Artykuły w czasopismach: „DisA protein”

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van Rijn, Piet A., Mieke A. Maris-Veldhuis, Massimo Spedicato, Giovanni Savini i René G. P. van Gennip. "Pentavalent Disabled Infectious Single Animal (DISA)/DIVA Vaccine Provides Protection in Sheep and Cattle against Different Serotypes of Bluetongue Virus". Vaccines 9, nr 10 (9.10.2021): 1150. http://dx.doi.org/10.3390/vaccines9101150.

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Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for “European” serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.

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van Rijn, Piet A., Mieke A. Maris-Veldhuis i René G. P. van Gennip. "The Bluetongue Disabled Infectious Single Animal (DISA) Vaccine Platform Based on Deletion NS3/NS3a Protein Is Safe and Protective in Cattle and Enables DIVA". Viruses 13, nr 5 (7.05.2021): 857. http://dx.doi.org/10.3390/v13050857.

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The bluetongue virus (BTV) is transmitted by Culicoides biting midges and causes bluetongue (BT), an OIE-notifiable disease of ruminants. At least 29 BTV serotypes are described as determined by the outer shell proteins VP2 and VP5. Vaccination is the most effective control measure. Inactivated and live-attenuated vaccines (LAVs) are currently available. These vaccines have their specific pros and cons, and both are not DIVA vaccines. The BT Disabled Infectious Single Animal (DISA) vaccine platform is based on LAV without nonessential NS3/NS3a expression and is applicable for many serotypes by the exchange of outer shell proteins. The DISA vaccine is effective and completely safe. Further, transmission of the DISA vaccine by midges is blocked (DISA principle). Finally, the DISA vaccine enables DIVA because of a lack of antibodies against the immunogenic NS3/NS3a protein (DIVA principle). The deletion of 72 amino acids (72aa) in NS3/NS3a is sufficient to block virus propagation in midges. Here, we show that a prototype DISA vaccine based on LAV with the 72aa deletion enables DIVA, is completely safe and induces a long-lasting serotype-specific protection in cattle. In conclusion, the in-frame deletion of 72-aa codons in the BT DISA/DIVA vaccine platform is sufficient to fulfil all the criteria for modern veterinary vaccines.

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Torres, Rubén, Carolina Gándara, Begoña Carrasco, Ignacio Baquedano, Silvia Ayora i Juan C. Alonso. "DisA Limits RecG Activities at Stalled or Reversed Replication Forks". Cells 10, nr 6 (31.05.2021): 1357. http://dx.doi.org/10.3390/cells10061357.

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The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c-di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

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Galati, Domenico F., Stephanie Bonney, Zev Kronenberg, Christina Clarissa, Mark Yandell, Nels C. Elde, Maria Jerka-Dziadosz, Thomas H. Giddings, Joseph Frankel i Chad G. Pearson. "DisAp-dependent striated fiber elongation is required to organize ciliary arrays". Journal of Cell Biology 207, nr 6 (22.12.2014): 705–15. http://dx.doi.org/10.1083/jcb.201409123.

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Cilia-organizing basal bodies (BBs) are microtubule scaffolds that are visibly asymmetrical because they have attached auxiliary structures, such as striated fibers. In multiciliated cells, BB orientation aligns to ensure coherent ciliary beating, but the mechanisms that maintain BB orientation are unclear. For the first time in Tetrahymena thermophila, we use comparative whole-genome sequencing to identify the mutation in the BB disorientation mutant disA-1. disA-1 abolishes the localization of the novel protein DisAp to T. thermophila striated fibers (kinetodesmal fibers; KFs), which is consistent with DisAp’s similarity to the striated fiber protein SF-assemblin. We demonstrate that DisAp is required for KFs to elongate and to resist BB disorientation in response to ciliary forces. Newly formed BBs move along KFs as they approach their cortical attachment sites. However, because they contain short KFs that are rotated, BBs in disA-1 cells display aberrant spacing and disorientation. Therefore, DisAp is a novel KF component that is essential for force-dependent KF elongation and BB orientation in multiciliary arrays.

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van de Water, Sandra G. P., René G. P. van Gennip, Christiaan A. Potgieter, Isabel M. Wright i Piet A. van Rijn. "VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV". Journal of Virology 89, nr 17 (10.06.2015): 8764–72. http://dx.doi.org/10.1128/jvi.01052-15.

