantha A; Cuny, Gregory D; Sherman, Woody; Glicksman, Marcie; Ray, Soumya S

    2013-03-12

    A number of well-known type II inhibitors (ATP-noncompetitive) that bind kinases in their DFG-out conformation were tested against wild-type LRRK2 and the most common Parkinson's disease-linked mutation, G2019S. We found that traditional type II inhibitors exhibit surprising variability in their inhibition mechanism between the wild type (WT) and the G2019S mutant of LRRK2. The type II kinase inhibitors were found to work in an ATP-competitive fashion against the G2019S mutant, whereas they appear to follow the expected noncompetitive mechanism against WT. Because the G2019S mutation lies in the DXG motif (DYG in LRRK2 but DFG in most other kinases) of the activation loop, we explored the structural consequence of the mutation on loop dynamics using an enhanced sampling method called metadynamics. The simulations suggest that the G2019S mutation stabilizes the DYG-in state of LRRK2 through a series of hydrogen bonds, leading to an increase in the conformational barrier between the active and inactive forms of the enzyme and a relative stabilization of the active form. The conformational bias toward the active form of LRRK2 mutants has two primary consequences. (1) The mutant enzyme becomes hyperactive, a known contributor to the Parkinsonian phenotype, as a consequence of being "locked" into the activated state, and (2) the mutation creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP-competitive fashion. Our results suggest that developing type II inhibitors, which are generally considered superior to type I inhibitors because of desirable selectivity profiles, might be especially challenging for the G2019S LRRK2 mutant.

  6. Behavioral modulation of neuronal calcium/calmodulin-dependent protein kinase II activity: differential effects on nicotine-induced spinal and supraspinal antinociception in mice.

    Science.gov (United States)

    Damaj, M Imad

    2007-10-15

    Recent studies have implicated the involvement of Ca(2+)-dependent mechanisms, in particular calcium/calmodulin-dependent protein kinase II (CaM kinase II) in nicotine-induced antinociception using the tail-flick test. The spinal cord was suggested as a possible site of this involvement. The present study was undertaken to investigate the hypothesis that similar mechanisms exist for nicotine-induced antinociception in the hot-plate test, a response thought to be centrally mediated. In order to assess these mechanisms, i.c.v. administered CaM kinase II inhibitors were evaluated for their effects on antinociception produced by either i.c.v. or s.c. administration of nicotine in both tests. In addition, nicotine's analgesic effects were tested in mice lacking half of their CaM kinase II (CaM kinase II heterozygous) and compare it to their wild-type counterparts. Our results showed that although structurally unrelated CaM kinase II inhibitors blocked nicotine's effects in the tail-flick test in a dose-related manner, they failed to block the hot-plate responses. In addition, the antinociceptive effects of systemic nicotine in the tail-flick but not the hot-plate test were significantly reduced in CaM kinase II heterozygous mice. These observations indicate that in contrast to the tail-flick response, the mechanism of nicotine-induced antinociception in the hot-plate test is not mediated primarily via CaM kinase II-dependent mechanisms at the supraspinal level.

  7. Hepatocyte Growth Factor Inhibits Apoptosis by the Profibrotic Factor Angiotensin II via Extracellular Signal-regulated Kinase 1/2 in Endothelial Cells and Tissue Explants

    Science.gov (United States)

    2010-12-01

    II via Extracellular Signal-regulated Kinase 1/2 in Endothelial Cells and Tissue Explants Young H. Lee, Ana P. Marquez , Ognoon Mungunsukh, and Regina...L., Gonzalez- Garcia , M., Page, C., Herrera, R., and Nunez, G. (1997). Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt... Marquez , A. P., and Day, R. M. (2010). Angiotensin-II-induced apoptosis requires regulation of nucleolin and Bcl-xL by SHP-2 in primary lung endothelial

  8. Investigation of Neuronal Cell Type-Specific Gene Expression of Ca2+/Calmodulin-dependent Protein Kinase II.

    Directory of Open Access Journals (Sweden)

    Mima Kazuko

    2002-01-01

    Full Text Available The promoter activity of the rat Ca2+/calmodulin-dependent protein kinase II gene was analyzed using the luciferase reporter gene in neuronal and non-neuronal cell lines. Neuronal cell type-specific promoter activity was found in the 5'-flanking region of &agr; and &bgr; isoform genes of the kinase. Silencer elements were also found further upstream of promoter regions. A brain-specific protein bound to the DNA sequence of the 5'-flanking region of the gene was found by gel mobility shift analysis in the nuclear extract of the rat brain, including the cerebellum, forebrain, and brainstem, but not in that of non-neuronal tissues, including liver, kidney and spleen. The luciferase expression system and gel shift analysis can be used as an additional and better index by which to monitor gene expression in most cell types.

  9. Development of casein microgels from cross-linking of casein micelles by genipin.

    Science.gov (United States)

    Silva, Naaman F Nogueira; Saint-Jalmes, Arnaud; de Carvalho, Antônio F; Gaucheron, Frédéric

    2014-09-02

    Casein micelles are porous colloidal particles, constituted of casein molecules, water, and minerals. The vulnerability of the supramolecular structure of casein micelles face to changes in the environmental conditions restrains their applications in other domains besides food. Thus, redesigning casein micelles is a challenge to create new functionalities for these biosourced particles. The objective of this work was to create stable casein microgels from casein micelles using a natural cross-linker, named genipin. Suspensions of purified casein micelles (25 g L(-1)) were mixed with genipin solutions to have final concentrations of 5, 10, and 20 mM genipin. Covalently linked casein microgels were formed via cross-linking of lysyl and arginyl residues of casein molecules. The reacted products exhibited blue color. The cross-linking reaction induced gradual changes on the colloidal properties of the particles. The casein microgels were smaller and more negatively charged and presented smoother surfaces than casein micelles. These results were explained based on the cross-linking of free NH2 present in an external layer of κ-casein. Light scattering and rheological measurements showed that the reaction between genipin and casein molecules was intramicellar, as one single population of particles was observed and the values of viscosity (and, consequently, the volume fraction of the particles) were reduced. Contrary to the casein micelles, the casein microgels were resistant to the presence of dissociating agents, e.g., citrate (calcium chelating) and urea, but swelled as a consequence of internal electrostatic repulsion and the disruption of hydrophobic interactions between protein chains. The casein microgels did not dissociate at the air-solution interface and formed solid-like interfaces rather than a viscoelastic gel. The potential use of casein microgels as adaptable nanocarriers is proposed in the article.

  10. Endogenous type II cGMP-dependent protein kinase exists as a dimer in membranes and can Be functionally distinguished from the type I isoforms

    NARCIS (Netherlands)

    A.B. Vaandrager (Arie); M.J. Edixhoven (Marcel); A.G. Bot (Alice); M.A. Kroos (Marian); T. Jarchau; S. Lohmann; H.G. Genieser; H.R. de Jonge (Hugo)

    1997-01-01

    textabstractIn mammalian tissues two types of cGMP-dependent protein kinase (cGK) have been identified. In contrast to the dimeric cGK I, cGK II purified from pig intestine was shown previously to behave as a monomer. However, recombinant rat cGK II was found to have hy

  11. Calmodulin Kinase II Interacts with the Dopamine Transporter C Terminus to Regulate Amphetamine-Induced Reverse Transport

    DEFF Research Database (Denmark)

    Fog, Jacob U; Khoshbouei, Habibeh; Holy, Marion

    2006-01-01

    Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the d...... in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux....

  12. Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport

    DEFF Research Database (Denmark)

    Fog, Jacob U; Khoshbouei, Habibeh; Holy, Marion

    2006-01-01

    Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the d...... in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux....

  13. Characterization and genetic analysis of bovine alpha S1-casein I variant.

    Science.gov (United States)

    Lühken, G; Caroli, A; Ibeagha-Awemu, E M; Erhardt, G

    2009-08-01

    The aim of this study was to identify the molecular genetic origin underlying the I variant of alpha(s1)-casein and to develop a DNA-based test for this polymorphism as a tool for genetic analyses independent of milk sample testing. All coding exons and flanking regions of the alpha(s1)-casein gene were sequenced in DNA samples from cattle of known alpha(s1)-casein genotypes (BI, CI, II, CC), determined by isoelectric focusing of milk samples. A nucleotide substitution (A>T) in exon 11 (g.19836A>T) leads to the exchange of Glu with Asp at amino acid position 84 of the mature protein (p.Glu84Asp) and perfectly co-segregated with the presence of the alpha(s1)-casein I variant in the milk of the analysed animals. Genotyping of a total of 680 DNA samples from 31 Bos taurus and Bos indicus cattle breeds and from Bos grunniens, Bison bison and Bison bonasus by restriction fragment length polymorphism analysis revealed the occurrence of Asp at position 84 at low frequencies in Bos taurus and Bos indicus breeds and established its origin from the alpha(s1)-casein C variant (p.Glu192Gly). Ten different intragenic haplotypes in the gene region from intron 8 to intron 12 were observed by sequencing, of which two occurred in Bison bison and one in Bison bonasus only. Using available casein gene complex information, an association of Asp at position 84 to beta-casein A(2) and kappa-casein B was shown in the Bos indicus breed Banyo Gudali. Taken together, we can postulate that the alpha(s1)-casein variant I is caused by a non-synonymous nucleotide substitution in exon 11 of the gene and that it originated within Bos indicus and spread to Bos taurus subsequently.

  14. Casein Micelle Dispersions under Osmotic Stress

    OpenAIRE

    Bouchoux, Antoine; Cayemitte, Pierre-Emerson; Jardin, Julien; Gésan-Guiziou, Geneviève; Cabane, Bernard

    2009-01-01

    Casein micelles dispersions have been concentrated and equilibrated at different osmotic pressures using equilibrium dialysis. This technique measured an equation of state of the dispersions over a wide range of pressures and concentrations and at different ionic strengths. Three regimes were found. i), A dilute regime in which the osmotic pressure is proportional to the casein concentration. In this regime, the casein micelles are well separated and rarely interact, whereas the osmotic press...

  15. Rheology and structural arrest of casein suspensions

    OpenAIRE

    Dahbi, Louisa; Alexander, M.; Trappe, Véronique; Dhont, J. K. G.; Schurtenberger, Peter

    2010-01-01

    The rheology of milk powder suspensions is investigated up to very high concentrations, where structural arrest occurs. The main component of the milk powder investigated is casein, so that the suspensions can be regarded as casein suspensions. Four concentration regimes are identified. For effective casein volume fractions less than 0.54 the concentration dependence of the zero-shear viscosity is similar to that of hard-sphere suspensions. However, due to the elastic deformation of the casei...

  16. Mitogen-activated protein kinase kinase 1/2 inhibition and angiotensin II converting inhibition in mice with cardiomyopathy caused by lamin A/C gene mutation

    Energy Technology Data Exchange (ETDEWEB)

    Muchir, Antoine, E-mail: a.muchir@institut-myologie.org [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Wu, Wei [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Sera, Fusako; Homma, Shunichi [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Worman, Howard J., E-mail: hjw14@columbia.edu [Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY (United States); Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY (United States)

    2014-10-03

    Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left

  17. Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells.

    Science.gov (United States)

    Lopes da Silva, Mafalda; O'Connor, Marie N; Kriston-Vizi, Janos; White, Ian J; Al-Shawi, Raya; Simons, J Paul; Mössinger, Julia; Haucke, Volker; Cutler, Daniel F

    2016-05-15

    Weibel-Palade bodies (WPBs) are endothelial storage organelles that mediate the release of molecules involved in thrombosis, inflammation and angiogenesis, including the pro-thrombotic glycoprotein von Willebrand factor (VWF). Although many protein components required for WPB formation and function have been identified, the role of lipids is almost unknown. We examined two key phosphatidylinositol kinases that control phosphatidylinositol 4-phosphate levels at the trans-Golgi network, the site of WPB biogenesis. RNA interference of the type II phosphatidylinositol 4-kinases PI4KIIα and PI4KIIβ in primary human endothelial cells leads to formation of an increased proportion of short WPB with perturbed packing of VWF, as exemplified by increased exposure of antibody-binding sites. When stimulated with histamine, these cells release normal levels of VWF yet, under flow, form very few platelet-catching VWF strings. In PI4KIIα-deficient mice, immuno-microscopy revealed that VWF packaging is also perturbed and these mice exhibit increased blood loss after tail cut compared to controls. This is the first demonstration that lipid kinases can control the biosynthesis of VWF and the formation of WPBs that are capable of full haemostatic function.

  18. Electrochemiluminescence resonance energy transfer between graphene quantum dots and graphene oxide for sensitive protein kinase activity and inhibitor sensing.

    Science.gov (United States)

    Liang, Ru-Ping; Qiu, Wei-Bin; Zhao, Hui-Fang; Xiang, Cai-Yun; Qiu, Jian-Ding

    2016-01-21

    Herein, a novel electrochemiluminescence resonance energy transfer (ECL-RET) biosensor using graphene quantum dots (GQDs) as donor and graphene oxide (GO) as acceptor for monitoring the activity of protein kinase was presented for the first time. Anti-phosphoserine antibody conjugated graphene oxide (Ab-GO) nonocomposite could be captured onto the phosphorylated peptide/GQDs modified electrode surface through antibody-antigen interaction in the presence of casein kinase II (CK2) and adenosine 5'-triphosphate (ATP), resulting in ECL from the GQDs quenching by closely contacting GO. This ECL quenching degree was positively correlated with CK2 activity. Therefore, on the basis of ECL-RET between GQDs and GO, the activity of protein kinase can be detected sensitively. This biosensor can also be used for quantitative analysis CK2 activity in serum samples and qualitative screening kinase inhibition, indicating the potential application of the developed method in biochemical fundamental research and clinical diagnosis.

  19. The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium.

    Directory of Open Access Journals (Sweden)

    Hyun-Gwan Lee

    Full Text Available Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine, is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3 with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII, but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg

  20. Myotonic dystrophy protein kinase (DMPK) prevents ROS-induced cell death by assembling a hexokinase II-Src complex on the mitochondrial surface.

    Science.gov (United States)

    Pantic, B; Trevisan, E; Citta, A; Rigobello, M P; Marin, O; Bernardi, P; Salvatori, S; Rasola, A

    2013-10-17

    The biological functions of myotonic dystrophy protein kinase (DMPK), a serine/threonine kinase whose gene mutations cause myotonic dystrophy type 1 (DM1), remain poorly understood. Several DMPK isoforms exist, and the long ones (DMPK-A/B/C/D) are associated with the mitochondria, where they exert unknown activities. We have studied the isoform A of DMPK, which we have found to be prevalently associated to the outer mitochondrial membrane. The kinase activity of mitochondrial DMPK protects cells from oxidative stress and from the ensuing opening of the mitochondrial permeability transition pore (PTP), which would otherwise irreversibly commit cells to death. We observe that DMPK (i) increases the mitochondrial localization of hexokinase II (HK II), (ii) forms a multimeric complex with HK II and with the active form of the tyrosine kinase Src, binding its SH3 domain and (iii) it is tyrosine-phosphorylated by Src. Both interaction among these proteins and tyrosine phosphorylation of DMPK are increased under oxidative stress, and Src inhibition selectively enhances death in DMPK-expressing cells after HK II detachment from the mitochondria. Down-modulation of DMPK abolishes the appearance of muscle markers in in vitro myogenesis, which is rescued by oxidant scavenging. Our data indicate that, together with HK II and Src, mitochondrial DMPK is part of a multimolecular complex endowed with antioxidant and pro-survival properties that could be relevant during the function and differentiation of muscle fibers.

  1. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L.; Hood, Molly M.; Lord, John W.; Lu, Wei-Ping; Miller, David F.; Patt, William C.; Smith, Bryan D.; Vogeti, Lakshminarayana; Kaufman, Michael D.; Petillo, Peter A.; Wise, Scott C.; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L. (Deciphera); (Emerald); (Cocrystal)

    2012-01-20

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

  2. Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

    Science.gov (United States)

    Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L; Hood, Molly M; Lord, John W; Lu, Wei-Ping; Miller, David F; Patt, William C; Smith, Bryan D; Vogeti, Lakshminarayana; Kaufman, Michael D; Petillo, Peter A; Wise, Scott C; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L

    2010-10-01

    Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

  3. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    Energy Technology Data Exchange (ETDEWEB)

    Souza, C.F. [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Carneiro, A.B.; Silveira, A.B. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); Laranja, G.A.T. [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); Silva-Neto, M.A.C. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); INCT, Entomologia Molecular (Brazil); Costa, S.C. Goncalves da [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Paes, M.C., E-mail: mcpaes@uerj.br [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); INCT, Entomologia Molecular (Brazil)

    2009-12-18

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  4. Milk Intolerance, Beta-Casein and Lactose.

    Science.gov (United States)

    Pal, Sebely; Woodford, Keith; Kukuljan, Sonja; Ho, Suleen

    2015-08-31

    True lactose intolerance (symptoms stemming from lactose malabsorption) is less common than is widely perceived, and should be viewed as just one potential cause of cows' milk intolerance. There is increasing evidence that A1 beta-casein, a protein produced by a major proportion of European-origin cattle but not purebred Asian or African cattle, is also associated with cows' milk intolerance. In humans, digestion of bovine A1 beta-casein, but not the alternative A2 beta-casein, releases beta-casomorphin-7, which activates μ-opioid receptors expressed throughout the gastrointestinal tract and body. Studies in rodents show that milk containing A1 beta-casein significantly increases gastrointestinal transit time, production of dipeptidyl peptidase-4 and the inflammatory marker myeloperoxidase compared with milk containing A2 beta-casein. Co-administration of the opioid receptor antagonist naloxone blocks the myeloperoxidase and gastrointestinal motility effects, indicating opioid signaling pathway involvement. In humans, a double-blind, randomized cross-over study showed that participants consuming A1 beta-casein type cows' milk experienced statistically significantly higher Bristol stool values compared with those receiving A2 beta-casein milk. Additionally, a statistically significant positive association between abdominal pain and stool consistency was observed when participants consumed the A1 but not the A2 diet. Further studies of the role of A1 beta-casein in milk intolerance are needed.

  5. Milk Intolerance, Beta-Casein and Lactose

    Directory of Open Access Journals (Sweden)

    Sebely Pal

    2015-08-01

    Full Text Available True lactose intolerance (symptoms stemming from lactose malabsorption is less common than is widely perceived, and should be viewed as just one potential cause of cows’ milk intolerance. There is increasing evidence that A1 beta-casein, a protein produced by a major proportion of European-origin cattle but not purebred Asian or African cattle, is also associated with cows’ milk intolerance. In humans, digestion of bovine A1 beta-casein, but not the alternative A2 beta-casein, releases beta-casomorphin-7, which activates μ-opioid receptors expressed throughout the gastrointestinal tract and body. Studies in rodents show that milk containing A1 beta-casein significantly increases gastrointestinal transit time, production of dipeptidyl peptidase-4 and the inflammatory marker myeloperoxidase compared with milk containing A2 beta-casein. Co-administration of the opioid receptor antagonist naloxone blocks the myeloperoxidase and gastrointestinal motility effects, indicating opioid signaling pathway involvement. In humans, a double-blind, randomized cross-over study showed that participants consuming A1 beta-casein type cows’ milk experienced statistically significantly higher Bristol stool values compared with those receiving A2 beta-casein milk. Additionally, a statistically significant positive association between abdominal pain and stool consistency was observed when participants consumed the A1 but not the A2 diet. Further studies of the role of A1 beta-casein in milk intolerance are needed.

  6. Focusing on the goat casein complex.

    Science.gov (United States)

    Caroli, A; Chiatti, F; Chessa, S; Rignanese, D; Bolla, P; Pagnacco, G

    2006-08-01

    The analysis of casein polymorphisms in goat species is rather difficult, because of a large number of mutations at each locus, and the tight linkage involving the 4 casein genes. Three goat breeds from Northern Italy, Orobica, Verzasca, and Frisa, were analyzed at the casein complex by milk isoelectrofocusing and analyses at the DNA level to identify the majority of all known polymorphisms. The casein gene structure of the 3 local breeds at alpha(S1)-casein (CSN1S1), beta-casein (CSN2), alpha(S2)-casein (CSN1S2), and kappa-casein (CSN3) was compared with that of Camosciata, a more widely distributed breed. A new allele was identified and characterized at CSN2 gene, which seemed to be specific to the Frisa breed. It was named CSN2*E, and was characterized by a transversion TCT --> TAT responsible for the amino acid exchange Ser(166) --> Tyr(166) in the mature protein. The casein haplotype structure is highly different among breeds. A total of 26 haplotypes showed a frequency higher than 0.01 in at least 1 of the 4 breeds considered, with 12, 3, 5, and 19 haplotypes in Frisa, Orobica, Verzasca, and Camosciata breeds, respectively. Only 13 haplotypes occurred at a frequency higher than 0.05 in at least 1 breed. With the molecular knowledge of each locus, the ancestral haplotype coding for CSN1S1*B, CSN2*A, CSN1S2*A, and CSN3*B protein variants can be postulated. A protein evolutionary model considering the whole casein haplotype is proposed.

  7. Dynamics of glucose-induced membrane recruitment of protein kinase C beta II in living pancreatic islet beta-cells.

    Science.gov (United States)

    Pinton, Paolo; Tsuboi, Takashi; Ainscow, Edward K; Pozzan, Tullio; Rizzuto, Rosario; Rutter, Guy A

    2002-10-01

    The mechanisms by which glucose may affect protein kinase C (PKC) activity in the pancreatic islet beta-cell are presently unclear. By developing adenovirally expressed chimeras encoding fusion proteins between green fluorescent protein and conventional (betaII), novel (delta), or atypical (zeta) PKCs, we show that glucose selectively alters the subcellular localization of these enzymes dynamically in primary islet and MIN6 beta-cells. Examined by laser scanning confocal or total internal reflection fluorescence microscopy, elevated glucose concentrations induced oscillatory translocations of PKCbetaII to spatially confined regions of the plasma membrane. Suggesting that increases in free cytosolic Ca(2+) concentrations ([Ca(2+)](c)) were primarily responsible, prevention of [Ca(2+)](c) increases with EGTA or diazoxide completely eliminated membrane recruitment, whereas elevation of cytosolic [Ca(2+)](c) with KCl or tolbutamide was highly effective in redistributing PKCbetaII both to the plasma membrane and to the surface of dense core secretory vesicles. By contrast, the distribution of PKCdelta.EGFP, which binds diacylglycerol but not Ca(2+), was unaffected by glucose. Measurement of [Ca(2+)](c) immediately beneath the plasma membrane with a ratiometric "pericam," fused to synaptic vesicle-associated protein-25, revealed that depolarization induced significantly larger increases in [Ca(2+)](c) in this domain. These data demonstrate that nutrient stimulation of beta-cells causes spatially and temporally complex changes in the subcellular localization of PKCbetaII, possibly resulting from the generation of Ca(2+) microdomains. Localized changes in PKCbetaII activity may thus have a role in the spatial control of insulin exocytosis.

  8. Hydrophobic motif site-phosphorylated protein kinaseII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

    Science.gov (United States)

    Das, Falguni; Ghosh-Choudhury, Nandini; Mariappan, Meenalakshmi M; Kasinath, Balakuntalam S; Choudhury, Goutam Ghosh

    2016-04-01

    PKCβII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCβII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCβII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCβII, dominant negative PKCβII, and PKCβII hydrophobic motif phosphorylation-deficient mutant, we found that PKCβII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCβII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCβII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCβII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCβII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCβII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.

  9. Neuronal calcium/calmodulin-dependent protein kinase II mediates nicotine reward in the conditioned place preference test in mice.

    Science.gov (United States)

    Jackson, Kia J; Muldoon, Pretal P; Walters, Carrie; Damaj, Mohamad Imad

    2016-02-01

    Several recent studies have indicated the involvement of calcium-dependent mechanisms, in particular the abundant calcium-activated kinase, calcium/calmodulin-dependent kinase II (CaMKII), in behaviors associated with nicotine dependence in mice. Behavioral and biochemical studies have shown that CaMKII is involved in acute and chronic nicotine behaviors and nicotine withdrawal; however, evidence of a role for CaMKII in nicotine reward is lacking. Thus, the goal of the current study was to examine the role of CaMKII in nicotine reward. Using pharmacological and genetic tools, we tested nicotine conditioned place preference (CPP) in C57Bl/6 mice after administration of CaMKII antagonists and in α-CaMKII wild-type (+/+) and heterozygote (±) mice. CaMKII antagonists blocked expression of nicotine CPP, and the preference score was significantly reduced in α-CaMKII ± mice compared with their +/+ counterparts. Further, we assessed CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), prefrontal cortex, and hippocampus after nicotine CPP and found significant increases in CaMKII activity in the mouse VTA and NAc that were blocked by CaMKII antagonists. The findings from this study show that CaMKII mediates nicotine reward and suggest that increases in CaMKII activity in the VTA and NAc are relevant to nicotine reward behaviors.

  10. Restriction fragment length polymorphism of ovine casein genes: close linkage between the alpha s1-, alpha s2-, beta- and kappa-casein loci.

    Science.gov (United States)

    Leveziel, H; Metenier, L; Guerin, G; Cullen, P; Provot, C; Bertaud, M; Mercier, J C

    1991-01-01

    Restriction fragment length polymorphism (RFLP) of ovine casein genes was investigated. Genomic DNA from 56 rams was digested with 10 restriction endonucleases and Southern blots probed with the four ovine casein cDNAs (alpha s1-, beta-, alpha s2- and kappa-Cn). Five enzymes, namely, BglI, PvuII, RsaI, TaqI and HindIII revealed nine different RFLPs. The inheritance of six of these polymorphisms was studied by segregation analysis of gametes in nine rams' families, and each of them could be related to the existence of alleles at the relevant casein locus. A close linkage between the four ovine casein genes was demonstrated since no recombination within the four pairs of loci examined, alpha s1-beta-Cn, alpha s1-kappa-Cn, beta-kappa-Cn and alpha s2-kappa-Cn, was observed in the progeny of double heterozygous rams. The casein genes are thus clustered in the ovine species as in the case of other mammals.

  11. Cloning and characterization of PAK5, a novel member of mammalian p21-activated kinase-II subfamily that is predominantly expressed in brain

    DEFF Research Database (Denmark)

    Pandey, A.; Dan, I.; Kristiansen, T.Z.;

    2002-01-01

    activates the JNK kinase pathway but not p38 or ERK pathways. PAK5 transcript is predominantly expressed in brain as revealed by Northern blot and in situ hybridization. The expression pattern of PAK5 is distinct from that of PAK4 and PAK6, suggesting a functional division among PAK-II subfamily kinases......The p21-activated kinase (PAK) family of protein kinases has recently attracted considerable attention as an effector of Rho family of small G proteins and as an upstream regulator of MAPK signalling pathways during cellular events such as re-arrangement of the cytoskeleton and apoptosis. We have...... that PAK5 preferentially binds to CDC42 in the presence of GTP and that CRIB motif is essential for this interaction. PAK5 is a functional protein kinase but unlike PAK-I family kinases (PAK1, 2, and 3), the kinase activity of PAK5 does not seem to require the binding of CDC42. Overexpression of PAK5...

  12. Stability of casein micelles in milk

    Science.gov (United States)

    Tuinier, R.; de Kruif, C. G.

    2002-07-01

    Casein micelles in milk are proteinaceous colloidal particles and are essential for the production of flocculated and gelled products such as yogurt, cheese, and ice-cream. The colloidal stability of casein micelles is described here by a calculation of the pair potential, containing the essential contributions of brush repulsion, electrostatic repulsion, and van der Waals attraction. The parameters required are taken from the literature. The results are expressed by the second osmotic virial coefficient and are quite consistent with experimental findings. It appears that the stability is mainly attributable to a steric layer of κ-casein, which can be described as a salted polyelectrolyte brush.

  13. cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

    NARCIS (Netherlands)

    A.B. Vaandrager (Arie); S.M. Lohmann (Suzanne); H.R. de Jonge (Hugo); W.C. Poller; B.C. Tilly (Bernard); A. Smolenski; S. Schneider-Rasp; A.G. Bot (Alice); M.J. Edixhoven (Marcel); B.J. Scholte (Bob); T. Jarchau; U. Walter

    1997-01-01

    textabstractIn order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was ana

  14. Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II

    NARCIS (Netherlands)

    P.J. French (Pim); J. Bijman (Jan); M.J. Edixhoven (Marcel); A.B. Vaandrager (Arie); B.J. Scholte (Bob); S.M. Lohmann (Suzanne); A.C. Nairn; H.R. de Jonge (Hugo)

    1995-01-01

    textabstractType II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type I alpha cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- channel in excis

  15. The calmodulin-dependent protein kinase II inhibitor KN-93 protects rat cerebral cortical neurons from N-methyl-D-aspartic acid-induced injury

    Institute of Scientific and Technical Information of China (English)

    Xuewen Liu; Cui Ma; Ruixian Xing; Weiwei Zhang; Buxian Tian; Xidong Li; Qiushi Li; Yanhui Zhang

    2013-01-01

    In this study, primary cultured cerebral cortical neurons of Sprague-Dawley neonatal rats were treated with 0.25, 0.5, and 1.0 μM calmodulin-dependent protein kinase II inhibitor KN-93 after 50 μM N-methyl-D-aspartic acid-induced injury. Results showed that, compared with N-methyl-Daspartic acid-induced injury neurons, the activity of cells markedly increased, apoptosis was significantly reduced, leakage of lactate dehydrogenase decreased, and intracellular Ca2+ concentrations in neurons reduced after KN-93 treatment. The expression of caspase-3, phosphorylated calmodulin-dependent protein kinase II and total calmodulin-dependent protein kinase II protein decreased after KN-93 treatment. And the effect was apparent at a dose of 1.0 μM KN-93. Experimental findings suggest that KN-93 can induce a dose-dependent neuroprotective effect, and that the underlying mechanism may be related to the down-regulation of caspase-3 and calmodulin- dependent protein kinase II expression.

  16. Extracellular signal regulated kinase and SMAD signaling both mediate the angiotensin II driven progression towards overt heart failure in homozygous TGR(mRen2)27

    NARCIS (Netherlands)

    de Boer, RA; Pokharel, S; Flesch, M; van Kampen, DA; Suurmeijer, AJH; Boomsma, F; van Gilst, WH; van Veldhuisen, DJ; Pinto, YM

    2004-01-01

    Angiotensin (Ang) II is a key player in left ventricular (LV) remodeling and cardiac fibrosis. Its effects are thought to be transferred at least in part by mitogen-activated protein kinases (MAPK), transforming growth factor (TGF) beta(1), and the Smad pathway. In this study we sought to elucidate

  17. Serotonin suppresses β-casein expression via PTP1B activation in human mammary epithelial cells.

    Science.gov (United States)

    Chiba, Takeshi; Maeda, Tomoji; Sanbe, Atsushi; Kudo, Kenzo

    2016-04-22

    Serotonin (5-hydroxytriptamine, 5-HT) has an important role in milk volume homeostasis within the mammary gland during lactation. We have previously shown that the expression of β-casein, a differentiation marker in mammary epithelial cells, is suppressed via 5-HT-mediated inhibition of signal transduction and activator of transcription 5 (STAT5) phosphorylation in the human mammary epithelial MCF-12A cell line. In addition, the reduction of β-casein in turn was associated with 5-HT7 receptor expression in the cells. The objective of this study was to determine the mechanisms underlying the 5-HT-mediated suppression of β-casein and STAT5 phosphorylation. The β-casein level and phosphorylated STAT5 (pSTAT5)/STAT5 ratio in the cells co-treated with 5-HT and a protein kinase A (PKA) inhibitor (KT5720) were significantly higher than those of cells treated with 5-HT alone. Exposure to 100 μM db-cAMP for 6 h significantly decreased the protein levels of β-casein and pSTAT5 and the pSTAT5/STAT5 ratio, and significantly increased PTP1B protein levels. In the cells co-treated with 5-HT and an extracellular signal-regulated kinase1/2 (ERK) inhibitor (FR180294) or Akt inhibitor (124005), the β-casein level and pSTAT5/STAT5 ratio were equal to those of cells treated with 5-HT alone. Treatment with 5-HT significantly induced PTP1B protein levels, whereas its increase was inhibited by KT5720. In addition, the PTP1B inhibitor sc-222227 increased the expression levels of β-casein and the pSTAT5/STAT5 ratio. Our observations indicate that PTP1B directly regulates STAT5 phosphorylation and that its activation via the cAMP/PKA pathway downstream of the 5-HT7 receptor is involved in the suppression of β-casein expression in MCF-12A cells.

  18. Effects of hydrolysed casein, intact casein and intact whey protein on energy expenditure and appetite regulation

    DEFF Research Database (Denmark)

    Bendtsen, Line Quist; Lorenzen, Janne Kunchel; Gomes, Sisse

    2014-01-01

    Casein and whey differ in amino acid composition and in the rate of absorption; however, the absorption rate of casein can be increased to mimic that of whey by exogenous hydrolysis. The objective of the present study was to compare the effects of hydrolysed casein (HC), intact casein (IC......) and intact whey (IW) on energy expenditure (EE) and appetite regulation, and thereby to investigate the influence of amino acid composition and the rate of absorption. In the present randomised cross-over study, twenty-four overweight and moderately obese young men and women consumed three isoenergetic...

  19. COMPARATIVE STUDY ON MILK CASEIN ASSAY METHODS

    Directory of Open Access Journals (Sweden)

    RODICA CĂPRIłĂ

    2013-12-01

    Full Text Available Casein, the main milk protein was determined by different assay methods: the gravimetric method, the method based on the neutralization of the NaOH excess used for the casein precipitate solving and the method based on the titration of the acetic acid used for the casein precipitation. The last method is the simplest one, with the fewer steps, and also with the lowest error degree. The results of the experiment revealed that the percentage of casein from the whole milk protein represents between 72.6–81.3% in experiment 1, between 73.6–81.3% in experiment 2 and between 74.3–81% in experiment 3.

  20. Monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca/sup 2 +/-calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    LeVine, H. III; Su, J.L.; Sahyoun, N.E.

    1988-08-23

    A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca/sup 2 +/ for the interaction. Other divalent cations such as Zn/sup 2 +/, Mn/sup 2 +/, Cd/sup 2 +/, Co/sup 2 +/, and Ni/sup 2 +/ will substitute for Ca/sup 2 +/, while Mg/sup 2 +/ and Ba/sup 2 +/ will not. The antibody reacts with both the ..cap alpha..- and ..beta..-subunits on Western blots in a similar Ca/sup 2 +/-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of /sup 125/I Bolton-Hunter-labeled kinase with unlabeled enzyme. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the K/sub m app/ for calmodulin and an increase in the V/sub max/ for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the M/sub r/ 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca/sup 2 +/-dependent conformational change. Thus, monoclonal antibodies can be useful tools in elucidating the mechanism by which Ca/sup 2 +/ and calmodulin act on the kinase molecule.

  1. Analysis of Casein Biopolymers Adsorption to Lignocellulosic Biomass as a Potential Cellulase Stabilizer

    Science.gov (United States)

    Eckard, Anahita Dehkhoda; Muthukumarappan, Kasiviswanathan; Gibbons, William

    2012-01-01

    Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM), fourier transform infrared spectroscopy (FT-IR), capillary electrophoresis (CE), and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively. PMID:23118515

  2. Analysis of Casein Biopolymers Adsorption to Lignocellulosic Biomass as a Potential Cellulase Stabilizer

    Directory of Open Access Journals (Sweden)

    Anahita Dehkhoda Eckard

    2012-01-01

    Full Text Available Although lignocellulosic materials have a good potential to substitute current feedstocks used for ethanol production, conversion of these materials to fermentable sugars is still not economical through enzymatic hydrolysis. High cost of cellulase has prompted research to explore techniques that can prevent from enzyme deactivation. Colloidal proteins of casein can form monolayers on hydrophobic surfaces that alleviate the de-activation of protein of interest. Scanning electron microscope (SEM, fourier transform infrared spectroscopy (FT-IR, capillary electrophoresis (CE, and Kjeldahl and BSA protein assays were used to investigate the unknown mechanism of action of induced cellulase activity during hydrolysis of casein-treated biomass. Adsorption of casein to biomass was observed with all of the analytical techniques used and varied depending on the pretreatment techniques of biomass. FT-IR analysis of amides I and II suggested that the substructure of protein from casein or skim milk were deformed at the time of contact with biomass. With no additive, the majority of one of the cellulase mono-component, 97.1 ± 1.1, was adsorbed to CS within 24 h, this adsorption was irreversible and increased by 2% after 72 h. However, biomass treatment with skim-milk and casein reduced the adsorption to 32.9% ± 6.0 and 82.8% ± 6.0, respectively.

  3. Casein micelles and their internal structure

    Energy Technology Data Exchange (ETDEWEB)

    De Kruif, Cornelis G [ORNL; Huppertz, Thom [NIZO Food Research; Urban, Volker S [ORNL; Petukhov, Andrei V [Van ' t Hoff laboratory for Physical and Colloid Chemistry, Utrecht University, The Netherlands

    2012-01-01

    The internal structure of casein micelles was studied by calculating the small-angle neutron and X-ray scattering and static light scattering spectrum (SANS, SAXS, SLS) as a function of the scattering contrast and composition. We predicted experimental SANS, SAXS, SLS spectra self consistently using independently determined parameters for composition size, polydispersity, density and voluminosity. The internal structure of the casein micelles, i.e. how the various components are distributed within the casein micelle, was modeled according to three different models advocated in the literature; i.e. the classical sub-micelle model, the nanocluster model and the dual binding model. In this paper we present the essential features of these models and combine new and old experimental SANS, SAXS, SLS and DLS scattering data with new calculations that predict the spectra. Further evidence on micellar substructure was obtained by internally cross linking the casein micelles using transglutaminase, which led to casein nanogel particles. In contrast to native casein micelles, the nanogel particles were stable in 6 M urea and after sequestering the calcium using trisodium citrate. The changed scattering properties were again predicted self consistently. An important result is that the radius of gyration is independent of contrast, indicating that the mass distribution within a casein micelle is homogeneous. Experimental contrast is predicted quite well leading to a match point at a D{sub 2}O volume fraction of 0.41 ratio in SANS. Using SANS and SAXS model calculations it is concluded that only the nanocluster model is capable of accounting for the experimental scattering contrast variation data. All features and trends are predicted self consistently, among which the 'famous' shoulder at a wave vector value Q = 0.35 nm{sup -1}. In the nanocluster model, the casein micelle is considered as a (homogeneous) matrix of caseins in which the colloidal calcium phosphate (CCP

  4. Early interference with p44/42 mitogen-activated protein kinase signaling in hypothalamic paraventricular nucleus attenuates angiotensin II-induced hypertension.

    Science.gov (United States)

    Yu, Yang; Xue, Bao-Jian; Zhang, Zhi-Hua; Wei, Shun-Guang; Beltz, Terry G; Guo, Fang; Johnson, Alan Kim; Felder, Robert B

    2013-04-01

    Blood-borne angiotensin II (ANG II) can upregulate p44/42 mitogen-activated protein kinase (MAPK) signaling and ANG II type-1 receptors in the hypothalamic paraventricular nucleus (PVN), a critical cardiovascular and autonomic center. We tested the hypothesis that brain p44/42 MAPK signaling contributes to the development of ANG II-induced hypertension. The ANG II infusion (120 ng/kg per min, subcutaneously) induced increases in phosphorylated p44/42 MAPK and ANG II type-1 receptors in the PVN after 1 week, before the onset of hypertension, that were sustained as hypertension developed during a 2- or 3-week infusion protocol. Bilateral PVN microinjections of small interfering RNAs for p44/42 MAPK, at the onset of the ANG II infusion or 1 week later, prevented the early increase in p44/42 MAPK activity. The early treatment normalized ANG II type-1 receptor expression in the PVN and attenuated the hypertensive response to the 2-week infusion of ANG II. The later small interfering RNA microinjections had a transient effect on ANG II type-1 receptor expression in PVN and no effect on the hypertensive response to the 3-week infusion of ANG II. The early treatment also normalized the pressure response to ganglionic blockade. The ANG II infusion induced increases in mRNA for proinflammatory cytokines that were not affected by either small interfering RNA treatment. These results suggest that the full expression of ANG II-induced hypertension depends on p44/42 MAPK-mediated effects. A potential role for p44/42 MAPK in modulating the ANG II-induced central inflammatory response might also be considered. MAPK signaling in PVN may be a novel target for early intervention in the progression of ANG II-dependent hypertension.

  5. Spatial memory deficits and motor coordination facilitation in cGMP-dependent protein kinase type II-deficient mice.

    Science.gov (United States)

    Wincott, Charlotte M; Kim, Seonil; Titcombe, Roseann F; Tukey, David S; Girma, Hiwot K; Pick, Joseph E; Devito, Loren M; Hofmann, Franz; Hoeffer, Charles; Ziff, Edward B

    2013-01-01

    Activity-dependent trafficking of AMPA receptors to synapses regulates synaptic strength. Activation of the NMDA receptor induces several second messenger pathways that contribute to receptor trafficking-dependent plasticity, including the NO pathway, which elevates cGMP. In turn, cGMP activates the cGMP-dependent protein kinase type II (cGKII), which phosphorylates the AMPA receptor subunit GluA1 at serine 845, a critical step facilitating synaptic delivery in the mechanism of activity-dependent synaptic potentiation. Since cGKII is expressed in the striatum, amygdala, cerebral cortex, and hippocampus, it has been proposed that mice lacking cGKII may present phenotypic differences compared to their wild-type littermates in emotion-dependent tasks, learning and memory, and drug reward salience. Previous studies have shown that cGKII KO mice ingest higher amounts of ethanol as well as exhibit elevated anxiety levels compared to wild-type (WT) littermates. Here, we show that cGKII KO mice are significantly deficient in spatial learning while exhibiting facilitated motor coordination, demonstrating a clear dependence of memory-based tasks on cGKII. We also show diminished GluA1 phosphorylation in the postsynaptic density (PSD) of cGKII KO prefrontal cortex while in hippocampal PSD fractions, phosphorylation was not significantly altered. These data suggest that the role of cGKII may be more robust in particular brain regions, thereby impacting complex behaviors dependent on these regions differently.

  6. Pavlovian fear conditioning regulates Thr286 autophosphorylation of Ca2+/calmodulin-dependent protein kinase II at lateral amygdala synapses.

    Science.gov (United States)

    Rodrigues, Sarina M; Farb, Claudia R; Bauer, Elizabeth P; LeDoux, Joseph E; Schafe, Glenn E

    2004-03-31

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a critical role in synaptic plasticity and memory formation in a variety of learning systems and species. The present experiments examined the role of CaMKII in the circuitry underlying pavlovian fear conditioning. First, we reveal by immunocytochemical and tract-tracing methods that alphaCaMKII is postsynaptic to auditory thalamic inputs and colocalized with the NR2B subunit of the NMDA receptor. Furthermore, we show that fear conditioning results in an increase of the autophosphorylated (active) form of alphaCaMKII in lateral amygdala (LA) spines. Next, we demonstrate that intra-amygdala infusion of a CaMK inhibitor, 1-[NO-bis-1,5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl-4-phenylpiperazine, KN-62, dose-dependently impairs the acquisition, but not the expression, of auditory and contextual fear conditioning. Finally, in electrophysiological experiments, we demonstrate that an NMDA receptor-dependent form of long-term potentiation at thalamic input synapses to the LA is impaired by bath application of KN-62 in vitro. Together, the results of these experiments provide the first comprehensive view of the role of CaMKII in the amygdala during fear conditioning.

  7. Anticancer Alkaloid Lamellarins Inhibit Protein Kinases

    Directory of Open Access Journals (Sweden)

    Laurent Meijer

    2008-10-01

    Full Text Available Lamellarins, a family of hexacyclic pyrrole alkaloids originally isolated from marine invertebrates, display promising anti-tumor activity. They induce apoptotic cell death through multi-target mechanisms, including inhibition of topoisomerase I, interaction with DNA and direct effects on mitochondria. We here report that lamellarins inhibit several protein kinases relevant to cancer such as cyclin-dependent kinases, dualspecificity tyrosine phosphorylation activated kinase 1A, casein kinase 1, glycogen synthase kinase-3 and PIM-1. A good correlation is observed between the effects of lamellarins on protein kinases and their action on cell death, suggesting that inhibition of specific kinases may contribute to the cytotoxicity of lamellarins. Structure/activity relationship suggests several paths for the optimization of lamellarins as kinase inhibitors.

  8. Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells.

    Science.gov (United States)

    Lipschutz, Joshua H; Li, Shixiong; Arisco, Amy; Balkovetz, Daniel F

    2005-02-01

    The tight junction of the epithelial cell determines the characteristics of paracellular permeability across epithelium. Recent work points toward the claudin family of tight junction proteins as leading candidates for the molecular components that regulate paracellular permeability properties in epithelial tissues. Madin-Darby canine kidney (MDCK) strain I and II cells are models for the study of tight junctions and based on transepithelial electrical resistance (TER) contain "tight" and "leaky" tight junctions, respectively. Overexpression studies suggest that tight junction leakiness in these two strains of MDCK cells is conferred by expression of the tight junction protein claudin-2. Extracellular signal-regulated kinase (ERK) 1/2 activation by hepatocyte growth factor treatment of MDCK strain II cells inhibited claudin-2 expression and transiently increased TER. This process was blocked by the ERK 1/2 inhibitor U0126. Transfection of constitutively active mitogen-activated protein kinase/extracellular signal-regulated kinase kinase into MDCK strain II cells also inhibited claudin-2 expression and increased TER. MDCK strain I cells have higher levels of active ERK 1/2 than do MDCK strain II cells. U0126 treatment of MDCK strain I cells decreased active ERK 1/2 levels, induced expression of claudin-2 protein, and decreased TER by approximately 20-fold. U0126 treatment also induced claudin-2 expression and decreased TER in a high resistance mouse cortical collecting duct cell line (94D). These data show for the first time that the ERK 1/2 signaling pathway negatively controls claudin-2 expression in mammalian renal epithelial cells and provide evidence for regulation of tight junction paracellular transport by alterations in claudin composition within tight junction complexes.

  9. Casein kinase 2 inhibitor reduces androgen receptor function and cell proliferation in prostate cancer cells%酪蛋白激酶2抑制剂降低前列腺癌雄激素受体功能和细胞增殖

    Institute of Scientific and Technical Information of China (English)

    尧凯; 高晓燕; 黄必军; 邹子君; 周芳坚; 李本义; 韩辉

    2012-01-01

    Objective To investigate the role of casein kinase 2 (CK2) selective inhibitor tribromoisocyanuric acid (TBCA) in androgen receptor (AR) transactivation,cell proliferation and viability in prostate cancer cell lines,and explore a new chemotherapy for prostate cancer.Methods Prostate cancer cell lines were prepared.With TBCA treatment,Alamar-blue assay was performed to assess cell proliferation and viability in C4-2 cells and flow cytometry was performed for cell cycle analysis in C4-2 and 22Rvl cells; while immunofluorescence staining was performed to determine AR nuclear translocationin in PC-3/AR cells with R1881 pretreatment,and luciferase gene reporter assay was performed to determine AR transactivation in LNCaP cells; Reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the mRNA level of prostate specific antigen (PSA).Results With TBCA treatment,Alamar-blue reading decreased in a dose-dependent manner,and the 50% inhibitory concentration ( IC50 ) was around 25 mol/L.Significant G2/M arrest was detected in C4-2 and 22Rvl cells.R1881-induced AR nuclear localization was reduced significantly ( P < 0.01 ).R1881 -stimulated ARE-LUC reporter activity in LNCaP cells decreased with reduced level of PSA mRNA,which was an AR endogenous target( P < 0.05 ).Conclusion The selective CK2 inhibitor TBCA can dramatically inhibite cell proliferation and cause a G2/M phase arrest in prostate cancer cells.%目的 观察人工合成酪蛋白激酶2(CK2)选择性抑制剂四溴肉桂酸(TBCA),对各种前列腺癌细胞株雄激素受体(AR)反式激活、细胞增殖和活力的效应.方法 TBCA和基因特异性小干扰RNA(siRNA)应用到前列腺癌细胞株,Alamar blue法检测细胞生长,流式细胞术检测细胞周期分布,荧光素酶基因报道分析检测AR反式激活,免疫荧光法检测AR核定位以及定量聚合酶链反应(PCR)检测AR介导的基因表达.结果 TBCA呈剂量依赖性抑制细胞增

  10. Production of superoxide from photosystem II-light harvesting complex II supercomplex in STN8 kinase knock-out rice mutants under photoinhibitory illumination.