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ABSTRACTAfrican horse sickness virus (AHSV) is a virus species in the genusOrbivirusof the familyReoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species ofCulicoidesbiting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate “synthetic” reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replicationin vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes.IMPORTANCEAfrican horse sickness virus is transmitted by species ofCulicoidesbiting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replicationin vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since thein vitrorelease of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

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Müller, Martina, Tobias Deimling, Karl-Peter Hopfner i Gregor Witte. "Structural analysis of the diadenylate cyclase reaction of DNA-integrity scanning protein A (DisA) and its inhibition by 3′-dATP". Biochemical Journal 469, nr 3 (23.07.2015): 367–74. http://dx.doi.org/10.1042/bj20150373.

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Structures of Thermotoga maritima DNA-integrity-scanning protein A (DisA) in different reaction states describe the diadenylate cyclase (DAC) reaction and the possibility of its inhibition. We conclude that the mechanisms of cyclic-di-AMP (c-di-AMP) synthesis and its inhibition are conserved among different DAC enzymes and bacterial species.

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Latoscha, Andreas, David Jan Drexler, Mahmoud M. Al-Bassam, Adrian M. Bandera, Volkhard Kaever, Kim C. Findlay, Gregor Witte i Natalia Tschowri. "c-di-AMP hydrolysis by the phosphodiesterase AtaC promotes differentiation of multicellular bacteria". Proceedings of the National Academy of Sciences 117, nr 13 (18.03.2020): 7392–400. http://dx.doi.org/10.1073/pnas.1917080117.

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Antibiotic-producing Streptomyces use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as Streptococcus pneumoniae and Mycobacterium tuberculosis. AtaC is monomeric in solution and binds Mn2+ to specifically hydrolyze c-di-AMP to AMP via the intermediate 5′-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-di-AMP promotes interaction between CpeA and the predicted cation/proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in Streptomyces, connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in Streptomyces.

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Manning, JC, i Staden J. Van. "The Development and Mobilisation of Seed Reserves in Some African Orchids". Australian Journal of Botany 35, nr 3 (1987): 343. http://dx.doi.org/10.1071/bt9870343.

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The development, final appearance and digestion (during germination) of seed reserves in a number of genera of the Orchidaceae (tribe Orchideae) has been studied comprehensively, using ultrastructural and histochemical techniques complemented by gas chromatographic analysis of free sugars. Mature seeds of Disa, Disperis and Huttonaea contain substantial reserves of lipid and protein in the embryo. The protodermal cells of Disperis also contain protein-carbohydrate bodies. Free sugars are present but starch occurs only in immature seeds. Glyoxysomes are absent and lipolysis does not occur in seeds incubated without an external source of sucrose, and although a little starch is formed it is apparently synthesised from endogenous sucrose reserves. In the presence of exogenous sucrose, however, proteins are hydrolysed and glyoxysomes appear. Substantial quantities of starch are formed in such seeds. From these observations it is apparent that orchid seeds are unable to utilise endogenous reserves of lipid unless simple sugars are supplied to the medium but can utilise the free sugars present in the embryo. Resultant conclusions on the role of mycorrhizae in the germination of orchid seeds are discussed.

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Welker, D. L. "The discoidin I gene family of Dictyostelium discoideum is linked to genes regulating its expression." Genetics 119, nr 3 (1.07.1988): 571–78. http://dx.doi.org/10.1093/genetics/119.3.571.

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Abstract The discoidin I protein has been studied extensively as a marker of early development in the cellular slime mold Dictyostelium discoideum. However, like most other developmentally regulated proteins in this system, no reliable information was available on the linkage of the discoidin genes to other known genes. Analysis of the linkage of the discoidin I genes by use of restriction fragment length polymorphisms revealed that all three discoidin I genes as well as a pseudogene are located on linkage group II. This evidence is consistent with the discoidin I genes forming a gene cluster that may be under the control of a single regulatory element. The discoidin I genes are linked to three genetic loci (disA, motA, daxA) that affect the expression of the discoidin I protein. Linkage of the gene family members to regulatory loci may be important in the coordinate maintenance of the gene family and regulatory loci. A duplication affecting the entire discoidin gene family is also linked to group II; this appears to be a small tandem duplication. This duplication was mapped using a DNA polymorphism generated by insertion of the Tdd-3 mobile genetic element into a Tdd-2 element flanking the gamma gene. A probe for Tdd-2 identified a restriction fragment length polymorphism in strain AX3K that was consistent with generation by a previously proposed Tdd-3 insertion event. A putative duplication or rearrangement of a second Tdd-2 element on linkage group IV of strain AX3K was also identified. This is the first linkage information available for mobile genetic elements in D. discoideum.

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Kinoshita, N., M. Goebl i M. Yanagida. "The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control". Molecular and Cellular Biology 11, nr 12 (grudzień 1991): 5839–47. http://dx.doi.org/10.1128/mcb.11.12.5839-5847.1991.

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The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis.

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Kinoshita, N., M. Goebl i M. Yanagida. "The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control." Molecular and Cellular Biology 11, nr 12 (grudzień 1991): 5839–47. http://dx.doi.org/10.1128/mcb.11.12.5839.

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The fission yeast mutant dis3-54 is defective in mitosis and fails in chromosome disjunction. Its phenotype is similar to that of dis2-11, a mutant with a mutation in the type 1 protein phosphatase gene. We cloned the dis3+ gene by transformation. Nucleotide sequencing predicts a coding region of 970 amino acids interrupted by a 164-bp intron at the 65th codon. The predicted dis3+ protein shares a weak but significant similarity with the budding yeast SSD1 or SRK1 gene product, the gene for which is a suppressor for the absence of a protein phosphatase SIT4 gene or the BCY1 regulatory subunit of cyclic AMP-dependent protein kinase. Anti-dis3 antibodies recognized the 110-kDa dis3+ gene product, which is part of a 250- to 350-kDa oligomer and is enriched in the nucleus. The cellular localization of the dis3+ protein is reminiscent of that of the dis2+ protein, but these two proteins do not form a complex. A type 1 protein phosphatase activity in the dis3-54 mutant extracts is apparently not affected. The dis3+ gene is essential for growth; gene disruptant cells do not germinate and fail in cell division. Increased dis3+ gene dosage reverses the Ts+ phenotype of a cdc25 wee1 strain, as does increased type 1 protein phosphatase gene dosage. Double mutant dis3 dis2 is lethal even at the permissive temperature, suggesting that the dis2+ and dis3+ genes may be functionally overlapped. The role of the dis3+ gene product in mitosis is unknown, but this gene product may be directly or indirectly involved in the regulation of mitosis.

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Zhang, Lei, i Zheng-Guo He. "Radiation-sensitive Gene A (RadA) Targets DisA, DNA Integrity Scanning Protein A, to Negatively Affect Cyclic Di-AMP Synthesis Activity inMycobacterium smegmatis". Journal of Biological Chemistry 288, nr 31 (10.06.2013): 22426–36. http://dx.doi.org/10.1074/jbc.m113.464883.

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Birhanu, Tewodros, Tesfaye Adiko i Ramesh Duraisamy. "Phytochemical Screening and Multivariate Analysis on Physicochemical and Nutraceutical Value of Kocho from False Banana (Enset)". International Journal of Food Science 2023 (9.03.2023): 1–11. http://dx.doi.org/10.1155/2023/6666635.

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Enset (Ensete ventricosum) is one of Ethiopia’s most important food crops. The objective of the present study is to evaluate (using multivariate analysis) the effect of fermentation time, varietal differences, and treatment with gammicho on the physicochemical and nutraceuticals of kocho obtained from false banana in highly cultivated areas such as Disa Kera and Koysha Gorta of Dawro zone, Loma Woreda, South Nations’ Nationalities People Regions, Ethiopia. The analyses were carried out for fresh and fermented (with and without local starter, gammicho) enset kocho varieties (Meazia and Katania) harvested in two locations. Statistical analysis of the acquired data was performed using Minitab software version 19. It was discovered that each factor influenced significantly ( p ≤ 0.05 ) the qualities of kocho independently and with interaction. After four months of fermentation with gammicho, various parameters such as fat (1.69 to 0.62%), fiber (11.46 to 2.79%), pH (6.50 to 3.00), and moisture were dramatically decreased (9.34 to 2.8%). On the other hand, some dietary elements in both kinds were reduced with increasing fermentation time, including ash (2.07 to 3.57%), protein (3.08 to 5.52%), and carbs (71.87 to 84.55%). The results of this study suggest that Meazia has superior physicochemical and nutritional qualities over Katania.

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Campos, S. S., J. R. Ibarra-Rodriguez, R. C. Barajas-Ornelas, F. H. Ramirez-Guadiana, A. Obregon-Herrera, P. Setlow i M. Pedraza-Reyes. "Interaction of Apurinic/Apyrimidinic Endonucleases Nfo and ExoA with the DNA Integrity Scanning Protein DisA in the Processing of Oxidative DNA Damage during Bacillus subtilis Spore Outgrowth". Journal of Bacteriology 196, nr 3 (15.11.2013): 568–78. http://dx.doi.org/10.1128/jb.01259-13.