    Science.gov (United States)

    Poudyal, Roshan Sharma; Nath, Krishna; Zulfugarov, Ismayil S; Lee, Choon-Hwan

    2016-09-01

    When phosphorylation of Photosystem (PS) II core proteins is blocked in STN8 knock-out mutants of rice (Oryza sativa) under photoinhibitory illumination, the mobilization of PSII supercomplex is prevented. We have previously proposed that more superoxide (O2(-)) is produced from PSII in the mutant (Nath et al., 2013, Plant J. 76, 675-686). Here, we clarify the type and site for the generation of reactive oxygen species (ROS). Using both histochemical and fluorescence probes, we observed that, compared with wild-type (WT) leaves, levels of ROS, including O2(-) and hydrogen peroxide (H2O2), were increased when leaves from mutant plants were illuminated with excess light. However, singlet oxygen production was not enhanced under such conditions. When superoxide dismutase was inhibited, O2(-) production was increased, indicating that it is the initial event prior to H2O2 production. In thylakoids isolated from WT leaves, kinase was active in the presence of ATP, and spectrophotometric analysis of nitrobluetetrazolium absorbance for O2(-) confirmed that PSII-driven superoxide production was greater in the mutant thylakoids than in the WT. This contrast in levels of PSII-driven superoxide production between the mutants and the WT plants was confirmed by conducting protein oxidation assays of PSII particles from osstn8 leaves under strong illumination. Those assays also demonstrated that PSII-LHCII supercomplex proteins were oxidized more in the mutant, thereby implying that PSII particles incur greater damage even though D1 degradation during PSII-supercomplex mobilization is partially blocked in the mutant. These results suggest that O2(-) is the major form of ROS produced in the mutant, and that the damaged PSII in the supercomplex is the primary source of O2(-).

  11. Structure-rheology relations in sodium caseinate containing systems

    NARCIS (Netherlands)

    Ruis, H.G.M.

    2007-01-01

    The general aim of the work described in this thesis was to investigate structure-rheologyrelations for dairy related products, focusing on model systems containing sodium caseinate. The acid inducedgelationof sodium caseinate, of sodium caseinate

  12. Rat vas deferens SERCA2 is modulated by Ca{sup 2+}/calmodulin protein kinase II-mediated phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, J.B.R.; Muzi-Filho, H. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Valverde, R.H.F. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Quintas, L.E.M. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Noel, F. [Programa de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Einicker-Lamas, M. [Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil); Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, Rio de Janeiro, RJ (Brazil); Cunha, V.M.N. [Programa de Farmacologia e Inflamação, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ (Brazil)

    2013-03-19

    Ca{sup 2+} pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca{sup 2+}-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca{sup 2+}-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca{sup 2+} (Ca{sub 0.5} = 780 nM) and a low sensitivity to vanadate (IC{sub 50} = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca{sup 2+} and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca{sup 2+} accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca{sup 2+} and CaM, possibly via CaMKII, in a process that results in stimulation of Ca{sup 2+} pumping activity.

  13. Thermodynamics of calmodulin trapping by Ca2+/calmodulin-dependent protein kinase II: subpicomolar Kd determined using competition titration calorimetry.

    Science.gov (United States)

    Tse, Joyce K Y; Giannetti, Anthony M; Bradshaw, J Michael

    2007-04-03

    Calmodulin (CaM) trapping by Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a phenomenon whereby the affinity of CaM for CaMKII increases >1000-fold following CaMKII autophosphorylation. The molecular basis of this effect is not entirely understood. Binding of CaM to the phosphorylated and the unphosphorylated states of CaMKII is well mimicked by the interaction of CaM with two different length peptides taken from the CaM-binding region of CaMKII, peptides we refer to as the long and intermediate peptides. To better understand the conformational change accompanying CaM trapping, we have used isothermal titration calorimetry (ITC) to compare the binding thermodynamics of CaM to these peptides as well as to a shorter CaMKII-based peptide. Calorimetric analysis revealed that the enthalpy, rather than the entropy, distinguished binding of these three peptides. Furthermore, the heat capacity change was found to be similar for the long and intermediate peptides but smaller in magnitude for the short peptide. Direct titration of CaM with peptide provided the Kd value for the short peptide (Kd = 5.9 +/- 2.4 microM), but a novel, two-phased competitive binding strategy was necessary to ascertain the affinities of the intermediate (Kd = 0.17 +/- 0.06 nM) and long (Kd = 0.07 +/- 0.04 pM) peptides. To our knowledge, the Kd for the long peptide is the most potent measured to date using ITC. Together, the findings reported here support a model whereby the final conformational change accompanying CaM trapping buries little additional surface area but does involve formation of new hydrogen bonds and van der Waals contacts that contribute to formation of the high-affinity, CaM-trapped state.

  14. Phosphorylation of calcium calmodulin-dependent protein kinase II following lateral fluid percussion brain injury in rats.

    Science.gov (United States)

    Folkerts, Michael M; Parks, Elizabeth A; Dedman, John R; Kaetzel, Marcia A; Lyeth, Bruce G; Berman, Robert F

    2007-04-01

    Traumatic brain injury (TBI) can dramatically increase levels of intracellular calcium ([Ca(2+)](i)). One consequence of increased [Ca(2+)](i) would be altered activity and function of calcium-regulated proteins, including calcium-calmodulin-dependent protein kinase II (CaMKII), which is autophosphorylated on Thr(286)(pCaMKII(286)) in the presence of calcium and calmodulin. Therefore, we hypothesized that TBI would result in increased levels of pCaMKII(286), and that such increases would occur early after injury in brain regions known to be damaged following lateral fluid percussion TBI (i.e., hippocampus and cortex). In order to test this hypothesis, immunostaining of CaMKII was examined in rat hippocampus and cortex after lateral fluid percussion (LFP) injury using an antibody directed against pCaMKII(286). LFP injury produced a marked increase in pCaMKII(286) immunostaining in the hippocampus and overlying cortex 30 min after TBI. The pattern of increased immunostaining was uneven, and unexpectedly absent in some hippocampal CA3 pyramidal neurons. This suggests that phosphatase activity may also increase following TBI, resulting in dephosphorylation of pCaMKII(286) in subpopulations of CA3 pyramidal neurons. Western blotting confirmed a rapid increase in levels of pCaMKII(286) at 10 and 30 min after brain injury, and that it was transient and no longer significantly elevated when examined at 3, 8, and 24 h. These results demonstrate that TBI alters the autophosphorylation state of CaMKII, an important neuronal regulator of critical cell functions, including enzyme activities, cell structure, gene expression, and neuronal plasticity, and provide a molecular mechanism that is likely to contribute to cell injury and impaired plasticity after TBI.

  15. Factors influencing casein micelle size in milk of individual cows: Genetic variants and glycosylation of k-casein

    NARCIS (Netherlands)

    Bijl, E.; Vries, de R.F.M.; Valenberg, van H.J.F.; Huppertz, T.; Hooijdonk, van A.C.M.

    2014-01-01

    The average casein micelle size varies widely between milk samples of individual cows. The factors that cause this variation in size are not known but could provide more insight into casein micelle structure and into the physiology of casein micelle formation. The objective of this research was ther

  16. Spermine stimulation of a nuclear NII kinase from pea plumules and its role in the phosphorylation of a nuclear polypeptide

    Science.gov (United States)

    Datta, N.; Schell, M. B.; Roux, S. J.

    1987-01-01

    We have previously demonstrated that spermine stimulates the phosphorylation of a 47 kilodalton nuclear polypeptide from pea plumules (N Datta, LK Hardison, SJ Roux 1986 Plant Physiol 82: 681-684). In this paper we report that spermine stimulates the activity of a cyclic AMP independent casein kinase, partially purified from a chromatin fraction of pea plumule nuclei. This effect of spermine was substrate specific; i.e. with casein as substrate, spermine stimulated the kinase activity, and with phosvitin as substrate, spermine completely inhibited the activity. The stimulation by spermine of the casein kinase was, in part, due to the lowering of the Mg2+ requirement of the kinase. Heparin could partially inhibit this casein kinase activity and spermine completely overcame this inhibition. By further purification of the casein kinase extract on high performance liquid chromatography, we fractionated it into an NI and an NII kinase. Spermine stimulated the NII kinase by 5- to 6-fold but had no effect on the NI kinase. Using [gamma-32P]GTP, we have shown that spermine promotes the phosphorylation of the 47 kilodalton polypeptide(s) in isolated nuclei, at least in part by stimulating an NII kinase.

  17. Casein - whey protein interactions in heated milk

    NARCIS (Netherlands)

    Vasbinder, Astrid Jolanda

    2003-01-01

    Heating of milk is an essential step in the processing of various dairy products, like for example yoghurt. A major consequence of the heat treatment is the denaturation of whey proteins, which either associate with the casein micelle or form soluble whey protein aggregates. By combination of enzyma

  18. Short communication: separation and quantification of caseins and casein macropeptide using ion-exchange chromatography.

    Science.gov (United States)

    Holland, B; Rahimi Yazdi, S; Ion Titapiccolo, G; Corredig, M

    2010-03-01

    The aim of this work was to improve an existing method to separate and quantify the 4 major caseins from milk samples (i.e., containing whey proteins) using ion-exchange chromatography. The separation process was carried out using a mini-preparative cation exchange column (1 or 5mL of column volume), using urea acetate as elution buffer at pH 3.5 with a NaCl gradient. All 4 major caseins were separated, and the purity of each peak was assessed using sodium dodecyl sulfate-PAGE. Purified casein fractions were also added to raw milk to confirm their elution volumes. The quantification was carried out using purified caseins in buffer as well as added directly to fresh skim milk. This method can also be employed to determine the decrease in kappa-casein and the release of the casein-macropeptide during enzymatic hydrolysis using rennet. In this case, the main advantage of using this method is the lack of organic solvents compared with the conventional method for separation of macropeptide (using reversed phase HPLC).

  19. Phosphorylation of acidic ribosomal proteins by ribosome-associated protein kinases of ``Saccharomyces cerevisiae`` and ``Schizosaccharomyces pombe``

    Energy Technology Data Exchange (ETDEWEB)

    Jakubowicz, T.; Cytrynska, M.; Kowalczyk, W.; Gasior, E. [Uniwersytet Marii Curie-Sklodowskiej, Lublin (Poland)

    1993-12-31

    Two proteins of 13 kDa and 38 kDa, the components of 60S ribosomal subunits, were identified as phosphorylation substrates for protein tightly associated with ``S. cerevisiae`` and ``Schizosaccharomyces pombe`` ribosomes. An enzyme with properties of multifunctional casein kinase II was detected in ribosome preparations from both yeast species. In S. cerevisiae another protein kinase with high substrate specificity toward those proteins was also identified. By using isoelectric focusing, the protein band of 13 kDa from ``S. cerevisiae`` and ``S. pombe`` was resolved respectively into three and four major forms of different charge. The same protein forms were phosphorylated in the in vivo {sup 32}P-labelling experiments. (author). 33 refs, 6 figs.

  20. Impact of Casein and Gluten Free Dietary Intervention on Selected Autistic Children

    Directory of Open Access Journals (Sweden)

    Veerappan Nishadevi

    2008-09-01

    Full Text Available Objective: Autism is a life long developmental disorder that emerges in early childhood and results in significant lifelong disability. The goal of treatment is to promote the childs social and language development and minimize behaviors that interfere with the childs functioning and learning. This study evaluated the impact of casein and gluten free diet among selected autistic children. Methods: Three private special schools in Salem District, Tamilnadu, India were selected. A total number of 50 autistic children 10 from SIMEC, 10 from MMIC and 30 from CSI comprised the study sample. Background information, clinical history and nutritional status, dietary pattern were collected from the 50 selected autistic children. Out of 50 autistic children 30 autistic children were selected for the dietary intervention. Diet counseling regarding casein free diet was imparted to Group I (n=10, gluten free diet to Group II (n=10 and both casein and gluten free diet for Group III (n=10. The diet was followed for a period of 2 months. The efficacy of the dietary exclusion of casein and gluten was evaluated using a food and behavior diary on a day to day basis, using observation method. Findings: Results about Group I autistic children who followed dietary exclusion of casein free diet showed that the mean scores before and after casein free dietary intervention depiticted these improvements as, 1 to 1.2 for attention, 2.8 to 2.9 for sleep, 1.1 to 1.3 for hyperactivity, 1.1 to 1.2 for anxiety/compulsion. For Group II autistic children who followed dietary exclusion of gluten free diet showed the improvements as 1.1 to 1.4 for attention 2.5 to 3 for sleep, 1.7 to 1.9 for hyperactivity, 1.1 to 1.2 for anxiety/compulsion. About Group III autistic children who followed dietary exclusion of both casein and gluten free diet showed the improvements as 1.1 to 1.3 for attention, 2.5 to 2.7 for sleep, 1.3 to 1.7 for hyperactivity, and 1.1 to 1.2 for anxiety

  1. Phosphorylation of the PCNA binding domain of the large subunit of replication factor C by Ca2+/calmodulin-dependent protein kinase II inhibits DNA synthesis

    DEFF Research Database (Denmark)

    Maga, G; Mossi, R; Fischer, R

    1997-01-01

    that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF......Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and DNA repair. It is a molecular matchmaker required for loading of the proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases...

  2. Protein kinase A type I activates a CRE-element more efficiently than protein kinase A type II regardless of C subunit isoform

    Directory of Open Access Journals (Sweden)

    Kvissel Anne-Katrine

    2011-02-01

    Full Text Available Abstract Background Protein kinase A type I (PKAI and PKAII are expressed in most of the eukaryotic cells examined. PKA is a major receptor for cAMP and specificity is achieved partly through tissue-dependent expression and subcellular localization of subunits with different biochemical properties. In addition posttranslational modifications help fine tune PKA activity, distribution and interaction in the cell. In spite of this the functional significance of two forms of PKA in one cell has not been fully determined. Here we have tested the ability of PKAI and PKAII formed by expression of the regulatory (R subunits RIα or RIIα in conjunction with Cα1 or Cβ2 to activate a co-transfected luciferace reporter gene, controlled by the cyclic AMP responsive element-binding protein (CREB in vivo. Results We show that PKAI when expressed at equal levels as PKAII was significantly (p Conclusion We suggest that differential effects of PKAI and PKAII in inducing Cre-luciferace activity depend on R and not C subunit identity.

  3. [Comparison of the function and conformation of human β-casein and bovine β-casein by spectroscopic study].

    Science.gov (United States)

    Liu, Wei; Li, Meng; Ren, Hao-wei; Liu, Ning

    2014-12-01

    β-Casein was the main component of human milk casein, but the content of β-casein in the bovine milk was less. The difference in β-casein content of the two samples was one of the reasons why human milk is more digestible than bovine milk. Studying the differences of structure and function in human and bovine milk β-casein can help us develop a new human milk simulated infant formula which will be more suitable for the infant gut. The UV spectrophotometer was used to study the solubility, sulfhydryl and emulsification of human milk β-casein and bovine milk β-casein, Fluorescence spectroscopy and the infrared spectroscopy were used to study the structural characteristics of human milk β-casein and bovine milk β-casein. The two samples shared a similar isoelectric point (pH 4.0~5.0), the solubility of human milk β-casein (10.83%) was lower than which in bovine milk β-casein (11.83%) near the pI, while it was higher when it deviated the pI. The emulsion ability (110~140 m2 · g(-1)) of human milk β-casein was higher than that in bovine milk β-casein (70~130 m2 · g(-1)) and surface sulfhydryl group (SH) of two kinds of milk protein were similar [(18.47±0.08) μmol · g(-1) and (18.67±0.17) μmol · g(-1)]. The total sulfhydryl group [(47.46±0.23) μmol · g(-1)] in bovine milk β-casein was more than that in human milk β-casein [(26.17±0.12) μmol · g(-1)]. Functional groups in two samples were similar and they both contained beta sheet, human milk β-casein had less H-bond and internal hydrophobic than bovine milk β-casein. The results showed that the two samples had similar functional groups, while human milk β-casein had much less secondary structure such as α-helix and β-sheet, a looser tertiary structure and a better interfacial activity.

  4. Olmesartan inhibits angiotensin II-Induced migration of vascular smooth muscle cells through Src and mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Kyotani, Yoji; Zhao, Jing; Tomita, Sayuko; Nakayama, Hitoshi; Isosaki, Minoru; Uno, Masayuki; Yoshizumi, Masanori

    2010-01-01

    Clinical studies have shown that angiotensin-receptor blockers (ARBs) reduce the risk of cardiovascular diseases in hypertensive patients. It is assumed that the reduction of the risk by ARBs may be attributed in part to the inhibition of angiotensin II (AII)-induced vascular smooth muscle cell (VSMC) migration associated with atherosclerosis. However, the effect of ARBs on AII-induced changes in intracellular signaling and resultant cell migration has not been well established. Here, we investigated the effect of olmesartan, an ARB, on AII-induced extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) activation and rat aortic smooth muscle cell (RASMC) migration. Olmesartan inhibited AII-induced ERK1/2 and JNK activation at lower concentrations (10 nM). On the other hand, PP2, a Src tyrosine kinase inhibitor, also inhibited AII-induced ERK1/2 and JNK activation, but its effect on ERK1/2 was less pronounced than that of olmesartan. Olmesartan, U0126 (an ERK1/2 inhibitor), SP600125 (a JNK inhibitor), and PP2 potently inhibited AII-induced RASMC migration. From these findings, it was inferred that angiotensin-receptor blockade by olmesartan results in the inhibition of AII-induced activation of Src, ERK1/2, and JNK in RASMC. Olmesartan may be a potent inhibitor of AII-induced VSMC migration, which may be involved in the progression of atherosclerosis.

  5. Phosphorylation and mRNA splicing of collapsin response mediator protein-2 determine inhibition of rho-associated protein kinase (ROCK) II function in carcinoma cell migration and invasion

    DEFF Research Database (Denmark)

    Morgan-Fisher, Marie; Couchman, John R; Yoneda, Atsuko

    2013-01-01

    migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition...... II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively......The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II...

  6. Structure of Ctk3, a subunit of the RNA polymerase II CTD kinase complex, reveals a noncanonical CTD-interacting domain fold.

    Science.gov (United States)

    Mühlbacher, Wolfgang; Mayer, Andreas; Sun, Mai; Remmert, Michael; Cheung, Alan C M; Niesser, Jürgen; Soeding, Johannes; Cramer, Patrick

    2015-10-01

    CTDK-I is a yeast kinase complex that phosphorylates the C-terminal repeat domain (CTD) of RNA polymerase II (Pol II) to promote transcription elongation. CTDK-I contains the cyclin-dependent kinase Ctk1 (homologous to human CDK9/CDK12), the cyclin Ctk2 (human cyclin K), and the yeast-specific subunit Ctk3, which is required for CTDK-I stability and activity. Here we predict that Ctk3 consists of a N-terminal CTD-interacting domain (CID) and a C-terminal three-helix bundle domain. We determine the X-ray crystal structure of the N-terminal domain of the Ctk3 homologue Lsg1 from the fission yeast Schizosaccharomyces pombe at 2.0 Å resolution. The structure reveals eight helices arranged into a right-handed superhelical fold that resembles the CID domain present in transcription termination factors Pcf11, Nrd1, and Rtt103. Ctk3 however shows different surface properties and no binding to CTD peptides. Together with the known structure of Ctk1 and Ctk2 homologues, our results lead to a molecular framework for analyzing the structure and function of the CTDK-I complex.

  7. Casein mediated green synthesis and decoration of reduced graphene oxide.

    Science.gov (United States)

    Maddinedi, Sireesh Babu; Mandal, Badal Kumar; Vankayala, Raviraj; Kalluru, Poliraju; Tammina, Sai Kumar; Kiran Kumar, H A

    2014-05-21

    This research is mainly focusing on one-step biosynthesis of graphene from graphene oxide and its stabilization using naturally occurring milk protein, casein. The synthesis of casein reduced graphene oxide (CRGO) was completed within 7h under reflux at 90°C with the formation of few layered fine graphene nanosheets. UV-Vis, XRD, XPS analysis data revealed the reduction process of the graphene oxide. Results of FT-IR, HPLC and TEM analysis have shown that the ensuing material consists of graphene decorated with casein molecules. Aspartic acid and glutamic acid residue present in casein molecules are responsible for the reduction of graphene oxide.

  8. Casein mediated green synthesis and decoration of reduced graphene oxide

    Science.gov (United States)

    Maddinedi, Sireesh Babu; Mandal, Badal Kumar; Vankayala, Raviraj; Kalluru, Poliraju; Tammina, Sai Kumar; Kiran Kumar, H. A.

    This research is mainly focusing on one-step biosynthesis of graphene from graphene oxide and its stabilization using naturally occurring milk protein, casein. The synthesis of casein reduced graphene oxide (CRGO) was completed within 7 h under reflux at 90 °C with the formation of few layered fine graphene nanosheets. UV-Vis, XRD, XPS analysis data revealed the reduction process of the graphene oxide. Results of FT-IR, HPLC and TEM analysis have shown that the ensuing material consists of graphene decorated with casein molecules. Aspartic acid and glutamic acid residue present in casein molecules are responsible for the reduction of graphene oxide.

  9. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells.

    Directory of Open Access Journals (Sweden)

    Cong Haolong

    Full Text Available Enterovirus 71 (EV71 is one of the main causative agents of foot, hand and mouth disease. Its infection usually causes severe central nervous system diseases and complications in infected infants and young children. In the present study, we demonstrated that EV71 infection caused the rearrangement of vimentin in human astrocytoma cells. The rearranged vimentin, together with various EV71 components, formed aggresomes-like structures in the perinuclear region. Electron microscopy and viral RNA labeling indicated that the aggresomes were virus replication sites since most of the EV71 particles and the newly synthesized viral RNA were concentrated here. Further analysis revealed that the vimentin in the virus factories was serine-82 phosphorylated. More importantly, EV71 VP1 protein is responsible for the activation of calmodulin-dependent protein kinase II (CaMK-II which phosphorylated the N-terminal domain of vimentin on serine 82. Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Collectively, this study identified the replication centers of EV71 in human astrocyte cells. This may help us understand the replication mechanism and pathogenesis of EV71 in human.

  10. Threonine-4 of mammalian RNA polymerase II CTD is targeted by Polo-like kinase 3 and required for transcriptional elongation.

    Science.gov (United States)

    Hintermair, Corinna; Heidemann, Martin; Koch, Frederic; Descostes, Nicolas; Gut, Marta; Gut, Ivo; Fenouil, Romain; Ferrier, Pierre; Flatley, Andrew; Kremmer, Elisabeth; Chapman, Rob D; Andrau, Jean-Christophe; Eick, Dirk

    2012-06-13

    Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5' and 3' regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo-like kinase 3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4-P levels in the 3' region of genes occurring subsequently to an increase of Ser2-P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells.

  11. Increased activity of rat liver nucleolar protein kinase following triiodothyronine administration.

    Science.gov (United States)

    Fugassa, E; Gallo, G; Pertica, M; Voci, A; Orunesu, M

    1977-12-08

    Triiodothyronine (T3) administration to thyroidectomized rats induces a significant increase in the nucleolus-associated protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) activity. The general properties of the protein kinase solubilized from liver nucleoli have been investigated. Mg2+ (20 mM) is essential for the reaction and an appropriate concentration of NaCl (100 mM) is required to achieve maximal phosphorylation rates. The optimal pH for casein phosphorylation is 7.6. The kinase phosphorylates casein more efficiently than phosvitin and displays an almost undetectable activity towards histones and protamine. No significant stimulation of the kinase activity by cyclic AMP has been detected. The apparent Km values for casein and ATP are 1.5 mg/ml and 1.5-10(-5) M, respectively, and are not affected by the hormone administration.

  12. Mangiferin, a natural xanthone, protects murine liver in Pb(II induced hepatic damage and cell death via MAP kinase, NF-κB and mitochondria dependent pathways.

    Directory of Open Access Journals (Sweden)

    Pabitra Bikash Pal

    Full Text Available One of the most well-known naturally occurring environmental heavy metals, lead (Pb has been reported to cause liver injury and cellular apoptosis by disturbing the prooxidant-antioxidant balance via oxidative stress. Several studies, on the other hand, reported that mangiferin, a naturally occurring xanthone, has been used for a broad range of therapeutic purposes. In the present study, we, therefore, investigated the molecular mechanisms of the protective action of mangiferin against lead-induced hepatic pathophysiology. Lead [Pb(II] in the form of Pb(NO32 (at a dose of 5 mg/kg body weight, 6 days, orally induced oxidative stress, hepatic dysfunction and cell death in murine liver. Post treatment of mangiferin at a dose of 100 mg/kg body weight (6 days, orally, on the other hand, diminished the formation of reactive oxygen species (ROS and reduced the levels of serum marker enzymes [alanine aminotranferase (ALT and alkaline phosphatase (ALP]. Mangiferin also reduced Pb(II induced alterations in antioxidant machineries, restored the mitochondrial membrane potential as well as mutual regulation of Bcl-2/Bax. Furthermore, mangiferin inhibited Pb(II-induced activation of mitogen-activated protein kinases (MAPKs (phospho-ERK 1/2, phosphor-JNK phospho- p38, nuclear translocation of NF-κB and apoptotic cell death as was evidenced by DNA fragmentation, FACS analysis and histological assessment. In vitro studies using hepatocytes as the working model also showed the protective effect of mangiferin in Pb(II induced cytotoxicity. All these beneficial effects of mangiferin contributes to the considerable reduction of apoptotic hepatic cell death induced by Pb(II. Overall results demonstrate that mangiferin exhibit both antioxidative and antiapoptotic properties and protects the organ in Pb(II induced hepatic dysfunction.

  13. Mangiferin, a Natural Xanthone, Protects Murine Liver in Pb(II) Induced Hepatic Damage and Cell Death via MAP Kinase, NF-κB and Mitochondria Dependent Pathways

    Science.gov (United States)

    Pal, Pabitra Bikash; Sinha, Krishnendu; Sil, Parames C.

    2013-01-01

    One of the most well-known naturally occurring environmental heavy metals, lead (Pb) has been reported to cause liver injury and cellular apoptosis by disturbing the prooxidant-antioxidant balance via oxidative stress. Several studies, on the other hand, reported that mangiferin, a naturally occurring xanthone, has been used for a broad range of therapeutic purposes. In the present study, we, therefore, investigated the molecular mechanisms of the protective action of mangiferin against lead-induced hepatic pathophysiology. Lead [Pb(II)] in the form of Pb(NO3)2 (at a dose of 5 mg/kg body weight, 6 days, orally) induced oxidative stress, hepatic dysfunction and cell death in murine liver. Post treatment of mangiferin at a dose of 100 mg/kg body weight (6 days, orally), on the other hand, diminished the formation of reactive oxygen species (ROS) and reduced the levels of serum marker enzymes [alanine aminotranferase (ALT) and alkaline phosphatase (ALP)]. Mangiferin also reduced Pb(II) induced alterations in antioxidant machineries, restored the mitochondrial membrane potential as well as mutual regulation of Bcl-2/Bax. Furthermore, mangiferin inhibited Pb(II)-induced activation of mitogen-activated protein kinases (MAPKs) (phospho-ERK 1/2, phosphor-JNK phospho- p38), nuclear translocation of NF-κB and apoptotic cell death as was evidenced by DNA fragmentation, FACS analysis and histological assessment. In vitro studies using hepatocytes as the working model also showed the protective effect of mangiferin in Pb(II) induced cytotoxicity. All these beneficial effects of mangiferin contributes to the considerable reduction of apoptotic hepatic cell death induced by Pb(II). Overall results demonstrate that mangiferin exhibit both antioxidative and antiapoptotic properties and protects the organ in Pb(II) induced hepatic dysfunction. PMID:23451106

  14. Theoretical investigation, biological evaluation and VEGFR2 kinase studies of metal(II) complexes derived from hydrotris(methimazolyl)borate.

    Science.gov (United States)

    Jayakumar, S; Mahendiran, D; Srinivasan, T; Mohanraj, G; Kalilur Rahiman, A

    2016-02-01

    The reaction of soft tripodal scorpionate ligand, sodium hydrotris(methimazolyl)borate with M(ClO4)2·6H2O [MMn(II), Ni(II), Cu(II) or Zn(II)] in methanol leads to the cleavage of B-N bond followed by the formation of complexes of the type [M(MeimzH)4](ClO4)2·H2O (1-4), where MeimzH=methimazole. All the complexes were fully characterized by spectro-analytical techniques. The molecular structure of the zinc(II) complex (4) was determined by X-ray crystallography, which supports the observed deboronation reaction in the scorpionate ligand with tetrahedral geometry around zinc(II) ion. The electronic spectra of complexes suggested tetrahedral geometry for manganese(II) and nickel(II) complexes, and square-planar geometry for copper(II) complex. Frontier molecular orbital analysis (HOMO-LUMO) was carried out by B3LYP/6-31G(d) to understand the charge transfer occurring in the molecules. All the complexes exhibit significant antimicrobial activity against Gram (-ve) and Gram (+ve) bacterial as well as fungal strains, which are quite comparable to standard drugs streptomycin and clotrimazole. The copper(II) complex (3) showed excellent free radical scavenging activity against DPPH in all concentration with IC50 value of 30μg/mL, when compared to the other complexes. In the molecular docking studies, all the complexes showed hydrophobic, π-π and hydrogen bonding interactions with BSA. The cytotoxic activity of the complexes against human hepatocellular liver carcinoma (HepG2) cells was assessed by MTT assay, which showed exponential responses toward increasing concentration of complexes.

  15. Influence of shear during enzymatic gelation of caseinate-water and caseinate-water-fat systems

    NARCIS (Netherlands)

    Manski, J.M.; Goot, van der A.J.; Boom, R.M.

    2007-01-01

    Solidification, emulsification and application of shear were combined to induce diversity and heterogeneity in the micro- and macrostructure of concentrated caseinate-based food matrices containing a dispersed fat phase. The products were evaluated with selected parameters from small-scale and large

  16. Origin of suppressed demixing in casein/xanthan mixtures

    NARCIS (Netherlands)

    van Gruijthuijsen, K.; Herle, V.; Tuinier, R.; Schurtenberger, P.; Stradner, A.

    2012-01-01

    We explore the properties of casein/xanthan mixtures for xanthan concentrations beyond those inducing phase separation. Previous work has successfully described the onset of demixing by depletion theory in the protein limit, where the xanthan polysaccharides, the polymers, are larger than the casein

  17. Is there a feeding strategy to increase milk casein content?

    Directory of Open Access Journals (Sweden)

    A. Formigoni

    2010-04-01

    Full Text Available Because more than 60% of milk produced in Italy is transformed into cheese, milk economical value strongly depends on cheese yield. Among the factors that influence cheese yield, milk casein and fat content plays a major role: when milk is converted into Grana Padano and Parmigiano reggiano, three grams of seasoned cheese are produced from one gram of milk casein.....

  18. Quantification of Melanoidin Concentration in Sugar-Casein Systems

    NARCIS (Netherlands)

    Brands, C.M.J.; Wedzicha, B.L.; Boekel, van M.A.J.S.

    2002-01-01

    Melanoidins are the final, brown, high molecular weight products of the Maillard reaction. The aim of the present study was to determine the average molar extinction coefficients of melanoidins formed in heated glucose-casein and fructose-casein systems. The value of the extinction coefficient can b

  19. Lactoferrin binding to transglutaminase cross-linked casein micelles

    NARCIS (Netherlands)

    Anema, S.G.; de Kruif, C.G.

    2012-01-01

    Casein micelles in skim milk were either untreated (untreated milk) or were cross-linked using transglutaminase (TGA-milk). Added lactoferrin (LF) bound to the casein micelles and followed Langmuir adsorption isotherms. The adsorption level was the same in both milks and decreased the micellar zeta

  20. Physical stability of caseinate stabilized emulsions during heating.

    NARCIS (Netherlands)

    Cruijsen, J.M.M.

    1996-01-01

    The physical stability of caseinate stabilized emulsions was studied during heating (80- 120°C). Coagulation, coalescence and phase separation of the caseinate emulsions was studied using objective heat stability tests. The physical changes were characterized by light microscopy, particle size measu

  1. Water holding capacity and swelling of casein hydrogels

    NARCIS (Netherlands)

    de Kruif, C; Anema, Skelte G.; Zhu, Changjun; Havea, Palatasa; Coker, Christina

    2015-01-01

    The water holding capacity of casein gels was investigated by measuring the swelling and de-swelling under a variety of conditions of temperature and salt concentration. Transglutaminase cross-linked sodium caseinate (15% w/w) gels will swell in good solvents or shrink in poor solvents until an equi

  2. Effect of Anacyclus pyrethrum on pentylenetetrazole-induced kindling, spatial memory, oxidative stress and rho-kinase II expression in mice.

    Science.gov (United States)

    Pahuja, Monika; Mehla, Jogender; Reeta, K H; Tripathi, Manjari; Gupta, Yogendra Kumar

    2013-03-01

    Anacyclus pyrethrum (A. pyrethrum) has been reported to exhibit anticonvulsant activity. In the present study, the effect of hydro-alcoholic extract of A. pyrethrum root (HEAP) on pentylenetetrazole (PTZ) induced kindling, spatial memory, oxidative stress and rho kinase (ROCK II) was assessed. Male albino mice (25-30 g) were used in the study. PTZ (35 mg/kg, i.p. on alternate days) was injected to induce kindling and PTZ (70 mg/kg, i.p) challenge was given 7 days post-kindling. HEAP was administered orally daily in the doses of 100, 250 and 500 mg/kg along with PTZ injections during the kindling process and continued till PTZ challenge post kindling. Spatial memory was assessed using Morris water maze test. Oxidative stress parameters [malondialdehyde (MDA) and reduced glutathione (GSH)] and ROCK II expression were estimated in whole brain at the end of the study. Pre-treatment with HEAP (250 and 500 mg/kg) showed significant increase in the myoclonic jerk latency and delay in the development of kindling. A significant decrease in mortality was observed at higher doses of HEAP (250 and 500 mg/kg). Pre-treatment with HEAP significantly increased the number of platform crossings and decreased the escape latency, as opposed to the PTZ group, thus showing protection against memory deficit. HEAP pre-treatment also attenuated the oxidative stress induced by PTZ kindling. PTZ induced kindling increased the ROCK II expression whereas, HEAP pre-treatment attenuated the increase in ROCK II expression. To conclude, HEAP pre-treatment showed antiepileptic effect and also showed protection against cognitive impairment by decreasing oxidative stress and ROCK II expression in PTZ kindled mice.

  3. AMP-activated protein kinase inhibits TGF-β-, angiotensin II-, aldosterone-, high glucose-, and albumin-induced epithelial-mesenchymal transition.

    Science.gov (United States)

    Lee, Jang Han; Kim, Ji Hyun; Kim, Ja Seon; Chang, Jai Won; Kim, Soon Bae; Park, Jung Sik; Lee, Sang Koo

    2013-03-15

    The epithelial-mesenchymal transition (EMT) is a novel mechanism that promotes renal fibrosis. Transforming growth factor-β (TGF-β), angiotensin II, aldosterone, high glucose, and urinary albumin are well-known causes of EMT and renal fibrosis. We examined whether and how activation of AMP-activated protein kinase (AMPK) suppressed EMT induced by the above agents in tubular epithelial cells. All experiments were performed using HK-2 cells. Protein expression was measured by Western blot analysis. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. Exposure of tubular cells to TGF-β (10 ng/ml), angiotensin II (1 μM), aldosterone (100 nM), high glucose (30 mM), and albumin (5 mg/ml) for 5 days induced EMT, as shown by upregulation of α-smooth muscle actin and downregulation of E-cadherin. ROS and NADPH oxidase 4 (Nox4) expression were increased, and antioxidants such as tiron and N-acetylcysteine inhibited EMT induction. Metformin (the best known clinical activator of AMPK) suppressed EMT induction through inhibition of ROS via induction of heme oxygenase-1 and endogenous antioxidant thioredoxin. An AMPK inhibitor (compound C) and AMPK small interfering RNA blocked the effect of metformin, and another AMPK activator [5-aminoimidazole-4-carboxamide-1β riboside (AICAR)] exerted the same effects as metformin. In conclusion, AMPK activation might be beneficial in attenuating the tubulointerstitial fibrosis induced by TGF-β, angiotensin II, aldosterone, high glucose, and urinary albumin.

  4. Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.

    Science.gov (United States)

    Yilmaz, Mehtap; Gangopadhyay, Samudra S; Leavis, Paul; Grabarek, Zenon; Morgan, Kathleen G

    2013-02-07

    CaMKII (Ca²⁺/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser²⁶, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser²⁶, we generated a phosphospecific Ser²⁶ antibody and demonstrated an increase in Ser²⁶ phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser²⁶ affects the kinase activity, we mutated Ser²⁶ to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr²⁸⁷ autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser²⁶ of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr²⁸⁷ most probably by blocking ATP binding. We propose that Ser²⁶ phosphorylation constitutes an important mechanism for switching off CaMKII activity.

  5. L-type calcium channels and calcium/calmodulin-dependent kinase II differentially mediate behaviors associated with nicotine withdrawal in mice.

    Science.gov (United States)

    Jackson, K J; Damaj, M I

    2009-07-01

    Smoking is a widespread health problem. Because the nicotine withdrawal syndrome is a major contributor to continued smoking and relapse, it is important to understand the molecular and behavioral mechanisms of nicotine withdrawal to generate more effective smoking cessation therapies. Studies suggest a role for calcium-dependent mechanisms, such as L-type calcium channels and calcium/calmodulin-dependent protein kinase II (CaMKII), in the effects of nicotine dependence; however, the role of these mechanisms in nicotine-mediated behaviors is unclear. Thus, the goal of this study was to elucidate the role of L-type calcium channels and CaMKII in nicotine withdrawal behaviors. Using both pharmacological and genetic methods, our results show that L-type calcium channels are involved in physical, but not affective, nicotine withdrawal behaviors. Although our data do provide evidence of a role for CaMKII in nicotine withdrawal behaviors, our pharmacological and genetic assessments yielded different results concerning the specific role of the kinase. Pharmacological data suggest that CaMKII is involved in somatic signs and affective nicotine withdrawal, and activity level is decreased after nicotine withdrawal, whereas the genetic assessments yielded results suggesting that CaMKII is involved only in the anxiety-related response, yet the kinase activity may be increased after nicotine withdrawal; thus, future studies are necessary to clarify the precise behavioral specifics of the relevance of CaMKII in nicotine withdrawal behaviors. Overall, our data show that L-type calcium channels and CaMKII are relevant in nicotine withdrawal and differentially mediate nicotine withdrawal behaviors.

  6. Phosphorylation of calcium/calmodulin-stimulated protein kinase II at T286 enhances invasion and migration of human breast cancer cells

    Science.gov (United States)

    Chi, Mengna; Evans, Hamish; Gilchrist, Jackson; Mayhew, Jack; Hoffman, Alexander; Pearsall, Elizabeth Ann; Jankowski, Helen; Brzozowski, Joshua Stephen; Skelding, Kathryn Anne

    2016-01-01

    Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional kinase that controls a range of cellular functions, including proliferation, differentiation and apoptosis. The biological properties of CaMKII are regulated by multi-site phosphorylation. However, the role that CaMKII phosphorylation plays in cancer cell metastasis has not been examined. We demonstrate herein that CaMKII expression and phosphorylation at T286 is increased in breast cancer when compared to normal breast tissue, and that increased CAMK2 mRNA is associated with poor breast cancer patient prognosis (worse overall and distant metastasis free survival). Additionally, we show that overexpression of WT, T286D and T286V forms of CaMKII in MDA-MB-231 and MCF-7 breast cancer cells increases invasion, migration and anchorage independent growth, and that overexpression of the T286D phosphomimic leads to a further increase in the invasive, migratory and anchorage independent growth capacity of these cells. Pharmacological inhibition of CaMKII decreases MDA-MB-231 migration and invasion. Furthermore, we demonstrate that overexpression of T286D, but not WT or T286V-CaMKII, leads to phosphorylation of FAK, STAT5a, and Akt. These results demonstrate a novel function for phosphorylation of CaMKII at T286 in the control of breast cancer metastasis, offering a promising target for the development of therapeutics to prevent breast cancer metastasis. PMID:27605043

  7. Ca²⁺/calmodulin-dependent protein kinase II contributes to hypoxic ischemic cell death in neonatal hippocampal slice cultures.

    Directory of Open Access Journals (Sweden)

    Qing Lu

    Full Text Available We have recently shown that p38MAP kinase (p38MAPK stimulates ROS generation via the activation of NADPH oxidase during neonatal hypoxia-ischemia (HI brain injury. However, how p38MAPK is activated during HI remains unresolved and was the focus of this study. Ca²⁺/calmodulin-dependent protein kinase II (CaMKII plays a key role in brain synapse development, neural transduction and synaptic plasticity. Here we show that CaMKII activity is stimulated in rat hippocampal slice culture exposed to oxygen glucose deprivation (OGD to mimic the condition of HI. Further, the elevation of CaMKII activity, correlated with enhanced p38MAPK activity, increased superoxide generation from NADPH oxidase as well as necrotic and apoptotic cell death. All of these events were prevented when CaMKII activity was inhibited with KN93. In a neonatal rat model of HI, KN93 also reduced brain injury. Our results suggest that CaMKII activation contributes to the oxidative stress associated with neural cell death after HI.

  8. Beta 2 subunit-containing nicotinic receptors mediate acute nicotine-induced activation of calcium/calmodulin-dependent protein kinase II-dependent pathways in vivo.

    Science.gov (United States)

    Jackson, K J; Walters, C L; Damaj, M I

    2009-08-01

    Nicotine is the addictive component of tobacco, and successful smoking cessation therapies must address the various processes that contribute to nicotine addiction. Thus, understanding the nicotinic acetylcholine receptor (nAChR) subtypes and subsequent molecular cascades activated after nicotine exposure is of the utmost importance in understanding the progression of nicotine dependence. One possible candidate is the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Substrates of this kinase include the vesicle-associated protein synapsin I and the transcription factor cAMP response element-binding protein (CREB). The goal of these studies was to examine these postreceptor mechanisms after acute nicotine treatment in vivo. We first show that administration of nicotine increases CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), and amygdala. In beta2 nAChR knockout (KO) mice, nicotine does not induce an increase in kinase activity, phosphorylated (p)Synapsin I, or pCREB. In contrast, alpha7 nAChR KO mice show nicotine-induced increases in CaMKII activity and pCREB, similar to their wild-type littermates. Moreover, we show that when animals are pretreated with the CaMKII inhibitors 4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl]phenyl isoquinolinesulfonic acid ester (KN-62) and N-[2-[[[3-(4-chlorophenyl)-2 propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide (KN-93), nicotine-induced increase in the kinase activity and pCREB was attenuated in the VTA and NAc, whereas pretreatment with (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate) (KN-92), the inactive analog, did not alter the nicotine-induced increase in pCREB. Taken together, these data suggest that the nicotine-induced increase in CaMKII activity may correlate with the nicotine-induced increase in pSynapsin I and pCREB in the VTA and NAc via beta2

  9. Central SDF-1/CXCL12 expression and its cardiovascular and sympathetic effects: the role of angiotensin II, TNF-α, and MAP kinase signaling.

    Science.gov (United States)

    Wei, Shun-Guang; Zhang, Zhi-Hua; Yu, Yang; Felder, Robert B

    2014-12-01

    The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptors are expressed by neurons and glial cells in cardiovascular autonomic regions of the brain, including the hypothalamic paraventricular nucleus (PVN), and contribute to neurohumoral excitation in rats with ischemia-induced heart failure. The present study examined factors regulating the expression of SDF-1 in the PVN and mechanisms mediating its sympatho-excitatory effects. In urethane anesthetized rats, a 4-h intracerebroventricular (ICV) infusion of angiotensin II (ANG II) or tumor necrosis factor-α (TNF-α) in doses that increase mean blood pressure (MBP) and sympathetic drive increased the expression of SDF-1 in PVN. ICV administration of SDF-1 increased the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK), JNK, and p38 MAPK in PVN, along with MBP, heart rate (HR), and renal sympathetic nerve activity (RSNA), but did not affect total p44/42 MAPK, JNK, and p38 MAPK levels. ICV pretreatment with the selective p44/42 MAPK inhibitor PD98059 prevented the SDF-1-induced increases in MBP, HR, and RSNA; ICV pretreatment with the selective JNK and p38 MAPK inhibitors attenuated but did not block these SDF-1-induced excitatory responses. ICV PD98059 also prevented the sympatho-excitatory response to bilateral PVN microinjections of SDF-1. ICV pretreatment with SDF-1 short-hairpin RNA significantly reduced ANG II- and TNF-α-induced phosphorylation of p44/42 MAPK in PVN. These findings identify TNF-α and ANG II as drivers of SDF-1 expression in PVN and suggest that the full expression of their cardiovascular and sympathetic effects depends upon SDF-1-mediated activation of p44/42 MAPK signaling.

  10. The Alpha Catalytic Subunit of Protein Kinase CK2 Is Required for Mouse Embryonic Development▿

    Science.gov (United States)

    Lou, David Y.; Dominguez, Isabel; Toselli, Paul; Landesman-Bollag, Esther; O'Brien, Conor; Seldin, David C.

    2008-01-01

    Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2α and/or CK2α′) and two CK2β regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2β gene is required for early embryonic development, while the CK2α′ subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2α gene in the mouse germ line. Embryos lacking CK2α have a marked reduction in CK2 activity in spite of the presence of the CK2α′ subunit. CK2α−/− embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2α appears to play an essential and uncompensated role in mammalian development. PMID:17954558

  11. Alginate-Casein Microspheres as Bioactive Vehicles for Nutrients

    Institute of Scientific and Technical Information of China (English)

    何志敏; 张茜青; 齐崴; 黄仁亮; 苏荣欣

    2015-01-01

    The aim of this work was to develop an alginate-casein composite microsphere as a bioactive vehicle for oral administration of nutrients by a simple extrusion dripping method. Riboflavin was selected as a model drug, and the microencapsulation efficiency was raised to 97.94%after optimizing the preparation conditions by response surface methodology. In vitro release studies showed that riboflavin was released completely from alginate-casein microspheres in simulated intestinal fluids. Meanwhile, the morphology, structure and interaction between alginate and casein were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectra.

  12. Aberrant calcium/calmodulin-dependent protein kinase II (CaMKII) activity is associated with abnormal dendritic spine morphology in the ATRX mutant mouse brain.

    Science.gov (United States)

    Shioda, Norifumi; Beppu, Hideyuki; Fukuda, Takaichi; Li, En; Kitajima, Isao; Fukunaga, Kohji

    2011-01-05

    In humans, mutations in the gene encoding ATRX, a chromatin remodeling protein of the sucrose-nonfermenting 2 family, cause several mental retardation disorders, including α-thalassemia X-linked mental retardation syndrome. We generated ATRX mutant mice lacking exon 2 (ATRX(ΔE2) mice), a mutation that mimics exon 2 mutations seen in human patients and associated with milder forms of retardation. ATRX(ΔE2) mice exhibited abnormal dendritic spine formation in the medial prefrontal cortex (mPFC). Consistent with other mouse models of mental retardation, ATRX(ΔE2) mice exhibited longer and thinner dendritic spines compared with wild-type mice without changes in spine number. Interestingly, aberrant increased calcium/calmodulin-dependent protein kinase II (CaMKII) activity was observed in the mPFC of ATRX(ΔE2) mice. Increased CaMKII autophosphorylation and activity were associated with increased phosphorylation of the Rac1-guanine nucleotide exchange factors (GEFs) T-cell lymphoma invasion and metastasis 1 (Tiam1) and kalirin-7, known substrates of CaMKII. We confirmed increased phosphorylation of p21-activated kinases (PAKs) in mPFC extracts. Furthermore, reduced protein expression and activity of protein phosphatase 1 (PP1) was evident in the mPFC of ATRX(ΔE2) mice. In cultured cortical neurons, PP1 inhibition by okadaic acid increased CaMKII-dependent Tiam1 and kalirin-7 phosphorylation. Together, our data strongly suggest that aberrant CaMKII activation likely mediates abnormal spine formation in the mPFC. Such morphological changes plus elevated Rac1-GEF/PAK signaling seen in ATRX(ΔE2) mice may contribute to mental retardation syndromes seen in human patients.