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van Rijn, Piet A., Mieke A. Maris-Veldhuis, Christiaan A. Potgieter i René G. P. van Gennip. "African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS Disabled Infectious Single Animal (DISA) vaccine platform". Vaccine 36, nr 15 (kwiecień 2018): 1925–33. http://dx.doi.org/10.1016/j.vaccine.2018.03.003.

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Lancero, Hope L., Schryl Castaneda, Nora B. Caberoy, Xiaoyuan Ma, Anthony G. Garza i Wenyuan Shi. "Analysing protein–protein interactions of the Myxococcus xanthus Dif signalling pathway using the yeast two-hybrid system". Microbiology 151, nr 5 (1.05.2005): 1535–41. http://dx.doi.org/10.1099/mic.0.27743-0.

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The dif operon is essential for fruiting body formation, fibril (exopolysaccharide) production and social motility of Myxococcus xanthus. The dif locus contains a gene cluster homologous to chemotaxis genes such as mcp (difA), cheW (difC), cheY (difD), cheA (difE) and cheC (difF), as well as an unknown ORF called difB. This study used yeast two-hybrid analysis to investigate possible interactions between Dif proteins, and determined that DifA, C, D and E interact in a similar fashion to chemotaxis proteins of Escherichia coli and Bacillus subtilis. It also showed that DifF interacted with DifD, and that the novel protein DifB did not interact with Dif proteins. Furthermore, DifA–F proteins were used to determine other possible protein–protein interactions in the M. xanthus genomic library. The authors not only confirmed the specific interactions among known Dif proteins, but also discovered two novel interactions between DifE and Nla19, and DifB and YidC, providing some new information about the Dif signalling pathway. Based on these findings, a model for the Dif signalling pathway is proposed.

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Zhang, Z., i A. R. Buchman. "Identification of a member of a DNA-dependent ATPase family that causes interference with silencing." Molecular and Cellular Biology 17, nr 9 (wrzesień 1997): 5461–72. http://dx.doi.org/10.1128/mcb.17.9.5461.

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DNA in eukaryotic cells is packed in tandem repeats of nucleosomes or higher-order chromatin structures, which present obstacles to many cellular processes that require protein-DNA interactions, such as transcription, DNA repair, and recombination. To find proteins that are involved in increasing the accessibility of specific DNA regions in yeast, we used a genetic approach that exploited transcriptional silencing normally occurring at HML and HMR loci. The silencing is mediated by cis-acting silencer elements and is thought to require the formation of a special chromatin structure that prevents accessibility to the silenced DNA. A previously uncharacterized gene, termed DIS1, was isolated from a screen for genes that interfere with silencing when overexpressed. DIS1 encodes a protein with conserved motifs that are present in a family of DNA-dependent ATPases, the SWI2/SNF2-like proteins. Overproduction of N-terminal half of DIS1 protein interfered specifically with ectopic silencing used in the screen as well as HMR E silencing. Two-hybrid studies revealed a specific interaction between the N terminus of DIS1 and the C-terminal half of SIR4, a protein essential for silencing. Cells with a dis1 knockout mutation had significantly lower mating-type switching rate. These results suggest that DIS1 may contribute to making the silenced DNA template at HM loci more accessible during the mating-type switching process.

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Martín-Galiano, Antonio J., María S. Escolano-Martínez, Bruno Corsini, Adela G. de la Campa i José Yuste. "Immunization with SP_1992 (DiiA) Protein of Streptococcus pneumoniae Reduces Nasopharyngeal Colonization and Protects against Invasive Disease in Mice". Vaccines 9, nr 3 (24.02.2021): 187. http://dx.doi.org/10.3390/vaccines9030187.

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Knowledge-based vaccinology can reveal uncharacterized antigen candidates for a new generation of protein-based anti-pneumococcal vaccines. DiiA, encoded by the sp_1992 locus, is a surface protein containing either one or two repeats of a 37mer N-terminal motif that exhibits low interstrain variability. DiiA belongs to the core proteome, contains several conserved B-cell epitopes, and is associated with colonization and pathogenesis. Immunization with DiiA protein via the intraperitoneal route induced a strong IgG response, including different IgG subtypes. Vaccination with DiiA increased bacterial clearance and induced protection against sepsis, conferring 70% increased survival at 48 h post-infection when compared to the adjuvant control. The immunogenic response and survival rates in mice immunized with a truncated DiiA version lacking 119 N-terminal residues were remarkably lower, confirming the relevance of the repeat zone in the immunoprotection by DiiA. Intranasal immunization of mice with the entire recombinant protein elicited mucosal IgG and IgA responses that reduced bacterial colonization of the nasopharynx, confirming that this protein might be a vaccine candidate for reducing the carrier rate. DiiA constitutes an example of how functionally unannotated proteins may still represent promising candidates that can be used in prophylactic strategies against the pneumococcal carrier state and invasive disease.

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Biyani, Neeta, i Nandkumar Dravid. "A Study of Urinary Protein Pattern Using Polyacrylamide Gel Disc Electrophoresis in Nephrotic Syndrome". Indian Journal of Pathology: Research and Practice 5, nr 3 (2016): 335–40. http://dx.doi.org/10.21088/ijprp.2278.148x.5316.16.

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Luton, Frédéric, Michael H. Cardone, Min Zhang i Keith E. Mostov. "Role of Tyrosine Phosphorylation in Ligand-induced Regulation of Transcytosis of the Polymeric Ig Receptor". Molecular Biology of the Cell 9, nr 7 (lipiec 1998): 1787–802. http://dx.doi.org/10.1091/mbc.9.7.1787.

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The polymeric Ig receptor (pIgR) transcytoses its ligand, dimeric IgA (dIgA), from the basolateral to the apical surface of epithelial cells. Although the pIgR is constitutively transcytosed in the absence of ligand, binding of dIgA stimulates transcytosis of the pIgR. We recently reported that dIgA binding to the pIgR induces translocation of protein kinase C, production of inositol triphosphate, and elevation of intracellular free calcium. We now report that dIgA binding causes rapid, transient tyrosine phosphorylation of several proteins, including phosphatidyl inositol-specific phospholipase C-γl. Protein tyrosine kinase inhibitors or deletion of the last 30 amino acids of pIgR cytoplasmic tail prevents IgA-stimulated protein tyrosine kinase activation, tyrosine phosphorylation of phospholipase C-γl, production of inositol triphosphate, and the stimulation of transcytosis by dIgA. Analysis of pIgR deletion mutants reveals that the same discrete portion of the cytoplasmic domain, residues 727–736 (but not the Tyr734), controls both the ability of pIgR to cause dIgA-induced tyrosine phosphorylation of the phospholipase C-γl and to undergo dIgA-stimulated transcytosis. In addition, dIgA transcytosis can be strongly stimulated by mimicking phospholipase C-γl activation. In combination with our previous results, we conclude that the protein tyrosine kinase(s) and phospholipase C-γl that are activated upon dIgA binding to the pIgR control dIgA-stimulated pIgR transcytosis.

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Rajasekaran, Shanmuganathan, Dilip Chand Raja Soundararajan, Chitraa Tangavel, Sri Vijay Anand K. S., Sharon Miracle Nayagam, Monica Steffi Matchado, Raveendran Muthurajan, Ajoy Prasad Shetty, Rishi Mugesh Kanna i K. Dharmalingam. "Proteomic Signature of Nucleus Pulposus in Fetal Intervertebral Disc". Asian Spine Journal 14, nr 4 (31.08.2020): 409–20. http://dx.doi.org/10.31616/asj.2019.0217.

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Study Design: Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD).Purpose: To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis.Overview of Literature: Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed.Methods: IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases.Results: A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins.Conclusions: This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.

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Donohue, P. J., M. R. Jahnke, J. D. Blaha i B. Caterson. "Characterization of link protein(s) from human intervertebral-disc tissues". Biochemical Journal 251, nr 3 (1.05.1988): 739–47. http://dx.doi.org/10.1042/bj2510739.

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Proteoglycan aggregates (A1) were prepared from the anulus fibrosus, nucleus pulposus and cartilage-endplate tissues of postnatal (0-6-month-old)-and young-adult (20-30-year-old)-human intervertebral discs. The A1 fractions from young-adult disc contained a greater proportion of non-aggregating proteoglycans than did postnatal tissues. After dissociative CsCl-density-gradient fractionation of the A1, more than 90% of the uronic acid was found in the postnatal A1D1, whereas only 60-80% of the hexuronate was present in the A1D1 isolated from young-adult disc tissues. These results indicated that more lower-buoyant-density proteoglycans occur in the young-adult disc. Link-protein-rich fractions (A1D3) were subjected to SDS/polyacrylamide-gel electrophoresis and immunolocation analyses using monoclonal antibodies specific for epitopes on link protein or proteoglycan. Under non-reducing conditions, the major link protein present in postnatal disc tissues was link protein 1. By contrast, all three link proteins (1, 2 and 3) were detected in young-adult tissues, with the smaller link protein 3 predominating. Analyses of the A1D3 fractions under reducing conditions also indicated the presence of link-protein-degradation peptides (Mr approx. 26,000) from young-adult disc tissues, but not from postnatal tissues. Sequential Sepharose CL-6B and Sephacryl S-300 chromatography in 4 M-guanidinium chloride was employed to separate the link proteins of the A1D3 fraction from protein-rich proteoglycan. Immunolocation analyses indicated that postnatal samples contained no detectable contaminating proteoglycan fragments. However, young-adult link-protein preparations could not be separated from hyaluronic acid-binding region and other proteoglycan fragments by means of these chromatographic procedures. The studies indicate that, compared with hyaline articular cartilage, degraded link protein and proteoglycan accumulate at an early age in young-adult disc tissues. These partially degraded proteoglycan aggregate components may significantly alter the biomechanical properties of disc tissues.