  13. Immune activation by casein dietary antigens in bipolar disorder

    NARCIS (Netherlands)

    Severance, E.G.; Dupont, D.; Dickerson, F.B.; Stallings, C.R.; Origoni, A.E.; Krivogorsky, B.; Yang, S.; Haasnoot, W.; Yolken, R.H.

    2010-01-01

    Objectives: Inflammation and other immune processes are increasingly linked to psychiatric diseases. Antigenic triggers specific to bipolar disorder are not yet defined. We tested whether antibodies to bovine milk caseins were associated with bipolar disorder, and whether patients recognized differe

  14. Examination of rheological properties of aqueous solutions of sodium caseinate

    Directory of Open Access Journals (Sweden)

    Jolanta Gawałek

    2012-12-01

    Full Text Available Application of sodium caseinate as a functional additive in manufacturing processes requires production of its concentrated aqueous solutions which, in industrial conditions, presents a number of difficulties. In order to develop an effective and optimal industrial process of mixing – manufacturing a concentrated solution of sodium caseinate, it is essential to know rheological properties in a definite range of concentrations changing in the course of the dissolving process. The material for investigations was typical commercial sodium caseinate in the form of dry powder manufactured in Poland from acid casein using the method of extrusion. The objective of the undertaken empirical studies was the assessment of the impact of the concentration on rheological properties of sodium caseinate concentrates. Investigations were carried out for five concentrates manufactured in a mixer equipped in a mechanical agitator at concentrations ranging X (% Î (2.5¸12.5 and changing mass proportions of sodium caseinate in the aqueous solution as follows: GS/G (kgS·kg-1 = 0.025. On the basis of the obtained research results, classical flow curves were plotted for individual concentrates. The determined values of viscosity and density of the examined solutions were correlated in the form of h = f(GS/G and r = f(GS/G dependencies which were used during the determination of classical characteristics of mixing forces essential for the assessment of energetic expenditures required to manufacture concentrates in a mixer equipped in a mechanical agitator. The density of the examined concentrates increased in a way directly proportional, while the dynamic viscosity coefficient increased exponentially together with the increase of sodium caseinate concentration. Sodium caseinate concentrates exhibited Newtonian character in the examined range of concentrations.

  15. Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kouchi, Zen, E-mail: zkouchi@toyaku.ac.jp [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan); Fujiwara, Yuki [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan); Yamaguchi, Hideki [Division of Metastasis and Invasion Signaling, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-city, Saitama 332-0012 (Japan); Nakamura, Yoshikazu; Fukami, Kiyoko [Laboratory of Genome and Biosignals, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-city, Tokyo 192-0392 (Japan)

    2011-05-20

    Highlights: {yields} We analyzed Phosphatidylinositol 5-phosphate kinase II{beta} (PIPKII{beta}) function in cancer. {yields} PIPKII{beta} is required for vitamin D receptor-mediated E-cadherin upregulation in SW480. {yields} PIPKII{beta} suppresses cellular motility through E-cadherin induction in SW480 cells. {yields} Nuclear PIP{sub 2} but not plasma membrane-localized PIP{sub 2} mediates E-cadherin upregulation. -- Abstract: Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1{alpha},25-dihydroxyvitamin D{sub 3} (1{alpha},25(OH){sub 2}D{sub 3}) has anti-cancer activity in several colon cancers. 1{alpha},25(OH){sub 2}D{sub 3} induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKII{beta}) but not PIPKII{alpha} is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLC{delta}1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P{sub 2}) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLC{delta}1 PHD inhibited 1{alpha},25(OH){sub 2}D{sub 3}-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P{sub 2} production mediates E-cadherin expression through PIPKII{beta} in a VDR-dependent manner. PIPKII{beta} is also involved in the suppression of the cell motility induced by 1{alpha},25(OH){sub 2}D{sub 3}. These results indicate that PIPKII{beta}-mediated PI(4,5)P{sub 2} signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.

  16. Na-caseinate/oil/water systems: emulsion morphology diagrams.

    Science.gov (United States)

    Tan, Hui Lin; McGrath, Kathryn M

    2012-09-01

    The concentrated (dispersed phase 50-70 wt%) composition space of Na-caseinate, a family of milk proteins, stabilised emulsions was investigated for three different oils: soybean oil, palm olein and tetradecane with pH 6.8 phosphate buffer continuous phase. The variation of emulsion stability and microstructure were explored using static light scattering, diffusion nuclear magnetic resonance, cryo-scanning electron microscopy, rheology and the time varying macroscopic phase separation of the emulsions. For soybean oil and palm olein a rich diversity of emulsion microstructures and stabilities are realised. Five emulsion domains, each having a different microstructure and macroscopic stability have been identified within the composition space probed. For the lowest concentrations of emulsifier bridging flocculation is evident and emulsions are of low stability. Increasing Na-caseinate concentration leads to an increased stability and the existence of distinct individual oil droplets, visualised using cryo-scanning electron microscopy. Further increases in Na-caseinate concentration reduce emulsion stability due to depletion flocculation. Na-caseinate self-assembly is then initiated. At sufficiently high Na-caseinate and/or oil concentrations the continuous phase of the emulsion is a three-dimensional protein network and emulsion stability is again enhanced. At the limits of the emulsion composition space a gel-like paste is formed. The diversity of emulsion microstructure is reduced when tetradecane is the discrete phase. Na-caseinate self-assembly is limited and there is no evidence for formation of a protein network.

  17. Theoretical calculations, DNA interaction, topoisomerase I and phosphatidylinositol-3-kinase studies of water soluble mixed-ligand nickel(II) complexes.

    Science.gov (United States)

    Gurumoorthy, Perumal; Mahendiran, Dharmasivam; Kalilur Rahiman, Aziz

    2016-03-25

    Eight water soluble mixed-ligand nickel(II) complexes of the type [NiL(1-4)(diimine)H2O]·(ClO4)2, (1-8) where L(1-4) = 2-((2-(piperazin-1-yl)ethylimino)methyl)-4-substituted phenols, and diimine = 2,2'-bipyridyl (bpy) or 1,10-phenanthroline (phen) were synthesized and characterized by elemental analysis and spectroscopic methods. The uncoordinated perchlorate anions was ascertained form IR spectra of the complexes, and the absorption spectra reveal the octahedron geometry around nickel(II) ion with tridentate Schiff base ligand, diimine and a coordinated water molecule. Cyclic voltammograms of the complexes indicate the one-electron irreversible processes in the cathodic and anodic region. In vitro antioxidant activity proved the significant radical scavenging activity of the complexes against DPPH radical. The groove/electrostatic binding nature of complexes with CT-DNA (calf thymus deoxyribonucleic acid) were affirmed by absorption, hydrodynamic and voltammetric titration experiments and docking analysis. All the complexes exhibit significant cleavage activity on plasmid DNA via hydrolytic and oxidatively, in which the oxidative mechanism involves hydroxyl radicals and supports the possibility of minor-groove binding. The complex 4 shows significant topoisomerase I (Topo-I) inhibitory activity. The molecular modeling analysis of complexes with phosphatidylinositol-3-kinase (PI3K) receptor indicate the hydrogen bonding with Met1039, Asp837 and Leu1027, and hydrophobic interactions with Ser488, Asn498, Asp500, Gln662, Lys668, Ile844, Ile847, Ile850, Val941, Leu942, Leu1020, Met1034, Leu1035, Thr1037, Met1039, Gln1041 and Ile1051 of subdomain IIA of BSA. The complexes show σ-π interaction between diimines and amino groups of Leu1030 and Arg839.

  18. cGMP-dependent protein kinase type II knockout mice exhibit working memory impairments, decreased repetitive behavior, and increased anxiety-like traits.

    Science.gov (United States)

    Wincott, Charlotte M; Abera, Sinedu; Vunck, Sarah A; Tirko, Natasha; Choi, Yoon; Titcombe, Roseann F; Antoine, Shannon O; Tukey, David S; DeVito, Loren M; Hofmann, Franz; Hoeffer, Charles A; Ziff, Edward B

    2014-10-01

    Neuronal activity regulates AMPA receptor trafficking, a process that mediates changes in synaptic strength, a key component of learning and memory. This form of plasticity may be induced by stimulation of the NMDA receptor which, among its activities, increases cyclic guanosine monophosphate (cGMP) through the nitric oxide synthase pathway. cGMP-dependent protein kinase type II (cGKII) is ultimately activated via this mechanism and AMPA receptor subunit GluA1 is phosphorylated at serine 845. This phosphorylation contributes to the delivery of GluA1 to the synapse, a step that increases synaptic strength. Previous studies have shown that cGKII-deficient mice display striking spatial learning deficits in the Morris Water Maze compared to wild-type littermates as well as lowered GluA1 phosphorylation in the postsynaptic density of the prefrontal cortex (Serulle et al., 2007; Wincott et al., 2013). In the current study, we show that cGKII knockout mice exhibit impaired working memory as determined using the prefrontal cortex-dependent Radial Arm Maze (RAM). Additionally, we report reduced repetitive behavior in the Marble Burying task (MB), and heightened anxiety-like traits in the Novelty Suppressed Feeding Test (NSFT). These data suggest that cGKII may play a role in the integration of information that conveys both anxiety-provoking stimuli as well as the spatial and environmental cues that facilitate functional memory processes and appropriate behavioral response.

  19. Ca2+/calmodulin-dependent protein kinase II alpha is required for the initiation and maintenance of opioid-induced hyperalgesia.

    Science.gov (United States)

    Chen, Yan; Yang, Cheng; Wang, Zaijie Jim

    2010-01-01

    Repeated administration of opioids not only leads to tolerance and dependence, but also results in nociceptive enhancement called opioid-induced hyperalgesia (OIH). Nociceptive mediators involved in OIH generation remain poorly understood. In the present study, we tested the hypothesis that Ca(2+)/calmodulin-depent protein kinase II (CaMKIIalpha) is critical for OIH. Opioid-induced hyperalgesia was produced by repeated morphine administration or pellet implantation in mice. Correlating with the development of tactile allodynia and thermal hyperalgesia, spinal CaMKIIalpha activity was significantly increased in OIH. KN93, a CaMKII inhibitor, dose- and time-dependently reversed OIH and CaMKII activation without impairing locomotor coordination. To elucidate the specific CaMKII isoform involved, we targeted CaMKIIalpha by using small interfering RNA and demonstrated that knockdown of spinal CaMKIIalpha attenuated OIH. Furthermore, morphine failed to induce OIH in CaMKIIalpha(T286A) point mutant mice, although wild-type littermate mice developed robust OIH after repeated treatments with morphine. These data implicate, for the first time, an essential role of CaMKIIalpha as a cellular mechanism leading to and maintaining opioid-induced hyperalgesia.

  20. Structural investigations of sodium caseinate micelles in complex environments

    Energy Technology Data Exchange (ETDEWEB)

    Huck Iriart, C.; Herrera, M.L.; Candal, R. [Universidad de Buenos Aires, Buenos Aires (Argentina); Oliveira, C.L.P. [Universidade de Sao Paulo (USP), Sao Paulo, SP (Brazil); Torriani, I. [Laboratorio Nacional de Luz Sincrotron (LNLS), Campinas, SP (Brazil)

    2012-07-01

    Full text: The most frequent destabilization mechanisms in Sodium Caseinate (NaCas) emulsions are creaming and flocculation. Coarse or fine emulsions with low protein con- tent destabilize mainly by creaming. If migration mechanism is suppressed, flocculation may become the main mechanism of destabilization. Small Angle X-Ray Scattering (SAXS) technique was applied to investigate sodium caseinate micelles structure in different environments. As many natural products, Sodium Caseinate samples have large polydisperse size distribution. The experimental data was analyzed using advanced modeling approaches. The Form Factor for the Caseinate micelle subunits was described by an ellipsoidal core shell model and the structure factor was split into two contributions, one corresponding to the particle-particle interactions and another one for the long range correlation of the subunits in the supramolecular structure. For the first term the hard sphere structure factor using the Percus-Yevick approximation for closure relation was used and for the second term a fractal model was applied. Three concentrations of sodium Caseinate (2, 5 and 7.5 %wt.) were measured in pure water, sugar solutions (20 %wt.) and in three different lipid phase emulsions containing 10 %wt. sunflower seed, olive and fish oils. Data analysis provided an average casein subunit radius of 4 nm, an average distance between the subunits of around 20nm and a fractal dimension value of around 3 for all samples. As indicated by the values of the correlation lengths for the set of studied samples, the casein aggregation is strongly affected by simple sugar additions and it is enhanced by emulsion droplets hydrophobic interaction. As will be presented, these nanoscale structural results provided by scattering experiments is consistent with macroscopic results obtained from several techniques, providing a new understanding of NaCas emulsions. (author)

  1. The role of casein in supporting the operation of surface bound kinesin

    Directory of Open Access Journals (Sweden)

    Hancock William O

    2008-10-01

    Full Text Available Abstract Microtubules and associated motor proteins such as kinesin are envisioned for applications such as bioseparation and molecular sorting to powering hybrid synthetic mechanical devices. One of the challenges in realizing such systems is retaining motor functionality on device surfaces. Kinesin motors adsorbed onto glass surfaces lose their functionality or ability to interact with microtubules if not adsorbed with other supporting proteins. Casein, a milk protein, is commonly used in microtubule motility assays to preserve kinesin functionality. However, the mechanism responsible for this preservation of motor function is unknown. To study casein and kinesin interaction, a series of microtubule motility assays were performed where whole milk casein, or its αs1 and αs2, β or κ subunits, were introduced or omitted at various steps of the motility assay. In addition, a series of epifluorescence and total internal reflection microscopy (TIRF experiments were conducted where fluorescently labeled casein was introduced at various steps of the motility assay to assess casein-casein and casein-glass binding dynamics. From these experiments it is concluded that casein forms a bi-layer which supports the operation of kinesin. The first tightly bound layer of casein mainly performs the function of anchoring the kinesin while the second more loosely bound layer of casein positions the head domain of the kinesin to more optimally interact with microtubules. Studies on individual casein subunits indicate that β casein was most effective in supporting kinesin functionality while κ casein was found to be least effective.

  2. Modulation of mice fecal microbiota by administration of casein glycomacropeptide

    Directory of Open Access Journals (Sweden)

    Qingsen Chen

    2012-12-01

    Full Text Available Casein glycomacropeptide (GMP is known to promote the in vitro growth of Bifidobacteria and Lactobacilli. In this paper, we used conven- tional culture techniques and fluorescent in situ hybridization (FISH techniques to investigate the effect of casein GMP on mice fecal microbiota. The population structure of the intestinal microbiota, including Lactobacillus, Bifidobacteria, Enterococcus, coliforms and Enterobacteriaceae, was tested and compared. After consecutive administration of casein GMP for 15 days, numbers of Lactobacillus and Bifidobacteria increased significantly (P<0.01, numbers of Enterobacteriaceae and Coliforms decreased significantly (P<0.05 while no significant changes were observed for Enterococcus. The detection limits of FISH technique were significantly lower (P<0.01 than the traditional culture method. These results suggested that consumption of casein GMP had a prebiotic effect on male BALB/c mice. Casein GMP helped establish a healthier intestinal microbiota. Additionally, FISH was proved to be a rapid and relatively low-cost detection method that can be used to further our understanding of human intestinal microbiota.

  3. A phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory mantle cell lymphoma

    NARCIS (Netherlands)

    F. Morschhauser; J.F. Seymour; H.C. Kluin-Nelemans (Hanneke); A. Grigg; M. Wolf; M. Pfreundschuh (Michael); H. Tilly (Herve); J. Raemaekers; M.B. van 't Veer (Mars); N. Milpied; G. Cartron; A. Pezzutto; A. Spencer; F. Reyes; M. Dreyling (Martin)

    2008-01-01

    textabstractBackground: Protein kinase C beta (PKCβ), a pivotal enzyme in B-cell signaling and survival, is overexpressed in most cases of mantle cell lymphoma (MCL). Activation of PI3K/AKT pathway is involved in pathogenesis of MCL. Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses

  4. A phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory mantle cell lymphoma

    NARCIS (Netherlands)

    Morschhauser, F.; Seymour, J. F.; Kluin-Nelemans, H. C.; Grigg, A.; Wolf, M.; Pfreundschuh, M.; Tilly, H.; Raemaekers, J.; van 't Veer, M. B.; Milpied, N.; Cartron, G.; Pezzutto, A.; Spencer, A.; Reyes, F.; Dreyling, M.

    2008-01-01

    Background: Protein kinase C beta (PKC beta), a pivotal enzyme in B-cell signaling and survival, is overexpressed in most cases of mantle cell lymphoma (MCL). Activation of PI3K/AKT pathway is involved in pathogenesis of MCL. Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signali

  5. Possible interaction of hippocampal nitric oxide and calcium/calmodulin-dependent protein kinase II on reversal of spatial memory impairment induced by morphine.

    Science.gov (United States)

    Farahmandfar, Maryam; Kadivar, Mehdi; Naghdi, Nasser

    2015-03-15

    The opioid system plays an important role in learning and memory by modulation of different molecules in the brain. The aim of the present study was to investigate the role of hippocampal nitric oxide and calcium/calmodulin-dependent protein kinase II (CaMKII) on the morphine-induced modulation of spatial memory consolidation in male rats. Spatial memory was assessed in Morris water maze task by a single training session of eight trials followed by a probe trial and visible test 24h later. Our data indicated that post-training administration of L-arginine, a nitric oxide precursor (6 and 9 µg/rat, intra-CA1) significantly decreased amnesia induced by morphine (10 mg/kg) in spatial memory consolidation. A reversal effect of L-arginine on morphine-induced amnesia prevented by KN-93 (N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl) phenyl]-N-[2-hydroxyethyl] methoxybenzenesulfnamide), CaMKII inhibitor, (10 nmol/0.5 µl/site). In addition, post-training injection of L-NAME, (NG-nitro-L-arginine methyl ester), a nitric oxide synthase (NOS) inhibitor (10 and 15 µg/rat) or KN-93 (10 nmol/0.5 µl/site) with lower dose of morphine (2.5 mg/kg), which did not induce amnesia by itself, caused inhibition of memory consolidation. We also showed that co-administration of L-arginine (9 µg/rat) and morphine (10 mg/kg) significantly increased CaMKII activity in the rat hippocampus. On the other hand, administration of L-NAME (10 µg/rat) led to a decrease in the haippocampal activity of CaMKII in morphine-treated (2.5mg/kg) animals. These results indicate that acute single exposure to morphine can modulate consolidation of spatial memory, which may be mediated by a hippocampal nitrergic system and CaMKII activity.

  6. Age-dependent targeting of protein phosphatase 1 to Ca2+/calmodulin-dependent protein kinase II by spinophilin in mouse striatum.

    Directory of Open Access Journals (Sweden)

    Anthony J Baucum

    Full Text Available Mechanisms underlying age-dependent changes of dendritic spines on striatal medium spiny neurons are poorly understood. Spinophilin is an F-actin- and protein phosphatase 1 (PP1-binding protein that targets PP1 to multiple downstream effectors to modulate dendritic spine morphology and function. We found that calcium/calmodulin-dependent protein kinase II (CaMKII directly and indirectly associates with N- and C-terminal domains of spinophilin, but F-actin can displace CaMKII from the N-terminal domain. Spinophilin co-localizes PP1 with CaMKII on the F-actin cytoskeleton in heterologous cells, and spinophilin co-localizes with synaptic CaMKII in neuronal cultures. Thr286 autophosphorylation enhances the binding of CaMKII to spinophilin in vitro and in vivo. Although there is no change in total levels of Thr286 autophosphorylation, maturation from postnatal day 21 into adulthood robustly enhances the levels of CaMKII that co-immunoprecipitate with spinophilin from mouse striatal extracts. Moreover, N- and C-terminal domain fragments of spinophilin bind more CaMKII from adult vs. postnatal day 21 striatal lysates. Total levels of other proteins that interact with C-terminal domains of spinophilin decrease during maturation, perhaps reducing competition for CaMKII binding to the C-terminal domain. In contrast, total levels of α-internexin and binding of α-internexin to the spinophilin N-terminal domain increases with maturation, perhaps bridging an indirect interaction with CaMKII. Moreover, there is an increase in the levels of myosin Va, α-internexin, spinophilin, and PP1 in striatal CaMKII immune complexes isolated from adult and aged mice compared to those from postnatal day 21. These changes in spinophilin/CaMKII interactomes may contribute to changes in striatal dendritic spine density, morphology, and function during normal postnatal maturation and aging.

  7. VEGFR tyrosine kinase inhibitor II (VRI) induced vascular insufficiency in zebrafish as a model for studying vascular toxicity and vascular preservation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Shang; Dang, Yuan Ye; Oi Lam Che, Ginny [State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao (China); Kwan, Yiu Wa [School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong (China); Chan, Shun Wan [State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong (China); Leung, George Pak Heng [Pharmacology and Pharmacy, Faculty of Medicine, The University of Hong Kong, Hong Kong (China); Lee, Simon Ming Yuen, E-mail: simonlee@umac.mo [State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao (China); Hoi, Maggie Pui Man, E-mail: maghoi@umac.mo [State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao (China)

    2014-11-01

    In ischemic disorders such as chronic wounds and myocardial ischemia, there is inadequate tissue perfusion due to vascular insufficiency. Besides, it has been observed that prolonged use of anti-angiogenic agents in cancer therapy produces cardiovascular toxicity caused by impaired vessel integrity and regeneration. In the present study, we used VEGFR tyrosine kinase inhibitor II (VRI) to chemically induce vascular insufficiency in zebrafish in vivo and human umbilical vein endothelial cells (HUVEC) in vitro to further study the mechanisms of vascular morphogenesis in these pathological conditions. We also explored the possibility of treating vascular insufficiency by enhancing vascular regeneration and repair with pharmacological intervention. We observed that pretreatment of VRI induced blood vessel loss in developing zebrafish by inhibiting angiogenesis and increasing endothelial cell apoptosis, accompanied by down-regulation of kdr, kdrl and flt-1 genes expression. The VRI-induced blood vessel loss in zebrafish could be restored by post-treatment of calycosin, a cardiovascular protective isoflavone. Similarly, VRI induced cytotoxicity and apoptosis in HUVEC which could be rescued by calycosin post-treatment. Further investigation of the underlying mechanisms showed that the PI3K/AKT/Bad cell survival pathway was a main contributor of the vascular regenerative effect of calycosin. These findings indicated that the cardiovascular toxicity in anti-angiogenic therapy was mainly caused by insufficient endothelial cell survival, suggesting its essential role in vascular integrity, repair and regeneration. In addition, we showed that VRI-induced blood vessel loss in zebrafish represented a simple and effective in vivo model for studying vascular insufficiency and evaluating cancer drug vascular toxicities. - Highlights: • In vivo VRI model • Rescue effects of calycosin • Calycosin EC survival pathways.

  8. Regulation of voltage-gated Ca(2+) currents by Ca(2+)/calmodulin-dependent protein kinase II in resting sensory neurons.

    Science.gov (United States)

    Kostic, Sandra; Pan, Bin; Guo, Yuan; Yu, Hongwei; Sapunar, Damir; Kwok, Wai-Meng; Hudmon, Andy; Wu, Hsiang-En; Hogan, Quinn H

    2014-09-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca(2+) channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca(2+) currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0μM) reduced depolarization-induced ICa by 16-30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total ICa. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent.

  9. Casein micelles: size distribution in milks from individual cows.

    Science.gov (United States)

    de Kruif, C G Kees; Huppertz, Thom

    2012-05-09

    The size distribution and protein composition of casein micelles in the milk of Holstein-Friesian cows was determined as a function of stage and number of lactations. Protein composition did not vary significantly between the milks of different cows or as a function of lactation stage. Differences in the size and polydispersity of the casein micelles were observed between the milks of different cows, but not as a function of stage of milking or stage of lactation and not even over successive lactations periods. Modal radii varied from 55 to 70 nm, whereas hydrodynamic radii at a scattering angle of 73° (Q² = 350 μm⁻²) varied from 77 to 115 nm and polydispersity varied from 0.27 to 0.41, in a log-normal distribution. Casein micelle size in the milks of individual cows was not correlated with age, milk production, or lactation stage of the cows or fat or protein content of the milk.

  10. Rheology and phase behavior of dense casein micelle dispersions

    Science.gov (United States)

    Bouchoux, A.; Debbou, B.; Gésan-Guiziou, G.; Famelart, M.-H.; Doublier, J.-L.; Cabane, B.

    2009-10-01

    Casein micelle dispersions have been concentrated through osmotic stress and examined through rheological experiments. In conditions where the casein micelles are separated from each other, i.e., below random-close packing, the dispersions have exactly the flow and dynamic properties of the polydisperse hard-sphere fluid, demonstrating that the micelles interact only through excluded volume effects in this regime. These interactions cause the viscosity and the elastic modulus to increase by three orders of magnitude approaching the concentration of random-close packing estimated at Cmax≈178 g/l. Above Cmax, the dispersions progressively turn into "gels" (i.e., soft solids) as C increases, with elastic moduli G' that are nearly frequency independent. In this second regime, the micelles deform and/or deswell as C increases, and the resistance to deformation results from the formation of bonds between micelles combined with the intrinsic mechanical resistance of the micelles. The variation in G' with C is then very similar to that observed with concentrated emulsions where the resistance to deformation originates from a set of membranes that separate the droplets. As in the case of emulsions, the G' values at high frequency are also nearly identical to the osmotic pressures required to compress the casein dispersions. The rheology of sodium caseinate dispersions in which the caseins are not structured into micelles is also reported. Such dispersions have the behavior of associative polymer solutions at all the concentrations investigated, further confirming the importance of structure in determining the rheological properties of casein micelle systems.

  11. Electrochemical immunosensor for the determination of β-casein

    Directory of Open Access Journals (Sweden)

    Judith Molinari

    2015-03-01

    Full Text Available An amperometric biosensor for the quantification of food allergens based on an inhibitory immunoassay is presented. As a proof of concept, the experimental conditions were optimized for the detection of β-casein in the 0-10 ppm range. Eight electro­che­mi­cal cells were integrated into a small-sized portable potentiostat controlled by a smart­phone via Bluetooth communication. The determination of β-casein in eight different samples can be measured with the electrochemical biosensor, which has the potential to be modified for the detection of multiple allergens.

  12. Horseradish peroxidase-catalyzed cross-linking of feruloylated arabinoxylans with β-casein

    NARCIS (Netherlands)

    Boeriu, C.G.; Oudgenoeg, G.; Spekking, W.T.J.; Berendsen, L.B.J.M.; Vancon, L.; Boumans, H.; Gruppen, H.; Berkel, W.J.H. van; Laane, C.; Voragen, A.G.J.

    2004-01-01

    Heterologous conjugates of wheat arabinoxylan and β-casein were prepared via enzymatic cross-linking, using sequential addition of the arabinoxylan to a mixture of β-casein, peroxidase, and hydrogen peroxide. The maximal formation of adducts between the β-casein and the feruloylated arabinoxylan was

  13. Indirect and direct determination of the casein content of milk by Kjeldahl nitrogen analysis: collaborative study.

    Science.gov (United States)

    Lynch, J M; Barbano, D M; Fleming, J R

    1998-01-01

    The classic method for determination of milk casein is based on precipitation of casein at pH 4.6. Precipitated milk casein is removed by filtration and the nitrogen content of either the precipitate (direct casein method) or filtrate (noncasein nitrogen; NCN) is determined by Kjeldahl analysis. For the indirect casein method, milk total nitrogen (TN; Method 991.20) is also determined and casein is calculated as TN minus NCN. Ten laboratories tested 9 pairs of blind duplicate raw milk materials with a casein range of 2.42-3.05% by both the direct and indirect casein methods. Statistical performance expressed in protein equivalents (nitrogen x 6.38) with invalid and outlier data removed was as follows: NCN method (wt%), mean = 0.762, sr = 0.010, sR = 0.016, repeatability relative standard deviation (RSDr) = 1.287%, reproducibility relative standard deviation (RSDr) = 2.146%; indirect casein method (wt%), mean = 2.585, repeatability = 0.015, reproducibility = 0.022, RSDr = 0.560%, RSDR = 0.841; direct casein method (wt%), mean = 2.575, sr = 0.015, sR = 0.025, RSDr = 0.597%, RSDR = 0.988%. Method performance was acceptable and comparable to similar Kjeldahl methods for determining nitrogen content of milk (Methods 991.20, 991.21, 991.22, 991.23). The direct casein, indirect casein, and noncasein nitrogen methods have been adopted by AOAC INTERNATIONAL.

  14. Phosphorylation of αS1-casein is regulated by different genes.

    Science.gov (United States)

    Bijl, E; van Valenberg, H J F; Huppertz, T; van Hooijdonk, A C M; Bovenhuis, H

    2014-11-01

    Casein phosphorylation is a posttranslational modification catalyzed by kinase enzymes that attach phosphate groups to specific AA in the protein sequence. This modification is one of the key factors responsible for the stabilization of calcium phosphate nanoclusters in casein micelles and for the internal structure of the casein micelles. α(S1)-Casein (α(s1)-CN) is of special interest because it constitutes up to 40% of the total casein fraction in milk, and it has 2 common phosphorylation states, with 8 (α(S1)-CN-8P) and 9 (α(S1)-CN-9P) phosphorylated serine residues. Factors affecting this variation in the degree of phosphorylation are not currently known. The objective of this research was to determine the genetic background of α(S1)-CN-8P and α(S1)-CN-9P. The genetic and phenotypic correlation between α(S1)-CN-8P and α(S1)-CN-9P was low (0.18 and 0.19, respectively). This low genetic correlation suggests a different genetic background. These differences were further investigated by means of a genome-wide association study, which showed that both α(S1)-CN-8P and α(S1)-CN-9P were affected by a region on Bos taurus autosome (BTA) 6, but only α(S1)-CN-8P was affected by a region on BTA11 that contains the gene that encodes for β-lactoglobulin (β-LG), and only α(S1)-CN-9P was affected by a region on BTA14 that contains the diacylglycerol acyltransferase 1 (DGAT1) gene. Estimated effects of β-LG protein genotypes showed that only α(S1)-CN-8P was associated with the β-LG A/B polymorphism (g.1772G>A and g.3054C>T); the AA genotype of β-LG was associated with a lower concentration of α(S1)-CN-8P (-0.32% wt/wt) than the BB genotype (+0.41% wt/wt). Estimated effects of DGAT1 K232A genotypes showed that only α(S1)-CN-9P was associated with the DGAT1 gene polymorphism; DGAT1 AA genotype was associated with a higher α(S1)-CN-9P concentration (+0.53% wt/wt) than the DGAT1 KK genotype (-0.44% wt/wt). The results give insight in phosphorylation of α(S1

  15. Protein kinase CK2 in health and disease: Protein kinase CK2: from structures to insights

    DEFF Research Database (Denmark)

    Niefind, K; Raaf, J; Issinger, Olaf-Georg

    2009-01-01

    Within the last decade, 40 crystal structures corresponding to protein kinase CK2 (former name 'casein kinase 2'), to its catalytic subunit CK2alpha and to its regulatory subunit CK2beta were published. Together they provide a valuable, yet by far not complete basis to rationalize the biochemical...... features of the enzyme, such as its constitutive activity, acidophilic substrate specificity, dual-cosubstrate specificity and its heterotetrameric quarternary structure. Comprehensive sets of structural superimpositions reveal that both CK2alpha and CK2beta are relatively rigid molecules. In CK2beta...... the critical region of CK2alpha recruitment is pre-formed in the unbound state. In CK2alpha the activation segment - a key element of protein kinase regulation - adapts invariably the typical conformation of the active enzymes. Recent structures of human CK2alpha revealed a surprising plasticity in the ATP...

  16. Casein Micelles: Size Distribution in Milks from Individual Cows

    NARCIS (Netherlands)

    de Kruif, C.G.; Huppertz, T.

    2012-01-01

    The size distribution and protein composition of casein micelles in the milk of Holstein-Friesian cows was determined as a function of stage and number of lactations. Protein composition did not vary significantly between the milks of different cows or as a function of lactation stage. Differences i

  17. Modification of Casein%酪素的改性

    Institute of Scientific and Technical Information of China (English)

    杨秦欢

    2013-01-01

    In this paper, the structure, characteristics, modification methods and application of casein are summarized. It is hoped that this can be helpful to related researchers.%  综述了酪素的结构、特性、改性方法和用途,希望能作为相关科技工作者的参考。

  18. Pressure-Aided Proteolysis of ß-Casein

    NARCIS (Netherlands)

    Bruins, M.E.; Creusot, N.P.; Gruppen, H.; Janssen, A.E.M.; Boom, R.M.

    2009-01-01

    ß-Casein, which is present in the form of micelles at atmospheric pressure, has been hydrolyzed during pressure treatment to improve the accessibility of the protein. Two proteolytic enzymes with different specificities were used. Trypsin was aimed at mainly hydrolyzing hydrophilic segments of ß-cas

  19. Modification of Casein by the Lipid Oxidation Product Malondialdehyde

    NARCIS (Netherlands)

    Adams, A.; Kimpe, de N.; Boekel, van T.

    2008-01-01

    The reaction of malondialdehyde with casein was studied in aqueous solution to evaluate the impact of this lipid oxidation product on food protein modification. By using multiresponse modeling, a kinetic model was developed for this reaction. The influence of temperature and pH on protein browning a

  20. Short communication: casein haplotype variability in sicilian dairy goat breeds.

    Science.gov (United States)

    Gigli, I; Maizon, D O; Riggio, V; Sardina, M T; Portolano, B

    2008-09-01

    In the Mediterranean region, goat milk production is an important economic activity. In the present study, 4 casein genes were genotyped in 5 Sicilian goat breeds to 1) identify casein haplotypes present in the Argentata dell'Etna, Girgentana, Messinese, Derivata di Siria, and Maltese goat breeds; and 2) describe the structure of the Sicilian goat breeds based on casein haplotypes and allele frequencies. In a sample of 540 dairy goats, 67 different haplotypes with frequency >or=0.01 and 27 with frequency >or=0.03 were observed. The most common CSN1S1-CSN2-CSN1S2-CSN3 haplotype for Derivata di Siria and Maltese was FCFB (0.17 and 0.22, respectively), whereas for Argentata dell'Etna, Girgentana and Messinese was ACAB (0.06, 0.23, and 0.10, respectively). According to the haplotype reconstruction, Argentata dell'Etna, Girgentana, and Messinese breeds presented the most favorable haplotype for cheese production, because the casein concentration in milk of these breeds might be greater than that in Derivata di Siria and Maltese breeds. Based on a cluster analysis, the breeds formed 2 main groups: Derivata di Siria, and Maltese in one group, and Argentata dell'Etna and Messinese in the other; the Girgentana breed was between these groups but closer to the latter.

  1. A dynamic model of interactions of Ca2+, calmodulin, and catalytic subunits of Ca2+/calmodulin-dependent protein kinase II.

    Directory of Open Access Journals (Sweden)

    Shirley Pepke

    2010-02-01

    Full Text Available During the acquisition of memories, influx of Ca2+ into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca2+influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity,including Ca2+/calmodulin-dependent protein kinase II (CaMKII, are regulated by calmodulin, a small protein that can bindup to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime,supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic

  2. Membrane actions of 1α,25(OH)2D3 are mediated by Ca(2+)/calmodulin-dependent protein kinase II in bone and cartilage cells.

    Science.gov (United States)

    Doroudi, Maryam; Plaisance, Marc C; Boyan, Barbara D; Schwartz, Zvi

    2015-01-01

    1α,25(OH)2D3 regulates osteoblasts and chondrocytes via its membrane-associated receptor, protein disulfide isomerase A3 (Pdia3) in caveolae. 1α,25(OH)2D3 binding to Pdia3 leads to phospholipase-A2 (PLA2)-activating protein (PLAA) activation, stimulating cytosolic PLA2 and resulting in prostaglandin E2 (PGE2) release and PKCα activation, subsequently stimulating differentiation. However, how PLAA transmits the signal to cPLA2 is unknown. Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) activation is required for PLA2 activation in vascular smooth muscle cells, suggesting a similar role in 1α,25(OH)2D3-dependent signaling. The aim of the present study is to evaluate the roles of CaM and CaMKII as mediators of 1α,25(OH)2D3-stimulated PLAA-dependent activation of cPLA2 and PKCα, and downstream biological effects. The results indicated that 1α,25(OH)2D3 and PLAA-peptide increased CaMKII activity within 9 min. Silencing Cav-1, Pdia3 or Plaa in osteoblasts suppressed this effect. Similarly, antibodies against Plaa or Pdia3 blocked 1α,25(OH)2D3-dependent CaMKII. Caveolae disruption abolished activation of CaMKII by 1α,25(OH)2D3 or PLAA. CaMKII-specific and CaM-specific inhibitors reduced cPLA2 and PKC activities, PGE2 release and osteoblast maturation markers in response to 1α,25(OH)2D3. Camk2a-silenced but not Camk2b-silenced osteoblasts showed comparable effects. Immunoprecipitation showed increased interaction of CaM and PLAA in response to 1α,25(OH)2D3. The results indicate that membrane actions of 1α,25(OH)2D3 via Pdia3 triggered the interaction between PLAA and CaM, leading to dissociation of CaM from caveolae, activation of CaMKII, and downstream PLA2 activation, and suggest that CaMKII plays a major role in membrane-mediated actions of 1α,25(OH)2D3.

  3. The Effect of Β-casein Nanoparticles on Bioavailability and Cellular Uptake of Platinum Complex as a Cancer Drug

    Directory of Open Access Journals (Sweden)

    M Razmi

    2013-12-01

    Full Text Available Abstract Background & aim: Due to the low solubility and high toxicity of drugs, treatment of cancers is problematic therefore, the encapsulation and targeted delivery of therapeutic effect is required. The aim of this study was to investigate the effect of nanoparticles on cellular uptake and bioavailability of beta-casein on platinum complexes as cancer drugs. Methods: In the present experimental study, the physicochemical properties of nanoparticles as drug carriers of beta-casein devices using dynamic light scattering (DLS and scanning electron microscopy (SEM were investigated. In order to evaluate the toxicity effects of platinum complexes, the colon cancer cells in the absence or presence of free platinum complex concentration and nanoparticle loaded with platinum complexes were incubated for 24 and 48 hours. LD50 Values (concentration of compound causing 50% mortality in the cells was determined using the MTT assay. Data were analyzed by ANOVA and post-hoc test. Results: At a concentration of 1 mg ml, beta-casein nanoparticle drug carriers were synthesized in the range of 100 to 300 µM. In addition, the mortality rate in cancer cells by the release of platinum complexes (without and with the capsule, were 70 and 26 in 24 hours, and 60 µM and 21 µM in 48 hours respectively, Conclusion: The study showed that the bioavailability of the encapsulated platinum complexes increases and new drug delivery system may be a good candidate for the treatment of cancer. Key words: Beta-casein, Pt (II Complex, Bioavalibility, Nanocarrier, Micelle

  4. On studying protein phosphorylation patterns using bottom-up LC-MS/MS: the case of human alpha-casein

    DEFF Research Database (Denmark)

    Kjeldsen, Frank; Savitski, Mikhail M; Nielsen, Michael L

    2007-01-01

    minerals to the new-born. Human alpha(s1)-casein has been reported to be much less phosphorylated than ruminant caseins, which may indicate a different function of caseins in humans. Revealing the phosphorylation pattern in human casein can thus shed light on its function. The current study found...

  5. Phosphatidylinositol 3-kinase in myogenesis.

    Science.gov (United States)

    Kaliman, P; Zorzano, A

    1997-08-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc.

  6. The increasing of enamel calcium level after casein phosphopeptideamorphous calcium phosphate covering

    Directory of Open Access Journals (Sweden)

    Widyasri Prananingrum

    2012-06-01

    Full Text Available Background: Caries process is characterized by the presence of demineralization. Demineralization is caused by organic acids as a result of carbohydrate substrate fermentation. Remineralization is a natural repair process for non-cavitated lesions. Remineralization occurs if there are Ca2+ and PO43- ions in sufficient quantities. Casein-amorphous calcium phosphate phosphopeptide (CPP-ACP is a paste material containing milk protein (casein, that actually contains minerals, such as calcium and phosphate. The casein ability to stabilize calcium phosphate and enhance mineral solubility and bioavailability confers upon CPP potential to be biological delivery vehicles for calcium and phosphate. Purpose: The aim of this study was to determine the calcium levels in tooth enamel after being covered with CPP-ACP 2 times a day for 3, 14 and 28 days. Methods: Sample were bovine incisors of 3 year old cows divided into 4 groups, namely group I as control group, group II, III and IV as treatment groups covered with CPP-ACP 2 times a day. All of those teeth were then immersed in artificial saliva. Group II was immersed for 3 days, while group III was immersed for 14 days, and group IV was immersed for 28 days. One drop of CPP-ACP was used to cover the entire labial surface of teeth. The measurement of the calcium levels was then conducted by using titration method. All data were analyzed by One- Way ANOVA test with 5% degree of confidence. Results: The results showed significant difference of the calcium levels in tooth enamel of those groups after covered with CPP-ACP 2 times a day for 3, 14 and 28 days (p = 0.001. There is also significant difference of the calcium levels in tooth enamel of those treatment groups and the control group (p = 0.001. Conclusion: The calcium levels of tooth enamel are increased after covered with CPP-ACP 2 times a day for 3, 14 and 28 days.Latar belakang: Proses terjadinya karies gigi ditandai oleh adanya demineralisasi

  7. From Sphingosine Kinase to Dihydroceramide Desaturase: A Structure-Activity Relationship (SAR) Study of the Enzyme Inhibitory and Anticancer Activity of 4-((4-(4-Chlorophenyl)thiazol-2-yl)amino)phenol (SKI-II).

    Science.gov (United States)

    Aurelio, Luigi; Scullino, Carmen V; Pitman, Melissa R; Sexton, Anna; Oliver, Victoria; Davies, Lorena; Rebello, Richard J; Furic, Luc; Creek, Darren J; Pitson, Stuart M; Flynn, Bernard L

    2016-02-11

    The sphingosine kinase (SK) inhibitor, SKI-II, has been employed extensively in biological investigations of the role of SK1 and SK2 in disease and has demonstrated impressive anticancer activity in vitro and in vivo. However, interpretations of results using this pharmacological agent are complicated by several factors: poor SK1/2 selectivity, additional activity as an inducer of SK1-degradation, and off-target effects, including its recently identified capacity to inhibit dihydroceramide desaturase-1 (Des1). In this study, we have delineated the structure-activity relationship (SAR) for these different targets and correlated them to that required for anticancer activity and determined that Des1 inhibition is primarily responsible for the antiproliferative effects of SKI-II and its analogues. In the course of these efforts, a series of novel SK1, SK2, and Des1 inhibitors have been generated, including compounds with significantly greater anticancer activity.

  8. Quantitative determination of casein genetic variants in goat milk: Application in Girgentana dairy goat breed.

    Science.gov (United States)

    Montalbano, Maria; Segreto, Roberta; Di Gerlando, Rosalia; Mastrangelo, Salvatore; Sardina, Maria Teresa

    2016-02-01

    The study was conducted to develop a high-performance liquid chromatographic (HPLC) method to quantify casein genetic variants (αs2-, β-, and κ-casein) in milk of homozygous individuals of Girgentana goat breed. For calibration experiments, pure genetic variants were extracted from individual milk samples of animals with known genotypes. The described HPLC approach was precise, accurate and highly suitable for quantification of goat casein genetic variants of homozygous individuals. The amount of each casein per allele was: αs2-casein A = 2.9 ± 0.8 g/L and F = 1.8 ± 0.4 g/L; β-casein C = 3.0 ± 0.8 g/L and C1 = 2.0 ± 0.7 g/L and κ-casein A = 1.6 ± 0.3 g/L and B = 1.1 ± 0.2 g/L. A good correlation was found between the quantities of αs2-casein genetic variants A and F, and β-casein C and C1 with other previously described method. The main important result was obtained for κ-casein because, till now, no data were available on quantification of single genetic variants for this protein.

  9. Digestibility of transglutaminase cross-linked caseinate versus native caseinate in an in vitro multicompartmental model simulating young child and adult gastrointestinal conditions

    NARCIS (Netherlands)

    Havenaar, R.; Jong, A. de; Koenen, M.E.; Bilsen, J. van; Janssen, A.M.; Labij, E.; Westerbeek, H.J.M.

    2013-01-01

    Aim of this study was to investigate the digestion of transglutaminase cross-linked caseinate (XLC) versus native caseinate (NC) in solution and in cheese spread under digestive conditions for adults and children mimicked in a gastrointestinal model. Samples were collected for gel electrophoresis an

  10. Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane.

    NARCIS (Netherlands)

    Bosch, R.R.; Bazuine, M.; Wake, M.M.; Span, P.N.; Olthaar, A.J.; Schurmann, A.; Maassen, J.A.; Hermus, A.R.M.M.; Willems, P.H.G.M.; Sweep, C.G.J.

    2003-01-01

    The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rat

  11. Characterization of the Casein/Keratin Self-Assembly Nanomicelles

    Directory of Open Access Journals (Sweden)

    Su Xiao-Zhou

    2014-01-01

    Full Text Available Complex nanomicelles were made from casein and keratin through electrostatic self-assembly and transglutaminase fixation that was proved to be harmless and green. The complex nanomicelles were characterized by dynamic light scattering, scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and steady-state florescence. The results show that the complex nanomcelles acquired at the neutral pH in the mass ratio of casein to keratin 4 : 1 exhibit an anomalous sphere shape with uniform size which the diameter is about 40–70 nm. The complex nanomicelles in solution possess excellent dilution and storage stability due to the fixation and their high ζ-potential (22.8 mV. The complex nanomicelles are relatively hydrophilic and have a good potential for industrial application.

  12. Geographic distribution of haplotype diversity at the bovine casein locus

    Directory of Open Access Journals (Sweden)

    Moazami-Goudarzi Katy

    2004-03-01

    Full Text Available Abstract The genetic diversity of the casein locus in cattle was studied on the basis of haplotype analysis. Consideration of recently described genetic variants of the casein genes which to date have not been the subject of diversity studies, allowed the identification of new haplotypes. Genotyping of 30 cattle breeds from four continents revealed a geographically associated distribution of haplotypes, mainly defined by frequencies of alleles at CSN1S1 and CSN3. The genetic diversity within taurine breeds in Europe was found to decrease significantly from the south to the north and from the east to the west. Such geographic patterns of cattle genetic variation at the casein locus may be a result of the domestication process of modern cattle as well as geographically differentiated natural or artificial selection. The comparison of African Bos taurus and Bos indicus breeds allowed the identification of several Bos indicus specific haplotypes (CSN1S1*C-CSN2*A2-CSN3*AI/CSN3*H that are not found in pure taurine breeds. The occurrence of such haplotypes in southern European breeds also suggests that an introgression of indicine genes into taurine breeds could have contributed to the distribution of the genetic variation observed.

  13. The cystic fibrosis transmembrane recruiter the alter ego of CFTR as a multi-kinase anchor.

    Science.gov (United States)

    Mehta, Anil

    2007-11-01

    This review focuses on a newly discovered interaction between protein kinases involved in cellular energetics, a process that may be disturbed in cystic fibrosis for unknown reasons. I propose a new model where kinase-mediated cellular transmission of energy provides mechanistic insight to a latent role of the cystic fibrosis transmembrane conductance regulator (CFTR). I suggest that CFTR acts as a multi-kinase recruiter to the apical epithelial membrane. My group finds that, in the cytosol, two protein kinases involved in cell energy homeostasis, nucleoside diphosphate kinase (NDPK) and AMP-activated kinase (AMPK), bind one another. Preliminary data suggest that both can also bind CFTR (function unclear). The disrupted role of this CFTR-kinase complex as 'membrane transmitter to the cell' is proposed as an alternative paradigm to the conventional ion transport mediated and CFTR/chloride-centric view of cystic fibrosis pathogenesis. Chloride remains important, but instead, chloride-induced control of the phosphohistidine content of one kinase component (NDPK, via a multi-kinase complex that also includes a third kinase, CK2; formerly casein kinase 2). I suggest that this complex provides the necessary near-equilibrium conditions needed for efficient transmission of phosphate energy to proteins controlling cellular energetics. Crucially, a new role for CFTR as a kinase controller is proposed with ionic concentration acting as a signal. The model posits a regulatory control relay for energy sensing involving a cascade of protein kinases bound to CFTR.

  14. Gluten- and casein-free diets for autistic spectrum disorder

    Science.gov (United States)

    Millward, Claire; Ferriter, Michael; Calver, Sarah J; Connell-Jones, Graham G

    2014-01-01

    Background It has been suggested that peptides from gluten and casein may have a role in the origins of autism and that the physiology and psychology of autism might be explained by excessive opioid activity linked to these peptides. Research has reported abnormal levels of peptides in the urine and cerebrospinal fluid of people with autism. Objectives To determine the efficacy of gluten and/or casein free diets as an intervention to improve behaviour, cognitive and social functioning in individuals with autism. Search methods The following electronic databases were searched: CENTRAL(The Cochrane Library Issue 2, 2007), MEDLINE (1966 to April 2007), PsycINFO (1971 to April 2007), EMBASE (1974 to April 2007), CINAHL (1982 to April 2007), ERIC (1965 to 2007), LILACS (1982 to April 2007), and the National Research register 2007 (Issue1). Review bibliographies were also examined to identify potential trials. Selection criteria All randomised controlled trials (RCT) involving programmes which eliminated gluten, casein or both gluten and casein from the diets of individuals diagnosed with an autistic spectrum disorder. Data collection and analysis Abstracts of studies identified in searches of electronic databases were assessed to determine inclusion by two independent authors The included trials did not share common outcome measures and therefore no meta-analysis was possible. Data are presented in narrative form. Main results Two small RCTs were identified (n = 35). No meta-analysis was possible. There were only three significant treatment effects in favour of the diet intervention: overall autistic traits, mean difference (MD) = −5.60 (95% CI −9.02 to −2.18), z = 3.21, p=0.001 (Knivsberg 2002) ; social isolation, MD = −3.20 (95% CI −5.20 to 1.20), z = 3.14, p = 0.002) and overall ability to communicate and interact, MD = 1.70 (95% CI 0.50 to 2.90), z = 2.77, p = 0.006) (Knivsberg 2003). In addition three outcomes showed no significant difference between the

  15. Tetrahydroindazoles as Interleukin-2 Inducible T-Cell Kinase Inhibitors. Part II. Second-Generation Analogues with Enhanced Potency, Selectivity, and Pharmacodynamic Modulation in Vivo.

    Science.gov (United States)

    Burch, Jason D; Barrett, Kathy; Chen, Yuan; DeVoss, Jason; Eigenbrot, Charles; Goldsmith, Richard; Ismaili, M Hicham A; Lau, Kevin; Lin, Zhonghua; Ortwine, Daniel F; Zarrin, Ali A; McEwan, Paul A; Barker, John J; Ellebrandt, Claire; Kordt, Daniel; Stein, Daniel B; Wang, Xiaolu; Chen, Yong; Hu, Baihua; Xu, Xiaofeng; Yuen, Po-Wai; Zhang, Yamin; Pei, Zhonghua

    2015-05-14

    The medicinal chemistry community has directed considerable efforts toward the discovery of selective inhibitors of interleukin-2 inducible T-cell kinase (ITK), given its role in T-cell signaling downstream of the T-cell receptor (TCR) and the implications of this target for inflammatory disorders such as asthma. We have previously disclosed a structure- and property-guided lead optimization effort which resulted in the discovery of a new series of tetrahydroindazole-containing selective ITK inhibitors. Herein we disclose further optimization of this series that resulted in further potency improvements, reduced off-target receptor binding liabilities, and reduced cytotoxicity. Specifically, we have identified a correlation between the basicity of solubilizing elements in the ITK inhibitors and off-target antiproliferative effects, which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized analogues were shown to reduce IL-2 and IL-13 production in vivo following oral or intraperitoneal dosing in mice.

  16. Synthesis and evaluation of the antiproliferative activity of novel pyrrolo[1,2-a]quinoxaline derivatives, potential inhibitors of Akt kinase. Part II.

    Science.gov (United States)

    Desplat, Vanessa; Moreau, Stephane; Gay, Aurore; Fabre, Solene Belisle; Thiolat, Denis; Massip, Stephane; Macky, Gregory; Godde, Frederic; Mossalayi, Djavad; Jarry, Christian; Guillon, Jean

    2010-04-01

    Attenuation of protein kinases by selective inhibitors is an extremely active field of activity in anticancer drug development. Therefore, Akt, a serine/threonine protein kinase, also known as protein kinase B (PKB), represents an attractive potential target for therapeutic intervention. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel inhibitors with various heterocycle scaffolds. Based on previous results obtained on the antiproliferative activities of new pyrrolo[1,2-a]quinoxalines, a novel series was designed and synthesized from various substituted phenyl-1H-pyrrole-2-carboxylic acid alkyl esters via a multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937, and HL60, and the breast cancer cell line MCF7. The first biological evaluation of our new substituted pyrrolo[1,2-a]quinoxalines showed antiproliferative activity against the tested cell lines. From a general SAR point of view, these preliminary biological results highlight the importance of substitution at the C-4 position of the pyrroloquinoxaline scaffold by a benzylpiperidinyl fluorobenzimidazole group, and also the need for a functionalization on the pyrrole ring.

  17. Characterization of casein and alpha lactalbumin of African elephant (Loxodonta africana) milk.

    Science.gov (United States)

    Madende, M; Osthoff, G; Patterton, H-G; Patterton, H E; Martin, P; Opperman, D J

    2015-12-01

    The current research reports partial characterization of the caseins and α-lactalbumin (α-LA) of the African elephant with proposed unique structure-function properties. Extensive research has been carried out to understand the structure of the casein micelles. Crystallographic structure elucidation of caseins and casein micelles is not possible. Consequently, several models have been developed in an effort to describe the casein micelle, specifically of cow milk. Here we report the characterization of African elephant milk caseins. The κ-caseins and β-caseins were investigated, and their relative ratio was found to be approximately 1:8.5, whereas α-caseins were not detected. The gene sequence of β-casein in the NCBI database was revisited, and a different sequence in the N-terminal region is proposed. Amino acid sequence alignment and hydropathy plots showed that the κ-casein of African elephant milk is similar to that of other mammals, whereas the β-casein is similar to the human protein, and displayed a section of unique AA composition and additional hydrophilic regions compared with bovine caseins. Elephant milk is destabilized by 62% alcohol, and it is speculated that the β-casein characteristics may allow maintenance of the colloidal nature of the casein micelle, a role that was previously only associated with κ-casein. The oligosaccharide content of milk was reported to be low in dairy animals but high in some other species such as humans and elephants. In the milk of the African elephant, lactose and oligosaccharides both occur at high levels. These levels are typically related to the content of α-LA in the mammary gland and thus point to a specialized carbohydrate synthesis, where the whey protein α-LA plays a role. We report the characterization of African elephant α-LA. Homology modeling of the α-LA showed that it is structurally similar to crystal structures of other mammalian species, which in turn may be an indication that its functional

  18. Characterization, subcellular localization and nuclear targeting of casein kinase 2 from Zea mays

    DEFF Research Database (Denmark)

    Peracchia, G; Jensen, A B; Culiáñez-Macià, F A

    1999-01-01

    by using in-frame fusions of the maize CK2alpha subunit to the reporter gene encoding beta-glucuronidase (GUS) which were assayed in transiently transformed onion epidermal cells. Analysis of chimeric constructs identified one region containing a nuclear localization signal (NLS) that is highly conserved...

  19. Assignment of casein kinase 2 alpha sequences to two different human chromosomes

    DEFF Research Database (Denmark)

    Boldyreff, B; Klett, C; Göttert, E;

    1992-01-01

    and one on 20p13. The existence of two separate chromosomal loci suggests that CK-2 alpha is a member of a gene family. Only the locus on chromosome 11 was confirmed by somatic cell hybrid analysis. The analysis was based on the presence of a CK-2-alpha-specific 20-kb fragment. However, the CK-2 alpha c...

  20. Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein

    OpenAIRE

    Homma, Miwako Kato; Li, Dongxia; Krebs, Edwin G.; Yuasa, Yasuhito; Homma, Yoshimi

    2002-01-01

    Mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in ...

  1. SOYBEAN AND CASEIN HYDROLYSATES INDUCE GRAPEVINE IMMUNE RESPONSES AND RESISTANCE AGAINST PLASMOPARA VITICOLA

    Directory of Open Access Journals (Sweden)

    Nihed eLachhab

    2014-12-01

    Full Text Available Plasmopara viticola, the causal agent of grapevine downy mildew, is one of the most devastating grape pathogen in Europe and North America. Although phytochemicals are used to control pathogen infections, the appearance of resistant strains and the concern for possible adverse effects on environment and human health are increasing the search for alternative strategies. In the present investigation, we successfully tested two protein hydrolysates from soybean (soy and casein (cas to trigger grapevine resistance against P. viticola. On Vitis vinifera cv. Marselan plants, the application of soy and cas reduced the infected leaf surface by 76 and 63%, as compared to the control, respectively. Since both hydrolysates might trigger the plant immunity, we investigated their ability to elicit grapevine defence responses. On grapevine cell suspensions, a different free cytosolic calcium signature was recorded for each hydrolysate, whereas a similar transient phosphorylation of two MAP kinases of 45 and 49 kDa was observed. These signalling events were followed by transcriptome reprogramming, including the up-regulation of defence genes encoding pathogenesis-related (PR proteins and the stilbene synthase enzyme responsible for the biosynthesis of resveratrol, the main grapevine phytoalexin. Liquid chromatography analyses confirmed the production of resveratrol and its dimer metabolites, δ- and ε-viniferins. Overall, soy effects were more pronounced as compared to the cas one. Both hydrolysates proved to act as elicitors to enhance grapevine immunity against pathogen attack.

  2. Stimulation of Leishmania tropica protein kinase CK2 activities by platelet-activating factor (PAF).

    Science.gov (United States)

    Dutra, Patricia M L; Vieira, Danielle P; Meyer-Fernandes, Jose R; Silva-Neto, Mario A C; Lopes, Angela H

    2009-09-01

    Leishmania tropica is one of the causative agents of cutaneous leishmaniasis. Platelet-activating factor (PAF) is a phospholipid mediator in diverse biological and pathophysiological processes. Here we show that PAF promoted a three-fold increase on ecto-protein kinase and a three-fold increase on the secreted kinase activity of L. tropica live promastigotes. When casein was added to the reaction medium, along with PAF, there was a four-fold increase on the ecto-kinase activity. When live L. tropica promastigotes were pre-incubated for 30 min in the presence of PAF-plus casein, a six-fold increase on the secreted kinase activity was observed. Also, a protein released from L. tropica promastigotes reacted with polyclonal antibodies for the mammalian CK2 alpha catalytic subunit. Furthermore, in vitro mouse macrophage infection by L. tropica was doubled when promastigotes were pre-treated for 2 h with PAF. Similar results were obtained when the interaction was performed in the presence of purified CK2 or casein. TBB and DRB, CK2 inhibitors, reversed PAF enhancement of macrophage infection by L. tropica. WEB 2086, a competitive PAF antagonist, reversed all PAF effects here described. This study shows for the first time that PAF promotes the activation of two isoforms of CK2, secreted and membrane-bound, correlating these activities to infection of mouse macrophages.

  3. Polymorphism of caprine milk alphas1-casein in relation to performance of four Polish goat breeds.

    Science.gov (United States)

    Barłowska, J; Litwińczuk, Z; Kedzierska-Matysek, M; Litwińczuk, A

    2007-01-01

    Polymorphism of goat milk alphas1-casein was determined and potential relations between genetic variants of this protein fraction and goat performance were evaluated. The investigations were performed on 598 goats assigned to of 4 breed groups (White improved 254 units, Coloured improved--124, White non-improved--146 and Coloured non-improved--74). For each goat, alphas1-casein polymorphism was determined in polyacrylamide gel by the PAGE-SDS method and percentage of milk alphas1-casein and gene frequency established. There was evaluated goat performance at successive lactations. In the goat population investigated, AA, AB, BB, AE, BE and EE alphas1-casein genotypes were identified. In all four breeds, alphas1-casein genotype EE clearly predominated (27.2-39.2%), recognized as "medium" and its share was higher in the groups of non-improved goats. It was conditioned by high frequency of gene E alphas1-casein (0.419-0.622). Generally, EE genotype percentage was higher in the non-improved goat groups. The improved goats, though, obtained higher productivity in each of the lactation studied. Analysis of relationships between alphas1-casein genetic variants and goats performance confirmed a significant influence on milk, protein and fat yields only in the Coloured improved goat group. There was revealed a more general tendency indicating a significant impact of "strong" alphas1-casein genotypes on a concentration of basic milk components, i.e. fat and protein, especially casein. In a group of goats producing milk of the highest casein content (over 2.4%) and protein (over 3.0%), the animals showing "strong" alphas1-casein variants dominated (85 and 70 %).

  4. A DIGITAL TOOL FOR CASEIN ISOLECTRIC POINT DETERMINATION

    Directory of Open Access Journals (Sweden)

    V. M.T. Trindade

    2015-08-01

    Full Text Available Introduction: Casein is a milk protein that has the same number of positive and negative charges in a pH around 4.7. This characteristic is called the isoelectric point (pI. The pI varies from protein to protein and it depends on the charges of the lateral chain of the constituent aminoacids. At this pH value the protein has its minimum solubility since the net charge is zero and the repulsion between molecules is decreased. Furthermore, the electrostatic interaction occurs between the protein molecules. Thus, they form clumps which tend to precipitate. On the other hand, when they are placed in a solution whose pH is above or below of its pI, the protein molecules have respectively a negative or positive net charge, with strong repulsion between themselves and great interaction with the solvent (water. Objective: This learning object presents a simulation of a laboratory practice for the determination of casein isoelectric point. Materials and Methods: Cartoons were planned in order to show the methodology procedures and biochemical fundamentals. Animations were developed with the aid of the Adobe ® Flash 8 software associated with logic programming. Results: The simulation consists of six steps that reproduce the activities performed in the laboratory. Among them, the user can observe different degrees of turbidity and / or precipitation of casein in solutions with different pH values, he can measure these values with paper indicator strips and then determine the pI of this protein. Conclusions: This learning object was tested by students of Biochemistry I, Pharmacy course since 2012/2. The navigation features, design, interaction, interactivity were considered excellent by 80% of students indicating that this object can be used as an interesting tool to assist the teaching and learning of basic biochemistry.  Available at:www.ufrgs.br/gcoeb/PontoIsoleletricoDaCaseina/PontoIsoleletricoDaCaseina.swf

  5. An antisense oligodeoxynucleotide targeted against the type II sub. beta. regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

    Energy Technology Data Exchange (ETDEWEB)

    Tortora, G.; Clair, T.; Cho-Chung, Y.S. (National Institutes of Health, Bethesda, MD (USA))

    1990-01-01

    The type II{sub {beta}} regulatory subunit of cAMP-dependent protein kinase (RII{sub {beta}}) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII{sub {beta}} antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII{sub {beta}} antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII{sub {beta}} protein. Exposure to RII{sub {beta}} sense, RI{sub {alpha}} and RII{sub {alpha}} antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII{sub {beta}} regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells.

  6. A DIGITAL TOOL FOR CASEIN ISOLECTRIC POINT DETERMINATION

    OpenAIRE

    V.M.T. Trindade; Santos, I. B.; Zanatta,G.; P. R. Arantes; Salbego, C. G.

    2015-01-01

    Introduction: Casein is a milk protein that has the same number of positive and negative charges in a pH around 4.7. This characteristic is called the isoelectric point (pI). The pI varies from protein to protein and it depends on the charges of the lateral chain of the constituent aminoacids. At this pH value the protein has its minimum solubility since the net charge is zero and the repulsion between molecules is decreased. Furthermore, the electrostatic interaction occurs between the prote...

  7. Monitoring the aggregation of single casein micelles using fluorescence microscopy

    DEFF Research Database (Denmark)

    Bomholt, Julie; Moth-Poulsen, Kasper; Harboe, Marianne

    2011-01-01

    The aggregation of casein micelles (CMs) induced by milk-clotting enzymes is a process of fundamental importance in the dairy industry for cheese production; however, it is not well characterized on the nanoscale. Here we enabled the monitoring of the kinetics of aggregation between single CMs (30......, suggesting significant differences in their composition or structure. Detailed size histograms of CMs during aggregation also revealed the presence of two distinct subpopulations with different growth amplitudes and kinetics. The dependence of these distinct nanoscale processes/parameters on aggregation...

  8. DNA polymorphism at the casein loci in sheep.

    Science.gov (United States)

    Di Gregorio, P; Rando, A; Pieragostini, E; Masina, P

    1991-01-01

    By using seven endonucleases and four bovine cDNA probes specific for alpha S1-, alpha S2-, beta-, and kappa-casein genes, nine restriction fragment length polymorphisms (RFLPs) have been found in the sheep orthologous DNA regions. In contrast to the low level of variation observed at the protein level, these DNA polymorphisms determine a high level of heterozygosity and, therefore, represent useful tools for genetic analyses since they can also be obtained without the need for gene expression. In fact, informative matings suggest that in sheep, as in cattle, the four loci are linked.

  9. Subunit and whole molecule specificity of the anti-bovine casein immune response in recent onset psychosis and schizophrenia

    NARCIS (Netherlands)

    Severance, E.G.; Dickerson, F.B.; Halling, M.; Krivogorsky, B.; Haile, L.; Yang, S.; Stallings, C.R.; Origoni, A.E.; Bossis, I.; Xiao, J.; Dupont, D.; Haasnoot, W.; Yolken, R.H.

    2010-01-01

    Previous studies show increased antibody levels to bovine casein in some individuals with schizophrenia. The immunogenicity of specific domains of bovine casein varies among people with milk sensitivities and thus could vary among different neuropsychiatric disorders. Using ELISAs and immunoblotting

  10. Lipoprotein(a) and dietary proteins: casein lowers lipoprotein(a) concentrations as compared with soy protein1-3

    DEFF Research Database (Denmark)

    Nilausen, Karin Johanne; Meinertz, H.

    1999-01-01

    Lipoprotein(a), plasma lipoproteins, dietary proteins, soy protein, casein, liquid-formula, coronary artery disease, men, Denmark......Lipoprotein(a), plasma lipoproteins, dietary proteins, soy protein, casein, liquid-formula, coronary artery disease, men, Denmark...

  11. Foams and surface rheological properties of b-casein, gliadin and glycinin

    NARCIS (Netherlands)

    Bos, M.A.; Dunnewind, B.; Vliet, van T.

    2003-01-01

    Interfacial rheological properties and their suitability for foam production and stability of two vegetable proteins were studied and compared to ß-casein. Proteins used ranged from flexible to rigid/globular in the order of ß-casein, gliadin and soy glycinin. Experiments were performed at pH 6.7. N

  12. Behaviour of casein micelles at conditions comparable to those in ice cream

    NARCIS (Netherlands)

    Jonkman, M.J.

    2000-01-01

    The physical properties of ice cream are mainly determined by the processing and the ingredients. Milk (powder) is one of the ingredients and ice cream thus contains casein, the major milk protein. A large proportion of casein in ice cream is present in the plasma phase of ice cream. Since the behav

  13. Foams and surface rheological properties of β-casein, gliadin and glycinin

    NARCIS (Netherlands)

    Bos, M.A.; Dunnewind, B.; Vliet, T. van

    2003-01-01

    Interfacial rheological properties and their suitability for foam production and stability of two vegetable proteins were studied and compared to β-casein. Proteins used ranged from flexible to rigid/globular in the order of β-casein, gliadin and soy glycinin. Experiments were performed at pH 6.7. N

  14. The association of low-molecular-weight hydrophobic compounds with native casein micelles in bovine milk.

    Science.gov (United States)

    Cheema, M; Mohan, M S; Campagna, S R; Jurat-Fuentes, J L; Harte, F M

    2015-08-01

    The agreed biological function of the casein micelles in milk is to carry minerals (calcium, magnesium, and phosphorus) from mother to young along with amino acids for growth and development. Recently, native and modified casein micelles were used as encapsulating and delivery agents for various hydrophobic low-molecular-weight probes. The ability of modified casein micelles to bind certain probes may derive from the binding affinity of native casein micelles. Hence, a study with milk from single cows was conducted to further elucidate the association of hydrophobic molecules into native casein micelles and further understand their biological function. Hydrophobic and hydrophilic extraction followed by ultraperformance liquid chromatography-high resolution mass spectrometry analysis were performed over protein fractions obtained from size exclusion fractionation of raw skim milk. Hydrophobic compounds, including phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, showed strong association exclusively to casein micelles as compared with whey proteins, whereas hydrophilic compounds did not display any preference for their association among milk proteins. Further analysis using liquid chromatography-tandem mass spectrometry detected 42 compounds associated solely with the casein-micelles fraction. Mass fragments in tandem mass spectrometry identified 4 of these compounds as phosphatidylcholine with fatty acid composition of 16:0/18:1, 14:0/16:0, 16:0/16:0, and 18:1/18:0. These results support that transporting low-molecular-weight hydrophobic molecules is also a biological function of the casein micelles in milk.

  15. Proteomic analysis and cross species comparison of casein fractions from the milk of dairy animals

    Science.gov (United States)

    Wang, Xiaxia; Zhao, Xiaowei; Huang, Dongwei; Pan, Xiaocheng; Qi, Yunxia; Yang, Yongxin; Zhao, Huiling; Cheng, Guanglong

    2017-01-01

    Casein micelles contribute to the physicochemical properties of milk and may also influence its functionality. At present, however, there is an incomplete understanding of the casein micelle associated proteins and its diversity among the milk obtained from different species. Therefore, milk samples were collected from seven dairy animals groups, casein fractions were prepared by ultracentrifugation and their constituent proteins were identified by liquid chromatography tandem mass spectrometry. A total of 193 distinct proteins were identified among all the casein micelle preparations. Protein interaction analysis indicated that caseins could interact with major whey proteins, including β-lactoglobulin, α-lactalbumin, lactoferrin, and serum albumin, and then whey proteins interacted with other proteins. Pathway analysis found that the peroxisome proliferator-activated receptor signaling pathway is shared among the studied animals. Additionally, galactose metabolism pathway is also found to be commonly involved for proteins derived from camel and horse milk. According to the similarity of casein micelle proteomes, two major sample clusters were classified into ruminant animals (Holstein and Jersey cows, buffaloes, yaks, and goats) and non-ruminants (camels and horses). Our results provide new insights into the protein profile associated with casein micelles and the functionality of the casein micelle from the studied animals. PMID:28240229

  16. Categorization of rheological scaling models for particle gels applied to casein gels

    NARCIS (Netherlands)

    Mellema, M.; Opheusden, van J.H.J.; Vliet, van T.

    2002-01-01

    Rennet-induced casein gels made from skim milk were studied rheologically. A scaling model or framework for describing the rheological behavior of gels is discussed and used for classification of the structure of casein gels. There are two main parameters in the model that describe the number of def

  17. Effect of boiling on the antidiabetic property of enzyme treated sheep milk casein

    Directory of Open Access Journals (Sweden)

    Farhath Jan

    2016-10-01

    Full Text Available Aim: Sheep milk-born bioactive peptides have been found to exhibit various physiological activities. The present work was designed with the aim to evaluate the effect of boiling on antidiabetic property of peptides derived from sheep milk caseinate on hydrolysis with three different proteases. Materials and Methods: In this investigation, casein prepared from raw and boiled sheep milk was hydrolyzed by three commercially available proteases (trypsin, pepsin, and chymotrypsin. These hydrolysates collected at different hydrolysis times (60, 120, 160, and 240 min were assayed for their antidiabetic activity. Results: Among the three different enzyme hydrolysates, casein treated with chymotrypsin shows the highest antidiabetic activity among other enzymes. While the antidiabetic effect of raw milk-derived casein hydrolysates always exceeds than effect shown by boiled milk casein hydrolysates. Conclusion: The result obtained hence shows that the effect of boiling on the properties of bioactive peptides released during different enzyme digestion depends largely on the enzymatic formulation used and treatment conditions. Chymotrypsin treatment of raw casein yields peptides with maximum antidiabetic activity as compared to pepsin and trypsin. Moreover, the peptides produced after enzymatic treatment of boiled casein show reduced antidiabetic properties. Therefore, enzymatically treated raw milk casein hydrolysates may be used as effective nutritional supplements for diabetic patients, as it causes a significant inhibition of α-amylase activity.

  18. Bioavailability of lysine for kittens in overheated casein is underestimated by the rat growth assay method.

    Science.gov (United States)

    Larsen, J A; Fascetti, A J; Calvert, C C; Rogers, Q R

    2010-10-01

    Growth assays were performed to determine lysine bioavailability for kittens and rats in untreated and heated casein; these values were compared with estimates obtained with an in vitro method. Body weight, food intake, nitrogen and dry matter digestibility, and plasma lysine were determined during an 80-day growth trial using kittens (n = 16). Body weight and food intake were determined during a 21-day growth trial using weanling rats (n = 80). The growth data showed bioavailable lysine to be 102.4% and 100.2% (for untreated casein) and 66.1% and 51.7% (for heated casein) for kittens and rats, respectively. There was no relationship between plasma lysine and dietary lysine concentrations for kittens. There were no significant differences in nitrogen or dry matter digestibility among diets for kittens. The chemically reactive lysine content of untreated casein was 99.6%, and of heated casein was 67.1%. Heat treatment of casein resulted in significantly decreased lysine bioavailability as estimated by all methods. For untreated casein, both growth assays showed good agreement with the in vitro method for available lysine. For heated casein, the rat growth assay significantly underestimated bioavailable lysine as determined in kittens while the in vitro method closely approximated this value for the cat.

  19. Orthokinetic flocculation of caseinate-stabilized emulsions : influence of calcium concentration, shear rate and protein content

    NARCIS (Netherlands)

    Schokker, E.P.; Dalgleish, D.G.

    2000-01-01

    Calcium-induced flocculation of caseinate-stabilized soybean oil-in- water emulsions in conditions of Couette flow was studied. A concentrated emulsion (20% oil, 0.5-2.0% sodium caseinate in 20 mM imidazole, pH 7) was diluted 20 times in buffer containing concentrations of CaCl2 between 9 and 17 mM

  20. Antimutagenic activity of casein against MNNG in the E. coli DNA repair host-mediated assay.

    NARCIS (Netherlands)

    Boekel, van M.A.J.S.; Goeptar, A.R.; Alink, G.M.

    1997-01-01

    The effect of caseinate and soy protein in the diet on the mutagenicity induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was assessed in-vivo and ex-vivo in the DNA-repair host-mediated assay and liquid suspension assay, respectively. Of the two proteins only casein showed a strong antimutagen

  1. Bovine Chymosin: A Computational Study of Recognition and Binding of Bovine κ-Casein

    DEFF Research Database (Denmark)

    Palmer, David S.; Christensen, Anders Uhrenholt; Sørensen, Jesper

    2010-01-01

    Bovine chymosin is an aspartic protease that selectively cleaves the milk protein κ-casein. The enzyme is widely used to promote milk clotting in cheese manufacturing. We have developed models of residues 97-112 of bovine κ-casein complexed with bovine chymosin, using ligand docking, conformational...

  2. Casein haplotypes and their association with milk production traits in the Finnish Ayrshire cattle.

    Science.gov (United States)

    Velmala, R; Vilkki, J; Elo, K; Mäki-Tanila, A

    1995-12-01

    Polymorphism of casein genes was studied in half-sib families of artificial insemination bulls of the Finnish Ayrshire dairy breed. Ten grandsires and 300 of their sons were genotyped for the following polymorphisms: alpha s1-casein (B, C), beta-casein (A1, A2), the microsatellite within the kappa-casein gene (ms5, ms4) and kappa-casein (A, B, E). Nine different combinations of these alleles, casein haplotypes, were found. Associations between casein haplotypes and milk production traits (milk and protein yield, fat and protein percentage and milking speed) were studied with ordinary least-squares analysis to find a direct effect of the haplotypes or an association within individual grandsire families using the granddaughter design. Estimated breeding values of sons were obtained from cow evaluation by animal model. No direct effect of the casein haplotypes on the traits was found. Within grandsire families, in one out of four families the chromosomal segment characterized by haplotype 3 (B-A2-ms4-A) was associated with an increase in milk yield (P < 0.01) and a decrease in fat percentage (P < 0.01) when contrasted with haplotype 8 (B-A1-ms4-E). The results provide evidence that in the Finnish Ayrshire breed at least one quantitative trait locus affecting the genetic variation in yields traits is segregating linked to either haplotype 3 (B-A2-ms4-A) or 8 (B-A1-ms4-E).

  3. Correlations between biochemical characteristics and foam-forming and -stabilizing ability of whey and casein hydrolysates

    NARCIS (Netherlands)

    Ven, van der C.; Bont, D.B.A.; Voragen, A.G.J.

    2002-01-01

    Whey protein and casein were hydrolyzed with 11 commercially available enzymes. Foam properties of 44 samples were measured and were related to biochemical properties of the hydrolysates using statistical data analysis. All casein hydrolysates formed high initial foam levels, whereas whey hydrolysat

  4. Dissolution and enzymatic hydrolysis of casein micelles studied by dynamic light scattering

    Institute of Scientific and Technical Information of China (English)

    LIU Rui; QI Wei; SU Rongxin; ZHANG Yubin; JIN Fengmin; HE Zhimin

    2007-01-01

    The effects of temperature,ionic strength,and enzymatic hydrolysis on the average hydrodynamic radius (Rh) of casein micelles in phosphate buffer were studied by using dynamic light scattering.The results showed that the average Rh value of casein mieelles decreased irreversibly during the heating,decreased with the increase of ionic strength in lower ionic strength solution (less than 0.05 tool/L),but opposite in higher ionic strength solution (above 0.1 tool/L).The Rh value of casein increased rapidly during the process of enzymatic hydrolysis,and the structural model of casein micelles in the enzymatic hydrolysis process was also proposed,i.e.the casein micelle changed from compact sphere into unfolded and regularly flocky peptides.

  5. Correlations between biochemical characteristics and foam-forming and -stabilizing ability of whey and casein hydrolysates.

    Science.gov (United States)

    van der Ven, Cornelly; Gruppen, Harry; de Bont, Dries B A; Voragen, Alphons G J

    2002-05-08

    Whey protein and casein were hydrolyzed with 11 commercially available enzymes. Foam properties of 44 samples were measured and were related to biochemical properties of the hydrolysates using statistical data analysis. All casein hydrolysates formed high initial foam levels, whereas whey hydrolysates differed in their foam-forming abilities. Regression analysis using the molecular weight distribution of whey hydrolysates as predictors showed that the hydrolysate fraction containing peptides of 3-5 kDa was most strongly related to foam formation. Foam stability of whey hydrolysates and of most casein hydrolysates was inferior to that of the intact proteins. The foam stability of casein hydrolysate foams was correlated to the molecular weight distribution of the hydrolysates; a high proportion of peptides >7 kDa, composed of both intact casein and high molecular weight peptides, was positively related to foam stability.

  6. Compound exocytosis of casein micelles in mammary epithelial cells.

    Science.gov (United States)

    Dylewski, D P; Keenan, T W

    1983-07-01

    Ultrastructure of lactating bovine and rat mammary epithelial cells was studied with emphasis on secretory vesicle interactions. In the apical zone of the cell, adjacent secretory vesicles formed ball and socket configurations at their points of apposition. Similar configurations were formed between plasma membrane and secretory vesicle membrane. These structures may be formed by the diffusion of water between vesicles with different osmotic potentials. Frequently, vesicular chains consisting of 10 or more linked secretory vesicles were observed. Prior to the exocytotic release of casein micelles, adjacent vesicles fused through fragmentation of the ball and socket membrane. These membrane fragments and the casein micelles appeared to be secreted into the alveolar lumen after passing from one vesicle into another and finally through a pore in the apical plasma membrane. Emptied vesicular chains appeared to collapse and fragmentation of their membrane was observed. Based on these observations, we suggest that most vesicular membrane does not directly contact or become incorporated into the plasma membrane during secretion of the nonfat phase of milk.

  7. Preparation of Ultra-High-Speed Casein Trade Mark Adhesive

    Institute of Scientific and Technical Information of China (English)

    Xiong-Zhi Wu; Yu-Jiao Li

    2005-01-01

    @@ 1Introduction In present trade mark adhesives for beer bottle labeling are mostly prepared based on starch[1] ,polyvinyl formal[2] and their derivants[3-5] ,but they can only be applied to the labeling speed of 20 000 bottle/h and their water proofness are not satisfactory, especially when the water temperature is 0℃ below. The application of metal foil requires that sealing and labeling must be finished at the same time, but different penetrating speed of water in metal foil and paper label causes different drying speed, and also, the labeling speed and need of iced beer are increasing now, so these changes result in more strict requirements for adhesive.In this paper the chelating casein adhesive was obtained with casein as main moldings, urea as dispersing agent, starch as auxiliary, and ZnSO4 as cross-linked agent. The effects of dispersing agent, cross-linked agent and starch on the performances of adhesive were investigated.

  8. A Milk Protein, Casein, as a Proliferation Promoting Factor in Prostate Cancer Cells

    Science.gov (United States)

    Park, Sung-Woo; Kim, Joo-Young; Kim, You-Sun; Lee, Sang Jin; Chung, Moon Kee

    2014-01-01

    Purpose Despite most epidemiologic studies reporting that an increase in milk intake affects the growth of prostate cancer, the results of experimental studies are not consistent. In this study, we investigated the proliferation of prostate cancer cells treated with casein, the main protein in milk. Materials and Methods Prostate cancer cells (LNCaP and PC3), lung cancer cells (A459), stomach cancer cells (SNU484), breast cancer cells (MCF7), immortalized human embryonic kidney cells (HEK293), and immortalized normal prostate cells (RWPE1) were treated with either 0.1 or 1 mg/mL of α-casein and total casein extracted from bovine milk. Treatments were carried out in serum-free media for 72 hours. The proliferation of each cell line was evaluated by an 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results α-Casein and total casein did not affect the proliferations of RWPE1, HEK293, A459, SNU484, MCF7, HEK293, or RWPE1 cells. However, PC3 cells treated with 1 mg/mL of α-casein and casein showed increased proliferation (228% and 166%, respectively), and the proliferation of LNCaP cells was also enhanced by 134% and 142%, respectively. The proliferation mechanism of α-casein in PC3 and LNCaP cells did not appear to be related to the induction of Insulin-like growth factor-1 (IGF-1), since the level of IGF-1 did not change upon the supplementation of casein. Conclusions The milk protein, casein, promotes the proliferation of prostate cancer cells such as PC3 and LNCaP. PMID:25237656

  9. Quantification of bio- and techno-functional peptides in tryptic bovine micellar casein and β-casein hydrolysates.

    Science.gov (United States)

    Holder, Aline; Thienel, Katharina; Klaiber, Iris; Pfannstiel, Jens; Weiss, Jochen; Hinrichs, Jörg

    2014-09-01

    Numerous peptides generated by triptic hydrolysis of micellar casein and β-casein from bovine milk have been reported to exhibit bio-functional or techno-functional activities. In this study, the focus was placed on the quantification of eleven potential functional peptides generated by enzymatic hydrolysis with trypsin. The identity of the target peptides was verified by LC-ESI-MS/MS and a reverse phase high performance liquid chromatography method (RP-HPLC) was established to quantify the peptides. The quantification was based on calibration curves drawn using synthesised purified peptides. The regression analysis comparing >30 standard measurements of each peptide standard showed a regression coefficient R(2)>0.9958. The inter-day repeatability of the quantification method was always within a relative error of 0.2-6.5%, while the relative error of the accuracy for reproducibility was in the range of 0.1-2.4%. The established method was successfully applied for the quantitative analysis of the eleven functional peptides in different dairy fractions generated by cross-flow ultrafiltration.

  10. Solution and film properties of sodium caseinate/glycerol and sodium caseinate/polyethylene glycol edible coating systems.

    Science.gov (United States)

    Siew, D C; Heilmann, C; Easteal, A J; Cooney, R P

    1999-08-01

    The aim of this study is to determine the effects of plasticizer hydrogen bonding capability and chain length on the molecular structure of sodium caseinate (NaCAS), in NaCAS/glycerol and NaCAS/polyethylene glycol 400 (PEG) systems. Both solution and film phases were investigated. Glycerol and PEG reduced the viscosity of aqueous NaCAS, with the latter having a greater effect. This was explained in terms of protein/plasticizer aggregate size and changes to the conformation of the caseinate chain. In the film phase, glycerol caused more pronounced changes to the film tensile strength compared with PEG. However, the effect of glycerol on film water vapor permeability was smaller. These observations are attributed to the differences in plasticizer size and hydrogen bonding strength that controls the protein-plasticizer and protein-protein interactions in the films. Glass transition calculations from the tensile strength data indicate that the distribution of bonding interactions is more homogeneous in NaCAS/PEG films than in NaCAS/glycerol films.

  11. Glucose-induced downregulation of angiotensin II and arginine vasopressin receptors in cultured rat aortic vascular smooth muscle cells. Role of protein kinase C.

    OpenAIRE

    Williams, B.; Tsai, P.; Schrier, R W

    1992-01-01

    Early diabetes mellitus is characterized by impaired responses to pressor hormones and pressor receptor downregulation. The present study examined the effect of elevated extracellular glucose concentrations on angiotensin II (AII) and arginine vasopressin (AVP) receptor kinetics in cultured rat vascular smooth muscle cells (VSMC). Scatchard analysis of [3H]AVP and 125I-AII binding to confluent VSMC showed that high glucose concentrations (20 mM) similarly depressed AVP and AII surface recepto...

  12. A novel method for separation of caseins from milk by phosphates precipitation.

    Science.gov (United States)

    Yen, Chon-Ho; Lin, Yin-Shen; Tu, Ching-Fu

    2015-01-01

    Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze-thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.

  13. Spectrofluoremetric and molecular docking study on the interaction of bisdemethoxycurcumin with bovine β-casein nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Mehranfar, Fahimeh [Department of Chemistry, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Bordbar, Abdol-Khalegh, E-mail: bordbar@chem.ui.ac.ir [Department of Chemistry, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Keyhanfar, Mehrnaz; Behbahani, Mandana [Faculty of Advanced Sciences and Technologies, Department of Biotechnology, University of Isfahan, Isfahan, 81746-73441 (Iran, Islamic Republic of)

    2013-11-15

    The interaction of bisdemethoxycurcumin (BDMC), as one of the main active component of turmeric (Curcuma longa L.), with bovine β-casein nanoparticle, as an efficient drug carrier system, was investigated using steady-state fluorescence spectroscopy and molecular docking calculations. Results of fluorescence quenching experiments, Forster energy transfer measurements and molecular docking calculations suggested that BDMC bind to the hydrophobic core of β-casein via formation of 3 hydrogen bonds and several vander Waals contacts that represented the encapsulation of BDMC in β-casein micelle nanoparticles. The binding parameters including number of substantive binding sites and the binding constants were evaluated by fluorescence quenching method. Additionally, the cytotoxicity of free BDMC and BDMC-β-casein complex in human breast cancer cell line MCF7 was evaluated in vitro. The study revealed the higher cytotoxic effects of encapsulated BDMC on MCF7 cells compared to equal dose of free BDMC. -- Highlights: • BDMC binds to the hydrophobic core of β-casein. • The effective encapsulation of BDMC in β-casein micelle nanoparticles was shown. • Enhanced cytotoxicity was observed for encapsulated BDMC in β-casein nanoparticles.

  14. Proteomic characterization of specific minor proteins in the human milk casein fraction.

    Science.gov (United States)

    Liao, Yalin; Alvarado, Rudy; Phinney, Brett; Lönnerdal, Bo

    2011-12-02

    Human milk contains many bioactive proteins that are likely to support the early development of the newborn. The aim of this study was to identify whether there are specific minor proteins associated with the human milk casein micelle prepared by the acid precipitation method. Protein identification was performed by liquid chromatography tandem mass spectrometry analysis. Eighty-two proteins were identified in the casein micelle, 18 of which are not present in their whey compartment. Thirty-two of these proteins specifically associated with the casein micelle have not previously been identified in human milk or colostrum. Proteins involved in immune function comprised the major part (28%) of total proteins, and another significant part is involved in metabolism/energy production (22%). Most of the proteins were of extracellular or cytoplasmic origin (accounting for 50 and 29%, respectively). This study indicates that various soluble proteins should be considered as part of the casein compartment, prepared by the acid precipitation method. The data provide new insight not only into the proteomic profile of the human milk casein micelle and its physiological significance, but also into the proper proportion of casein and casein-associated proteins to use in infant formula.

  15. Association of triclosan to casein proteins through solvent-mediated high-pressure homogenization.

    Science.gov (United States)

    Roach, A; Dunlap, J; Harte, F

    2009-03-01

    The association of triclosan (TCS), a widely used hydrophobic compound, to the bovine casein micelle is investigated in this study. The use of high-pressure homogenization (HPH) at 0, 100, 200, and 300 MPa was introduced as a method for the dissociation of casein micelles in a skim milk/ethanol solution (1: 1, v/v) in the presence of TCS at 20, 80, and 160 mg/L where ethanol evaporation served as the final step for TCS association to caseins. The majority of TCS (over 80%) was associated with the caseins regardless of initial TCS concentration or applied pressure. TCS association to caseins was enhanced by 30% with continued pressurization to 300 MPa. Micellar dissociation and reassociation was found to be an irreversible process as evidenced by microscopy images. Pressurization to 300 MPa resulted in the formation of an integrated protein network of casein proteins and noncovalently linked whey proteins where the solubility of TCS was enhanced up to 40 times its reported water solubility at the highest initial TCS level of 160 mg/L. Reformed micelles exhibited Newtonian flow behavior at all pressure levels. This study provides evidence for the solubility enhancing quality of TCS through the solvent-mediated pressure/shear-induced dissociation of casein proteins.

  16. Nutritional evaluation of caseins and whey proteins and their hydrolysates from Protamex

    Institute of Scientific and Technical Information of China (English)

    SINDAYIKENGERA Séverin; XIA Wen-shui

    2006-01-01

    Whey protein concentrate (WPC 80) and sodium caseinate were hydrolyzed by Protamex to 5%, 10%, 15%, and 20% degree of hydrolysis (DH). WPC 80, sodium caseinate and their hydrolysates were then analyzed, compared and evaluated for their nutritional qualities. Their chemical composition, protein solubility, amino acid composition, essential amino acid index (EAA index), biological value (BV), nutritional index (NI), chemical score, enzymic protein efficiency ratio (E-PER) and in vitro protein digestibility (IVPD) were determined. The results indicated that the enzymatic hydrolysis of WPC 80 and sodium caseinate by Protamex improved the solubility and IVPD of their hydrolysates. WPC 80, sodium caseinate and their hydrolysates were high-quality proteins and had a surplus of essential amino acids compared with the FAO/WHO/UNU (1985) reference standard.The nutritive value of WPC 80 and its hydrolysates was superior to that of sodium caseinate and its hydrolysates as indicated by some nutritional parameters such as the amino acid composition, chemical score, EAA index and predicted BV. However, the E-PER was lower for the WPC hydrolysates as compared to unhydrolyzed WPC 80 but sodium caseinate and its hydrolysates did not differ significantly. The nutritional qualities of WPC 80, sodium caseinate and their hydrolysates were good and make them appropriate for food formulations or as nutritional supplements.

  17. Nutritional evaluation of caseins and whey proteins and their hydrolysates from Protamex.

    Science.gov (United States)

    Sindayikengera, Séverin; Xia, Wen-shui

    2006-02-01

    Whey protein concentrate (WPC 80) and sodium caseinate were hydrolyzed by Protamex to 5%, 10%, 15%, and 20% degree of hydrolysis (DH). WPC 80, sodium caseinate and their hydrolysates were then analyzed, compared and evaluated for their nutritional qualities. Their chemical composition, protein solubility, amino acid composition, essential amino acid index (EAA index), biological value (BV), nutritional index (NI), chemical score, enzymic protein efficiency ratio (E-PER) and in vitro protein digestibility (IVPD) were determined. The results indicated that the enzymatic hydrolysis of WPC 80 and sodium caseinate by Protamex improved the solubility and IVPD of their hydrolysates. WPC 80, sodium caseinate and their hydrolysates were high-quality proteins and had a surplus of essential amino acids compared with the FAO/WHO/UNU (1985) reference standard. The nutritive value of WPC 80 and its hydrolysates was superior to that of sodium caseinate and its hydrolysates as indicated by some nutritional parameters such as the amino acid composition, chemical score, EAA index and predicted BV. However, the E-PER was lower for the WPC hydrolysates as compared to unhydrolyzed WPC 80 but sodium caseinate and its hydrolysates did not differ significantly. The nutritional qualities of WPC 80, sodium caseinate and their hydrolysates were good and make them appropriate for food formulations or as nutritional supplements.

  18. Mammalian Target of Rapamycin (mTOR) Tagging Promotes Dendritic Branch Variability through the Capture of Ca2+/Calmodulin-dependent Protein Kinase II α (CaMKIIα) mRNAs by the RNA-binding Protein HuD.

    Science.gov (United States)

    Sosanya, Natasha M; Cacheaux, Luisa P; Workman, Emily R; Niere, Farr; Perrone-Bizzozero, Nora I; Raab-Graham, Kimberly F

    2015-06-26

    The fate of a memory, whether stored or forgotten, is determined by the ability of an active or tagged synapse to undergo changes in synaptic efficacy requiring protein synthesis of plasticity-related proteins. A synapse can be tagged, but without the "capture" of plasticity-related proteins, it will not undergo long lasting forms of plasticity (synaptic tagging and capture hypothesis). What the "tag" is and how plasticity-related proteins are captured at tagged synapses are unknown. Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) is critical in learning and memory and is synthesized locally in neuronal dendrites. The mechanistic (mammalian) target of rapamycin (mTOR) is a protein kinase that increases CaMKIIα protein expression; however, the mechanism and site of dendritic expression are unknown. Herein, we show that mTOR activity mediates the branch-specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. mTOR inhibition decreased the dendritic levels of CaMKIIα protein and mRNA by shortening its poly(A) tail. Overexpression of the RNA-stabilizing protein HuD increased CaMKIIα protein levels and preserved its selective expression in one daughter branch over the other when mTOR was inhibited. Unexpectedly, deleting the third RNA recognition motif of HuD, the domain that binds the poly(A) tail, eliminated the branch-specific expression of CaMKIIα when mTOR was active. These results provide a model for one molecular mechanism that may underlie the synaptic tagging and capture hypothesis where mTOR is the tag, preventing deadenylation of CaMKIIα mRNA, whereas HuD captures and promotes its expression in a branch-specific manner.

  19. Mammalian Target of Rapamycin (mTOR) Tagging Promotes Dendritic Branch Variability through the Capture of Ca2+/Calmodulin-dependent Protein Kinase II α (CaMKIIα) mRNAs by the RNA-binding Protein HuD*

    Science.gov (United States)

    Sosanya, Natasha M.; Cacheaux, Luisa P.; Workman, Emily R.; Niere, Farr; Perrone-Bizzozero, Nora I.; Raab-Graham, Kimberly F.