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Benson, Andrew, Josh Steed, Mia Malloy i Adam J. Davis. "Quantitative Protein Analysis of ZPB2, ZPB1 and ZPC in the Germinal Disc and a Non-Germinal Disc Region of the Inner Perivitelline Layer in Two Genetic Lines of Turkey Hens That Differ in Fertility". Animals 12, nr 13 (29.06.2022): 1672. http://dx.doi.org/10.3390/ani12131672.

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The avian inner perivitelline layer (IPVL), containing the zona pellucida (ZP) family of proteins, surrounds the ovulated ovum. In mammalian species, ZP proteins serve as key component(s) in binding sperm and initiating the acrosome reaction. Sperm binding at the germinal disc (GD) region of the IPVL initiates fertilization in avian species, and the amount of sperm binding at the GD reflects female fertility. The current research determined whether reported differences in mRNA expression in two genetic lines of turkey hens (E, high fertility and F, low fertility) translated to the protein level. ZPB2 in the IPVL is greater in the GD region compared with the nongerminal disc (NGD) region, as indicated by both mRNA and protein expression. However, protein expressions of ZPB1 and ZPC in the IPVL of E- and F-line turkey hens was in contrast to previously reported mRNA expression. The results indicate that the mRNA expression of ZP proteins at their site of synthesis in E- and F-line hens often does not directly correlate with the IPVL abundance of these proteins. The greater protein concentration of ZPB2 in the GD region compared with the NGD regions suggests that this protein may be critical for sperm binding at the GD region.

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Berthoux, Lionel, Christine Péchoux i Jean-Luc Darlix. "Multiple Effects of an Anti-Human Immunodeficiency Virus Nucleocapsid Inhibitor on Virus Morphology and Replication". Journal of Virology 73, nr 12 (1.12.1999): 10000–10009. http://dx.doi.org/10.1128/jvi.73.12.10000-10009.1999.

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ABSTRACT Human immunodeficiency virus type 1 nucleocapsid protein is a major structural component of the virion core and a key factor involved in proviral DNA synthesis and virus formation. 2,2′-Dithiobenzamides (DIBA-1) and related compounds that are inhibitors of NCp7 are thought to eject zinc ions from NCp7 zinc fingers, inhibiting the maturation of virion proteins. Here, we show that the presence of DIBA-1 at the time of virus formation causes morphological malformations of the virus and reduces proviral DNA synthesis. Thus, it seems that DIBA-1 is responsible for a “core-freezing effect,” as shown by electron microscopy analyses. DIBA-1 can also directly interfere with the fate of the newly made proviral DNA in a manner independent of its effects on virion core formation. These data strongly suggest that nucleocapsid protein is a prime target for new compounds aimed at inhibiting human immunodeficiency virus and other retroviruses.

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Zhang, Guangzhi, Lei Li, Zhili Yang, Cangyu Zhang i Xuewen Kang. "TMT-Based Proteomics Analysis of Senescent Nucleus Pulposus from Patients with Intervertebral Disc Degeneration". International Journal of Molecular Sciences 24, nr 17 (26.08.2023): 13236. http://dx.doi.org/10.3390/ijms241713236.