    2015-01-01

    The fate of a memory, whether stored or forgotten, is determined by the ability of an active or tagged synapse to undergo changes in synaptic efficacy requiring protein synthesis of plasticity-related proteins. A synapse can be tagged, but without the “capture” of plasticity-related proteins, it will not undergo long lasting forms of plasticity (synaptic tagging and capture hypothesis). What the “tag” is and how plasticity-related proteins are captured at tagged synapses are unknown. Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) is critical in learning and memory and is synthesized locally in neuronal dendrites. The mechanistic (mammalian) target of rapamycin (mTOR) is a protein kinase that increases CaMKIIα protein expression; however, the mechanism and site of dendritic expression are unknown. Herein, we show that mTOR activity mediates the branch-specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. mTOR inhibition decreased the dendritic levels of CaMKIIα protein and mRNA by shortening its poly(A) tail. Overexpression of the RNA-stabilizing protein HuD increased CaMKIIα protein levels and preserved its selective expression in one daughter branch over the other when mTOR was inhibited. Unexpectedly, deleting the third RNA recognition motif of HuD, the domain that binds the poly(A) tail, eliminated the branch-specific expression of CaMKIIα when mTOR was active. These results provide a model for one molecular mechanism that may underlie the synaptic tagging and capture hypothesis where mTOR is the tag, preventing deadenylation of CaMKIIα mRNA, whereas HuD captures and promotes its expression in a branch-specific manner. PMID:25944900

  20. β-casein as a functional food component

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria

    Milk is a complex mixture of various components which include proteins, carbohydrates, lipids and minerals. Milk proteins can be divided into two classes: whey proteins (20%) and caseins (80% of total milk protein). As thermal treatment is a traditional method of food preservation, interactions...... which can improve stability and safety of food. Traditional methods of food preservation and sterilization require high temperature, which may result in undesirable changes such as change of color or texture. Development of novel food processing methods, which allow to create new milk-based products...... with novel textures, is highly valuable to the dairy industry. Understanding interactions that undergo during food processing is necessary for proper design of process. For example control of aggregation is required to achieve desired texture of the final product....

  1. Detecting β-Casein Variation in Bovine Milk

    Directory of Open Access Journals (Sweden)

    Anna Maria Caroli

    2016-01-01

    Full Text Available In bovine species, β-casein (β-CN is characterized by genetic polymorphism. The two most common protein variants are β-CN A2 (the original one and A1, differing from A2 for one amino acid substitution (Pro67 to His67. Several bioactive peptides affecting milk nutritional properties can originate from β-CN. Among them, β-casomorphin-7 (BCM7 ranging from amino acid 60 to 66 can be released more easily from β-CN variants carrying His67 (A1 type instead of Pro67 (A2 type. Nowadays, “A2 milk” is produced in different countries claiming its potential benefits in human health. The aim of this study was to further develop and apply an isoelectric focusing electrophoresis (IEF method to bulk and individual milk samples in order to improve its use for β-CN studies. We succeeded in identifying A2 milk samples correctly and quantifying the percentage of A2, A1, and B variants in bulk samples not derived from A2 milk as well as in individual milk samples. The method allows us to quantify the relative proportion of β-CN variants in whole milk without eliminating whey protein by acid or enzymatic precipitation of caseins. The aim of this study was also to study the different behavior of β-CN and β-lactoglobulin (β-LG in the presence of trichloroacetic acid (TCA. The higher sensitivity of β-CN to TCA allows quantifying β-CN variants after TCA fixation because β-LG is not visible. Monitoring β-CN variation in cattle breeds is important in order to maintain a certain balance between Pro67 and His67 in dairy products. Overall, the debate between A1 and A2 milk needs further investigation.

  2. Comparative screening of single nucleotide polymorphisms in β-casein and κ-casein gene in different livestock breeds of India.

    Science.gov (United States)

    Singh, Lakshya Veer; Jayakumar, S; Sharma, Anurodh; Gupta, Shishir Kumar; Dixit, S P; Gupta, Neelam; Gupta, S C

    2015-06-01

    The most polymorphic milk protein gene is β-casein; 13 protein variants are known in cattle. Milk protein genetic polymorphism has received considerable research interest in recent years because of possible associations between milk protein and economically important traits in livestock. The present study was undertaken to explore the genetic polymorphisms in exon 7 of β-casein and exon 4 of κ-casein genes in Arunachali yaks (Bos grunniens), Sahiwal (Bos indicus) cattle, malpura sheep (Ovis aries) and Gaddi goat (Capra hircus). Results of the study revealed presence of 11 SNP variants in all livestock species. Four SNPs were observed in Bos indicus; two SNPs in Bos grunniens; three SNPs in Ovis aries and three SNPs in Capra hircus. These variations are found to be synonymous in nature as these variations do not result in their corresponding amino acids. A total of five polymorphic sites have been described at the κ-casein (CSN3) locus in the Indian domestic Gaddi goat (Capra hircus) when compared with exotic goat (X60763) while sequence analysis of κ-casein gene in sheep showed three novel nucleotide changes in malpura sheep when compared with the exotic sheep (AY237637). These results highlight the importance of taking into consideration the CSN3 SNPs when performing selection for milk composition in dairy livestock breeds.

  3. Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1 promoter and its regulation by Sp1

    Directory of Open Access Journals (Sweden)

    Botella Luisa M

    2010-06-01

    Full Text Available Abstract Background Activin receptor-like kinase 1 (ALK1 is a Transforming Growth Factor-β (TGF-β receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (ACVRL1 give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of ACVRL1. Here, we have studied the different origins of ACVRL1 transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of ACVRL1. Results We have performed a 5'Rapid Amplification of cDNA Ends (5'RACE of ACVRL1 transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of ACVRL1 (-1,035/+210 was analyzed in silico, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, ACVRL1 promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of ACVRL1 transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of ACVRL1 in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in ACVRL1 transcription, whereas in vitro hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of ACVRL1. Conclusions Our results describe two new transcriptional start sites in ACVRL1 gene, and indicate that Sp1 is a key regulator of ACVRL1 transcription, providing new insights into

  4. Hydrolyzed casein reduces diet-induced obesity in male C57BL/6J mice

    DEFF Research Database (Denmark)

    Lillefosse, Haldis Haukås; Tastesen, Hanne Sørup; Du, Zhen-Yu;

    2013-01-01

    The digestion rate of dietary protein is a regulating factor for postprandial metabolism both in humans and animal models. However, few data exist about the habitual consumption of proteins with different digestion rates with regard to the development of body mass and diet-induced obesity. Here, we...... used a factorial ANOVA design to investigate the effects of protein form (intact vs. hydrolyzed casein) and protein level (16 vs. 32 energy percent protein) on body mass gain and adiposity in obesity-prone male C57BL/6J mice fed Western diets with 35 energy percent fat. Mice fed the hydrolyzed casein...... by hydrolyzed casein ingestion translated into decreased body and adipose tissue masses. We conclude that chronic consumption of extensively hydrolyzed casein reduces body mass gain and diet-induced obesity in male C57BL/6J mice....

  5. Spectroscopic Evaluation of Effects of Heat Treatments on the Structures and Emulsifying Properties of Caseins

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hao; WANG Peng-jie; LEI Xin-gen; YANG Hong-ju; ZHANG Lu-da; REN Fa-zheng; ZHENG Li-min

    2013-01-01

    The effects of heat treatment(heating temperature and pH) on the structures and emulsifying properties of caseins were systematically studied by spectroscopy.Heat treatment from 60 to 100 ℃ resulted in an increase in their fluorescence intensity,hydrodynamic diameter,turbidity and emulsifying activity index,but decreased the size polydispersity of caseins.In the pH range of 5.5 to 7.0,the fluorescence intensity,hydrodynamic diameter,turbidity and emulsifying properties decreased with increased heating pH,but the size polydispersity of caseins increased with increased pH.The relationship between the surface fluorescence intensity and emulsifying activity was also investigated,revealing a correlation coefficient of 0.90.These results suggested that heat treatment could be used to modify the structures and emulsifying properties of caseins by appropriately selecting heating conditions.

  6. Skim Milk, Whey, and Casein Increase Body Weight and Whey and Casein Increase the Plasma C-Peptide Concentration in Overweight Adolescents12

    DEFF Research Database (Denmark)

    Arnberg, Karina; Mølgaard, Christian; Michaelsen, Kim Fleischer

    2012-01-01

    insulin, and insulin secretion estimated as the plasma C-peptide concentration in overweight adolescents. Overweight adolescents (n = 203) aged 12–15 y with a BMI of 25.4 ± 2.3 kg/m2 (mean ± SD) were randomized to 1 L/d of skim milk, whey, casein, or water for 12 wk. All milk drinks contained 35 g protein....... Outcomes were BMI-for-age Z-scores (BAZs), waist circumference, plasma insulin, homeostatic model assessment, and plasma C-peptide. We found no change in BAZ in the pretest control and water groups, whereas it was greater at 12 wk in the skim milk, whey, and casein groups compared with baseline...... and with the water and pretest control groups. The plasma C-peptide concentration increased from baseline to wk 12 in the whey and casein groups and increments were greater than in the pretest control (P milk or water group. These data suggest...

  7. Electrochemical Sensing of Casein Based on the Interaction between Its Phosphate Groups and a Ruthenium(III) Complex.

    Science.gov (United States)

    Inaba, Iku; Kuramitz, Hideki; Sugawara, Kazuharu

    2016-01-01

    A reaction to casein, along with β-lactoglobulin, is a main cause of milk allergies, and also is a useful indicator of protein in allergic analyses. In the present study, a simple casein sensor was developed based on the interaction between a phosphate group of casein and electroactive [Ru(NH3)6](3+). We evaluated the voltammetric behavior of a casein-[Ru(NH3)6](3+) complex using a glassy carbon electrode. When the ruthenium(III) complex was combined with the phosphate groups of casein, the structure of the casein was changed. Since the hydrophobicity of casein was increased due to the binding, the casein was adsorbed onto the electrode. Furthermore, we modified an electrode with a ruthenium(III) ions/collagen film. When the sensor was applied to the detection of the casein contained in milk, the values coincided with those indicated by the manufacturer. Accordingly, this electrode could be a powerful sensor for the determination of casein in several foods.

  8. Effect of boiling on the antidiabetic property of enzyme treated sheep milk casein

    OpenAIRE

    Farhath Jan; Santosh Kumar; Richa Jha

    2016-01-01

    Aim: Sheep milk-born bioactive peptides have been found to exhibit various physiological activities. The present work was designed with the aim to evaluate the effect of boiling on antidiabetic property of peptides derived from sheep milk caseinate on hydrolysis with three different proteases. Materials and Methods: In this investigation, casein prepared from raw and boiled sheep milk was hydrolyzed by three commercially available proteases (trypsin, pepsin, and chymotrypsin). These hydrol...

  9. Systematic Characterization of the Immune Response to Gluten and Casein in Autism Spectrum Disorders. Revision

    Science.gov (United States)

    2013-08-01

    of the Immune Response to Gluten and Casein in Autism Spectrum Disorders PRINCIPAL INVESTIGATOR: Armin Alaedini, PhD CONTRACTING... Gluten and Casein in Autism Spectrum Disorders 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH 10-1-0887 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...heterogeneity within this spectrum and the lack of biomarkers to characterize disease phenotypes and to understand treatment outcome. Dietary gluten

  10. TTBK2: A Tau Protein Kinase beyond Tau Phosphorylation

    Directory of Open Access Journals (Sweden)

    Jung-Chi Liao

    2015-01-01

    Full Text Available Tau tubulin kinase 2 (TTBK2 is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1 kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions.

  11. Adsorption of hydrophobin/β-casein mixtures at the solid-liquid interface.

    Science.gov (United States)

    Tucker, I M; Petkov, J T; Penfold, J; Thomas, R K; Cox, A R; Hedges, N

    2016-09-15

    The adsorption behaviour of mixtures of the proteins β-casein and hydrophobin at the hydrophilic solid-liquid surface have been studied by neutron reflectivity. The results of measurements from sequential adsorption and co-adsorption from solution are contrasted. The adsorption properties of protein mixtures are important for a wide range of applications. Because of competing factors the adsorption behaviour of protein mixtures at interfaces is often difficult to predict. This is particularly true for mixtures containing hydrophobin as hydrophobin possesses some unusual surface properties. At β-casein concentrations ⩾0.1wt% β-casein largely displaces a pre-adsorbed layer of hydrophobin at the interface, similar to that observed in hydrophobin-surfactant mixtures. In the composition and concentration range studied here for the co-adsorption of β-casein-hydrophobin mixtures the adsorption is dominated by the β-casein adsorption. The results provide an important insight into how the competitive adsorption in protein mixtures of hydrophobin and β-casein can impact upon the modification of solid surface properties and the potential for a wide range of colloid stabilisation applications.

  12. Micellisation and immunoreactivities of dimeric beta-caseins.

    Science.gov (United States)

    Yousefi, Reza; Gaudin, Jean-Charles; Chobert, Jean-Marc; Pourpak, Zahra; Moin, Mostafa; Moosavi-Movahedi, Ali Akbar; Haertle, Thomas

    2009-12-01

    Bovine beta-casein (beta-CN) is a highly amphiphilic micellising phospho-protein showing chaperone-like activity in vitro. Recently, existence of multiple sequential epitopes on beta-CN polypeptide chain in both hydrophilic-polar (psi) and hydrophobic-apolar domains (phi) has been evidenced. In order to clarify specific contribution of polar and apolar domains in micellisation process and in shaping immunoreactivity of beta-CN, its dimeric/bi-amphiphilic "quasi palindromic" forms covalently connected by a disulfide bond linking either N-terminal (C4 beta-CND) or C-terminal domain (C208 beta-CND) were produced and studied. Depending on the C- or N-terminal position of inserted cysteine, each dimeric beta-CN contains one polar/apolar region at the centre and two external hydrophobic/hydrophilic ends. Consequently, such casein dimers have radically different polarities/hydrophobicities on their outside surfaces. Dynamic light scattering (DLS) measurements indicate that these dimeric casein molecules form micelles of different sizes depending on arrangement of polar fragments of the beta-CN mutants in their constrained dimers. Non-aggregated dimers have different hydrodynamic diameters that could be explained by their different geometries. Measurements of fluorescence showed more hydrophobic environment of Trp residues of C208 beta-CND, while in similar experimental conditions Trp residues of C4 beta-CND and native beta-CN were more exposed to the polar medium. Both fluorescence and DLS studies showed greater propensity for micellisation of the dimeric beta-CNs, suggesting that the factors inducing the formation of micelles are stronger in the bi-amphiphilic dimers. 1-anilino-naphthalene-8-sulfonate (ANS) binding studies showed different binding of ANS by these dimers as well as different exposition of ANS binding (hydrophobic) regions in the micellar states. The differences in fluorescence resonance energy transfer (FRET) profiles of C4 beta-CND and C208 beta-CND can

  13. Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jung-Hyun; Lee, Sangmi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Cho, Du-Hyong [Department of Neuroscience, School of Medicine, Konkuk University, Seoul 143-701 (Korea, Republic of); Park, Young Mi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Kang, Duk-Hee [Division of Nephrology, Department of Internal Medicine, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Jo, Inho, E-mail: inhojo@ewha.ac.kr [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of)

    2013-07-12

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

  14. [Effects of glycerol on the spectral properties of sodium caseinate].

    Science.gov (United States)

    Li, Yan; Chang, Fen-fen; Gao, Huan-yuan; Cao, Qing; Jin, Li-e

    2015-01-01

    Although the immigration of water molecule, and diffusion and traversing of oxygen can be prevented by the edible film prepared through sodium caseinate, which plays a good protection role for the food, the strong hydrophilicity makes its watertightness and mechanical properties become inferior. Because the toughness and water resistance of SC films can be enhanced by glycerol (G) as an additive, it is necessary to elucidate the interaction between G and SC through the spectral characteristics such as fluorescence spectra, infrared spectra and UV spectra. The results show that the fluorescence intensity of SC decreases due to the addition of G. The binding constant obtained by the double logarithmic regression curve analysis is 1. 127 x 10(3) L . mol-1 and the number of binding sites reaches 1. 161. It indicates that the weak chemical bond is primary between G and SC molecules; From IR the absorption peaks of SC are almost the same before and after adding G. However, there is a certain difference among their absorption intensities. It reveals that the secondary structure of SC is affected, β folding length decreases, α helix, random coil structure, β angle structure increases, and the intermolecular hydrogen bond is strengthened; From UV the peptide bond structure of SC is not changed after the addition of G, but the polymer with larger molecular weight, which is formed by non-covalent bond, makes the peak intensity decrease. The research gives the mode of G and SC from the molecular level.

  15. Casein/pectin nanocomplexes as potential oral delivery vehicles.

    Science.gov (United States)

    Luo, Yangchao; Pan, Kang; Zhong, Qixin

    2015-01-01

    Delivery systems prepared with natural biopolymers are of particular interests for applications in food, pharmaceutics and biomedicine. In this study, nanocomplex particles of sodium caseinate (NaCas) and pectin were fabricated and investigated as potential oral delivery vehicles. Nanocomplexes were prepared with three mass ratios of NaCas/pectin by acidification using glucono-δ-lactone and thermal treatment. NaCas/pectin at 1:1 mass ratio resulted in dispersions with the lowest turbidity and the smallest and most uniform nanocomplexes. Thermal treatment at 85 °C for 30 min facilitated the formation of stable, compact, and spherical nanocomplexes. Heating not only greatly increased the yield of nanocomplexes but also significantly improved the encapsulation capability of rutin studied as a model compound. Pectin in nanocomplexes delayed the hydrolysis of NaCas by pepsin at gastric conditions and enabled the controlled release of most rutin in simulated intestinal conditions. The nanocomplexes based on food-sourced biopolymers have promising features for oral delivery of nutrients and medicines.

  16. Investigating cold gelation properties of recombined highly concentrated micellar casein concentrate and cream for use in cheese making.

    Science.gov (United States)

    Lu, Y; McMahon, D J; Vollmer, A H

    2016-07-01

    Highly concentrated micellar casein concentrate (HC-MCC), a potential ingredient for cheese making, contains ~20% casein with ~70% of serum proteins removed by microfiltration and diafiltration of skim milk, followed by vacuum evaporation. Our objective was to investigate cold gelation properties of recombined concentrated milk (RCM) by mixing thawed frozen HC-MCC and cream under different casein levels, pH, and protein-to-fat ratios, and with addition of sodium citrate or calcium. The HC-MCC was recombined with cream using low shear at 50°C for 30 min, and rheological measurements were conducted. Cold-gelling temperature [the temperature at which storage modulus (G')=loss modulus (G″)] was linearly correlated with casein levels from 8.6 to 11.5% (R(2)=0.71), pH from 6.6 to 7.0 (R(2)=0.96), and addition of sodium citrate from 0 to 0.36mmol/g of casein (R(2)=0.80). At pH 7.0, gelation occurred at 12, 26, and 38°C with 9, 10, and 11% casein, respectively. At pH 6.6, 6.8, and 7.0, RCM with 12% casein gelled at a mean temperature of 12, 26, and 37°C, respectively. Adding calcium chloride at 0.17mmol/g of casein significantly increased cold-gelling temperature from 18 to ≥50°C, whereas no significant change was observed at levels up to 0.12mmol/g of casein. Different protein to fat ratios ranging from 0.8 to 1.2 did not significantly influence gelling temperature. In transmission electron micrographs of RCM with 12% casein, casein micelles were nonspherical and partially dissociated into small protein strands. Upon addition of calcium chloride at 0.21mmol/g of casein, casein micelles were more spherical and retained colloidal structure with the presence of aggregated casein micelles. These gelation processes of RCM with or without addition of trisodium citrate were both reversible. We propose that cold gelation of RCM occurs when protein strands that have been partially released from the casein micelles entangle, restrict their mobility, and form a fine

  17. Moleculer analysis of genotype kappa casein and composition of goat milk Etawah grade, Saanen and their crossbred

    OpenAIRE

    Yayu Zurriyati; R. R. Noor; R. R. A. Maheswari

    2011-01-01

    Polymorphism of goat casein gene closely linked to the quality of milk protein. κ-casein is one of the casein fractions that influence the shape and stability of grain milk. This study is aimed to identify the variation of genotype κ-casein and related with milk quality from Etawah grade, Saanen and their crossbreed (PE-SA). The number of dairy goats used in this study was 150 animals consisted of Etawah grade (48 animals), Saanen (51 animals) and PE-SA (51 animals). Steps of experiment were:...

  18. Effect of cross-linking on microstructure and physical performance of casein protein.

    Science.gov (United States)

    Ghosh, Arun; Ali, M Azam; Dias, George J

    2009-07-13

    The development of advanced materials from biorenewable protein biopolymers requires the generation of more exogenous bonds to maintain the microstructure and durability in the final products. Casein is the main protein of milk, representing about 80% of the total protein. In the present investigation the casein protein was solubilized and/or emulsified in aqueous alkaline solutions, and 2D films and 3D matrices were produced. The effects of silane (3-aminopropyl triethoxy silane), DL-glyceraldehyde and glutaraldehyde on tensile properties and water swelling/absorption of 2D casein films and also the microstructure of the freeze-dried 3D matrices were analyzed. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that there were no significant changes in the molecular weight (19-23.9 kDa) of the casein proteins on exposure to alkaline solutions of sodium hydroxide and silane. The casein films produced without glycerol plasticizer and with heat treatment (130 °C for 18 h) were fragile. However, the fragile films were transformed into ductile and tough materials on exposure to moisture (i.e., conditioned for one week at 50 ± 2% relative humidity and 22 ± 2 °C) and showed a maximum average tensile strength of 49-52 MPa and modulus of 1107-1391 MPa. The chemical cross-linkers (i.e., DL-glyceraldehyde and glutaraldehyde) improved the microstructure of glycerol plasticized casein protein, when analyzed under scanning electron microscope (SEM). Furthermore, these chemical cross-linking agents enhanced the mechanical properties and water resistant properties of casein films.

  19. Characterization of the casein gene complex in West African goats and description of a new alpha(s1)-casein polymorphism.

    Science.gov (United States)

    Caroli, A; Chiatti, F; Chessa, S; Rignanese, D; Ibeagha-Awemu, E M; Erhardt, G

    2007-06-01

    The analysis of casein polymorphisms was carried out in West Africa goat populations: Red Sokoto (n = 57), West African Dwarf Nigeria (n = 27), West African Dwarf Cameroon (n = 39), and Borno (n = 37). The 4 casein genes alpha(s1) (CSN1S1), beta (CSN2), alpha(s2) (CSN1S2), and kappa (CSN3) were typed at the DNA level. No null alleles were found in any of the genes analyzed. A PCR single-strand conformation polymorphism method was implemented for the identification of CSN1S1*F allele simultaneously with A/0(1), B/E, N and the new allele. The allele differed from CSN1S1*B by a synonymous transversion TCG-->TCT in the codon corresponding to Ser(66) of the mature protein. The new allele, named CSN1S1*B', occurred at a high frequency in all the populations, ranging from 0.295 (West African Dwarf Cameroon) to 0.405 (Borno). A greater frequency was found for alleles associated with high alpha(s1)-casein quantity, as has already been observed in the goat populations from the Mediterranean area. The intermediate E allele occurred only in the Red Sokoto and at a low frequency. The faint F allele occurred in 3 populations at frequencies lower than 0.03. Linkage disequilibrium occurred in all the populations, with highly significant differences in Borno, Red Sokoto, and West Africa Dwarf Nigeria, and significant differences in West Africa Dwarf Cameroon. Only 10 haplotypes showed frequencies > or =0.05 in at least 1 of the 4 populations considered, and the overall frequency was >0.1 only for 4 haplotypes: BAAB, B'ACA, ACAB, and BACA (in the order CSN1S1-CSN2-CSN1S2-CSN3). Haplotype BAAB, postulated as an ancestral haplotype in previous studies, was the most common haplotype in all breeds except Borno, where B'ACA was predominant. The results obtained are of considerable significance given that very little information exists on the subject for African goats. The high frequency of strong alleles in the calcium-sensitive caseins as well as the high linkage disequilibrium found

  20. Crystal structure of pyridoxal kinase from the Escherichia coli pdxK gene: implications for the classification of pyridoxal kinases.

    Science.gov (United States)

    Safo, Martin K; Musayev, Faik N; di Salvo, Martino L; Hunt, Sharyn; Claude, Jean-Baptiste; Schirch, Verne

    2006-06-01

    The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-A resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4' substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4' of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.

  1. Skim milk, whey, and casein increase body weight and whey and casein increase the plasma C-peptide concentration in overweight adolescents.

    Science.gov (United States)

    Arnberg, Karina; Mølgaard, Christian; Michaelsen, Kim Fleischer; Jensen, Signe Marie; Trolle, Ellen; Larnkjær, Anni

    2012-12-01

    In adults, dietary protein seems to induce weight loss and dairy proteins may be insulinotropic. However, the effect of milk proteins in adolescents is unclear. The objective was to test whether milk and milk proteins reduce body weight, waist circumference, homeostatic model assessment, plasma insulin, and insulin secretion estimated as the plasma C-peptide concentration in overweight adolescents. Overweight adolescents (n = 203) aged 12-15 y with a BMI of 25.4 ± 2.3 kg/m(2) (mean ± SD) were randomized to 1 L/d of skim milk, whey, casein, or water for 12 wk. All milk drinks contained 35 g protein/L. Before randomization, a subgroup of adolescents (n = 32) was studied for 12 wk before the intervention began as a pretest control group. The effects of the milk-based test drinks were compared with baseline (wk 0), the water group, and the pretest control group. Diet and physical activity were registered. Outcomes were BMI-for-age Z-scores (BAZs), waist circumference, plasma insulin, homeostatic model assessment, and plasma C-peptide. We found no change in BAZ in the pretest control and water groups, whereas it was greater at 12 wk in the skim milk, whey, and casein groups compared with baseline and with the water and pretest control groups. The plasma C-peptide concentration increased from baseline to wk 12 in the whey and casein groups and increments were greater than in the pretest control (P milk or water group. These data suggest that high intakes of skim milk, whey, and casein increase BAZs in overweight adolescents and that whey and casein increase insulin secretion. Whether the effect on body weight is primary or secondary to the increased insulin secretion remains to be elucidated.

  2. Beta2-containing nicotinic acetylcholine receptors mediate calcium/calmodulin-dependent protein kinase-II and synapsin I protein levels in the nucleus accumbens after nicotine withdrawal in mice.

    Science.gov (United States)

    Jackson, Kia J; Imad Damaj, M

    2013-02-15

    Nicotinic acetylcholine receptors are calcium-permeable and the initial targets for nicotine. Studies suggest that calcium-dependent mechanisms mediate some behavioral responses to nicotine; however, the post-receptor calcium-dependent mechanisms associated with chronic nicotine and nicotine withdrawal remain unclear. The proteins calcium/calmodulin-dependent protein kinase II (CaMKII) and synapsin I are essential for neurotransmitter release and were shown to be involved in drug dependence. In the current study, using pharmacological techniques, we sought to (a) complement previously published behavioral findings from our lab indicating a role for calcium-dependent signaling in nicotine dependence and (b) expand on previously published acute biochemical and pharmacological findings indicating the relevance of calcium-dependent mechanisms in acute nicotine responses by evaluating the function of CaMKII and synapsin I after chronic nicotine and withdrawal in the nucleus accumbens, a brain region implicated in drug dependence. Male mice were chronically infused with nicotine for 14 days, and treated with the β2-selective antagonist dihydro-β-erythroidine (DHβE), or the α7 antagonist, methyllycaconitine citrate (MLA) 20min prior to dissection of the nucleus accumbens. Results show that phosphorylated and total CaMKII and synapsin I protein levels were significantly increased in the nucleus accumbens after chronic nicotine infusion, and reduced after treatment with DHβE, but not MLA. A spontaneous nicotine withdrawal assessment also revealed significant reductions in phosphorylated CaMKII and synapsin I levels 24h after cessation of nicotine treatment. Our findings suggest that post-receptor calcium-dependent mechanisms associated with nicotine withdrawal are mediated through β2-containing nicotinic receptors.

  3. Active Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII Regulates NMDA Receptor Mediated Postischemic Long-Term Potentiation (i-LTP by Promoting the Interaction between CaMKII and NMDA Receptors in Ischemia

    Directory of Open Access Journals (Sweden)

    Ning Wang

    2014-01-01

    Full Text Available Active calcium/calmodulin-dependent protein kinase II (CaMKII has been reported to take a critical role in the induction of long-term potentiation (LTP. Changes in CaMKII activity were detected in various ischemia models. It is tempting to know whether and how CaMKII takes a role in NMDA receptor (NMDAR-mediated postischemic long-term potentiation (NMDA i-LTP. Here, we monitored changes in NMDAR-mediated field excitatory postsynaptic potentials (NMDA fEPSPs at different time points following ischemia onset in vitro oxygen and glucose deprivation (OGD ischemia model. We found that 10 min OGD treatment induced significant i-LTP in NMDA fEPSPs, whereas shorter (3 min or longer (25 min OGD treatment failed to induce prominent NMDA i-LTP. CaMKII activity or CaMKII autophosphorylation displays a similar bifurcated trend at different time points following onset of ischemia both in vitro OGD or in vivo photothrombotic lesion (PT models, suggesting a correlation of increased CaMKII activity or CaMKII autophosphorylation with NMDA i-LTP. Disturbing the association between CaMKII and GluN2B subunit of NMDARs with short cell-permeable peptides Tat-GluN2B reversed NMDA i-LTP induced by OGD treatment. The results provide support to a notion that increased interaction between NMDAR and CaMKII following ischemia-induced increased CaMKII activity and autophosphorylation is essential for induction of NMDA i-LTP.

  4. Antioxidant, DNA interaction, VEGFR2 kinase, topoisomerase I and in vitro cytotoxic activities of heteroleptic copper(II) complexes of tetrazolo[1,5-a]pyrimidines and diimines.

    Science.gov (United States)

    Haleel, A; Mahendiran, D; Veena, V; Sakthivel, N; Rahiman, A Kalilur

    2016-11-01

    A series of heteroleptic mononuclear copper(II) complexes of the type [Cu(L(1-3))(diimine)]ClO4 (1-6) containing three tetrazolo[1,5-a]pyrimidine core ligands, ethyl 5-methyl-7-(2-hydroxyphenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(1)), ethyl 5-methyl-7-(4-diethylamino-2-hydroxyphenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(2)) or ethyl 5-methyl-7-(2-hydroxy-4-nitrophenyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (HL(3)), and two diimine coligands, 2,2'-bipyridyl (bpy) or 1,10-phenanthroline (phen) have been synthesized and characterized by spectral methods. The geometry of complexes have been determined with the help of electronic absorption and EPR splitting patterns, which suggest four coordinated square planar geometry around copper(II) ion. The lowering of HOMO-LUMO band gap value of complex 4 implies its higher biological activity compared to other complexes. Antioxidant studies revealed that the complexes possess considerable radical scavenging potency against DPPH. The binding studies of the complexes with calf thymus DNA (CT-DNA) revealed groove mode of binding, which was further supported by docking simulation. The complexes 3 and 4 strongly inhibit the topoisomerase I, and also strongly interact with VEGFR2 kinase receptor via π-π, σ-π and hydrogen bonding interaction. Gel electrophoresis experiments demonstrated the ability of the complexes to cleave plasmid DNA in the absence of activators. In vitro cytotoxic activities of the complexes were examined on three cancerous cell lines such as human lung (A549), cervical (HeLa) and colon (HCT-15), and two normal cells such as human embryonic kidney (HEK) and peripheral blood mononuclear cells (PBMCs). The live cell and fluorescent imaging of cancer cells were observed with acridine orange/ethidium bromide staining assay. All encouraging chemical and biological findings indicate that the complex 4 is a suitable candidate for drug target.

  5. Milk and growth in children: effects of whey and casein.

    Science.gov (United States)

    Mølgaard, Christian; Larnkjær, Anni; Arnberg, Karina; Michaelsen, Kim F

    2011-01-01

    Consumption of cow's milk is recommended in many countries. Observational and intervention studies show that cow's milk most likely has a positive influence on growth in children. The strongest evidence comes from observational studies and intervention studies in low-income countries, but there are also observational studies from high-income countries showing positive associations between milk intake and growth. Milk seems thus to have a specific stimulating effect on linear growth, not only in developing countries with high rates of malnutrition, but also in industrialized countries. However, it is not known which components in milk stimulate growth. Possible components are proteins, minerals, vitamins or combinations of these. Cow's milk proteins have a high protein quality, and whey has a slightly higher quality than casein, according to some indices based on amino acid composition. Studies, mainly from sport medicine, have suggested that whey protein also has the potential to increase muscle mass. Whether whey improves body composition to a larger extent than other milk proteins is not clear. The mechanism behind a possible growth-stimulating effect of milk and milk components is likely to be through a stimulation of insulin-like growth factor-I synthesis and maybe insulin secretion. In conclusion, there is strong evidence that milk stimulates linear growth. The mechanism is not yet clear, and more intervention studies are needed to understand which components in milk are responsible for the growth stimulation. The effects of milk on linear growth and adult height may have both positive and negative long-term implications.

  6. How to squeeze a sponge: casein micelles under osmotic stress, a SAXS study.

    Science.gov (United States)

    Bouchoux, Antoine; Gésan-Guiziou, Geneviève; Pérez, Javier; Cabane, Bernard

    2010-12-01

    By combining the osmotic stress technique with small-angle x-ray scattering measurements, we followed the structural response of the casein micelle to an overall increase in concentration. When the aqueous phase that separates the micelles is extracted, they behave as polydisperse repelling spheres and their internal structure is not affected. When they are compressed, the micelles lose water and shrink to a smaller volume. Our results indicate that this compression is nonaffine, i.e., some parts of the micelle collapse, whereas other parts resist deformation. We suggest that this behavior is consistent with a spongelike casein micelle having a triple hierarchical structure. The lowest level of the structure consists of the CaP nanoclusters that serve as anchors for the casein molecules. The intermediate level consists of 10- to 40-nm hard regions that resist compression and contain the nanoclusters. Those regions are connected and/or partially merged with each other, thus forming a continuous and porous material. The third level of structure is the casein micelle itself, with an average size of 100 nm. In our view, such a structure is consistent with the observation of 10- to 20-nm casein particles in the Golgi vesicles of lactating cells: upon aggregation, those particles would rearrange, fuse, and/or swell to form the spongelike micelle.

  7. The pressure-induced, lactose-dependent changes in the composition and size of casein micelles.

    Science.gov (United States)

    Wang, Pengjie; Jin, Shaoming; Guo, Huiyuan; Zhao, Liang; Ren, Fazheng

    2015-04-15

    The effects of lactose on the changes in the composition and size of casein micelles induced by high-pressure treatment and the related mechanism of action were investigated. Dispersions of ultracentrifuged casein micelle pellets with 0-10% (w/v) lactose were subjected to high pressure (400 MPa) at 20 °C for 40 min. The results indicated that the level of non-sedimentable caseins was positively related to the amount of lactose added prior to pressure treatment, and negatively correlated to the size. A mechanism for the pressure-induced, lactose-dependent changes in the casein micelles is proposed. Lactose inhibits the hydrophobic interactions between the micellar fragments during or after pressure release, through the hydrophilic layer formed by their hydrogen bonds around the micellar fragments. In addition, lactose does not favour the association between calcium and the casein aggregates after pressure release. Due to these two functions, lactose inhibited the formation of larger micelles after pressure treatment.

  8. Analysis of polymorphism in the bovine casein genes by use of the polymerase chain reaction.

    Science.gov (United States)

    Pinder, S J; Perry, B N; Skidmore, C J; Savva, D

    1991-01-01

    Methods have been devised for detecting polymorphisms in the bovine beta- and kappa-casein genes using the polymerase chain reaction (PCR) followed either by restriction enzyme digestion (to reveal a restriction fragment length polymorphism (RFLP] or by hybridization of an allele-specific oligonucleotide. These methods, as well as being faster and more sensitive than traditional RFLP methods, are of more general applicability since they can detect any change in DNA sequence. They require only a small sample of blood or semen and are applicable to animals of any age or sex. These methods make possible large-scale screening and thus selection for alleles at these loci. Typing of blood DNA can give erroneous results when the animal concerned is a twin; however, this can be overcome by retesting using milk or semen. Analysis of the kappa-casein genotype of Holstein-Friesian bulls gives frequencies for the A and B alleles of 0.80 and 0.20 respectively. Selection in favour of the B allele, which is superior for cheese production, could thus have a large effect. The A3 and B alleles at the beta-casein locus have been shown to be rare in the Holstein-Friesian population. Linkage disequilibrium exists between beta-casein B and kappa-casein B.

  9. Function of ram spermatozoa frozen in diluents supplemented with casein and vegetable oils.

    Science.gov (United States)

    Del Valle, I; Souter, A; Maxwell, W M C; Muiño-Blanco, T; Cebrián-Pérez, J A

    2013-05-01

    The aim of this study was to assess biologically safer components as alternatives to egg yolk for the frozen storage of ram semen using casein, coconut or palm oil in either Salamon's diluent (S) or a swim-up medium (SU). Ejaculates were frozen as pellets and sperm motility (subjectively) and acrosome integrity (FITC-PNA/PI) by flow cytometry were assessed at 0, 3 and 6h after thawing and incubation at 37°C. Three experiments were done: different concentrations of palm oil (5%, 10% and 20%); casein added as emulsifier and protective agent; and differences between egg yolk, coconut and palm oil in S and SU. 20% of oil added to SU accounted for a lesser percentage (Poil levels on viable cells. When casein was added to diluents containing 5% of palm oil, no differences were found between palm or casein (P>0.05). No differences were found when S and SU were compared neither as groups nor between S alone and containing coconut or palm oil; however, SU alone yielded less motility than SU 5% coconut. However, in both groups, S and SU, egg yolk accounted for the greatest values in both bases. These results indicate that none of biologically safer media components (casein, palm or coconut oil) used in this study maintained the function of ram spermatozoa after freeze-thawing better than S-containing egg yolk. The application of vegetable oils as substitutes for egg yolk in diluents for the cryopreservation of ram spermatozoa requires further research.

  10. The membrane-associated form of α(s1-casein interacts with cholesterol-rich detergent-resistant microdomains.

    Directory of Open Access Journals (Sweden)

    Annabelle Le Parc

    Full Text Available Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1-casein. These experiments reveal that the insolubility of α(s1-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells.

  11. Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

    OpenAIRE

    Marialuisa Moccia; Qingsong Liu; Teresa Guida; Giorgia Federico; Annalisa Brescia; Zheng Zhao; Hwan Geun Choi; Xianming Deng; Li Tan; Jinhua Wang; Marc Billaud; Gray, Nathanael S.; Francesca Carlomagno; Massimo Santoro

    2015-01-01

    Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-media...

  12. Effect of Fatty acids and beeswax addition on properties of sodium caseinate dispersions and films.

    Science.gov (United States)

    Fabra, M J; Jiménez, A; Atarés, L; Talens, P; Chiralt, A

    2009-06-08

    Edible films based on sodium caseinate and different saturated fatty acids, oleic acid, or beeswax were formulated. Film-forming emulsions were characterized in terms of particle size distribution, rheological behavior and surface tension. In order to evaluate the influence of lipids on sodium caseinate matrices, mechanical, optical, and water vapor barrier properties were studied, taking into account the effect of water content and film structure on such properties. Saturated fatty acids affected the film properties in a particular way due to the formation of bilayer structures which limited water vapor permeability, giving rise to nonflexible and more opaque films. Oleic acid and beeswax were less effective as water vapor barriers, although the former imparted more flexibility to the caseinate films and did not reduce the film transparency notably.

  13. First inactive conformation of CK2 alpha, the catalytic subunit of protein kinase CK2

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Issinger, Olaf-Georg; Niefind, Karsten

    2009-01-01

    (EPKs). To function as regulatory key components, EPKs normally exist in inactive ground states and are activated only upon specific signals. Typically, this activation is accompanied by large conformational changes in helix alpha C and in the activation segment, leading to a characteristic arrangement......The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2beta, noncatalytic subunit of CK2). CK2alpha belongs to the superfamily of eukaryotic protein kinases...... of catalytic key elements. For CK2alpha, however, no strict physiological control of activity is known. Accordingly, CK2alpha was found so far exclusively in the characteristic conformation of active EPKs, which is, in this case, additionally stabilized by a unique intramolecular contact between the N...

  14. Converting potent indeno[1,2-b]indole inhibitors of protein kinase CK2 into selective inhibitors of the breast cancer resistance protein ABCG2.

    Science.gov (United States)

    Jabor Gozzi, Gustavo; Bouaziz, Zouhair; Winter, Evelyn; Daflon-Yunes, Nathalia; Aichele, Dagmar; Nacereddine, Abdelhamid; Marminon, Christelle; Valdameri, Glaucio; Zeinyeh, Waël; Bollacke, Andre; Guillon, Jean; Lacoudre, Aline; Pinaud, Noël; Cadena, Silvia M; Jose, Joachim; Le Borgne, Marc; Di Pietro, Attilio

    2015-01-08

    A series of indeno[1,2-b]indole-9,10-dione derivatives were synthesized as human casein kinase II (CK2) inhibitors. The most potent inhibitors contained a N(5)-isopropyl substituent on the C-ring. The same series of compounds was found to also inhibit the breast cancer resistance protein ABCG2 but with totally different structure-activity relationships: a N(5)-phenethyl substituent was critical, and additional hydrophobic substituents at position 7 or 8 of the D-ring or a methoxy at phenethyl position ortho or meta also contributed to inhibition. The best ABCG2 inhibitors, such as 4c, 4h, 4i, 4j, and 4k, behaved as very weak inhibitors of CK2, whereas the most potent CK2 inhibitors, such as 4a, 4p, and 4e, displayed limited interaction with ABCG2. It was therefore possible to convert, through suitable substitutions of the indeno[1,2-b]indole-9,10-dione scaffold, potent CK2 inhibitors into selective ABCG2 inhibitors and vice versa. In addition, some of the best ABCG2 inhibitors, which displayed a very low cytotoxicity, thus giving a high therapeutic ratio, and appeared not to be transported, constitute promising candidates for further investigations.

  15. Chaperone-Like Activity of ß-Casein and Its Effect on Residual in Vitro Activity of Food Enzymes

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria

    Agaricus bisporus and equine cytochrome c. Only for the first target β-casein was acting as a molecular chaperone i.e. its presence resulted in higher residual activity (higher degree of the function preservation). β-Casein did not have any influence on the residual activity of tyrosinase. Surprisingly...

  16. Identification of casein as the major allergenic and antigenic protein of cow's milk.

    Science.gov (United States)

    Docena, G H; Fernandez, R; Chirdo, F G; Fossati, C A

    1996-06-01

    The objective of this study was to analyze both the allergenicity and immunogenicity of cow's milk proteins. To this end, 80 milk-atopic patients were selected on the basis of the presence of cow's milk-specific IgE antibodies in serum and compatible clinical history. Fifteen patients allergic to other allergens and 10 nonatopic subjects were studied as controls. The specificity of serum IgG and IgE antibodies was determined by immunoblotting, employing both cow's milk and milk components, i.e., alpha- and beta-casein, beta-lactoglobulin, and alpha-lactalbumin separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The experiments showed that casein-specific IgE antibodies were present in all (80/80) sera examined; 10/80 showed reactivity to beta-lactoglobulin, and 5/80 showed reactivity to alpha-lactalbumin. None of the 25 negative control sera analyzed showed the presence of specific IgE antibodies against milk proteins. These results were similar to those corresponding to the detection, by the radioallergosorbent test, of IgE antibodies against the milk components coupled to paper disks. All sera from milk-atopic patients also showed IgE reactivity against a high-molecular-mass fraction that hardly enters the gel. This fraction, after separation by gel filtration and treatment with beta-mercaptoethanol and urea, was shown by SDS-PAGE analysis to be formed by casein monomers. All sera analyzed by immunoblotting reacted against the components corresponding to casein monomers. Inhibition of immunoblotting by adsorption with different milk components confirmed that those high-molecular-mass aggregates are formed by casein components. The results presented here strongly suggest that casein is the major allergenic component of cow's milk.

  17. Complexation of sodium caseinate with gum tragacanth: Effect of various species and rheology of coacervates.

    Science.gov (United States)

    Ghorbani Gorji, Sara; Ghorbani Gorji, Elham; Mohammadifar, Mohammad Amin; Zargaraan, Azizollaah

    2014-06-01

    We investigated complex coacervation of sodium caseinate/Astragalus rahensis (A.r) as a function of pH with light scattering, spectrophotometry, and viscosity measurements. Interestingly, sodium caseinate/A.r displayed five structural transitions; pH 7.00 to pH ∼5.40: no interaction occurred, pH ∼5.40 to pH ∼4.80: initiation of the formation of primary soluble complexes, pH ∼4.80 to ∼4.30: formation of interpolymer complexes, pH ∼4.30 to ∼4.02: optimum coacervation and pH ∼4.02 to ∼2.50: suppression of coacervation. In addition, rheological properties of sodium caseinate/A.r coacervates were studied at various pH values. A much higher storage modulus (G') than loss modulus (G″) for all sodium caseinate/A.r coacervates suggests the formation of highly interconnected gel-like network structures with mainly elastic behaviour. Moreover, sodium caseinate/A.r coacervates at all pH values exhibited a shear thinning behaviour across the entire shear rate range investigated. Effects of different species of gum tragacanth on the interactions with sodium caseinate have been scarcely studied. Our study showed that systems containing various species (A.r, soluble fraction of A.r and Astragalus gossypinus (A.g)) had different critical pH values and particle sizes during complex coacervation, which could be due to different ratio of soluble to insoluble fractions and uronic acid content of various species.

  18. Analysis of beta-casein gene (CSN2 polymorphism in different breeds of cattle

    Directory of Open Access Journals (Sweden)

    Martina Miluchová

    2014-11-01

    Full Text Available Normal 0 21 false false false SK X-NONE X-NONE MicrosoftInternetExplorer4 The goal of work was identification of b - casein gene polymorphism in different breeds of cow. The beta - casein constitutes up to 45 % of the casein of bovine milk. The most common forms of beta-casein in dairy cattle breeds are A1 and A2, while B is less common. The b-casein A1 variant was associated with the incidence of diabetes mellitus 1st type, coronary heart disease and autism. The A2 variant reduces serum cholesterol. The material involved 287 cows (Simmental breed – 111 cows, Pinzgau breed – 89 cows, Holstein breed – 87 cows. Bovine genomic DNA was extracted from whole blood by using commercial kit and used in order to estimate b - casein genotypes by means of PCR-RFLP method. In the populations included in the study were detected all three genotypes – homozygote genotype A1A1, heterozygote genotype A1A2 and homozygote genotype A2A2 with frequencies 0.1261, 0.3333 and 0.5405 in Simmental breed; 0.1379, 0.4598 and 0.4023 in Holstein breed, 0.3034, 0.5168 and 0.1798 in Pinzgau breed. In population of Simmental breed and Holstein breed was higher frequency of allele A2 (0.7072 and 0.6322. In opposite, in population of Pinzgau breed was present higher frequency of the allele A1 (0.5618.

  19. Whey or Casein Hydrolysate with Carbohydrate for Metabolism and Performance in Cycling.

    Science.gov (United States)

    Oosthuyse, T; Carstens, M; Millen, A M E

    2015-07-01

    The protein type most suitable for ingestion during endurance exercise is undefined. This study compared co-ingestion of either 15 g/h whey or casein hydrolysate with 63 g/h fructose: maltodextrin (0.8:1) on exogenous carbohydrate oxidation, exercise metabolism and performance. 2 h postprandial, 8 male cyclists ingested either: carbohydrate-only, carbohydrate-whey hydrolysate, carbohydrate-casein hydrolysate or placebo-water in a crossover, double-blind design during 2 h of exercise at 60%W max followed by a 16-km time trial. Data were evaluated by magnitude-based inferential statistics. Exogenous carbohydrate oxidation, measured from (13)CO2 breath enrichment, was not substantially influenced by co-ingestion of either protein hydrolysate. However, only co-ingestion of carbohydrate-casein hydrolysate substantially decreased (98% very likely decrease) total carbohydrate oxidation (mean±SD, 242±44; 258±47; 277±33 g for carbohydrate-casein, carbohydrate-whey and carbohydrate-only, respectively) and substantially increased (93% likely increase) total fat oxidation (92±14; 83±27; 73±19 g) compared with carbohydrate-only. Furthermore, only carbohydrate-casein hydrolysate ingestion resulted in a faster time trial (-3.6%; 90% CI: ±3.2%) compared with placebo-water (95% likely benefit). However, neither protein hydrolysate enhanced time trial performance when compared with carbohydrate-only. Under the conditions of this study, ingesting carbohydrate-casein, but not carbohydrate-whey hydrolysate, favourably alters metabolism during prolonged moderate-strenuous cycling without substantially altering cycling performance compared with carbohydrate-only.

  20. Treatment of post-orthodontic white spot lesions with casein phosphopeptide-stabilised amorphous calcium phosphate

    DEFF Research Database (Denmark)

    Bröchner, Ann; Christensen, Carsten; Kristensen, Bjarne;

    2010-01-01

    This study aims to investigate the effect of topical applications of 10% casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on white spot lesions (WSL) detected after treatment with fixed orthodontic appliances. Sixty healthy adolescents with >/=1 clinically visible WSL at debonding were...... findings were largely reflected by the clinical scores. No side effects were reported. Topical treatment of white spot lesions after debonding of orthodontic appliances with a casein phosphopeptide-stabilised amorphous calcium phosphate agent resulted in significantly reduced fluorescence and a reduced...

  1. Casein addition to a whey-based formula has limited effects on gut function in preterm pigs

    DEFF Research Database (Denmark)

    Thymann, T.; Støy, Ann Cathrine Findal; Bering, S. B.

    2012-01-01

    followed by 30 h of enteral feeding with whey [protein fraction of milk formula based on whey (WHEY); n = 11] or casein and/or whey [protein fraction of milk formula based on a combination of casein and whey (CASEIN); n = 9]-based formulas. Sugar absorptive function was investigated at 6 and 30 h after......Preterm infants are susceptible to necrotizing enterocolitis (NEC). Using preterm pigs, we determined whether a whey–casein-based formula would be superior to a formula based on whey protein alone. Twenty cesarean-derived preterm pigs (92% gestation) were given total parenteral nutrition for 36 h...... studied in gut contents. Severity of NEC lesions was similar between diet groups but galactose absorption was markedly higher in CASEIN than in WHEY (P

  2. Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Ermakowa, I;

    2001-01-01

    subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit......The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic...... as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure....

  3. Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.

    Science.gov (United States)

    Li, Y S; Meng, X Y; Zhou, Y; Zhang, Y Y; Meng, X M; Yang, L; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Wang, X R

    2015-04-15

    In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb-β-casein-McAb-AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL(-1). The limit of detection (LOD) was 4.8ngmL(-1) which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6-7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R(2)=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen-antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples.

  4. Linoleic acid-induced expression of inducible nitric oxide synthase and cyclooxygenase II via p42/44 mitogen-activated protein kinase and nuclear factor-kappaB pathway in retinal pigment epithelial cells.

    Science.gov (United States)

    Fang, I-Mo; Yang, Chang-Hao; Yang, Chung-May; Chen, Muh-Shy

    2007-11-01

    High linoleic acid (LA) intake is known to correlate with age-related macular degeneration (AMD), but the molecular mechanisms remain unclear. This study was conducted to investigate the effects of LA on expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) and their associated signaling pathways in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were treated with different concentrations of LA. Expressions of iNOS and COX-2 were examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Concentrations of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in the culture medium were determined by enzyme-link immunosorbent assay (ELISA). Activation of p42/44, p38, JNK mitogen-activated protein kinase (MAPK) and nuclear factors (NF)-kappaB were evaluated by Western blot analysis and electrophoretic mobility shift assay (EMSA). We found that LA induced expression of iNOS and COX-2 in RPE cells at the mRNA and protein levels in a time-and dose-dependent manner. Upregulation of iNOS and COX-2 resulted in increased production of NO and PGE(2). Moreover, LA caused degradation of IkappaB and increased NF-kappaB DNA binding activity. Effects of LA-induced iNOS and COX-2 expression were inhibited by a NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). LA activated p42/44, but not p38 or JNK MAPK. Inhibition of p42/44 activity by PD98059 significantly reduced LA-induced activation of NF-kappaB. Linoleic acid-induced expression of iNOS and COX-2 as well as PGE(2) and NO release in RPE cells were sequentially mediated through activation of p42/p44, MAPK, then NF-kappaB. These results may provide new insights into both mechanisms of LA action on RPE cells and pathogenesis of age-related macular degeneration.

  5. The Ca²⁺-calmodulin-Ca²⁺/calmodulin-dependent protein kinase II signaling pathway is involved in oxidative stress-induced mitochondrial permeability transition and apoptosis in isolated rat hepatocytes.

    Science.gov (United States)

    Toledo, Flavia D; Pérez, Leonardo M; Basiglio, Cecilia L; Ochoa, Justina E; Sanchez Pozzi, Enrique J; Roma, Marcelo G

    2014-09-01

    Oxidative stress (OS) is a common event in most hepatopathies, leading to mitochondrial permeability transition pore (MPTP) formation and further exacerbation of both OS from mitochondrial origin and cell death. Intracellular Ca²⁺ increase plays a permissive role in these events, but the underlying mechanisms are poorly known. We examined in primary cultured rat hepatocytes whether the Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling pathway is involved in this process, by using tert-butyl hydroperoxide (tBOOH) as a pro-oxidant, model compound. tBOOH (500 μM, 15 min) induced MPTP formation, as assessed by measuring mitochondrial membrane depolarization as a surrogate marker, and increased lipid peroxidation in a cyclosporin A (CsA)-sensitive manner, revealing the involvement of MPTPs in tBOOH-induced radical oxygen species (ROS) formation. Intracellular Ca²⁺ sequestration with BAPTA/AM, CaM blockage with W7 or trifluoperazine, and CaMKII inhibition with KN-62 all fully prevented tBOOH-induced MPTP opening and reduced tBOOH-induced lipid peroxidation to a similar extent to CsA, suggesting that Ca²⁺/CaM/CaMKII signaling pathway fully mediates MPTP-mediated mitochondrial ROS generation. tBOOH-induced apoptosis, as shown by flow cytometry of annexin V/propidium iodide, mitochondrial release of cytochrome c, activation of caspase-3 and increase in the Bax-to-Bcl-xL ratio, and the Ca²⁺/CaM/CaMKII signaling antagonists fully prevented these effects. Intramitochondrial CaM and CaMKII were partially involved in tBOOH-induced MPTP formation, since W7 and KN-62 both attenuated the tBOOH-induced, MPTP-mediated swelling of isolated mitochondria. We concluded that Ca²⁺/CaM/CaMKII signaling pathway is a key mediator of OS-induced MPTP formation and the subsequent exacerbation of OS from mitochondrial origin and apoptotic cell death.

  6. Mitotic Activation of a Novel Histone Deacetylase 3-Linker Histone H1.3 Protein Complex by Protein Kinase CK2.

    Science.gov (United States)

    Patil, Hemangi; Wilks, Carrie; Gonzalez, Rhiannon W; Dhanireddy, Sudheer; Conrad-Webb, Heather; Bergel, Michael

    2016-02-12

    Histone deacetylase 3 (HDAC3) and linker histone H1 are involved in both chromatin compaction and the regulation of mitotic progression. However, the mechanisms by which HDAC3 and H1 regulate mitosis and the factors controlling HDAC3 and H1 activity during mitosis are unclear. Furthermore, as of now, no association between class I, II, or IV (non-sirtuin) HDACs and linker histones has been reported. Here we describe a novel HDAC3-H1.3 complex containing silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor 1 (N-CoR) that accumulated in synchronized HeLa cells in late G2 phase and mitosis. Nonetheless, the deacetylation activity by HDAC3 in the complex was evident only in mitotic complexes. HDAC3 associated with H1.3 was highly phosphorylated on Ser-424 only during mitosis. Isolation of inactive HDAC3-H1.3 complexes from late G2 phase cells, and phosphorylation of HDAC3 in the complexes at serine 424 by protein kinase CK2 (also known as casein kinase 2) activated the HDAC3 in vitro. In vivo, CK2α and CK2α' double knockdown cells demonstrated a significant decrease in HDAC3 Ser-424 phosphorylation during mitosis. HDAC3 and H1.3 co-localized in between the chromosomes, with polar microtubules and spindle poles during metaphase through telophase, and partially co-localized with chromatin during prophase and interphase. H1 has been reported previously to associate with microtubules and, therefore, could potentially function in targeting HDAC3 to the microtubules. We suggest that phosphorylation of HDAC3 in the complex by CK2 during mitosis activates the complex for a dual role: compaction of the mitotic chromatin and regulation of polar microtubules dynamic instability.

  7. Identification of four genomic loci highly related to casein-kinase-2-alpha cDNA and characterization of a casein kinase-2-alpha pseudogene within the mouse genome

    DEFF Research Database (Denmark)

    Boldyreff, B; Wehr, K; Hecht, R;

    1992-01-01

    positive clone was further analyzed by sequencing a 3.1 kb XbaI fragment. This clone displays the characteristics of a pseudogene, i.e. lack of introns and several nucleotide insertions and deletions. In its 3' region it contains a 91 bp large CT-rich stretch which consists of (CCTT) and (CT) repeats...

  8. Thymidine kinases in archaea

    DEFF Research Database (Denmark)

    Clausen, A.R.; Matakos, A.; Sandrini, Michael;

    2006-01-01

    Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea...... that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell....

  9. Stimulation of muscle protein synthesis by whey and caseinate ingestion after resistance exercise in elderly individuals

    DEFF Research Database (Denmark)

    Dideriksen, K J; Reitelseder, S; Petersen, S G;

    2011-01-01

    before exercise (CasPre), caseinate intake immediately after exercise (CasPost), whey intake immediately after exercise (Whey), or intake of a non-caloric control drink (Control). Muscle myofibrillar and collagen fractional synthesis rates (FSR) were measured by a primed continuous infusion of L-[1...... protein synthesis (MPS) to intakes of whey and caseinate after heavy resistance exercise in healthy elderly individuals, and, furthermore, to compare the timing effect of caseinate intake. Twenty-four elderly men and women (mean ± SEM; 68 ± 1 years) were randomized to one of four groups: caseinate intake......-(13) C]leucine using labeled proteins during a 6-h recovery period. No differences were observed in muscle myofibrillar and collagen FSR with Whey (0.09 ± 0.01%/h) compared with CasPost (0.09 ± 0.003%/h), and it did not differ between CasPre (0.10 ± 0.01%/h) and CasPost. MPS does not differ with whey...

  10. The balance between caseins and whey proteins in cow's milk determines its allergenicity.

    Science.gov (United States)

    Lara-Villoslada, F; Olivares, M; Xaus, J

    2005-05-01

    Cow's milk allergy is quite common in the first years of human life. Protein composition plays an important role in this pathology, particularly the casein/whey protein ratio. It is known that milks from different species have different sensitization capacities although their protein sources are quite similar. Thus, the objective of this work was to compare the allergenicity of native cow's milk and milk with a modified ratio of casein and whey proteins in a murine model of atopy. Twenty-four Balb/c mice were orally sensitized to native cow's milk or modified cow's milk with a casein/whey protein ratio of 40:60. During the sensitization period, the number of mice suffering from diarrhea was significantly higher in the native cow's milk-sensitized group than in the modified milk-sensitized group. Once mice were killed, plasma histamine levels were shown to be significantly higher in native cow's milk-sensitized mice. In addition, cow's milk proteins induced a higher lymphocyte sensitization in the native milk-sensitized mice, with a significant increase in the specific proliferation ratio of these cells. These results suggest that the balance between caseins and whey proteins plays an important role in the sensitization capacity of cow's milk, and its modification might be a way to reduce the allergenicity of cow's milk.

  11. Acid-induced gelation behavior of casein/whey protein solutions assessed by oscillatory rheology.

    Science.gov (United States)

    Sadeghi, Mahboubeh; Madadlou, Ashkan; Khosrowshahi, Asghar; Mohammadifar, Mohammadamin

    2014-09-01

    Gelation process of acid-induced casein gels was studied using response surface method (RSM). Ratio of casein to whey proteins, incubation and heating temperatures were independent variables. Final storage modulus (G') measured 200 min after the addition of glucono-δ-lactone and the gelation time i.e. the time at which G' of gels became greater than 1 Pa were the parameters studied. Incubation temperature strongly affected both parameters. The higher the incubation temperature, the lower was the G' and the shorter the gelation time. Increased heating temperature however, increased the G' but again shortened the gelation time. Increase in G' was attributed to the formation of disulphide cross-linkages between denatured whey proteins and casein chains; whilst the latter was legitimized by considering the higher isoelectric pH of whey proteins. Maximum response (G' = 268.93 Pa) was obtained at 2.7 % w/w, 25 °C and 90 °C for casein content, incubation and heating temperatures, respectively.

  12. The adsorption of lysozyme and chymosin onto emulsion droplets and their association with caseins

    NARCIS (Netherlands)

    Roos, de A.

    1999-01-01

    The proteolytic action of proteases present in cheese plays a major role in the ripening of cheese. These proteases originate from the rennet, the starter cultures and from the milk itself. The proteolysis in cheese results in the degradation of the casein proteins into smaller peptides and free ami

  13. Trichostatin A inhibits beta-casein expression in mammary epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pujuguet, Philippe; Radisky, Derek; Levy, Dinah; Lacza, Charlemagne; Bissell, Mina J.

    2002-02-22

    Many aspects of cellular behavior are affected by information derived from association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. Previously, we defined the minimal ECM- and Prl-responsive enhancer element BCE-1 from the upstream region of the beta-casein gene. We also found that BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous b-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide evidence that the differing response of b-casein and BCE-1 to TSA is neither due to an unusual effect of TSA on mammary epithelial cells, nor to secondary consequences from the expression of a separate gene, nor to a particular property of the BCE-1 construct. As a component of this investigation, we also showed that ECM could mediate rapid histone deacetylation in mammary epithelial cells. These results are discussed in combination with previous work showing that TSA mediates the differentiation of many types of cancer cells but inhibits differentiation of some nonmalignant cell types.

  14. Casein and soybean protein-based thermoplastics and composites as alternative biodegradable polymers for biomedical applications

    NARCIS (Netherlands)

    Vaz, C.M.; Fossen, M.; Tuil, van R.F.; Graaf, de L.A.; Reis, R.L.; Cunha, A.M.

    2003-01-01

    This work reports on the development and characterization of novel meltable polymers and composites based on casein and soybean proteins. The effects of inert (Al2O3) and bioactive (tricalcium phosphate) ceramic reinforcements over the mechanical performance, water absorption, and bioactivity behavi

  15. Effects of structural rearrangements on the rheology of rennet-induced casein particle gels

    NARCIS (Netherlands)

    Mellema, M.; Walstra, P.; Opheusden, van J.H.J.; Vliet, van T.

    2002-01-01

    During ageing of casein or skim milk gels, structural changes take place that affect gel parameters, such as pore size and storage modulus. These changes can be explained in terms of rearrangements of the gel network at various length scales. In this paper, rheological experiments on rennet-induced

  16. The influence of casein and urea as nitrogen sources on in vitro equine caecal fermentation

    NARCIS (Netherlands)

    Santos, A.S.; Ferreira, L.M.M.; Martin-Rosset, W.; Cotovio, M.; Silva, F.; Bennett, R.N.; Cone, J.W.; Bessa, R.J.B.; Rodrigues, M.A.M.

    2012-01-01

    To access the fermentative response of equine caecal microbial population to nitrogen availability, an in vitro study was conducted using caecal contents provided with adequate energy sources and nitrogen as limiting nutrient. Two nitrogen (N) sources were provided, protein (casein) and non-protein

  17. Ultrasound effects on the assembly of casein micelles in reconstituted skim milk.

    Science.gov (United States)

    Liu, Zheng; Juliano, Pablo; Williams, Roderick P W; Niere, Julie; Augustin, Mary Ann

    2014-05-01

    Reconstituted skim milks (10 % w/w total solids, pH 6·7-8·0) were ultrasonicated (20, 400 or 1600 kHz at a specific energy input of 286 kJ/kg) at a bulk milk temperature of <30 °C. Application of ultrasound to milk at different pH altered the assembly of the casein micelle in milk, with greater effects at higher pH and lower frequency. Low frequency ultrasound caused greater disruption of casein micelles causing release of protein from the micellar to the serum phase than high frequency. The released protein re-associated to form aggregates of smaller size but with surface charge similar to the casein micelles in the original milk. Ultrasound may be used as a physical intervention to alter the size of the micelles and the partitioning of caseins between the micellar and serum phases in milk. The altered protein equilibria induced by ultrasound treatment may have potential for the development of milk with novel functionality.

  18. Divergence at the casein haplotypes in dairy and meat goat breeds.

    Science.gov (United States)

    Küpper, Julia; Chessa, Stefania; Rignanese, Daniela; Caroli, Anna; Erhardt, Georg

    2010-02-01

    Casein genes have been proved to have an influence on milk properties, and are in addition appropriate for phylogeny studies. A large number of casein polymorphisms exist in goats, making their analysis quite complex. The four casein loci were analyzed by molecular techniques for genetic polymorphism detection in the two dairy goat breeds Bunte Deutsche Edelziege (BDE; n=96), Weisse Deutsche Edelziege (WDE; n=91), and the meat goat breed Buren (n=75). Of the 35 analyzed alleles, 18 were found in BDE, and 17 in Buren goats and WDE. In addition, a new allele was identified at the CSN1S1 locus in the BDE, showing a frequency of 0.05. This variant, named CSN1S1*A', is characterized by a t-->c transversion in intron 9. Linkage disequilibrium was found at the casein haplotype in all three breeds. A total of 30 haplotypes showed frequencies higher than 0.01. In the Buren breed only one haplotype showed a frequency higher than 0.1. The ancestral haplotype B-A-A-B (in the order: CSN1S1-CSN2-CSN1S2-CSN3) occurred in all three breeds, showing a very high frequency (>0.8) in the Buren.

  19. The Gluten-Free, Casein-Free Diet and Autism: Limited Return on Family Investment

    Science.gov (United States)

    Hurwitz, Sarah

    2013-01-01

    The gluten-free, casein-free (GFCF) diet is widely used by families of children with autism spectrum disorders (ASD). Despite its popularity, there is limited evidence in support of the diet. The purpose of this article was to identify and evaluate well-controlled studies of the GFCF diet that have been implemented with children with ASD. A review…

  20. Properties of acid casein gels made by acidification with Glucono-δ-lactone. 1. Rheological properties

    NARCIS (Netherlands)

    Lucey, J.A.; Vliet, van T.; Grolle, K.; Geurts, T.; Walstra, P.

    1997-01-01

    The effects of gelation temperature (20, 30 or 40 °C), assay temperature, concentration of glucono-δ-lactone (GDL) added, and NaCl concentration on the rheological properties of acid casein gels were studied at small and large deformations. Gels prepared at a high incubation temperature had very low

  1. Caseins from bovine colostrum and milk strongly bind piscidin-1, an antimicrobial peptide from fish.

    Science.gov (United States)

    Kütt, Mary-Liis; Stagsted, Jan

    2014-09-01

    A model system of bovine colostrum and piscidin, a fish-derived antimicrobial peptide, was developed to study potential interactions of antimicrobial peptides in colostrum. We did not detect any antimicrobial activity of colostrum using the radial plate diffusion assay; in fact colostrum completely abrogated activity of added piscidin. This could not be explained by degradation of piscidin by colostrum, which was less than ten percent. We found that colostrum even protected piscidin against degradation by added proteases. We further observed that colostrum and milk rapidly quenched the fluorescence of fluorescein-piscidin but not that of fluorescein. This effect was not seen with BSA and the specific quenching of fluorescein-piscidin by colostrum was saturably inhibited with unlabeled piscidin. Size exclusion chromatography indicated that fluorescein-piscidin bound to casein micelles with no apparent binding to IgG or whey proteins. Further, addition of pure caseins was able to quench fluorescence of fluorescein-piscidin and to inhibit the antimicrobial activity of piscidin. The interaction between caseins and piscidin could be dissociated by guanidine hydrochloride and recovered piscidin had antimicrobial activity against bacteria. Based on our results we propose that caseins could be carriers for antimicrobial peptides in colostrum and milk.

  2. Analysis of the endogenous peptide profile of milk: identification of 248 mainly casein-derived peptides.

    Science.gov (United States)

    Baum, Florian; Fedorova, Maria; Ebner, Jennifer; Hoffmann, Ralf; Pischetsrieder, Monika

    2013-12-06

    Milk is an excellent source of bioactive peptides. However, the composition of the native milk peptidome has only been partially elucidated. The present study applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) directly or after prefractionation of the milk peptides by reverse-phase high-performance liquid chromatography (RP-HPLC) or OFFGEL fractionation for the comprehensive analysis of the peptide profile of raw milk. The peptide sequences were determined by MALDI-TOF/TOF or nano-ultra-performance liquid chromatography-nanoelectrospray ionization-LTQ-Orbitrap-MS. Direct MALDI-TOF-MS analysis led to the assignment of 57 peptides. Prefractionation by both complementary methods led to the assignment of another 191 peptides. Most peptides originate from α(S1)-casein, followed by β-casein, and α(S2)-casein. κ-Casein and whey proteins seem to play only a minor role as peptide precursors. The formation of many, but not all, peptides could be explained by the activity of the endogenous peptidases, plasmin or cathepsin D, B, and G. Database searches revealed the presence of 22 peptides with established physiological function, including those with angiotensin-converting-enzyme (ACE) inhibitory, immunomodulating, or antimicrobial activity.

  3. Interfacial properties, thin film stability and foam stability of casein micelle dispersions

    NARCIS (Netherlands)

    Chen, Min; Sala, G.; Meinders, M.B.J.; Valenberg, van H.J.F.; Linden, van der E.; Sagis, L.M.C.

    2017-01-01

    Foam stability of casein micelle dispersions (CMDs) strongly depends on aggregate size. To elucidate the underlying mechanism, the role of interfacial and thin film properties was investigated. CMDs were prepared at 4 °C and 20 °C, designated as CMD4 °C and CMD20 °C. At equal protein concentrations,

  4. Protein kinase CK2: a newcomer in the 'druggable kinome'.

    Science.gov (United States)

    Pagano, M A; Cesaro, L; Meggio, F; Pinna, L A

    2006-12-01

    The acronym CK2 (derived from the misnomer 'casein kinase' 2) denotes one of the most pleiotropic members of the eukaryotic protein kinase superfamily, characterized by an acidic consensus sequence in which a carboxylic acid (or pre-phosphorylated) side chain at position n+3 relative to the target serine/threonine residue plays a crucial role. The latest repertoire of CK2 substrates includes approx. 300 proteins, but the analysis of available phosphopeptide databases from different sources suggests that CK2 alone may be responsible for the generation of a much larger proportion (10-20%) of the eukaryotic phosphoproteome. Although for the time being CK2 is not included among protein kinases whose inhibitors are in clinical practice or in advanced clinical trials, evidence is accumulating that elevated CK2 constitutive activity co-operates to induce a number of pathological conditions, including cancer, infectious diseases, neurodegeneration and cardiovascular pathologies. The development and usage of cell-permeant, selective inhibitors discloses a scenario whereby CK2 plays a global anti-apoptotic role, which under special circumstances may lead to untimely and pathogenic cell survival.

  5. Relative efficacy of casein or soya protein combined with palm or safflower-seed oil on hyperuricaemia in rats.

    Science.gov (United States)

    Lo, Hui-Chen; Wang, Yao-Horng; Chiou, Hue-Ying; Lai, Shan-Hu; Yang, Yu

    2010-07-01

    Diets that ameliorate the adverse effects of uric acid (UA) on renal damage deserve attention. The effects of casein or soya protein combined with palm or safflower-seed oil on various serum parameters and renal histology were investigated on hyperuricaemic rats. Male Wistar rats administered with oxonic acid and UA to induce hyperuricaemia were fed with casein or soya protein plus palm- or safflower-seed oil-supplemented diets. Normal rats and hyperuricaemic rats with or without allopurinol treatment (150 mg/l in drinking water) were fed with casein plus maize oil-supplemented diets. After 8 weeks, allopurinol treatment and soya protein plus safflower-seed oil-supplemented diet significantly decreased serum UA in hyperuricaemic rats (one-way ANOVA; P soya protein and casein attenuated hyperuricaemia-induced decreases in serum albumin and insulin, respectively (two-way ANOVA; P soya protein significantly decreased renal NO and nitrotyrosine and palm oil significantly decreased renal nitrotyrosine, TNF-alpha and interferon-gamma and increased renal transforming growth factor-beta. Casein with safflower-seed oil significantly attenuated renal tubulointerstitial nephritis, crystals and fibrosis. Comparing casein v. soya protein combined with palm or safflower-seed oil, the results support that casein with safflower-seed oil may be effective in attenuating hyperuricaemia-associated renal damage, while soya protein with safflower-seed oil may be beneficial in lowering serum UA and TAG.

  6. Characterization of casein gene complex and genetic diversity analysis in Indian goats.

    Science.gov (United States)

    Rout, P K; Kumar, A; Mandal, A; Laloe, D; Singh, S K; Roy, R

    2010-04-01

    Milk protein polymorphism plays an important role in genetic diversity analysis, phylogenetic studies, establishing geographical diversity, conservation decision, and improving breeding goals. Milk protein polymorphism in Indian goat breeds has not been well studied; therefore, an investigation was carried out to analyze the genetic structure of the casein gene and milk protein diversity at six milk protein loci in nine Indian goat breeds/genetic groups from varied agro-climatic zones. Milk protein genotyping was carried out in 1098 individual milk samples by SDS-PAGE at alphaS1-CN (CSN1S1), beta-CN (CSN2), alphaS2-CN (CSN1S2), kappa-CN (CSN3), beta-LG, and alpha-LA loci. Indian goats exhibited alphaS1-casein A allele in higher frequency in the majority of breeds except Ganjam and local goats. The alphaS1-casein A allele frequencies varied from 0.45 to 0.77. A total of 16 casein haplotypes were observed in seven breeds and breed specific haplotypes were observed with respect to geographic region. The average number of alleles was lowest in Ganjam (1.66 +/- 0.81) and highest in Sirohi goats (2.50 +/- 1.05). Expected heterozygosity at six different loci demonstrated genetic diversity and breed fragmentation. Neighbor-Joining tree was built basing on Nei's distance. There was about 16.95% variability due to differences between breeds, indicating a strong subdivision. Principal component analysis was carried out to highlight the relationship among breeds. The variability among goat breeds was contributed by alphaS2-CN, beta-LG and alphaS1-CN. The Indian goats exhibited alphaS1-CN (CSN1S1) A allele in higher frequency in all the breeds indicating the higher casein yield in their milk.

  7. Casein kinase 2 regulates the active uptake of the organic osmolyte taurine in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Jacobsen, Jack H; Clement, Christian A; Friis, Martin B;

    2008-01-01

    T to ER but has no detectable effect on TauT protein expression. On the other hand, CK2 inhibition increases the affinity of TauT towards Na(+ )and reduces the Na(+)/taurine stoichiometry for active taurine uptake. It is suggested that CK2 controls the cellular taurine uptake in unperturbated NIH3T3 cells...

  8. Distribution of protein kinase Mzeta and the complete protein kinase C isoform family in rat brain

    DEFF Research Database (Denmark)

    Naik, M U; Benedikz, Eirikur; Hernandez, I

    2000-01-01

    Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region o...

  9. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A;

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  10. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H; Speichermann, N

    1980-01-01

    identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6...

  11. Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

    DEFF Research Database (Denmark)

    Bischoff, Nils; Olsen, Birgitte; Raaf, Jennifer;

    2011-01-01

    the limited biochemical knowledge about the second paralog (CK2α'), we developed a well-soluble catalytically active full-length mutant of human CK2α', characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity......Protein kinase CK2 (formerly "casein kinase 2") is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement...

  12. Effect of Fluoride, Casein Phosphopeptide–Amorphous Calcium Phosphate and Casein Phosphopeptide–Amorphous Calcium Phosphate Fluoride on Enamel Surface Microhardness After Microabrasion: An In Vitro Study

    Directory of Open Access Journals (Sweden)

    Ghazaleh Ahmadi Zenouz

    2016-03-01

    Full Text Available Objectives: This study aimed to assess the effect of applying casein phosphopeptide–amorphous calcium phosphate (CPP-ACP paste, casein phosphopeptide–amorphous calcium phosphate fluoride (CPP-ACPF paste and sodium fluoride gel on surface microhardness of enamel after microabrasion.Materials and Methods: Thirty freshly extracted human premolars were selected. All samples were subjected to hardness indentations made with the Vickers hardness machine and the average value was recorded as the initial surface microhardness. The specimens were then randomly divided into three groups (n=10 of CPP-ACPF, fluoride and CPP-ACP. The teeth were micro-abraded with Opalustre. Microhardness test was performed to assess the post-abrasion hardness. Three remineralization modalities were performed on samples of each group. The enamel surface microhardness measurements were performed. To compare the difference between groups, the rehardening and softening values were defined. One-way ANOVA and Tukey’s post hoc test at a significance level of 5% were used for statistical analysis.Results: The mean microhardness value (MMV had a significant decrease after microabrasion from baseline. The MMV had a significant increase after remineralization in all groups. The MMV of CPP-ACPF group was significantly more than that of fluoride group (P=0.027. The rehardening value of fluoride group was significantly more than that of other groups (P<0.001.Conclusion: All the remineralizing agents were effective for rehardening the enamel after microabrasion. The CPP-ACP and CPP-ACPF pastes are effective, but to a lesser extent than neutral sodium fluoride gel in remineralizing enamel surface. Incorporation of fluoride to CPP-ACP formulation does not provide any additional remineralizing potential.Keywords: Casein phosphopeptide-amorphous calcium phosphate nanocomplex; Enamel Microabrasion; Hardness; Sodium Fluoride

  13. Effect of spray drying on the sensory and physical properties of hydrolysed casein using gum arabic as the carrier.

    Science.gov (United States)

    Subtil, S F; Rocha-Selmi, G A; Thomazini, M; Trindade, M A; Netto, F M; Favaro-Trindade, C S

    2014-09-01

    This study was aimed at spray drying hydrolysed casein using gum Arabic as the carrier agent, in order to decrease the bitter taste. Three formulations with differing proportions of hydrolysed casein: gum Arabic (10:90, 20:80 and 30:70) were prepared and characterized. They were evaluated for their moisture content, water activity, hygroscopicity, dispersibility in water and in oil, particle size and distribution, particle morphology, thermal behaviour (DSC) and bitter taste by a trained sensory panel using a paired-comparison test (free samples vs. spray dried samples). The proportion of hydrolysed casein did not affect the morphology of the microspheres. The spray drying process increased product stability and modified the dissolution time, but had no effect on the ability of the material to dissolve in either water or oil. The sensory tests showed that the spray drying process using gum Arabic as the carrier was efficient in attenuating or masking the bitter taste of the hydrolysed casein.

  14. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Boura, Evzen, E-mail: boura@uochb.cas.cz; Nencka, Radim, E-mail: nencka@uochb.cas.cz

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  15. Inhibitory role of peroxiredoxin II (Prx II) on cellular senescence.

    Science.gov (United States)

    Han, Ying-Hao; Kim, Hyun-Sun; Kim, Jin-Man; Kim, Sang-Keun; Yu, Dae-Yeul; Moon, Eun-Yi

    2005-08-29

    Reactive oxygen species (ROS) were generated in all oxygen-utilizing organisms. Peroxiredoxin II (Prx II) as one of antioxidant enzymes may play a protective role against the oxidative damage caused by ROS. In order to define the role of Prx II in organismal aging, we evaluated cellular senescence in Prx II(-/-) mouse embryonic fibroblast (MEF). As compared to wild type MEF, cellular senescence was accelerated in Prx II(-/-) MEF. Senescence-associated (SA)-beta-galactosidase (Gal)-positive cell formation was about 30% higher in Prx II(-/-) MEF. N-Acetyl-l-cysteine (NAC) treatment attenuated SA-beta-Gal-positive cell formation. Prx II(-/-) MEF exhibited the higher G2/M (41%) and lower S (1.6%) phase cells as compared to 24% and 7.3% [corrected] in wild type MEF, respectively. A high increase in the p16 and a slight increase in the p21 and p53 levels were detected in PrxII(-/-) MEF cells. The cellular senescence of Prx II(-/-) MEF was correlated with the organismal aging of Prx II(-/-) mouse skin. While extracellular signal-regulated kinase (ERK) and p38 activation was detected in Prx II(-/-) MEF, ERK and c-Jun N-terminal kinase (JNK) activation was detected in Prx II(-/-) skin. These results suggest that Prx II may function as an enzymatic antioxidant to prevent cellular senescence and skin aging.

  16. Casein addition to a whey-based formula has limited effects on gut function in preterm pigs.

    Science.gov (United States)

    Thymann, T; Støy, C A F; Bering, S B; Mølbak, L; Sangild, P T

    2012-12-01

    Preterm infants are susceptible to necrotizing enterocolitis (NEC). Using preterm pigs, we determined whether a whey-casein-based formula would be superior to a formula based on whey protein alone. Twenty cesarean-derived preterm pigs (92% gestation) were given total parenteral nutrition for 36 h followed by 30 h of enteral feeding with whey [protein fraction of milk formula based on whey (WHEY); n = 11] or casein and/or whey [protein fraction of milk formula based on a combination of casein and whey (CASEIN); n = 9]-based formulas. Sugar absorptive function was investigated at 6 and 30 h after initiation of enteral feeding using bolus feedings with galactose and mannitol. Pigs were killed after the last in vivo sugar absorption test and evaluated for NEC and the mid intestine was used for ex vivo measurements of hexose absorption. Microbiota profile and short chain fatty acid (SCFA) levels were studied in gut contents. Severity of NEC lesions was similar between diet groups but galactose absorption was markedly higher in CASEIN than in WHEY (P SCFA concentration. Casein may transiently stimulate intestinal sugar absorption but has limited effects on gut structure, microbiota, and NEC in preterm pigs.

  17. Moleculer analysis of genotype kappa casein and composition of goat milk Etawah grade, Saanen and their crossbred

    Directory of Open Access Journals (Sweden)

    Yayu Zurriyati

    2011-03-01

    Full Text Available Polymorphism of goat casein gene closely linked to the quality of milk protein. κ-casein is one of the casein fractions that influence the shape and stability of grain milk. This study is aimed to identify the variation of genotype κ-casein and related with milk quality from Etawah grade, Saanen and their crossbreed (PE-SA. The number of dairy goats used in this study was 150 animals consisted of Etawah grade (48 animals, Saanen (51 animals and PE-SA (51 animals. Steps of experiment were: blood and milk sampling collection, DNA amplification by PCR and the product digestion using Pst1 enzyme, κ-casein gene sequencing and analyzing the quality of fresh milk. The results showed that κ-casein gene is monomorphic by PCR-RFLP (Pst1 for all the goat breeds, but DNA sequencing indicated 38 point of mutation. Observation on goat milk quality showed that Etawah grade milk had highest (P < 0.05 density value (1.033 ± 0.002 and solid non fat (9.577 ± 0.704% than those of Saanen and PE-SA fresh milk goat.

  18. Estimation of whey protein in casein coprecipitate and milk powder by high-performance liquid chromatography quantification of cysteine.

    Science.gov (United States)

    Ballin, Nicolai Z

    2006-06-14

    An analytical high-performance liquid chromatography (HPLC)-fluorescence method for indirect measuring of whey protein in casein coprecipitate and milk powder was developed. Samples were hydrolyzed with HCl, and cysteyl residues were derivatized with 3,3'-dithiodipropionic acid and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The cysteine content was used to calculate the percentage of whey protein in commercial samples with use of European Union Regulation cysteine reference values in both casein and whey protein. Method validation studies were performed for caseinates and milk powder, and results indicate that the present HPLC approach can be applied as a fast method with a standard deviation of repeatability between 3.3 and 9.5%. Applicability was studied by analysis of 40 commercial caseinate samples, and all complied to European legislation with a content of whey protein not exceeding 5%. Finally, an approach used to estimate the cysteine amount in pure casein by comparison of calculated and experimental values questions the generally accepted cysteine reference value in casein, which is most likely an overestimation.

  19. Interfacial properties, thin film stability and foam stability of casein micelle dispersions.

    Science.gov (United States)

    Chen, Min; Sala, Guido; Meinders, Marcel B J; van Valenberg, Hein J F; van der Linden, Erik; Sagis, Leonard M C

    2017-01-01

    Foam stability of casein micelle dispersions (CMDs) strongly depends on aggregate size. To elucidate the underlying mechanism, the role of interfacial and thin film properties was investigated. CMDs were prepared at 4°C and 20°C, designated as CMD4°C and CMD20°C. At equal protein concentrations, foam stability of CMD4°C (with casein micelle aggregates) was markedly higher than CMD20°C (without aggregates). Although the elastic modulus of CMD4°C was twice as that of CMD20°C at 0.005Hz, the protein adsorbed amount was slightly higher for CMD20°C than for CMD4°C, which indicated a slight difference in interfacial composition of the air/water interface. Non-linear surface dilatational rheology showed minor differences between mechanical properties of air/water interfaces stabilized by two CMDs. These differences in interfacial properties could not explain the large difference in foam stability between two CMDs. Thin film analysis showed that films made with CMD20°C drained to a more homogeneous film compared to films stabilized by CMD4°C. Large casein micelle aggregates trapped in the thin film of CMD4°C made the film more heterogeneous. The rupture time of thin films was significantly longer for CMD4°C (>1h) than for CMD20°C (foam stability decreased significantly. In conclusion, the increased stability of foam prepared with CMD4°C appears to be the result of entrapment of casein micelle aggregates in the liquid films of the foam.

  20. How to Squeeze a Sponge: Casein Micelles under Osmotic Stress, a SAXS Study

    OpenAIRE

    Bouchoux, Antoine; Gésan-Guiziou, Geneviève; Pérez, Javier; Cabane, Bernard

    2010-01-01

    By combining the osmotic stress technique with small-angle x-ray scattering measurements, we followed the structural response of the casein micelle to an overall increase in concentration. When the aqueous phase that separates the micelles is extracted, they behave as polydisperse repelling spheres and their internal structure is not affected. When they are compressed, the micelles lose water and shrink to a smaller volume. Our results indicate that this compression is nonaffine, i.e., some p...

  1. Gluten-free and casein-free diets in the treatment of autism

    OpenAIRE

    Andreas Reissmann; Joachim Hauser; Ewelina Makulska-Gertruda; Loredana Tomsa; Lange, Klaus W.

    2014-01-01

    Background: Autism is a complex psychiatric disorder characterized by three core symptoms, i.e. impairments in social interaction, restricted patterns of behavior and impairments in communication. In the framework of the “opioid excess theory”, the disorder symptoms are compared to the behavioral effects of opiates. Based on this, a possible nutritional basis of autism has been proposed, hypothesizing that certain food proteins such as gluten and casein can be transformed ...

  2. A review of gluten- and casein-free diets for treatment of autism: 2005–2015

    OpenAIRE

    Elder, Jennifer; Kreider, Consuelo; Schaefer, Nancy; deLaosa,Mary

    2015-01-01

    Jennifer Harrison Elder,1 Consuelo Maun Kreider,2 Nancy M Schaefer,3 Mary B de Laosa4 1Department of Family and Community Health Nursing Science, 2Department of Occupational Therapy, 3Health Science Center Library, 4Department of Psychology, University of Florida, Gainesville, FL, USA Background: The gluten-free, casein-free (GFCF) diet is heralded by strong anecdotal parental reports to greatly improve and even "cure" symptoms of autism spectrum disorders (ASDs). Yet, to d...

  3. A review of gluten- and casein-free diets for treatment of autism: 2005–2015

    OpenAIRE

    Elder JH; Kreider CM; Schaefer NM; de Laosa MB

    2015-01-01

    Jennifer Harrison Elder,1 Consuelo Maun Kreider,2 Nancy M Schaefer,3 Mary B de Laosa4 1Department of Family and Community Health Nursing Science, 2Department of Occupational Therapy, 3Health Science Center Library, 4Department of Psychology, University of Florida, Gainesville, FL, USA Background: The gluten-free, casein-free (GFCF) diet is heralded by strong anecdotal parental reports to greatly improve and even "cure" symptoms of autism spectrum disorders (ASDs). Yet, to date, lit...

  4. Kappa-casein gene study in Iranian Sistani cattle breed (Bos indicus) using PCR-RFLP.

    Science.gov (United States)

    Rohallah, Alinaghizadeh; Mohammadreza, Mohammad Abadi; Shahin, Moradnasab Badrabadi

    2007-12-01

    In cattle, caseins are subdivided into four main groups: alphaS1-, alphaaS2-, beta- and kappa-caseins. kappa-caseins (CSN2) considerably differ from other caseins in structure and other properties. Testing the A and B alleles is of practical importance, because the milk of cows that carry the B allele of CSN3 has a better thermal resistance and shorter coagulation time, better curdles and contains micelles of different sizes. Iranian Sistani cattle (Bos indicus) are a heavy built breed and used as dual-purpose cattle breed in Eastern Iran. This breed is a genetic resource that shows special features of adaptation to rustic environments. One of the most distinctive features of Sistani cattle is its great capability to resist diseases which makes it a potential reservoir of germplasm useful for future crosses. Our main goal was to study DNA-polymorphism of the CSN3 gene in the Iranian Sistani native cattle (Bos indicus) and analyze the information value of CSN3 gene polymorphism as a genetic marker. We genotyped and analyzed 65 animals of this breed using PCR-RFLP. The frequencies of A and B alleles were 0.6385 and 0.3615 and those of AA, AB and BB genotypes were 0.4000, 0.4769 and 0.1231, respectively. In the Sistani Zebu breed, frequency of B allele is higher than other Zebu breeds, indicating that intensive selection for dairy production have been done and indirectly influenced CSN3 allele frequencies.

  5. Short communication: genetic variability in the predicted microRNA target sites of caprine casein genes.

    Science.gov (United States)

    Zidi, A; Amills, M; Tomás, A; Vidal, O; Ramírez, O; Carrizosa, J; Urrutia, B; Serradilla, J M; Clop, A

    2010-04-01

    The main goal of the current work was to identify single nucleotide polymorphisms (SNP) that might create or disrupt microRNA (miRNA) target sites in the caprine casein genes. The 3' untranslated regions of the goat alpha(S1)-, alpha(S2)-, beta-, and kappa-casein genes (CSN1S1, CSN1S2, CSN2, and CSN3, respectively) were resequenced in 25 individuals of the Murciano-Granadina, Cashmere, Canarian, Saanen, and Sahelian breeds. Five SNP were identified through this strategy: c.175C>T at CSN1S1; c.109T>C, c.139G>C, and c.160T>C at CSN1S2; and c.216C>T at CSN2. Analysis with the Patrocles Finder tool predicted that all of these SNP are located within regions complementary to the seed of diverse miRNA sequences. These in silico results suggest that polymorphism at miRNA target sites might have some effect on casein expression. We explored this issue by genotyping the c.175C>T SNP (CSN1S1) in 85 Murciano-Granadina goats with records for milk CSN1S1 concentrations. This substitution destroys a putative target site for miR-101, a miRNA known to be expressed in the bovine mammary gland. Although TT goats had higher levels (6.25 g/L) of CSN1S1 than their CT (6.05 g/L) and CC (6.04 g/L) counterparts, these differences were not significant. Experimental confirmation of the miRNA target sites predicted in the current work and performance of additional association analyses in other goat populations will be an essential step to find out if polymorphic miRNA target sites constitute an important source of variation in casein expression.

  6. K-酪蛋白研究进展%Research on k-casein: A review

    Institute of Scientific and Technical Information of China (English)

    刘文静; 郑玉才; 钟光辉

    2001-01-01

    对k-酪蛋白(kappa-casein,k-CN)的基本特征,遗传多态性及其与乳成分、凝乳性能的关系,以及k-CN定型技术作了综述;并探讨了k-CN遗传多态性在乳酪生产和牦牛育种中的应用前景.

  7. Galactose content of legumes, caseinates, and some hard cheeses: implications for diet treatment of classic galactosemia.

    Science.gov (United States)

    Van Calcar, Sandra C; Bernstein, Laurie E; Rohr, Frances J; Yannicelli, Steven; Berry, Gerard T; Scaman, Christine H

    2014-02-12

    There are inconsistent reports on the lactose and/or galactose content of some foods traditionally restricted from the diet for classic galactosemia. Therefore, samples of cheeses, caseinates, and canned black, pinto, kidney, and garbanzo beans were analyzed for free galactose content using HPLC with refractive index or pulsed amperometric detection. Galactose concentrations in several hard and aged cheeses and three mild/medium Cheddars, produced by smaller local dairies, was galactosemia.

  8. Studying Kinetochore Kinases

    NARCIS (Netherlands)

    Saurin, Adrian T; Kops, Geert J P L

    2016-01-01

    Mitotic kinetochores are signaling network hubs that regulate chromosome movements, attachment error-correction, and the spindle assembly checkpoint. Key switches in these networks are kinases and phosphatases that enable rapid responses to changing conditions. Describing the mechanisms and dynamics

  9. Pkh-kinases control eisosome assembly and organization

    OpenAIRE

    Walther, TC; Aguilar, PS; Fröhlich, F.; Chu, F.; Moreira, K.; Burlingame, Al; WALTER, P.

    2007-01-01

    Eisosomes help sequester a subgroup of plasma membrane proteins into discrete membrane domains that colocalize with sites of endocytosis. Here we show that the major eisosome component Pil1 in vivo is a target of the long-chain base (LCB, the biosynthetic precursors to sphingolipids)-signaling pathway mediated by the Pkh-kinases. Eisosomes disassemble if Pil1 is hyperphosphorylated (i) upon overexpression of Pkh-kinases, (ii) upon reducing LCB concentrations by inhibiting serine-palmitoyl tra...

  10. Bovine kappa-casein gene polymorphism and its association with milk production traits

    Directory of Open Access Journals (Sweden)

    Satyanarayana Rachagani

    2008-12-01

    Full Text Available Point mutations in exon IV of the bovine κ-casein (CSN3 gene determine two allelic variants, A and B. These variants were distinguished by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP analysis in the indigenous Sahiwal and Tharparkar cattle breeds. DNA samples (252 Sahiwal and 56 Tharparkar were analyzed for allelic variants of the CSN3 gene. Polymorphism was detected by digestion of PCR-amplified products with HindIII, HhaI and HaeIII restriction enzymes, followed by separation on 3% agarose gels, and resolved by ethidium bromide staining. Allele A of the κ-casein gene occurred at a higher frequency than allele B, in both Sahiwal and Tharparkar breeds. The genotypic frequencies of AA, AB, and BB in the Sahiwal and Tharparkar breeds were 0.758, 0.230 and 0.012, and 0.0.732, 0.250 and 0.018, respectively. The frequencies of alleles A and B in the Sahiwal and Tharparkar breeds were 0.873 and 0.127, and 0.857 and 0.143, respectively. Genotype BB of the kappa-casein gene had more influence on the monthly milk yield, 305-days milk yield, monthly solids-not-fat (SNF yield, and monthly protein yield, in the Sahiwal cattle.

  11. Casein peptization, functional properties, and sensory acceptance of processed cheese spreads made with different emulsifying salts.

    Science.gov (United States)

    Cunha, Clarissa R; Viotto, Walkiria H

    2010-01-01

    "Requeijão cremoso" is a traditional Brazilian processed cheese spread, showing ample acceptance on the national market. Emulsifying salts (ES) are an important factor influencing the characteristics of processed cheeses, but the literature presents conflicting results about their action on cheese functionality. Requeijão cremoso obtained from anhydrous ingredients allows the study of the influence of each type of ES on the cheese properties, since it can be treated as a model system where the variables are limited and well known. The objective of this study was to evaluate the effect of different types of ES (TSC-sodium citrate, SHMP-sodium hexametaphosphate, STPP-sodium tripolyphosphate, and TSPP-tetrasodium pyrophosphate) on the sensory and functional characteristics of requeijão cremoso-processed cheeses obtained from anhydrous ingredients. The physicochemical composition, degree of casein dissociation, fat particle size, melting index, color, texture profile, and sensory acceptance of the cheeses were determined. The functional behavior of processed cheeses was strongly influenced by the type of ES and its physicochemical properties including its ability to bind Ca, the casein dispersion during cooking, and the possible creation of cross-links with casein during cooling. The cheese made with SHMP was the one most differentiated from the others, presenting lower melting index, whiter color, and higher values for hardness, gumminess, and adhesiveness. The differences in texture had an impact on sensory acceptance: with the exception of the sample manufactured with sodium hexametaphosphate, all the samples presented good sensory acceptance.