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Lower back pain, a leading cause of disability worldwide, is associated with intervertebral disc degeneration (IDD) in approximately 40% of cases. Although nucleus pulposus (NP) cell senescence is a major contributor to IDD, the underlying mechanisms remain unclear. We collected NP samples from IDD patients who had undergone spinal surgery. Healthy and senescent NP tissues (n = 3) were screened using the Pfirrmann grading system combined with immunohistochemistry, as well as hematoxylin and eosin, Safranin O, Alcian blue, and Masson staining. Differentially expressed proteins (DEPs) were identified using quantitative TMT-based proteomics technology. Bioinformatics analyses included gene ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein–protein interaction (PPI) analyses. In addition, immunofluorescence was used to verify protein expression. In total, 301 DEPs were identified in senescent NP tissues, including 92 upregulated and 209 downregulated proteins. In GO, DEPs were primarily associated with NF-kappaB transcription factor, extracellular regions, cellular protein metabolic processes, and post-translational protein modification. The enriched KEGG pathways included TGF-β, Wnt, RAP1, interleukin-17, extracellular matrix-receptor adhesion, and PI3K/Akt signaling pathways. PPI analysis demonstrated interactions between multiple proteins. Finally, immunofluorescence verified the expressions of MMP3, LUM, TIMP1, and CDC42 in senescent NP cells. Our study provides valuable insights into the mechanisms underlying senescent NP tissues in IDD patients. DEPs provide a basis for further investigation of the effects of senescent factors on IDD.

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Nosala, Christopher, Kari D. Hagen, Nicholas Hilton, Tiffany M. Chase, Kelci Jones, Rita Loudermilk, Kristofer Nguyen i Scott C. Dawson. "Disc-associated proteins mediate the unusual hyperstability of the ventral disc in Giardia lamblia". Journal of Cell Science 133, nr 16 (13.07.2020): jcs227355. http://dx.doi.org/10.1242/jcs.227355.

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ABSTRACTGiardia lamblia, a widespread parasitic protozoan, attaches to the host gastrointestinal epithelium by using the ventral disc, a complex microtubule (MT) organelle. The ‘cup-like’ disc is formed by a spiral MT array that scaffolds numerous disc-associated proteins (DAPs) and higher-order protein complexes. In interphase, the disc is hyperstable and has limited MT dynamics; however, it remains unclear how DAPs confer these properties. To investigate mechanisms of hyperstability, we confirmed the disc-specific localization of over 50 new DAPs identified by using both a disc proteome and an ongoing GFP localization screen. DAPs localize to specific disc regions and many lack similarity to known proteins. By screening 14 CRISPRi-mediated DAP knockdown (KD) strains for defects in hyperstability and MT dynamics, we identified two strains – DAP5188KD and DAP6751KD –with discs that dissociate following high-salt fractionation. Discs in the DAP5188KD strain were also sensitive to treatment with the MT-polymerization inhibitor nocodazole. Thus, we confirm here that at least two of the 87 known DAPs confer hyperstable properties to the disc MTs, and we anticipate that other DAPs contribute to disc MT stability, nucleation and assembly.

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27

Lee, Sarah C., i Naomi L. Pollock. "Membrane proteins: is the future disc shaped?" Biochemical Society Transactions 44, nr 4 (15.08.2016): 1011–18. http://dx.doi.org/10.1042/bst20160015.

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The use of styrene maleic acid lipid particles (SMALPs) for the purification of membrane proteins (MPs) is a rapidly developing technology. The amphiphilic copolymer of styrene and maleic acid (SMA) disrupts biological membranes and can extract membrane proteins in nanodiscs of approximately 10 nm diameter. These discs contain SMA, protein and membrane lipids. There is evidence that MPs in SMALPs retain their native structures and functions, in some cases with enhanced thermal stability. In addition, the method is compatible with biological buffers and a wide variety of biophysical and structural analysis techniques. The use of SMALPs to solubilize and stabilize MPs offers a new approach in our attempts to understand, and influence, the structure and function of MPs and biological membranes. In this review, we critically assess progress with this method, address some of the associated technical challenges, and discuss opportunities for exploiting SMA and SMALPs to expand our understanding of MP biology.

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Knöll, Ralph, Byambajav Buyandelger i Max Lab. "The Sarcomeric Z-Disc and Z-Discopathies". Journal of Biomedicine and Biotechnology 2011 (2011): 1–12. http://dx.doi.org/10.1155/2011/569628.

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The sarcomeric Z-disc defines the lateral borders of the sarcomere and has primarily been seen as a structure important for mechanical stability. This view has changed dramatically within the last one or two decades. A multitude of novel Z-disc proteins and their interacting partners have been identified, which has led to the identification of additional functions and which have now been assigned to this structure. This includes its importance for intracellular signalling, for mechanosensation and mechanotransduction in particular, an emerging importance for protein turnover and autophagy, as well as its molecular links to the t-tubular system and the sarcoplasmic reticulum. Moreover, the discovery of mutations in a wide variety of Z-disc proteins, which lead to perturbations of several of the above-mentioned systems, gives rise to a diverse group of diseases which can be termed Z-discopathies. This paper provides a brief overview of these novel aspects as well as points to future research directions.