  12. Genetic parameters for casein and urea content in the Italian Brown Swiss dairy cattle

    Directory of Open Access Journals (Sweden)

    O. Pedron

    2010-04-01

    Full Text Available A total of 137,753 test day records of 20,745 Italian Brown Swiss dairy cows from 26 provinces of Italy were used to estimate heritability for casein and urea content in milk and their genetic correlations with other production traits and milk somatic cell score. Milk component values were obtained by Fourier Transformed Infrared (IR Spectroscopy from milk samples collected during national routine recording and were analysed using test day repeatability animal models. Fixed effects included 1,001 levels of herd-test date, 15 classes of days in milk, and 13 classes of age at calving within parity. The variation among cows was large for most of the traits. The heritability value for casein content was 0.31, as for protein content, and genetic and phenotypic correlations between these two traits were large (0.99 and 0.97 respectively. Milk urea content had a heritability of 0.17 and a positive genetic relationship with fat (0.12, null with protein (0.03 and casein (0.002 content and a negative genetic correlation with milk yield (-0.17 suggesting that the genetic improvement for milk urea content would be possible, but genetic gain would be affected by other traits included as selection criteria in the economic index and by their relative economic emphasis.

  13. Influence of ultrasound on chemically induced gelation of micellar casein systems.

    Science.gov (United States)

    Chandrapala, Jayani; Zisu, Bogdan; Kentish, Sandra; Ashokkumar, Muthupandian

    2013-05-01

    Gelation is a significant operation in dairy processing. Protein gelation can be affected by several factors such as temperature, pH, or enzyme addition. Recently, the use of ultrasonication has been shown to have a significant impact on the formation of whey protein gels. In this work, the effect of ultrasonication on the gelation of casein systems was investigated. Gels were formed by the addition of 7.6 mm Tetra Sodium Pyro Phosphate (TSPP) to 5 wt% micellar casein (MC) solutions. Sonication at 20 KHz and 31 W for up to 30 min changed the surface hydrophobicity of the proteins, whereas surface charge was unaltered. Sonication before the addition of TSPP formed a firm gel with a fine protein network and low syneresis. Conversely, sonication after TSPP addition led to an inconsistent weak-gel-like structure with high syneresis. Gel strength in both cases increased significantly after short sonication times, while the viscoelastic properties were less affected. Overall, the results showed that ultrasonication can have a significant effect on the final gel properties of casein systems.

  14. Kinetics of N(ε)-(carboxymethyl)lysine formation in aqueous model systems of sugars and casein.

    Science.gov (United States)

    Nguyen, Ha T; van der Fels-Klerx, H J; van Boekel, Martinus A J S

    2016-02-01

    This study investigated the formation of N(ε)-carboxymethyllysine (CML) in two caseinate solutions containing: (1) glucose, (2) lactose, each heated at 120 °C and 130 °C. At both heating temperatures, CML concentration in lactose-caseinate solution was higher than in glucose-caseinate solution. In both solutions, more CML was formed at 130 °C than at 120 °C. Using multiresponse modelling, two degradation routes for the sugars were confirmed: (1) isomerisation of glucose or lactose and subsequent degradation via Lobry de Bruyn-Alberda van Ekenstein (LA) arrangement; (2) the Maillard reaction between the reducing sugar and lysine residues. Modelling results suggested that CML was not formed from oxidation of the reducing sugars, but from the Maillard reaction via the Amadori rearrangement product. Since CML appeared to be thermally unstable under the current study conditions, it may not be a perfect indicator for heat damage of processed foods. This is the first study in which CML formation was linked to available information on the Maillard reaction via multiresponse modelling.

  15. Incorporation of radiolabeled whey proteins into casein micelles by heat processing

    Energy Technology Data Exchange (ETDEWEB)

    Noh, B.; Richardson, T. (Univ. of California, Davis (USA))

    1989-07-01

    Skim milk was heated at .70, 95, and 140{degree}C to simulate the processes of pasteurization, forewarming, and UHT sterilization, and the specific interactions between {alpha}-lactalbumin or {beta}-lactoglobulin and the caseins studied using tracer amounts of added {sup 14}C-labeled whey protein. Radioactivities of the whey and of the washed casein pellets from renneted skim milk were measured and the extent of the interaction estimated. Upon heating skim milk at 70{degree}C for 45 s, less than 2% {beta}-lactoglobulin and less than .3% {alpha}-lactalbumin were incorporated into the curd. Heating at 95{degree}C for .5 to 20 min resulted in 58 to 85% of the {beta}-lactoglobulin and 8 to 55% of the {alpha}-lactalbumin becoming associated with the curd. Heating at 140{degree}C for 2 and 4 s caused 43 and 54% of the {beta}-lactoglobulin and 9 and 12% of the {alpha}-lactalbumin, respectively, to be bound to the curd fraction. The radiolabeling technique is very sensitive and useful for tracing low levels of interaction between whey proteins and casein in heated milk systems.

  16. Enhanced dispersibility and bioactivity of curcumin by encapsulation in casein nanocapsules.

    Science.gov (United States)

    Pan, Kang; Zhong, Qixin; Baek, Seung Joon

    2013-06-26

    In this work, a novel encapsulation method was studied by spray-drying a warm aqueous ethanol solution with codissolved sodium caseinate (NaCas) and lipophilic food components, using curcumin as a model compound. The encapsulation caused the loss of crystallinity of curcumin. After hydration of spray-dried powder and centrifugation, 137 μg/mL curcumin was dispersed in the transparent dispersion, which was 4 decades higher than its water solubility. Dynamic light scattering and atomic force microscopy results showed that curcumin-loaded casein nanoparticles were bigger than those of NaCas processed at encapsulation conditions but were smaller than those of the native NaCas. The increased nanoparticle dimension, together with fluorescence and FTIR spectroscopy results, suggested that curcumin was entrapped in the nanoparticle core through hydrophobic interactions. The curcumin encapsulated in casein nanoparticles had higher biological activity, as assessed by antioxidant and cell proliferation assays, than pristine curcumin, likely due to the improved dispersibility. This simple approach may be applied to encapsulate various lipophilic bioactive compounds.

  17. Calcium, vitamin D, casein and whey protein intakes and periodontitis among Danish adults

    DEFF Research Database (Denmark)

    Adegboye, Amanda Ra; Boucher, Barbara J; Kongstad, Johanne

    2016-01-01

    OBJECTIVE: To investigate whether intakes of Ca, vitamin D, casein and whey are associated with periodontitis and to investigate the possibility of interactions between them. DESIGN: Cross-sectional study. An Internet-based, 267-item FFQ was used to assess dietary intake. Intakes of casein (32.0 g....../d), whey proteins (9.6 g/d) and vitamin D (5.8 μg/d) were classified as within v. above the 50th percentile. Ca intake was classified as within v. below age-specific recommendations. Severe periodontitis was defined as having ≥2 inter-proximal sites with clinical attachment loss ≥6 mm (not on the same...... study of DANHES 2007-2008. RESULTS: Intakes of Ca within recommendations (OR=0.76; 95% CI 0.58, 0.99), whey ≥9.6 g/d (OR=0.75; 95% CI 0.58, 0.97) and casein ≥32 g/d (OR=0.75 95% CI 0.58, 0.97) were associated with lower likelihood of severe periodontitis after adjustment for age, gender, education...

  18. Stimulation of muscle protein synthesis by whey and caseinate ingestion after resistance exercise in elderly individuals

    DEFF Research Database (Denmark)

    Dideriksen, Kasper; Reitelseder, Søren; Petersen, S.G.

    2011-01-01

    -(13) C]leucine using labeled proteins during a 6-h recovery period. No differences were observed in muscle myofibrillar and collagen FSR with Whey (0.09 ± 0.01%/h) compared with CasPost (0.09 ± 0.003%/h), and it did not differ between CasPre (0.10 ± 0.01%/h) and CasPost. MPS does not differ with whey......Sarcopenia is a well-known phenomenon in elderly individuals and resistance exercise together with sufficient amino acid (AA) availability has proved to be a counteractive implement. However, the source of AA and supplement timing require further investigation. The objective was to compare muscle...... protein synthesis (MPS) to intakes of whey and caseinate after heavy resistance exercise in healthy elderly individuals, and, furthermore, to compare the timing effect of caseinate intake. Twenty-four elderly men and women (mean ± SEM; 68 ± 1 years) were randomized to one of four groups: caseinate intake...

  19. Biosurfactant Production by Cultivation of Bacillus atrophaeus ATCC 9372 in Semidefined Glucose/Casein-Based Media

    Science.gov (United States)

    Das Neves, Luiz Carlos Martins; de Oliveira, Kátia Silva; Kobayashi, Márcio Junji; Vessoni Penna, Thereza Christina; Converti, Attilio

    Biosurfactants are proteins with detergent, emulsifier, and antimicrobial actions that have potential application in environmental applications such as the treatment of organic pollutants and oil recovery. Bacillus atrophaeus strains are nonpathogenic and are suitable source of biosurfactants, among which is surfactin. The aim of this work is to establish a culture medium composition able to stimulate biosurfactants production by B. atrophaeus ATCC 9372. Batch cultivations were carried out in a rotary shaker at 150 rpm and 35°C for 24 h on glucose- and/or casein-based semidefined culture media also containing sodium chloride, dibasic sodium phosphate, and soy flour. The addition of 14.0 g/L glucose in a culture medium containing 10.0 g/L of casein resulted in 17 times higher biosurfactant production (B max=635.0 mg/L). Besides, the simultaneous presence of digested casein (10.0 g/L), digested soy flour (3.0 g/L), and glucose (18.0 g/L) in the medium was responsible for a diauxic effect during cell growth. Once the diauxie started, the average biosurfactants concentration was 16.8% less than that observed before this phenomenon. The capability of B. atrophaeus strain to adapt its own metabolism to use several nutrients as energy sources and to preserve high levels of biosurfactants in the medium during the stationary phase is a promising feature for its possible application in biological treatments.

  20. Gluten- and casein-free dietary intervention for autism spectrum conditions.

    Directory of Open Access Journals (Sweden)

    Paul eWhiteley

    2013-01-01

    Full Text Available Dietary intervention as a tool for maintaining and improving physical health and wellbeing is a widely researched and discussed topic. Speculation that diet may similarly affect mental health and wellbeing particularly in cases of psychiatric and behavioural symptomatology opens up various avenues for potentially improving quality of life. We examine evidence suggestive that a gluten-free, casein-free or combined gluten- and casein-free diet can ameliorate core and peripheral symptoms and improve developmental outcome in some cases of autism spectrum conditions. Although not wholly affirmative, the majority of published studies indicate statistically significant positive changes to symptom presentation following dietary intervention. In particular, changes to areas of communication, attention and hyperactivity are detailed, despite the presence of various methodological shortcomings. Specific characteristics of best- and non-responders to intervention have not been fully elucidated; neither has the precise mode of action for any universal effect outside of known individual cases of food-related co-morbidity. With the publication of controlled medium- and long-term group studies of a gluten- and casein-free diet alongside more consolidated biological findings potentially linked to intervention, the appearance of a possible diet-related autism phenotype seems to be emerging supportive of a positive dietary effect in some cases. Further debate on whether such dietary intervention should form part of best practice guidelines for autism spectrum conditions and onward representative of an autism dietary-sensitive enteropathy is warranted.

  1. Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei

    Science.gov (United States)

    Li, H.; Dauwalder, M.; Roux, S. J.

    1991-01-01

    Almost all the Ca(2+)-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca2+ and reaches half-maximal activation at about 3 x 10(-7) molar free Ca2+, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca(2+)-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca(2+)-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.

  2. Serine phosphorylation of NPM-ALK, which is dependent on the auto-activation of the kinase activation loop, contributes to its oncogenic potential.

    Science.gov (United States)

    Wang, Peng; Wu, Fang; Zhang, Jingdong; McMullen, Todd; Young, Leah C; Ingham, Robert J; Li, Liang; Lai, Raymond

    2011-02-01

    It is well established that the tumorigenic potential of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, is dependent on its tyrosine phosphorylation. Using tandem affinity purification-mass spectrometry, we found evidence of phosphorylation of three serine residues of NPM-ALK (Serine¹³⁵, Serine¹⁶⁴ and Serine⁴⁹⁷) ectopically expressed in GP293 cells. Using a specific anti-phosphoserine antibody and immunoprecipitation, we confirmed the presence of serine phosphorylation of NPM-ALK in all three NPM-ALK-expressing cell lines examined. Similar to the tyrosine phosphorylation, phosphorylation of these serine residues was dependent on the activation status of the kinase activation loop of ALK. All of these three serine residues are biologically important as mutation of any one of these residues resulted in a significant reduction in the tumorigenicity of NPM-ALK (assessed by cell viability and clonogenic assay), which correlated with a substantial reduction in the phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinase and signal transducer and activator of transcription 6. Serine phosphorylation of NPM-ALK appears to be regulated by multiple serine kinases since it was markedly reduced by pharmacologic inhibitors for glycogen synthase kinase-3, casein kinase I or mitogen-activated protein kinases. In summary, our study is the first to identify serine phosphorylation of NPM-ALK and to provide evidence that it enhances the tumorigenic potential of this oncogenic protein.

  3. Protein Kinase CK2α Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2α Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma.

    Science.gov (United States)

    Zhou, Bingying; Ritt, Daniel A; Morrison, Deborah K; Der, Channing J; Cox, Adrienne D

    2016-08-19

    The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies.

  4. Optimization of caseinate-coated simvastatin-zein nanoparticles: improved bioavailability and modified release characteristics

    Directory of Open Access Journals (Sweden)

    Ahmed OA

    2015-01-01

    Full Text Available Osama AA Ahmed,1,2 Khaled M Hosny,1,3 Majid M Al-Sawahli,1,4 Usama A Fahmy11Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; 2Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Minia University, Minia, Egypt; 3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Beni Suef University, Beni Suef, Egypt; 4Holding Company for Biological Products & Vaccines (VACSERA, Cairo, EgyptAbstract: The current study focuses on utilization of the natural biocompatible polymer zein to formulate simvastatin (SMV nanoparticles coated with caseinate, to improve solubility and hence bioavailability, and in addition, to modify SMV-release characteristics. This formulation can be utilized for oral or possible depot parenteral applications. Fifteen formulations were prepared by liquid–liquid phase separation method, according to the Box–Behnken design, to optimize formulation variables. Sodium caseinate was used as an electrosteric stabilizer. The factors studied were: percentage of SMV in the SMV-zein mixture (X1, ethanol concentration (X2, and caseinate concentration (X3. The selected dependent variables were mean particle size (Y1, SMV encapsulation efficiency (Y2, and cumulative percentage of drug permeated after 1 hour (Y3. The diffusion of SMV from the prepared nanoparticles specified by the design was carried out using an automated Franz diffusion cell apparatus. The optimized SMV-zein formula was investigated for in vivo pharmacokinetic parameters compared with an oral SMV suspension. The optimized nanosized SMV-zein formula showed a 131 nm mean particle size and 89% encapsulation efficiency. In vitro permeation studies displayed delayed permeation characteristics, with about 42% and 85% of SMV cumulative amount released after 12 and 48 hours, respectively. Bioavailability estimation in rats revealed an augmentation in SMV bioavailability

  5. Proving the adulteration of ewe and goat cheeses with cow milk using the reference method of isoelectric focusing of γ-casein

    OpenAIRE

    Nataša Mikulec; Dijana Plavljanić; Jasminka Špoljarić; Biljana Radeljević; Jasmina Havranek; Neven Antunac

    2013-01-01

    The aim of this study was to introduce a reference method for the detection of cow milk in ewe and goat cheeses (EC 273/08) in order to protect manufacturers and consumers from adulterations and imitations, and to ensure alignment with the demands of domestic and EU markets. The method includes isolation of casein from cheese, isoelectric focusing of γ2- and γ3-casein originating from the hydrolysis of β-casein by plasmin, the detection and quantitative determination of γ-casein in cow, ewe a...

  6. 酪蛋白胶束结构研究方法综述%A review on the studying means of casein micelle structure

    Institute of Scientific and Technical Information of China (English)

    史莹; 杨敏; 梁琪; 乔海军; 张卫兵

    2012-01-01

    Caseins,the main components of milk protein, represent about 80% of the total protein in the milk.Owing to the importance of casein and casein micelles for the functional behavior of dairy products, and widely application of caseins, the study of casein is always drawing more attention.Especially, the exact structure of casein micelles is still under debate.Many scholars have proposed various theoretic models for casein micelle structure. This paper reviewed kinds means of casein micelles structure,dissociation and aggregation in order to provide a reference for studying casein in the future.%酪蛋白是乳中蛋白质的主要成分,约占其80%。由于酪蛋白胶束对乳制品的功能特性起重要作用,因此其研究一直备受关注,但酪蛋白胶束的内部结构至今没有确切的定义,而是许多学者提出了各种理论模型。文章综述了酪蛋白胶束结构、解离与聚集的多种研究方法,为以后学者研究酪蛋白提供借鉴。

  7. Studies on the photophysical properties of 1,8-naphthalimide derivative and aggregation induced emission recognition for casein

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yang, E-mail: 66160692@qq.com [Department of Chemistry and Chemical Engineering, Xi' an University of Arts and Science, No. 168, Taibai South Road, Xi' an, Shaanxi 710065 (China); Liang, Xuhua; Fan, Jun [School of Chemical Engineering, Northwest University, No. 229, Taibai North Road, Xi' an, Shaanxi 710069 (China); Han, Quan, E-mail: xahanq@hotmail.com [Department of Chemistry and Chemical Engineering, Xi' an University of Arts and Science, No. 168, Taibai South Road, Xi' an, Shaanxi 710065 (China)

    2013-09-15

    A novel water-soluble 1,8-naphthalimide derivative 1, bearing two acetic carboxylic groups, exhibited fluorescent turn-on recognition for casein micelle based on the aggregation induced emission (AIE) character. The photophysical properties of 1 consisting of donor and acceptor units were investigated by the solvation effect. Changing from polar to non-polar solvent increased the solvent interaction; both the excitation and emission spectra were shifted to shorter wavelength and intensity decreased through taking advantage of twisted intramolecular charge transfer (TICT) and self-association fluorescence emission. Moreover, the red-shift and quenching in protic solvent were caused by the excited-state hydrogen bond strengthening effect. The density functional theory (DFT) and time dependent density functional theory (TDDFT) were used to obtain the most stable structure, electronic excitation energy, dipole moments and charge distribution. The AIE mechanism of 1 with casein micelle was due to 1 docked in the hydrophobic cavity between sub-micelles and bound with amino acid residues, resulting in the aggregation of 1 on the casein micelle surface and emission enhancement, based on which, a novel casein assay method was developed. The proposed method exhibited a good linear range from 0.1 to 10.5 μg mL{sup −1}, with the detection limit of 3.0 ng mL{sup −1}. Satisfactory reproducibility, reversibility and a short response time were realized. This method was applied for the determination of casein in milk powder samples, avoiding the interferences from other components and illegal additives in milk. -- Highlights: • A water-soluble 1,8-naphthalimide-based fluorescent probe 1 was synthesized. • Photophysical characterization of 1 was studied. • Aggregation induced emission enhancement of 1 with casein was investigated. • A novel casein quantification method was developed.

  8. Short communication: retinoic acid plus prolactin to synergistically increase specific casein gene expression in MAC-T cells.

    Science.gov (United States)

    Lee, H Y; Heo, Y T; Lee, S E; Hwang, K C; Lee, H G; Choi, S H; Kim, N H

    2013-06-01

    Mammary alveolar (MAC-T) cells, an established bovine mammary epithelial cell line, are frequently used to investigate differentiation. A lactogenic phenotype in these cells is induced by treatment with a combination of hydrocortisone, insulin, and prolactin (PRL). The effect of the vitamin A derivative retinoic acid (RA), which induces differentiation in many cells, has not been studied in MAC-T cells. The objective of this study was to evaluate the differentiation potential of RA (1 μM) in MAC-T cells and to examine the effect of combined treatment with RA (1 μM) and PRL (5 μg/mL). Although RA treatment alone inhibited MAC-T cell proliferation, co-treatment of RA with PRL increased cell growth compared with the control group (treated with 1 μg/mL hydrocortisone and 5 μg/mL insulin). The ratio of Bcl to Bax mRNA was decreased in the RA treatment compared with RA+PRL or control. Retinoic acid-induced differentiation of MAC-T cells was associated with an increase in the mRNA expression of αS1-casein (3.9-fold), αS2-casein (4.5-fold), and β-casein (4.4-fold) compared with the control group. Expression of αS1-casein, αS2-casein, and β-casein was increased 12.9-fold, 11.9-fold, and 19.3-fold, respectively, following treatment with RA and PRL combined compared with the control group. These results demonstrate that RA induces differentiation of MAC-T cells and acts synergistically with PRL to increase specific casein gene expression.

  9. Enterococcus faecalis phosphomevalonate kinase.

    Science.gov (United States)

    Doun, Stephanie S; Burgner, John W; Briggs, Scott D; Rodwell, Victor W

    2005-05-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.

  10. Separation and characterization of mares' milk alpha(s1)-, beta-, kappa-caseins, gamma-casein-like, and proteose peptone component 5-like peptides.

    Science.gov (United States)

    Egito, A S; Miclo, L; López, C; Adam, A; Girardet, J M; Gaillard, J L

    2002-04-01

    The equine alpha(s1)- and beta-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The alpha(s1)-, beta-, and kappa-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. Kappa-casein was characterized after electrophoresis by glycoprotein-specific staining. To identify alpha(s1)-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified alpha(s1)-CN. These sequences, that could be estimated to correspond to 62% of the full protein, presented strong identities with regions of alpha(s1)-CN primary structures of other species. In particular, 51, 48, 43, and 40% identities were obtained with corresponding regions of sow, dromedary, cow, and human alpha(s1)-CN, respectively. On the other hand, trace amounts of equine gamma-CN-like and proteose peptone component 5-like peptides were found in the whole CN. They were identified by microsequencing and corresponded to beta-CN peptides generated by plasmin action on the whole CN. The equine alpha(s1), beta-, and kappa-CN were separated by bidimensional electrophoresis in numerous isoelectric variants with apparent isoelectric points distributed between pH 4.4 to 6.3, 4.4 to 5.9, and 3.5 to 5.5, respectively. The beta- and kappa-CN displayed a more acidic character in the mare than in the cow.

  11. Inhibitors of p21-activated kinases (PAKs).

    Science.gov (United States)

    Rudolph, Joachim; Crawford, James J; Hoeflich, Klaus P; Wang, Weiru

    2015-01-08

    The p21-activated kinase (PAK) family of serine/threonine protein kinases plays important roles in cytoskeletal organization, cellular morphogenesis, and survival, and members of this family have been implicated in many diseases including cancer, infectious diseases, and neurological disorders. Owing to their large and flexible ATP binding cleft, PAKs, particularly group I PAKs (PAK1, -2, and -3), are difficult to drug; hence, few PAK inhibitors with satisfactory kinase selectivity and druglike properties have been reported to date. Examples are a recently discovered group II PAK (PAK4, -5, -6) selective inhibitor series based on a benzimidazole core, a group I PAK selective series based on a pyrido[2,3-d]pyrimidine-7-one core, and an allosteric dibenzodiazepine PAK1 inhibitor series. Only one compound, an aminopyrazole based pan-PAK inhibitor, entered clinical trials but did not progress beyond phase I trials. Clinical proof of concept for pan-group I, pan-group II, or PAK isoform selective inhibition has yet to be demonstrated.

  12. Marked effect of beta-lactoglobulin polymorphism on the ratio of casein to total protein in milk.

    Science.gov (United States)

    Lundén, A; Nilsson, M; Janson, L

    1997-11-01

    The relationship between genetic variants for milk protein and the composition of milk was analyzed on 4475 repeated milk samples from individual cows; 371 dairy cows of the Swedish Red and White breed and 204 cows of the Swedish Holstein breed were used. The registrations included percentages of casein, protein, fat, and lactose in combination with milk yield and SCC. The genotype of individual cows for alpha(s1)-CN, beta-CN, kappa-CN, and beta-LG was determined by alkaline and acidic PAGE. A mixed animal model was used for the analysis; beta-LG and aggregate casein genotypes were included simultaneously as separate fixed effects in the statistical model. The results suggest a positive additive effect of the beta-LG B allele on casein content and on the ratio of casein to total protein. For the latter trait, the beta-LG genotype accounted for a relatively large part of the phenotypic variance, corresponding to a reduction in residual variance of 11% when included in the model. The corresponding value for casein content was 0.5%. The lack of unfavorable associations between milk protein variants and the traits included in this study makes the beta-LG gene an obvious candidate when the breeding objective is improved conversion of milk protein into cheese.

  13. Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli.

    Science.gov (United States)

    Yoo, Gu; Saenger, Thorsten; Bong, Ji-Hong; Jose, Joachim; Kang, Min-Jung; Pyun, Jae-Chul

    2015-12-01

    In this work, two proteins, Z-domains and bovine casein, were auto-displayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different auto-display vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-auto-displayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-auto-displayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-auto-displayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-auto-displayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with auto-displayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.

  14. Comparative analysis on the structural features of the 5' flanking region of κ-casein genes from six different species

    Directory of Open Access Journals (Sweden)

    Whitelaw C Bruce A

    2002-01-01

    Full Text Available Abstract κ-casein plays an essential role in the formation, stabilisation and aggregation of milk micelles. Control of κ-casein expression reflects this essential role, although an understanding of the mechanisms involved lags behind that of the other milk protein genes. We determined the 5'-flanking sequences for the murine, rabbit and human κ-casein genes and compared them to the published ruminant sequences. The most conserved region was not the proximal promoter region but an approximately 400 bp long region centred 800 bp upstream of the TATA box. This region contained two highly conserved MGF/STAT5 sites with common spacing relative to each other. In this region, six conserved short stretches of similarity were also found which did not correspond to known transcription factor consensus sites. On the contrary to ruminant and human 5' regulatory sequences, the rabbit and murine 5'-flanking regions did not harbour any kind of repetitive elements. We generated a phylogenetic tree of the six species based on multiple alignment of the κ-casein sequences. This study identified conserved candidate transcriptional regulatory elements within the κ-casein gene promoter.

  15. Update on Aurora Kinase Targeted Therapeutics in Oncology

    Science.gov (United States)

    Green, Myke R.; Woolery, Joseph E.

    2011-01-01

    Introduction Mammalian cells contain three distinct serine/threonine protein kinases with highly conserved catalytic domains, including aurora A and B kinases that are essential regulators of mitotic entry and progression. Overexpression of aurora A and/or B kinase is associated with high proliferation rates and poor prognosis, making them ideal targets for anti-cancer therapy. Disruption of mitotic machinery is a proven anti-cancer strategy employed by multiple chemotherapeutic agents. Numerous small molecule inhibitors of the aurora kinases have been discovered and tested in vivo and in vitro, with a few currently in phase II testing. Areas covered This review provides the reader with updated results from both preclinical and human studies for each of the aurora kinase inhibitors (AKI) that are currently being investigated. The paper also covers in detail the late breaking and phase I data presented for AKIs thereby allowing the reader to compare and contrast individual and classrelated effects of AKIs. Expert opinion While the successful development and approval of an AKI for anti-cancer therapy remains unresolved, pre-clinical identification of resistant mechanisms would help design better early phase clinical trials where relevant combinations may be evaluated prior to phase II testing. The authors believe that aurora kinases are important anti-cancer targets that operate in collaboration with other oncogenes intimately involved in uncontrolled tumor proliferation and by providing a unique, targeted and complimentary anti-cancer mechanism, expand the available armamentarium against cancer. PMID:21556291

  16. Kappa-Casein Genotypic Frequencies in Holstein-Friesian Dairy Cattle in West Java Province

    Directory of Open Access Journals (Sweden)

    A. Anggraeni

    2010-08-01

    Full Text Available Kappa casein (ĸ-cn gene as one of the four casein genes commonly has two variant alleles. B allele is positively correlated with milk proteins, so potentially be used as MAS to improve milk protein content. This study aimed to identify genetic variation of the ĸ-casein gene in Holstein-Friesian (HF from several dairy regions in West Java. Blood samples were taken through jugular vein of lactating heifers and cows, from intensive managements under the Local Government Dairy Breeding Station - Cikole (BBPT Cikole-SP (82 hds, Embryo Transfer Station (BET (50 hds, and Eco Farm of Animal Science Faculty of Bogor Agricultural University (EcoFarm (20 hds; as well as from semi-intensive managements of small dairy farmers under the supervision of the North Lembang Cooperative Unit (KPSBU Lembang in two villages of Cilumber (98 hds and Pasar Kemis (92 hds. Blood samples were also taken via cocsigalis vein of AI (active and non active HF bulls at the two National AI Stations in Lembang (25 hds and Singosari (32 hds. Identification of genetic variation was by applying PCR-RFLP method. Three genotypes were identified, namely AA, AB, and BB, resulting two alleles of A and B. Results entirely showed that the frequencies of the BB HF female were very low, with the range of 0%-6% (vs AA genotype= 10%-54% and AB genotype= 46%-85%, despite of the relatively high frequency of the B allele over the A allele (23%-48% vs. 52%-77%. The low frequency of the observed BB females might be due to the limited AI active BB bulls used for services by the two national AI stations (0%-4%.

  17. Casein haplotypes and their association with milk production traits in Norwegian Red cattle.

    Science.gov (United States)

    Nilsen, Heidi; Olsen, Hanne Gro; Hayes, Ben; Sehested, Erling; Svendsen, Morten; Nome, Torfinn; Meuwissen, Theo; Lien, Sigbjørn

    2009-02-20

    A high resolution SNP map was constructed for the bovine casein region to identify haplotype structures and study associations with milk traits in Norwegian Red cattle. Our analyses suggest separation of the casein cluster into two haplotype blocks, one consisting of the CSN1S1, CSN2 and CSN1S2 genes and another one consisting of the CSN3 gene. Highly significant associations with both protein and milk yield were found for both single SNPs and haplotypes within the CSN1S1-CSN2-CSN1S2 haplotype block. In contrast, no significant association was found for single SNPs or haplotypes within the CSN3 block. Our results point towards CSN2 and CSN1S2 as the most likely loci harbouring the underlying causative DNA variation. In our study, the most significant results were found for the SNP CSN2_67 with the C allele consistently associated with both higher protein and milk yields. CSN2_67 calls a C to an A substitution at codon 67 in beta-casein gene resulting in histidine replacing proline in the amino acid sequence. This polymorphism determines the protein variants A1/B (CSN2_67 A allele) versus A2/A3 (CSN2_67 C allele). Other studies have suggested that a high consumption of A1/B milk may affect human health by increasing the risk of diabetes and heart diseases. Altogether these results argue for an increase in the frequency of the CSN2_67 C allele or haplotypes containing this allele in the Norwegian Red cattle population by selective breeding.

  18. Application of Asian pumpkin (Cucurbita ficifolia) serine proteinase for production of biologically active peptides from casein.

    Science.gov (United States)

    Dąbrowska, Anna; Szołtysik, Marek; Babij, Konrad; Pokora, Marta; Zambrowicz, Aleksandra; Chrzanowska, Józefa

    2013-01-01

    The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

  19. Multiple host kinases contribute to Akt activation during Salmonella infection.

    Science.gov (United States)

    Roppenser, Bernhard; Kwon, Hyunwoo; Canadien, Veronica; Xu, Risheng; Devreotes, Peter N; Grinstein, Sergio; Brumell, John H

    2013-01-01

    SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

  20. Multiple host kinases contribute to Akt activation during Salmonella infection.

    Directory of Open Access Journals (Sweden)

    Bernhard Roppenser

    Full Text Available SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4 P2/PI(3-5 P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4 P2/PI(3-5 P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

  1. Plant phosphatidylinositol 3-kinase

    NARCIS (Netherlands)

    Lee, Y.; Munnik, T.; Munnik, T.

    2010-01-01

    Phosphatidylinositol 3-kinase (PI3K) phosphorylates the D-3 position of phosphoinositides. In Arabidopsis, only one PI3K exists, which belongs to the class-III PI3K subfamily which makes phosphatidylinositol 3-phosphate (PtdIns3P). The single AtPI3K gene is essential for survival, since loss of its

  2. The effect of sucrose on unfrozen water and syneresis of acidified sodium caseinate-xanthan gels.

    Science.gov (United States)

    Braga, A L M; Cunha, R L

    2005-07-01

    The influence of the ingredients of acidified Na caseinate-xanthan-sucrose gels on thermophysical properties and syneresis of the gels was studied. Sucrose concentration affected all of the gel equilibrium properties and the rate of syneresis. The positive effect of sucrose on syneresis and unfrozen water (UFW) values was attributed to different effects. The amount of UFW was governed mainly by the colligative properties of sucrose whereas the equilibrium syneresis behaviour was associated with the changes in network dynamics caused by the kosmotropic properties of sucrose. The latter could enhance xanthan-sucrose association or favour xanthan-protein interactions.

  3. Differential effects of casein versus whey on fasting plasma levels of insulin, IGF-1 and IGF-1/IGFBP-3

    DEFF Research Database (Denmark)

    Hoppe, Camilla; Mølgaard, Christian; Dalum, Cathrine

    2009-01-01

    to the identification of which components in milk that stimulate growth, we have performed an intervention study with 57 eight-year-old boys in which we examined the effects of the two major milk protein fractions, whey and casein, and milk minerals (Ca and P) in a 2x2 factorial design on IGFs and glucose......–insulin metabolism. The amounts of whey and casein were identical to the content in 1.5 l skim milk. The amounts of Ca and P were similar to 1.5 l skim milk in the high-mineral drinks, whereas the amounts of Ca and P were reduced in the low-mineral drinks. Results: There were no interactions between milk mineral...... groups (high, low) and milk protein groups (whey, casein). Serum IGF-1 increased by 15% (P...

  4. Quantification of αS1-casein in breast milk using a targeted mass spectrometry-based approach.

    Science.gov (United States)

    Altendorfer, Irina; König, Simone; Braukmann, Achim; Saenger, Thorsten; Bleck, Ellen; Vordenbäumen, Stefan; Kubiak, Anna; Schneider, Matthias; Jose, Joachim

    2015-01-25

    The caseins comprise a milk protein fraction of high nutritional value and, as more recently discovered, of immunologic relevance. In particular, αS1-casein (CSN1S1) is of interest being a potential autoantigen. So far, the concentration of caseins in human milk was primarily determined by indirect methods. The aim of this study was to directly measure the CSN1S1 content in breast milk using mass spectrometry (MS). The quantification was based on tryptic CSN1S1 peptides with the best response in liquid chromatography (LC)-MS/MS analysis. Targeted experiments allowed both specific and sensitive detection at the low fmol level. For this pilot study, twenty breast milk samples of the first week post-partum were analyzed and contained between 3 and 540μg/ml CSN1S1. Limitations of CSN1S1 quantification are discussed.

  5. Chaperone-like activity of β-casein and its effect on residual in vitro activity of horseradish peroxidase

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria; Olsen, Karsten; Sørensen, Jens Christian

    2014-01-01

    , as similar experiment with bovine serum albumin resulted in residual activity of horseradish peroxidase that was significantly lower than without any addition. The effect of β-casein on HRP disappears when pH is below the isoelectric point of β-casein. It was also proven by light scattering studies that β...

  6. RNA-sequencing data analysis of uterus in ovariectomized rats fed with soy protein isolate,17B-estradiol and casein

    Science.gov (United States)

    This data file describes the bioinformatics analysis of uterine RNA-seq data comparing genome wide effects of feeding soy protein isolate compared to casein to ovariectomized female rats age 64 days relative to treatment of casein fed rats with 5 ug/kg/d estradiol and relative to rats treated with e...

  7. Cross-linking of bovine and caprine caseins by microbial transglutaminase and their use as microencapsulating agents for n-3 fatty acids

    Science.gov (United States)

    Bovine and caprine caseins were cross-linked with microbial transglutaminase (mTG). The mTG-cross-linked bovine or caprine casein dispersion, mixed with 14.5% maltodextrin (DE = 40), was used to prepare emulsions with 10.5% algae oil. Oxidative stability of emulsions was evaluated by peroxide valu...

  8. Do different casein concentrations increase the adverse effect of rutin on the biology of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)?

    Science.gov (United States)

    Salvador, Mariana C; Boiça, Arlindo L; de Oliveira, Maria C N; da Graça, José P; da Silva, Débora M; Hoffmann-Campo, Clara B

    2010-01-01

    The flavonoid rutin is recognized as playing an important role in the protection of plants against lepidopterans. Bioassays with this compound are generally carried out using artificial diets. Proteins of high energy value, such as casein, are important ingredients of insect artificial diets as a source of essential amino acids. However, such proteins can generally increase the allelochemical activity. Our objective was to evaluate the effects of rutin on larvae of the velvetbean caterpillar Anticarsia gemmatalis Hübner by incorporating this allelochemical into diets with different concentrations of casein. Three casein concentrations (0, 7 g, or 14 g) combined with none, 0.65%, or 1.30% of rutin were added to the rearing diet and offered to the larvae from hatching to pupation. Rutin negatively affected larval development, the amount of food consumed, and pupal weight of A. gemmatalis. These negative effects were clearly seen in insects fed on diets with 7 g of casein to which any concentration of rutin was added. The effects of rutin when added to the diets without casein were stronger than in diets containing a suitable amount of casein (14 g). The greater negative effects of rutin in diets containing suboptimal concentrations of casein indicate that casein can increase the effects of rutin only when the diets are nutritionally unsuitable for insect development.

  9. Modification of Casein and its Application in Leather Finishing%酪素的改性方法及其在皮革涂饰中的应用

    Institute of Scientific and Technical Information of China (English)

    唐瑶; 付柯; 赵丹; 张哲

    2015-01-01

    简述了酪素的性质,综述了酪素的改性方法及在皮革涂饰中的应用。%The properties of casein were briefly introduced. Modification methods of casein and its applica-tion in synthesizing leather finishing agents were reviewed.

  10. Effects of soy vs. casein protein on body weight and glycemic control in female monkeys and their offspring.

    Science.gov (United States)

    Wagner, Janice D; Jorgensen, Matthew J; Cline, J Mark; Lees, Cynthia J; Franke, Adrian A; Zhang, Li; Ayers, Melissa R; Schultz, Carrie; Kaplan, Jay R

    2009-09-01

    Nutritional interventions are important for reducing obesity and related conditions. Soy is a good source of protein and also contains isoflavones that may affect plasma lipids, body weight, and insulin action. Described here are data from a monkey breeding colony in which monkeys were initially fed a standard chow diet that is low fat with protein derived from soy. Monkeys were then randomized to a defined diet with a fat content similar to the typical American diet (TAD) containing either protein derived from soy (TAD soy) or casein-lactalbumin (TAD casein). The colony was followed for over two years to assess body weight, and carbohydrate and lipid measures in adult females (n=19) and their offspring (n=25). Serum isoflavone concentrations were higher with TAD soy than TAD casein, but not as high as when monkey chow was fed. Offspring consuming TAD soy had higher serum isoflavone concentrations than adults consuming TAD soy. Female monkeys consuming TAD soy had better glycemic control, as determined by fructosamine concentrations, but no differences in lipids or body weight compared with those consuming diets with TAD casein. Offspring born to dams consuming TAD soy had similar body weights at birth but over a two-year period weighed significantly less, had significantly lower triglyceride concentrations, and like adult females, had significantly lower fructosamine concentrations compared to TAD casein. Glucose tolerance tests in adult females were not significantly different with diet, but offspring eating TAD soy had increased glucose disappearance with overall lower glucose and insulin responses to the glucose challenge compared with TAD casein. Potential reasons for the additional benefits of TAD soy observed in offspring but not in adults may be related to higher serum isoflavone concentrations in offspring, presence of the diet differences throughout more of their lifespan (including gestation), or different tissue susceptibilities in younger animals.

  11. Immediate and residual effects of heat stress and restricted intake on milk protein and casein composition and energy metabolism.

    Science.gov (United States)

    Cowley, F C; Barber, D G; Houlihan, A V; Poppi, D P

    2015-04-01

    The effects of heat stress on dairy production can be separated into 2 distinct causes: those effects that are mediated by the reduced voluntary feed intake associated with heat stress, and the direct physiological and metabolic effects of heat stress. To distinguish between these, and identify their effect on milk protein and casein concentration, mid-lactation Holstein-Friesian cows (n = 24) were housed in temperature-controlled chambers and either subjected to heat stress [HS; temperature-humidity index (THI) ~78] or kept in a THIheat-stressed cows (TN-R) for 7 d. A control group of cows was kept in a THIheat stress. Heat stress reduced the milk protein concentration, casein number, and casein concentration and increased the urea concentration in milk beyond the effects of restriction of intake. Under HS, the proportion in total casein of αS1-casein increased and the proportion of αS2-casein decreased. Because no effect of HS on milk fat or lactose concentration was found, these effects appeared to be the result of specific downregulation of mammary protein synthesis, and not a general reduction in mammary activity. No residual effects were found of HS or TN-R on milk production or composition after THIHeat-stressed cows had elevated blood concentrations of urea and Ca, compared with TN-R and TN-AL. Cows in TN-R had higher serum nonesterified fatty acid concentrations than cows in HS. It was proposed that HS and TN-R cows may mobilize different tissues as endogenous sources of energy.

  12. Rennet-induced gelation of concentrated milk in the presence of sodium caseinate: differences between milk concentration using ultrafiltration and osmotic stressing.

    Science.gov (United States)

    Krishnankutty Nair, P; Corredig, M

    2015-01-01

    Concentrating milk is a common unit operation in the dairy industry. With the reduction of water, the particles interact more frequently with each other and the functionality of the casein micelles may depend on the interactions occurring during concentration. The objective of this research was to investigate the effect of concentration on the renneting properties of the casein micelles by comparing 2 concentration methods: ultrafiltration and osmotic stressing. Both methods selectively concentrate the protein fraction of milk, while the composition of the soluble phase is unaltered. To evaluate possible differences in the rearrangements of the casein micelles during concentration, renneting properties were evaluated with or without the addition of soluble caseins, added either before or after concentration. The results indicate that casein micelles undergo rearrangements during concentration and that shear during membrane filtration may play a role in affecting the final properties of the milk.

  13. Dystroglycan loss disrupts polarity and beta-casein induction inmammary epithelial cells by perturbing laminin anchoring

    Energy Technology Data Exchange (ETDEWEB)

    Weir, M. Lynn; Oppizzi, Maria Luisa; Henry, Michael D.; Onishi,Akiko; Campbell, Kevin P.; Bissell, Mina J.; Muschler, John L.

    2006-02-17

    Precise contact between epithelial cells and their underlying basement membrane is critical to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG expression in cultured mammary epithelial cells (MECs). Strikingly, DG loss disrupted laminin-111-induced polarity and {beta}-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. DG re-expression restored these deficiencies. Investigations of mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when the entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in MECs that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity, and {beta}-casein induction. The observed loss of laminin-111 assembly and signaling in DG-/-MECs provides insights into the signaling changes occurring in breast carcinomas and other cancers, where DG's laminin-binding function is frequently defective.

  14. Novel hierarchical microparticles super-assembled from nanoparticles with the induction of casein micelles

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Xiaopeng, E-mail: xpxiong@xmu.edu.cn; Duan, Jiangjiang; Wang, Yong; Yu, Zhaoju [Xiamen University, Department of Materials Science and Engineering, College of Materials (China)

    2013-08-15

    We have demonstrated a solution-based synthesis of novel waxberry-like hierarchical ZnO microparticles in the presence casein micelles under mild conditions. The microstructures of the sub-micrometer-sized hierarchical microparticles were characterized, and the synthesis conditions were optimized. The formation mechanism of the hierarchical microparticle was analyzed through control experiments. The hierarchical ZnO microparticles are found to be super-assemblies of 30-70 nm ZnO nanoparticles, which are thought to be based on casein micelle induction followed by Ostwald ripening. In the same manner, copper-based hierarchical microparticles with a similar morphology have also been successfully synthesized. By controlling the synthetic time or temperature, solid or hollow microparticles can be fabricated. The narrowly distributed ZnO microparticles have a high specific surface area, exhibiting great potential application in fields such as photocatalytic and energy conversion. Our findings may meanwhile open a new bottom-up strategy in order to construct structurally sophisticated nanomaterials.

  15. Encapsulation of antioxidant peptide enriched casein hydrolysate using maltodextrin-gum arabic blend.

    Science.gov (United States)

    Rao, Priyanka Singh; Bajaj, Rajesh Kumar; Mann, Bimlesh; Arora, Sumit; Tomar, S K

    2016-10-01

    Antioxidant peptide enriched casein hydrolysate (AO-CH) are receiving increasing attention due to their potential as functional ingredient. Encapsulation of AO-CH using maltodextrin-gum arabic (MD/GA) as wall material could represent an attractive approach to overcome the problems related to their direct application. Encapsulation parameter were optimized using different ratio of core to coat and proportion of coating material (10:0, 8:2, 6:4) under varying pH (2-8) for encapsulation efficiency (EE).The preparation P3 resulted in maximum EE (87%) using core to coat ratio 1:20, at pH 6.0 with 8:2 MD/GA ratio. The encapsulated preparation showed reduced bitterness (p < 0.05) compared to the casein hydrolysate together with maximum retention of antioxidant activity (93%). Further, the narrow range of particle size, indicates their better stability and represents a promising food additive for incorporation in food.

  16. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    Energy Technology Data Exchange (ETDEWEB)

    Medina, D.; Oborn, C.J. (Baylor College of Medicine, Houston, TX (USA)); Li, M.L.; Bissell, M.J. (Univ. of California, Berkeley (USA))

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  17. How does oil type determine emulsion characteristics in concentrated Na-caseinate emulsions?

    Science.gov (United States)

    Tan, Hui Lin; McGrath, Kathryn M

    2013-08-01

    Macroscopic properties and ensemble average diffusion of concentrated (dispersed phase 50-60 wt%) Na-caseinate-stabilised emulsions for three different oils (soybean oil, palm olein and tetradecane) were explored. On a volume fraction basis, pulsed gradient stimulated echo (PGSTE)-NMR data show that droplet dynamics for all three systems are similar within a region of the emulsion morphology diagram. The exact limits of the emulsion space depend however on which oil is considered. The reduced solubility of tetradecane in water, and Na-caseinate in tetradecane, result in the stabilisation of flocs during formulation. Floc formation is not observed when soybean oil or palm olein is used under identical emulsion formulation conditions. Linear rheology experiments provide indirect evidence that the local structure and the properties of the thin film interfacial domain of tetradecane emulsions vary from those of soybean oil and palm olein emulsions. Collectively these data indicate that protein/oil interactions within a system dominate over specific oil droplet structure and size distribution, which are similar in the three systems.

  18. Genetic polymorphism of β-lactoglobulin and κ-casein of cattle breeds in Croatia

    Directory of Open Access Journals (Sweden)

    Ante Ivanković

    2011-12-01

    Full Text Available Profitable milk production respects the interests of producers, processing industries, consumer requirements and welfare of animals. Development of new methods of direct gene analysis responsible for milk proteins polymorphism provide new tools to raise the profitability of milk production and dairy products through implementation of breed genetic profile in breeding program. Because of necessity to determinate genetic profiles of cattle breeds in Croatia using new analytical methods, the ratio of dominant allelic polymorphic variants of beta-lactoglobulin (β-Lg and kappa-casein (κ-CN is defined. The share of beta-lactoglobulin B variant is dominant in all investigated cattle breeds (>52.9 %. Kappa- casein allelic variant A is dominant in selected cattle breeds (60.7-76.4 %, while the share of B variant is significantly more presented in autochthonous cattle breeds (48.2-84.1 %. Knowledge about genetic profile of breeds due to studied polymorphic variants of milk proteins is useful in further breeding development and economic reaffirmation of cattle breeds, especially autochthonous ones.

  19. A sialic acid assay in isolation and purification of bovine k-casein glycomacropeptide: a review.

    Science.gov (United States)

    Nakano, Takuo; Ozimek, Lech

    2014-01-01

    Sialic acid is a carbohydrate moiety of k-casein glycomacropeptide (GMP), which is a 64 amino acid residue C-terminal sialylated phosphorylated glycopeptide released from k-casein by the action of chymosin during cheese making. GMP lacks aromatic amino acids including phenylalanine, tyrosine, and tryptophan. Because of its unique amino acid composition and various biological activities, GMP is thought to be a potential ingredient for dietetic foods (e.g., a food for PKU patients) and pharmaceuticals. Thus, increased attention has been given to the development of techniques to purify GMP. In this review, techniques of GMP purification described in patents and scientific research papers were introduced. A sialic acid assay is the important method to track GMP isolation and purification processes, for which the thiobarbituric acid reaction with 1-propanol as a chromophore extracting solvent is an inexpensive, practical and specific technique. Sephacryl S-200 gel filtration chromatography, cellulose acetate electrophoresis, and sodium dodecyl sulfate polyacrylamide gel electrophoresis are the major techniques to identify sialic acid specific to GMP. Sephacryl S-200 chromatography and cellulose acetate electrophoresis are also used to detect GMP sialic acid in whey pearmeate and whey added commercial margarine samples. Future research includes development of an economical industrial scale method to produce high purity GMP.

  20. Interfacial and foaming interactions between casein glycomacropeptide (CMP) and propylene glycol alginate.

    Science.gov (United States)

    Martinez, María J; Pizones Ruiz-Henestrosa, Víctor M; Carrera Sánchez, Cecilio; Rodríguez Patino, Juan M; Pilosof, Ana M R

    2012-06-15

    Proteins and polysaccharides are widely used in food formulation. While most of the proteins are surface active, only few polysaccharides can adsorb at the air-water interface; this is the case of propylene glycol alginates (PGA). It is known that casein glycomacropeptide (CMP), a bioactive polypeptide derived from κ-casein by the action of chymosin, presents a great foaming capacity but provides unstable foams. So, the objective of this work was to analyze the impact of mixing CMP and a commercial variety of PGA, Kelcoloid O (KO), on the interfacial and foaming properties at pH 7.0. It was determined the surface pressure isotherm, the dynamics of adsorption and the foaming properties for CMP, KO and the mixed system CMP-KO. CMP dominated the surface pressure of CMP-KO mixed system. The presence of KO synergistically improved the viscoelastic properties of surface film. The foaming capacity of CMP was altered by KO. KO foam presented a higher stability than CMP foam and it controlled the stability against drainage and the initial collapse in the mixed foam.

  1. Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey.

    Science.gov (United States)

    Baieli, María F; Urtasun, Nicolás; Martinez, María J; Hirsch, Daniela B; Pilosof, Ana M R; Miranda, María V; Cascone, Osvaldo; Wolman, Federico J

    2017-01-01

    Casein glycomacropeptide (CMP) is a 64- amino acid peptide found in cheese whey, which is released after κ-casein specific cleavage by chymosin. CMP lacks aromatic amino acids, a characteristic that makes it usable as a nutritional supplement for people with phenylketonuria. CMP consists of two nonglycosylated isoforms (aCMP A and aCMP B) and its different glycosylated forms (gCMP A and gCMP B). The most predominant carbohydrate of gCMP is N-acetylneuraminic acid (sialic acid). Here, we developed a CMP purification process based on the affinity of sialic acid for wheat germ agglutinin (WGA). After formation of chitosan beads and adsorption of WGA, the agglutinin was covalently attached with glutaraldehyde. Two matrices with different WGA density were assayed for CMP adsorption. Maximum adsorption capacities were calculated according to the Langmuir model from adsorption isotherms developed at pH 7.0, being 137.0 mg/g for the matrix with the best performance. In CMP reduction from whey, maximum removal percentage was 79% (specifically 33.7% of gCMP A and B, 75.8% of aCMP A, and 93.9% of aCMP B). The CMP was recovered as an aggregate with an overall yield of 64%. Therefore, the matrices developed are promising for CMP purification from cheese whey. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:171-180, 2017.

  2. Impact of cationic surfactant on the self-assembly of sodium caseinate.

    Science.gov (United States)

    Vinceković, Marko; Curlin, Marija; Jurašin, Darija

    2014-08-27

    The impact of a cationic surfactant, dodecylammonium chloride (DDACl), on the self-assembly of sodium caseinate (SC) has been investigated by light scattering, zeta potential, and rheological measurements as well as by microscopy (transmission electron and confocal laser scanning microscopy). In SC dilute solutions concentration-dependent self-assembly proceeds through the formation of spherical associates and their aggregation into elongated structures composed of connected spheres. DDACl interacts with SC via its hydrophilic and hydrophobic groups, inducing changes in SC self-assembled structures. These changes strongly depend on the surfactant aggregation states (monomeric or micellar) as well as concentration ratio of both components, leading to the formation of soluble and insoluble complexes of nano- to microdimensions. DDACl monomers interact with SC self-assembled entities in a different way compared to their micelles. Surfactant monomers form soluble complexes (similar to surfactant mixed micelles) at lower SC concentration but insoluble gelatinous complexes at higher SC concentration. At surfactant micellar concentration soluble complexes with casein chains wrapped around surfactant micelles are formed. This study suggests that the use of proper cationic surfactant concentration will allow modification and control of structural changes of SC self-assembled entities.

  3. Treatment of post-orthodontic white spot lesions with casein phosphopeptide-stabilised amorphous calcium phosphate.

    Science.gov (United States)

    Bröchner, Ann; Christensen, Carsten; Kristensen, Bjarne; Tranæus, Sofia; Karlsson, Lena; Sonnesen, Liselotte; Twetman, Svante

    2011-06-01

    This study aims to investigate the effect of topical applications of 10% casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) on white spot lesions (WSL) detected after treatment with fixed orthodontic appliances. Sixty healthy adolescents with ≥1 clinically visible WSL at debonding were recruited and randomly allocated to a randomised controlled trial with two parallel groups. The intervention group was instructed to topically apply a CPP-ACP -containing agent (Tooth Mousse, GC Europe) once daily and the subjects of the control group brushed their teeth with standard fluoride toothpaste. The intervention period was 4 weeks and the endpoints were quantitative light-induced fluorescence (QLF) on buccal surfaces of the upper incisors, cuspids and first premolars and visual scoring from digital photos. The attrition rate was 15%, mostly due to technical errors, and 327 lesions were included in the final evaluation. A statistically significant (p white spot lesions after debonding of orthodontic appliances with a casein phosphopeptide-stabilised amorphous calcium phosphate agent resulted in significantly reduced fluorescence and a reduced area of the lesions after 4 weeks as assessed by QLF. The improvement was however not superior to the "natural" regression following daily use of fluoride toothpaste.

  4. Is gluten free and casein free (GFCF diet effective for individuals with autism?

    Directory of Open Access Journals (Sweden)

    Indah S. Widyahening

    2011-05-01

    Full Text Available Background: Even though the etiology of autistic spectrum disorder (ASD has not been clearly known, various types of therapies have been offered. One of the most popular therapies is Gluten Free and Casein Free Diet – GFCF diet.Methods: A structured internet literature search was conducted using Pubmed and Cochrane library. Titles and abstracts were screened using predetermined selection criteria. Critical appraisal was conducted based on standard criteria for relevance, validity, and levels of evidence.Results: Based on relevance, only four out of twelve selected articles can be used; one is a systematic review and three randomized controlled trials (RCTs. Several methodological problems which could lead to over-estimation of the effect were found in the studies including poor randomization and selective drop-out. Risk of under-estimation of report due to small number of participants and in-adequate duration of intervention was also found. Small to moderate improvement were found in some outcome measured including overall autistic traits, social isolation, and the ability in communication and interaction. However, insignificant differences also found in some area such as individual’s behavior, cognitive and motor function.Conclusion: Available studies fail to provide sufficiently credible and strong evidence to recommend the practice of GFCF diet in treating autism. (Med J Indones 2011; 20:114-8Keywords: autism, gluten free and casein free diet

  5. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

    Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

  6. Enterococcus faecalis phosphomevalonate kinase

    OpenAIRE

    Doun, Stephanie S.; Burgner, John W.; Briggs, Scott D.; Rodwell, Victor W.

    2005-01-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone D...

  7. Oncoprotein protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Lin, Anning (La Jolla, CA); Davis, Roger (Princeton, MA); Derijard, Benoit (Shrewsbury, MA)

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  8. Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)2] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38MAPK/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment.

    Science.gov (United States)

    Filomeni, Giuseppe; Cardaci, Simone; Da Costa Ferreira, Ana Maria; Rotilio, Giuseppe; Ciriolo, Maria Rosa

    2011-08-01

    We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N']copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

  9. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  10. Probing the characteristics of casein as green binder for non-aqueous electrochemical double layer capacitors' electrodes

    Science.gov (United States)

    Varzi, Alberto; Raccichini, Rinaldo; Marinaro, Mario; Wohlfahrt-Mehrens, Margret; Passerini, Stefano

    2016-09-01

    Casein from bovine milk is evaluated in this work as binding agent for electrochemical double layer capacitors (EDLCs) electrodes. It is demonstrated that casein provides excellent adhesion strength to the current collector (1187 kPa compared to 51 kPa achieved with PVdF), thus leading to mechanically stable electrodes. At the same time, it offers high thermal stability (above 200 °C) and electrochemical stability in organic electrolytes. Apparently though, the casein-based electrodes offer lower electronic conductivity than those based on other state-of-the-art binders, which can limit the rate performance of the resulting EDLC. In the attempt of improving the electrochemical performance, it is found that the application of a pressing step can solve this issue, leading to excellent rate capability (up to 84% capacitance retention at 50 mA cm-2) and cycling stability (96.8% after 10,000 cycles at 10 mA cm-2) in both PC- and ACN-based electrolytes. Although the adhesive power casein is known since ancient times, this report presents the first proof of concept of its employment in electrochemical power sources.

  11. Possible mechanisms behind the differential effects of soy protein and casein feedings on colon cancer biomarkers in the rat

    NARCIS (Netherlands)

    Vis, E.H.; Geerse, G.J.; Klaassens, E.S.; Boekel, van M.A.J.S.; Alink, G.M.

    2005-01-01

    In the present studies, several hypotheses were tested to explain previously reported differential effects of soy and casein on colon cancer biomarkers like cell proliferation, fecal fat, fecal bile acid, alkaline phosphatase, and magnesium excretion in rats. In Study 1, the effect of methionine, a

  12. Nutritional Impact of a Gluten-Free Casein-Free Diet in Children with Autism Spectrum Disorder

    Science.gov (United States)

    Marí-Bauset, Salvador; Llopis-González, Agustín; Zazpe, Itziar; Marí-Sanchis, Amelia; Suárez-Varela, María Morales

    2016-01-01

    We compared anthropometric values, nutrient intake, the Healthy Eating Index and food variety in children with autism spectrum disorder (ASD), 20 on a gluten-free casein-free (GFCF) diet and 85 on a regular diet in Valencia (Spain) using 3-days food diaries. Those on the GFCF diet had a lower weight, body mass index, and total energy, pantothenic…

  13. EFFECT OF CASEIN-BASED SEMISYNTHETIC FOOD ON RENAL ACID EXCRETION AND ACID-BASE STATE OF BLOOD IN DOGS

    NARCIS (Netherlands)

    ZIJLSTRA, WG; LANGBROEK, AJM; KRAAN, J; RISPENS, P; NIJMEIJER, A

    1995-01-01

    Urinary acid excretion and blood acid-base stare were determined in dogs fed a casein-based semi-synthetic food (SSF), to which different amounts of salts had been added, in comparison with feeding normal dog food. Net acid excretion (NAE) and inorganic acid excretion (IAE) increased during SSF feed

  14. Influence of calcium chelators on concentrated micellar casein solutions : from micellar structure to viscosity and heat stability

    NARCIS (Netherlands)

    Kort, de E.J.P.

    2012-01-01

    In practice it is challenging to prepare a concentrated medical product with high heat stability
    and low viscosity. Calcium chelators are often added to dairy products to improve heat stability,
    but this may increase viscosity through interactions with the casein proteins. The aim of thi

  15. Whey Protein Delays Gastric Emptying and Suppresses Plasma Fatty Acids and Their Metabolites Compared to Casein, Gluten, and Fish Protein

    DEFF Research Database (Denmark)

    Stanstrup, Jan; Schou, Simon S; Holmer-Jensen, Jens

    2014-01-01

    Whey protein has been demonstrated to improve fasting lipid and insulin response in overweight and obese individuals. To establish new hypotheses for this effect and to investigate the impact of stomach emptying, we compared plasma profiles after intake of whey isolate (WI), casein, gluten (GLU...

  16. Gluten-Free and Casein-Free Diets in the Treatment of Autism Spectrum Disorders: A Systematic Review

    Science.gov (United States)

    Mulloy, Austin; Lang, Russell; O'Reilly, Mark; Sigafoos, Jeff; Lancioni, Giulio; Rispoli, Mandy

    2010-01-01

    This paper systematically reviews research on the effects of gluten-free and/or casein-free (GFCF) diets in the treatment of ASD. Database, hand, and ancestry searches identified 15 articles for review. Each study was analyzed and summarized in terms of (a) participants, (b) specifics of the intervention, (c) dependent variables, (d) results, and…

  17. Addendum to "Gluten-Free and Casein-Free Diets in Treatment of Autism Spectrum Disorders: A Systematic Review"

    Science.gov (United States)

    Mulloy, Austin; Lang, Russell; O'Reilly, Mark; Sigafoos, Jeff; Lancioni, Giulio; Rispoli, Mandy

    2011-01-01

    Shortly after the publication of our literature review on gluten-free and casein-free (GFCF) diets in the treatment of autism spectrum disorders (ASD; Mulloy et al., 2010), Whiteley et al. (2010) published the results of experimental evaluation of a GFCF diet. To update our previous literature review, we herein provide an analysis of the Whiteley…

  18. The effects of transglutaminase catalysis on the functional and immunoglobulin binding properties of peanut flour dispersions containing casein.

    Science.gov (United States)

    The functionality of light roasted peanut flour (PF) dispersions containing supplemental casein (CN), was altered after polymerization with microbial transglutaminase (TGase). The formation of high molecular weight covalent cross-links was observed with potential formation of PF-PF, PF-CN, and CN-CN...

  19. Enzymatic hydrolysis of whey and casein protein- effect on functional, rheological, textural and sensory properties of breads.

    Science.gov (United States)

    Gani, Adil; Broadway, A A; Masoodi, Farooq Ahmad; Wani, Ali Abas; Maqsood, Sajid; Ashwar, Bilal Ahmad; Shah, Asima; Rather, Sajad Ahmad; Gani, Asir

    2015-12-01

    Milk proteins were hydrolyzed by papain and their effect on the rheological, textural and sensory properties of bread were investigated. Water absorption capacity, emulsification capacity, foam volume, foam stability and solubility of Whey and casein protein concentrates and their hydrolysates were determined. The farinograph parameters of wheat flour and blends of wheat flour with casein and whey protein and their hydrolysates were determined to evaluate changes in water absorption capacity, dough development time, dough stability time and mixing tolerance index. The incorporation of WPC, casein and their hydrolysates up to the level of 5 % showed dough properties comparable to control. It was also found that 5 % level incorporation of milk proteins and their hydrolysates have no drastic effect on physical and sensory attributes of bread. The pasting properties showed significant decrease (p ≤ 0.05) when compared with wheat flour at all levels of addition of whey and casein protein concentrates and hydrolysates. Scanning electron microscopy of bread samples shows disruption in the well-defined protein - starch complex of wheat flour bread and the structure of gluten was weak as the concentration of whey protein increases in the wheat flour bread.

  20. Diacylglycerol Kinase Inhibition and Vascular Function.

    Science.gov (United States)

    Choi, Hyehun; Allahdadi, Kyan J; Tostes, Rita C A; Webb, R Clinton

    2009-01-01

    Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction.

  1. Kinase inhibitors: a new class of antirheumatic drugs

    Directory of Open Access Journals (Sweden)

    Kyttaris VC

    2012-09-01

    Full Text Available Vasileios C KyttarisDivision of Rheumatology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USAAbstract: The outlook for patients with rheumatoid arthritis has improved significantly over the last three decades with the use of disease-modifying antirheumatic drugs. However, despite the use of methotrexate, cytokine inhibitors, and molecules targeting T and B cells, a percentage of patients do not respond or lose their response over time. The autoimmune process in rheumatoid arthritis depends on activation of immune cells, which utilize intracellular kinases to respond to external stimuli such as cytokines, immune complexes, and antigens. In the past decade, small molecules targeting several kinases, such as p38 MAPK, Syk, and JAK have been developed. Several p38 MAPK inhibitors proved ineffective in treating rheumatoid arthritis. The Syk inhibitor, fostamatinib, proved superior to placebo in Phase II trials and is currently under Phase III investigation. Tofacitinib, a JAK1/3 inhibitor, was shown to be efficacious in two Phase III trials, while VX-509, a JAK3 inhibitor, showed promising results in a Phase II trial. Fostamatinib and tofacitinib were associated with increased rates of infection, elevation of liver enzymes, and neutropenia. Moreover, fostamatinib caused elevations of blood pressure and diarrhea, while tofacitinib was associated with an increase in creatinine and elevation of lipid levels.Keywords: rheumatoid arthritis, kinase inhibitors, mitogen-activated phosphokinase p38, spleen tyrosine kinase, Janus kinases

  2. Fission yeast Cdk7 controls gene expression through both its CAK and C-terminal domain kinase activities.

    Science.gov (United States)

    Devos, Maxime; Mommaerts, Elise; Migeot, Valerie; van Bakel, Harm; Hermand, Damien

    2015-05-01

    Cyclin-dependent kinase (Cdk) activation and RNA polymerase II transcription are linked by the Cdk7 kinase, which phosphorylates Cdks as a trimeric Cdk-activating kinase (CAK) complex, and serine 5 within the polymerase II (Pol II) C-terminal domain (CTD) as transcription factor TFIIH-bound CAK. However, the physiological importance of integrating these processes is not understood. Besides the Cdk7 ortholog Mcs6, fission yeast possesses a second CAK, Csk1. The two enzymes have been proposed to act redundantly to activate Cdc2. Using an improved analogue-sensitive Mcs6-as kinase, we show that Csk1 is not a relevant CAK for Cdc2. Further analyses revealed that Csk1 lacks a 20-amino-acid sequence required for its budding yeast counterpart, Cak1, to bind Cdc2. Transcriptome profiling of the Mcs6-as mutant in the presence or absence of the budding yeast Cak1 kinase, in order to uncouple the CTD kinase and CAK activities of Mcs6, revealed an unanticipated role of the CAK branch in the transcriptional control of the cluster of genes implicated in ribosome biogenesis and cell growth. The analysis of a Cdc2 CAK site mutant confirmed these data. Our data show that the Cdk7 kinase modulates transcription through its well-described RNA Pol II CTD kinase activity and also through the Cdc2-activating kinase activity.

  3. Fat digestion in veal calves fed milk replacers low or high in calcium and containing either casein or soy protein isolate.

    Science.gov (United States)

    Yuangklang, C; Wensing, Th; Van den Broek, L; Jittakhot, S; Beynen, A C

    2004-04-01

    The hypothesis tested was that the inhibitory effect of dietary soy protein versus casein on fat digestion in veal calves would be smaller when diets were fed with high instead of low calcium content. Male calves, 1 wk of age, were fed 1 of 4 experimental milk replacers in a 2 x 2 factorial design. There were 19 animals per dietary group. The milk replacers contained either casein or soy protein isolate as variable protein source and were either low or high in calcium. Body weight gain was not significantly affected by the experimental diets. Soy protein isolate versus casein significantly reduced apparent fat digestibility. High versus low calcium intake also depressed fat digestion. The protein effect was smaller (2.9% units) for the high than the low calcium diets (3.6% units), but the interaction did not reach statistical significance. Soy protein isolate versus casein raised fecal bile acid excretion and so did high versus low calcium intake. The difference in bile acid excretion between the soy and casein containing diets was significantly greater for the high than low calcium diets. The absorption of phosphorus, calcium, and magnesium was higher for the casein diets than for the soy-containing diets. This study shows for the first time that soy protein isolate versus casein depressed fat digestion and raised fecal bile acid excretion in veal calves.

  4. Using self-consistent-field theory to understand enhanced steric stabilization by casein-like copolymers at low surface coverage in mixed protein layers.

    Science.gov (United States)

    Parkinson, Emma L; Ettelaie, Rammile; Dickinson, Eric

    2005-01-01

    We present a statistical mechanical approach to predicting the properties of mixed copolymer layers using the Scheutjens-Fleer self-consistent-field theory. Our model copolymers are based on the primary structures of the major bovine casein monomers, alpha(s1)-casein and beta-casein. Numerical calculations have been carried out to determine the polymer segment density profiles at an isolated hydrophobic surface and the interaction forces as a pair of polymer-coated surfaces is brought to close interlayer separation. For a copolymer model containing hydrophilic and hydrophobic segments, we show how the steric stabilizing capacity of a casein-like macromolecule at very low surface coverage is enhanced in the presence of a thin dense layer of shorter tethered amphiphilic chains. Using a more refined protein model, which also distinguishes between the charged and uncharged hydrophilic segments along the chain, we clearly demonstrate that the enhanced steric repulsion from beta-casein exceeds that from alpha(s1)-casein. These calculations explain how the replacement of just a few percent of beta-lactoglobulin by casein can inhibit the heat-induced thickening and flocculation behavior observed experimentally with some whey protein-stabilized oil-in-water emulsions.

  5. Viability of the microencapsulation of a casein hydrolysate in lipid microparticles of cupuacu butter and stearic acid

    Directory of Open Access Journals (Sweden)

    Samantha Cristina Pinho

    2013-04-01

    Full Text Available Normal 0 21 false false false PT-BR X-NONE X-NONE Solid lipid microparticles produced with a mixture of cupuacu butter and stearic acid were used to microencapsulate a commercial casein hydrolysate (Hyprol 8052. The composition of the lipid matrix used for the production of the lipid microparticles was chosen according to data on the wide angle X-ray diffraction (WAXD and differential scanning calorimetry (DSC of bulk lipid mixtures, which indicated that the presence of 10 % cupuacu butter was sufficient to significantly change the crystalline arrangement of pure stearic acid. Preliminary tests indicated that a minimum proportion of 4 % of surfactant (polysorbate 80 was necessary to produce empty spherical lipid particles with average diameters below 10 mm. The lipid microparticles were produced using 20 % cupuacu butter and 80 % stearic acid and then stabilized with 4 % of polysorbate 80, exhibiting an encapsulation efficiency of approximately 74 % of the casein hydrolysate. The melting temperature of the casein hydrolysate-loaded lipid microparticles was detected at 65.2 °C, demonstrating that the particles were solid at room temperature as expected and indicating that the incorporation of peptides had not affected their thermal behavior. After 25 days of storage, however, there was a release of approximately 30 % of the initial amount of encapsulated casein hydrolysate. This release was not thought to have been caused by the liberation of encapsulated casein hydrolysate. Instead, it was attributed to the possible desorption of the adsorbed peptides present on the surface of the lipid microparticles.

  6. Composite β-κ-casein genotypes and their effect on composition and coagulation of milk from Estonian Holstein cows.

    Science.gov (United States)

    Vallas, M; Kaart, T; Värv, S; Pärna, K; Jõudu, I; Viinalass, H; Pärna, E

    2012-11-01

    The objective of this study was to estimate the effect of composite β-κ-CN genotypes on milk coagulation and composition traits, and on the additive genetic variation of these traits in Estonian Holstein dairy cattle. A total of 23,970 milk samples, repeated measurements from the first to third lactation from 2,859 Estonian Holstein cows from 78 herds across the country, were analyzed for milk yield, milk fat and protein percentages, somatic cell count, and milk coagulation properties (milk coagulation time and curd firmness). Each cow had at least 3 measurements per lactation. Two single-trait random regression animal models were fitted for the traits studied. The first model considered fixed effects of year-season of sampling and year-season of calving, calving age (nested within lactation), sample age (only for milk coagulation traits) and days in milk, and random herd, additive genetic, and permanent environmental effects. The animal and permanent environmental effects were modeled over the lactation period by using Legendre polynomials. The second model had the additional fixed β-κ-casein effect in the form of a third-order Legendre polynomial. The 2 most frequent β-κ-casein composite genotypes were A2A2AA and A1A2AA, both with prevalence greater than 20%. Percentages of the remaining 31 genotypes were less than 8%, including 20 genotypes with percentages less than 1%. The β-κ-casein genotype-specific lactation curves were significantly different for milk coagulation traits and milk protein percentage. The B variant of κ-casein showed a favorable effect on both milk coagulation traits, whereas the IB haplotype had an increasing effect on curd firmness and protein percentage. Inclusion of the β-κ-casein genotype effects in the model resulted in decreases in the mean additive genetic variations for milk coagulation time and curd firmness of 12.9 and 51.1%, respectively.

  7. Casein Films: The Effects of Formulation, Environmental Conditions and the Addition of Citric Pectin on the Structure and Mechanical Properties

    Directory of Open Access Journals (Sweden)

    Laetitia M. Bonnaillie

    2014-07-01

    Full Text Available Thin casein films for food packaging applications reportedly possess good strength and low oxygen permeability, but low elasticity and high sensitivity to moisture. Modifying the films to target specific behaviors depending on environmental conditions can enable a variety of commercial applications for casein-based films. The mechanical properties of solvent-cast (15% solids calcium-caseinate/glycerol films (CaCas:Gly ratio of 3:1 were characterized as a function of processing and environmental conditions, including film thickness, solution formulation and ambient humidity (from 22% to 70% relative humidity (RH at ~20 °C. At constant RH, the elongation at break (EAB had a strong positive dependence on the film thickness. When RH increased, the tensile strength (TS and modulus (E decreased approximately linearly, while EAB increased. From 0.05% to 1% (w/w of citric pectin (CP was then incorporated into CaCas/Gly films following seven different formulations (mixing sequences, to alter the protein network and to evaluate the effects of CP on the tensile properties of CaCas/Gly/CP films. At constant film thickness and ~60% RH, the addition of 0.1% or 1.0% CP to the films considerably increased or decreased EAB, TS and E in different directions and to different extents, depending on the formulation, while optical micrographs also showed vastly differing network configurations, suggesting complex formulation- and stoichiometry-dependent casein-pectin interactions within the dried films. Depending on the desired film properties and utilization conditions, pectin may be a useful addition to casein film formulations for food packaging applications.

  8. Small intestinal digestion of raw cornstarch in cattle consuming a soybean hull-based diet is improved by duodenal casein infusion.

    Science.gov (United States)

    Brake, D W; Titgemeyer, E C; Bailey, E A; Anderson, D E

    2014-09-01

    Six duodenally and ileally cannulated steers were used in 3 sequential studies to measure 1) basal nutrient flows from a soybean hull-based diet, 2) small intestinal digestibility of raw cornstarch continuously infused into the duodenum, and 3) responses of small intestinal starch digestion to duodenal infusion of 200 or 400 g/d casein. Our objective was to evaluate responses in small intestinal starch digestion in cattle over time and to measure responses in small intestinal starch digestion to increasing amounts of MP. On average, cattle consumed 3.7 kg/d DM, 68 g/d dietary N, and 70 g/d dietary starch. Starch flow to the duodenum was small (38 g/d), and N flow was 91 g/d. Small intestinal digestibility of duodenal N was 57%, and small intestinal digestion of duodenal starch flow was extensive (92%). Small intestinal starch digestibility was 34% when 1.5 kg/d raw cornstarch was continuously infused into the duodenum. Subsequently, cattle were placed in 1 of 2 replicated Latin squares that were balanced for carryover effects to determine response to casein infusions and time required for adaptation. Duodenal infusion of casein linearly increased (P ≤ 0.05) small intestinal starch digestibility, and small intestinal starch digestion adapted to infusion of casein in 6 d. Ethanol-soluble starch and unpolymerized glucose flowing to the ileum increased linearly (P ≤ 0.05) with increasing infusion of casein. Plasma cholecystokinin was not affected by casein infusion, but circulating levels of glucose were increased by casein supplementation (P ≤ 0.05). Responses in small intestinal starch digestion in cattle adapted to casein within 6 d, and increases in duodenal supply of casein up to 400 g/d increased small intestinal starch digestion in cattle.

  9. Quantitative analysis of complex casein hydrolysates based on chromatography and membrane

    Institute of Scientific and Technical Information of China (English)

    Qi Wei; Yu Yanjun; He Zhimin

    2006-01-01

    The enzymatic hydrolysates of casein are so complex that there is no effective method to do quantitative analysis.The common techniques,such as high performance chromatography and SDS-PAGE,can only carry out qualitative analysis.On the basis of membrane separation and high performance size exclusion chromatography (HPSEC),standard peptides with different molecular mass range were prepared,and the linear relationships between mass concentration of the standard peptides and the ultraviolet absorption of corresponding peak areas were established.Consequently,mass concentration of the different hydrolysates at different reaction times could be accurately calculated.The combination of chromatography and membrane separation is of great importance to the quantitative analysis of the complex hydrolysates,which can also be applied to the other macromolecular systems,such as carbohydrates.

  10. The gluten-free, casein-free diet in autism: an overview with clinical implications.

    Science.gov (United States)

    Elder, Jennifer Harrison

    The prevalence of classic autism and autism spectrum disorder (ASD) appears to be on the rise, and to date, there remains no clear etiology or cure. Out of desperation, many families are turning to new therapies and interventions discovered through various media sources and anecdotal reports from other parents. Unfortunately, many of these newer, well-publicized interventions have little empirical support. One of the most popular yet currently scientifically unproven interventions for ASD is the gluten-free, casein-free (GFCF) diet. Clinicians working with families of individuals with ASD are often asked for advice and find themselves unable to offer the most up-to-date and scientifically credible information. This article provides an overview of ASD and the GFCF diet, a summary and critique of current research findings, recommendations for future research, and practical advice for families to use in deciding if a trial of the GFCF diet is in the best interest of their child and family.

  11. Casein improves brachial and central aortic diastolic blood pressure in overweight adolescents: a randomised, controlled trial

    DEFF Research Database (Denmark)

    Arnberg, Karina; Larnkjær, Anni; Michaelsen, Kim F.

    2013-01-01

    of water, skimmed milk, whey or casein for 12 weeks. The milk-based test drinks contained 35 g protein/l. The effects were compared with the water group and a pretest control group consisting of thirty-two of the adolescents followed 12 weeks before the start of the intervention. Outcomes were brachial......Arterial stiffness, blood pressure (BP) and blood lipids may be improved by milk in adults and the effects may be mediated via proteins. However, limited is known about the effects of milk proteins on central aortic BP and no studies have examined the effects in children. Therefore, the present...... trial examined the effect of milk and milk proteins on brachial and central aortic BP, blood lipids, inflammation and arterial stiffness in overweight adolescents. A randomised controlled trial was conducted in 193 overweight adolescents aged 12–15 years. They were randomly assigned to drink 1 litre...

  12. Effect of Xanthan Gum on the Rheological Behavior and Microstructure of Sodium Caseinate Acid Gels

    Directory of Open Access Journals (Sweden)

    María E. Hidalgo

    2016-09-01

    Full Text Available The aim of this work was to study the effect of xanthan gum (XG on the gelation process of bovine sodium caseinate (NaCAS induced by acidification with glucono-δ-lactone (GDL and on the mixed acid gel microstructure. Before GDL addition, segregative phase separation was observed in all the NaCAS-XG mixtures evaluated. The gelation process was analyzed by using a fractional factorial experimental design. The images of the microstructure of the mixed acid gels were obtained by conventional optical microscopy and the mean diameter of the interstices was determined. Both the elastic character and the microstructure of the gels depended on the concentrations of XG added. As XG concentration increased, the kinetics of the gelation process was modified and the degree of compactness and elasticity component of the gel network increased. The microstructure of gels depends on the balance among thermodynamic incompatibility, protein gelation and NaCAS-XG interactions.

  13. Heterogeneity of caprine beta-casein elucidated by RP-HPLC/MS: genetic variants and phosphorylations.

    Science.gov (United States)

    Neveu, Carole; Mollé, Daniel; Moreno, Javier; Martin, Patrice; Léonil, Joëlle

    2002-11-01

    Casein variants occurring in milks from goats homozygous at the alpha(s1)-Cn locus were separated and identified by an RP-HPLC/ESI-MS method. Preferential haplotypes arose as well as some particularities in posttranslational modifications. A new variant of caprine beta-Cn, named C, as well as the phosphorylations pattern of the protein were characterized by the combined use of peptide mass fingerprinting and sequencing by tandem mass spectrometry. The molecular mass of the new variant in its 6P form was measured as 23854 Da and it differs in a mono amino acid substitution, A177 --> V177, from the variant A. The phosphorylation pattern of caprine beta-Cn is homologous to bovine beta-Cn concerning the 5P located on Ser15, 17, 18, 19, 35 but it presents a specific additional phosphorylation site on Thr12 that is comparable to human beta-Cn phosphorylation located on Thr3.

  14. Drying Technology of Angiotensin Converting Enzyme Inhibitory Peptide Derived from Bovine Casein

    Institute of Scientific and Technical Information of China (English)

    JIANG Zhanmei; Hue Guicheng; TIAN Be

    2009-01-01

    Drying technology of angiotensin converting enzyme (ACE) inhibitory peptides derived from bovine casein was investigated. No significance was observed on ACE inhibitory activity of products prepared by spay drying and freeze drying (P>0.05). Spay drying was the best drying process for practical industry production. The inlet temperature ranged from 140℃ to 160℃ and the exit temperature ranged from 70℃ to 90℃ during the spay drying process. Under the optimal eonditious, scale-up of angiotensin converted enzyme inhibitory peptide from 1 L to 10 L and the experiment was successively conducted. Peptide yield was 29% and half inhibitory concentration(IC50) was 0.53g·L-1.

  15. Genetic polymorphism of the K-casein (CSN3 gene in goats reared in Southern Italy

    Directory of Open Access Journals (Sweden)

    Fabio Pilla

    2010-01-01

    Full Text Available K-casein (K-CN represents one of the most important proteins determining the manufacturing properties of milk,because of its essential role in micelle formation and stabilisation.Several genetic variants of K-CN have been described in goats. To investigate the occurrence of seven alleles and theirdistribution among breeds, a total of 170 animals, from six different breeds reared in Italy (Cilentana Nera, Derivata diSiria, Maltese, Jonica, Garganica and Cashmere, have been analysed in this paper by the primer extension method.Alleles A and B were found to be the most represented in all the analysed breeds; allele D is present only in Maltese andCashmere animals with a very low frequency; while allele G has been found in all but two (Garganica and Cashmerebreeds. Alleles C, E and F were not present in the material used for this study.

  16. IDENTIFICATION OF BETA-LACTOGLOBULIN AND KAPPA-CASEIN GENOTYPES IN CATTLE

    Directory of Open Access Journals (Sweden)

    TESIO C.D.

    2007-01-01

    Full Text Available Beta-lactoglobulin (b-Lg and kappa-casein (k-Cn are two of the most importantproteins in the mammals’ milk synthesized by the epithelial cells of the mammaryglands. They play a crucial role in the milk quality and coagulation process(production of cheese and butter. The PCR-RFLP test was performed to distinguishthe different alleles in a population of Romanian Black Spotted cattle, a dairy breed.Genetic polymorphism was detected by digestion with the endonucleases Hae III (b-Lg and Hinf I (k-Cn, followed by electrophoresis in agarose high resolution gelstained with ethidium bromide. Fifty DNA samples from Romanian Black Spottedbreed were analyzed for A and B variants. This simple PCR-RFLP test makesfeasible the inclusion of b-Lg and k-Cn genotypes in breeding plans and cattleselection.

  17. Localisation of Protein Kinase C in Apoptosis and Neurite Outgrowth

    OpenAIRE

    Schultz, Anna

    2005-01-01

    Protein kinase C (PKC) is a family of serine/threonine kinases, which are subgrouped into classical (a, bI, bII, g), novel (d, e, h, q) and atypical (z, i/l) isoforms. One major aim of this thesis work was to investigate if altered levels of PKC isoforms influence the apoptotic responses of malignant cell-lines. We show that overexpression of PKCd or PKCq renders SK-N-BE(2) neuroblastoma cells sensitive to apoptosis induced by phorbol esters or C2-ceramide. Moreover, overexpression of PKCa, P...

  18. Main: 1DAW [RPSD[Archive

    Lifescience Database Archive (English)

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  11. Formulation and characterization of nanoencapsulated curcumin using sodium caseinate and its incorporation in ice cream.

    Science.gov (United States)

    Kumar, Deep Diyuti; Mann, Bimlesh; Pothuraju, Ramesh; Sharma, Rajan; Bajaj, Rajesh; Minaxi

    2016-01-01

    In the present investigation, the preparation and characterization of a curcumin nanoemulsion with milk protein (sodium caseinate) and its incorporation into ice cream were undertaken. Among the different combinations, the most stable formulation was observed using milk fat (8%), medium chain triglycerides (2%), curcumin (0.24%) and sodium caseinate (6%) with a mean particle size of 333.8 ± 7.18 nm, a zeta potential of -44.1 ± 0.72 mV and an encapsulation efficiency of 96.9 ± 0.28%. The effect of different processing conditions (heating, pH and ionic strength) on the particle size distribution and zeta potential of the nanoemulsion was evaluated. During heat treatment, the particle size of the nanoemulsion was increased from 333.8 ± 7.18 to 351.1 ± 4.04 nm. The nanoemulsion was destabilized at pH 4.6 and the particle size increased above and below pH 5.0. However, there was a slight increase in the particle size with a change in the ionic concentration. The release kinetics data suggested that in simulated gastro-intestinal digestion, the nanoemulsion was stable against pepsin digestion (a 5.25% release of curcumin), while pancreatic action led to a 16.12% release of curcumin from the nanoemulsion. Finally, our formulation was successfully incorporated into ice cream and the sensory attributes were evaluated. No significant difference was observed in the scores of the sensory attributes between the control and ice cream prepared with a curcumin nanoemulsion. Moreover, the encapsulation efficiency of the curcumin incorporated into the ice cream was 93.7%, which indicates that it can withstand the processing conditions. The findings suggest that ice cream is a suitable dairy product for the delivery of lipophilic bioactive components (curcumin) which can be used for therapeutic purposes.

  12. Novel ionically crosslinked casein nanoparticles for flutamide delivery: formulation, characterization, and in vivo pharmacokinetics

    Directory of Open Access Journals (Sweden)

    Elzoghby AO

    2013-05-01

    Full Text Available Ahmed O Elzoghby,1 Maged W Helmy,2 Wael M Samy,1,3 Nazik A Elgindy11Department of Industrial Pharmacy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt; 2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Pharos University, Alexandria, Egypt; 3Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Beirut Arab University, Beirut, LebanonAbstract: A novel particulate delivery matrix based on ionically crosslinked casein (CAS nanoparticles was developed for controlled release of the poorly soluble anticancer drug flutamide (FLT. Nanoparticles were fabricated via oil-in-water emulsification then stabilized by ionic crosslinking of the positively charged CAS molecules below their isoelectric point, with the polyanionic crosslinker sodium tripolyphosphate. With the optimal preparation conditions, the drug loading and incorporation efficiency achieved were 8.73% and 64.55%, respectively. The nanoparticles exhibited a spherical shape with a size below 100 nm and a positive zeta potential (+7.54 to +17.3 mV. FLT was molecularly dispersed inside the nanoparticle protein matrix, as revealed by thermal analysis. The biodegradability of CAS nanoparticles in trypsin solution could be easily modulated by varying the sodium tripolyphosphate crosslinking density. A sustained release of FLT from CAS nanoparticles for up to 4 days was observed, depending on the crosslinking density. After intravenous administration of FLT-CAS nanoparticles into rats, CAS nanoparticles exhibited a longer circulation time and a markedly delayed blood clearance of FLT, with the half-life of FLT extended from 0.88 hours to 14.64 hours, compared with drug cosolvent. The results offer a promising method for tailoring biodegradable, drug-loaded CAS nanoparticles as controlled, long-circulating drug delivery systems of hydrophobic anticancer drugs in aqueous vehicles.Keywords: casein nanoparticles, ionic crosslinking, biodegradability

  13. Autoantibodies to αS1-casein are induced by breast-feeding.

    Directory of Open Access Journals (Sweden)

    Klaudia Petermann

    Full Text Available BACKGROUND: The generation of antibodies is impaired in newborns due to an immature immune system and reduced exposure to pathogens due to maternally derived antibodies and placental functions. During nursing, the immune system of newborns is challenged with multiple milk-derived proteins. Amongst them, caseins are the main constituent. In particular, human αS1-casein (CSN1S1 was recently shown to possess immunomodulatory properties. We were thus interested to determine if auto-antibodies to CSN1S1 are induced by breast-feeding and may be sustained into adulthood. METHODS: 62 sera of healthy adult individuals who were (n = 37 or were not (n = 25 breast-fed against human CSN1S1 were investigated by a new SD (surface display-ELISA. For cross-checking, these sera were tested for anti Epstein-Barr virus (EBV antibodies by a commercial ELISA. RESULTS: IgG-antibodies were predominantly detected in individuals who had been nursed. At a cut-off value of 0.4, the SD-ELISA identified individuals with a history of having been breast-fed with a sensitivity of 80% and a specificity of 92%. Under these conditions, 35 out of 37 sera from healthy donors, who where breast-fed, reacted positively but only 5 sera of the 25 donors who were not breast-fed. The duration of breast-feeding was of no consequence to the antibody reaction as some healthy donors were only short term breast-fed (5 days minimum until 6 weeks maximum, but exhibited significant serum reaction against human CSN1S1 nonetheless. CONCLUSION: We postulate that human CSN1S1 is an autoantigen. The antigenicity is orally determined, caused by breast-feeding, and sustained into adulthood.

  14. Commitment to the CD4 lineage mediated by extracellular signal-related kinase mitogen-activated protein kinase and lck signaling.

    Science.gov (United States)

    Sharp, L L; Hedrick, S M

    1999-12-15

    The development of T cells results in a concordance between the specificity of the TCR for MHC class I and class II molecules and the expression of CD8 and CD4 coreceptors. Based on analogy to simple metazoan models of organ development and lineage commitment, we sought to determine whether extracellular signal-related kinase (Erk) mitogen-activated protein (MAP) kinase pathway signaling acts as an inductive signal for the CD4 lineage. Here, we show that, by altering the intracellular signaling involving the Erk/MAP kinase pathway, T cells with specificity for MHC class I can be diverted to express CD4, and, conversely, T cells with specificity for MHC class II can be diverted to express CD8. Furthermore, we find that activation of the src-family tyrosine kinase, p56lck is an upstream mediator of lineage commitment. These results suggest a simple mechanism for lineage commitment in T cell development.

  15. Phosphorylation of tau by death-associated protein kinase 1 antagonizes the kinase-induced cell apoptosis.

    Science.gov (United States)

    Duan, Dong-Xiao; Chai, Gao-Shang; Ni, Zhong-Fei; Hu, Yu; Luo, Yu; Cheng, Xiang-Shu; Chen, Ning-Ning; Wang, Jian-Zhi; Liu, Gong-Ping

    2013-01-01

    The intracellular accumulation of hyperphosphorylated tau plays a crucial role in neurodegeneration of Alzheimer's disease (AD), but the mechanism is not fully understood. From the observation that tau hyperphosphorylation renders cells more resistant to chemically-induced cell apoptosis, we have proposed that tau-involved apoptotic abortion may be the trigger of neurodegeneration. Here, we further studied whether this phenomenon is also applicable for the cell death induced by constitutively expressed factors, such as death-associated protein kinase 1 (DAPK1). We found that DAPK1 was upregulated and accumulated in the brain of human tau transgenic mice. Overexpression of DAPK1 in HEK293 and N2a cells decreased cell viability with activation of caspase-3, whereas simultaneous expression of tau antagonized DAPK1-induced apoptotic cell death. Expression of DAPK1 induced tau hyperphosphorylation at Thr231, Ser262, and Ser396 with no effects on protein phosphatase 2A, glycogen synthase kinase-3β, protein kinase A, calcium/calmodulin dependent protein kinase II, cell division cycle 2, or cyclin dependent protein kinase 5. The phosphorylation level of microtubule affinity-regulating kinase 2 (MARK2) was increased by expression of DAPK1, but simultaneous downregulation of MARK2 did not affect the DAPK1-induced tau hyperphosphorylation. DAPK1 was co-immunoprecipitated with tau proteins both in vivo and in vitro, and expression of the kinase domain-truncated DAPK1 did not induce tau hyperphosphorylation. These data suggest that tau hyperphosphorylation at Thr231, Ser262, and Ser396 by DAPK1 renders the cells more resistant to the kinase-induced apoptotic cell death, providing new insights into the tau-involved apoptotic abortion in the course of chronic neurodegeneration.

  16. The transcription factor YY1 is a novel substrate for Aurora B kinase at G2/M transition of the cell cycle.

    Directory of Open Access Journals (Sweden)

    Ari Kassardjian

    Full Text Available Yin Yang 1 (YY1 is a ubiquitously expressed and highly conserved multifunctional transcription factor that is involved in a variety of cellular processes. Many YY1-regulated genes have crucial roles in cell proliferation, differentiation, apoptosis, and cell cycle regulation. Numerous mechanisms have been shown to regulate the function of YY1, such as DNA binding affinity, subcellular localization, and posttranslational modification including phosphorylation. Polo-like kinase 1(Plk1 and Casein kinase 2α (CK2 α were the first two kinases identified to phosphorylate YY1. In this study, we identify a third kinase. We report that YY1 is a novel substrate of the Aurora B kinase both in vitro and in vivo. Serine 184 phosphorylation of YY1 by Aurora B is cell cycle regulated and peaks at G2/M and is rapidly dephosphorylated, likely by protein phosphatase 1 (PP1 as the cells enter G1. Aurora A and Aurora C can also phosphorylate YY1 in vitro, but at serine/threonine residues other than serine 184. We present evidence that phosphorylation of YY1 in the central glycine/alanine (G/A-rich region is important for DNA binding activity, with a potential phosphorylation/acetylation interplay regulating YY1 function. Given their importance in mitosis and overexpression in human cancers, Aurora kinases have been identified as promising therapeutic targets. Increasing our understanding of Aurora substrates will add to the understanding of their signaling pathways.

  17. Specific MAP-kinase blockade protects against renal damage in homozygous TGR(mRen2)27 rats

    NARCIS (Netherlands)

    de Borst, MH; Navis, G; de Boer, RA; Vis, LM; van Gilst, WH; van Goor, H

    2003-01-01

    Angiotensin II (AngII) plays an important role in renal damage by acting on hemodynamics, cell-growth, proliferation, and fibrosis, mainly by effects on the AngII type 1 (AT,) receptor. The AT, receptor activates several intracellular signaling molecules such as mitogen-activated protein kinases ext

  18. Whey and casein labelled with L-[1-13C]-leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion

    DEFF Research Database (Denmark)

    Reitelseder, Søren; Agergaard, Jakob; Doessing, Simon

    2011-01-01

    to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass......), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments......, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after...

  19. Rapid quantification of casein in skim milk using Fourier transform infrared spectroscopy, enzymatic perturbation, and multiway partial least squares: Monitoring chymosin at work

    DEFF Research Database (Denmark)

    Baum, Andreas; Hansen, P. W.; Nørgaard, Lars

    2016-01-01

    least squares regression. Because we observed casein-specific kinetic changes, the retrieved models were independent of the chemical background matrix and were therefore robust against possible covariance effects. We tested the robustness of the models by spiking the milk solutions with whey, calcium......In this study, we introduce enzymatic perturbation combined with Fourier transform infrared (FTIR) spectroscopy as a concept for quantifying casein in subcritical heated skim milk using chemometric multiway analysis. Chymosin is a protease that cleaves specifically caseins. As a result...... of hydrolysis, all casein proteins clot to form a creamy precipitate, and whey proteins remain in the supernatant. We monitored the cheese-clotting reaction in real time using FTIR and analyzed the resulting evolution profiles to establish calibration models using parallel factor analysis and multiway partial...

  20. Study on genetic variability at αS1 and αS2 casein loci in autochthonous goat populations of the Lombardy Alps

    Directory of Open Access Journals (Sweden)

    P. Crepaldi

    2011-03-01

    Full Text Available In cattle, sheep and goats (Hayes et al., 1993 the casein genes form a cluster, on the chromosome 6, of four tightly linked genes, whose sequence is: αS1-β-αS2-k (Threadgill and Womack, 1990; Ferretti et al., 1990; Gallagher et al., 1994; Rijnkles et al., 1997. For this reason the alleles at these loci are generally inherited all together (Di Stasio and Mariani, 2000, with low recombination frequency. Thus it is worth noting the importance of describing the casein haplotypes namely the inherited combination of closely linked alleles (King and Stansfield, 1985; Meuwissen and Goddard, 2001. The knowledge of the casein allele associations could be important for the selection. In goat milk the casein alleles are related to important differences in the level of synthesis of the respective gene products and in milk chemical-physical properties........