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Wadmore, Kirsty, Amar J. Azad i Katja Gehmlich. "The Role of Z-disc Proteins in Myopathy and Cardiomyopathy". International Journal of Molecular Sciences 22, nr 6 (17.03.2021): 3058. http://dx.doi.org/10.3390/ijms22063058.

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The Z-disc acts as a protein-rich structure to tether thin filament in the contractile units, the sarcomeres, of striated muscle cells. Proteins found in the Z-disc are integral for maintaining the architecture of the sarcomere. They also enable it to function as a (bio-mechanical) signalling hub. Numerous proteins interact in the Z-disc to facilitate force transduction and intracellular signalling in both cardiac and skeletal muscle. This review will focus on six key Z-disc proteins: α-actinin 2, filamin C, myopalladin, myotilin, telethonin and Z-disc alternatively spliced PDZ-motif (ZASP), which have all been linked to myopathies and cardiomyopathies. We will summarise pathogenic variants identified in the six genes coding for these proteins and look at their involvement in myopathy and cardiomyopathy. Listing the Minor Allele Frequency (MAF) of these variants in the Genome Aggregation Database (GnomAD) version 3.1 will help to critically re-evaluate pathogenicity based on variant frequency in normal population cohorts.

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Moen, Aurora, Anne-Li Lind, Måns Thulin, Masood Kamali-Moghaddam, Cecilie Røe, Johannes Gjerstad i Torsten Gordh. "Inflammatory Serum Protein Profiling of Patients with Lumbar Radicular Pain One Year after Disc Herniation". International Journal of Inflammation 2016 (2016): 1–8. http://dx.doi.org/10.1155/2016/3874964.

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Earlier studies suggest that lumbar radicular pain following disc herniation may be associated with a local or systemic inflammatory process. In the present study, we investigated the serum inflammatory protein profile of such patients. All 45 patients were recruited from Oslo University Hospital, Ullevål, Norway, during the period 2007–2009. The new multiplex proximity extension assay (PEA) technology was used to analyze the levels of 92 proteins. Interestingly, the present data showed that patients with radicular pain 12 months after disc herniation may be different from other patients with regard to many measurable serum cytokines. Given a false discovery rate (FDR) of 0.10 and 0.05, we identified 41 and 13 proteins, respectively, which were significantly upregulated in the patients with severe pain one year after disc herniation. On the top of the list ranked by estimated increase we found C-X-C motif chemokine 5 (CXCM5; 217% increase), epidermal growth factor (EGF; 142% increase), and monocyte chemotactic protein 4 (MCP-4; 70% increase). Moreover, a clear overall difference in the serum cytokine profile between the chronic and the recovered patients was demonstrated. Thus, the present results may be important for future protein serum profiling of lumbar radicular pain patients with regard to prognosis and choice of treatment. We conclude that serum proteins may be measurable molecular markers of persistent pain after disc herniation.

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Белых, Evgeniy Belykh, Бардонова, Lyudmila Bardonova, Бывальцев i Vadim Byvaltsev. "BONE MORPHOGENETIC PROTEIN-2 INFLUENCE ON METABOLIC ACTIVITY AND PROTEOGLYCAN SYNTHESIS BY INTERVERTEBRAL DISC CELLS". Бюллетень Восточно-Сибирского научного центра Сибирского отделения Российской академии медицинских наук 1, nr 4 (28.11.2016): 99–103. http://dx.doi.org/10.12737/22977.

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Modern therapeutic strategies for intervertebral disc repair mainly focus on targeting molecular pathways of extracel-lular matrix degeneration. Anabolic strategies for regeneration are aimed to increase production of major extracellular molecules. Members of TGF-b superfamily proteins, particularly the bone morphogenetic proteins (BMP) have a high regenerative potential regarding the mesenchymal cells. The goal of this study is to study production of proteoglycans by the intervertebral disc cells under the influence of bone morphogenetic protein 2.Material and methods. The experiment was carried out on the cell cultures derived from the annulus fibrosis cells and nucleus pulposus cells of the human intervertebral disc. We studied cell livability, metabolic activity and proteoglycan expression. Cell livability was assessed using the trypan blue staining. Alamar blue test was used for the estimation of metabolic activity. Amount of sulfated glycosaminoglycans was assessed using the assay based on the reaction with 1,9-Dimethylmethylene Blue.Results. Cultivation with bone morphogenetic protein 2 in different concentrations did not decrease livability of the cells. Study cell cultures with application of bone morphogenetic protein 2 in different concentrations showed significant increase in metabolic activity and proteoglycan synthesis by the annulus fibrosis cells. Despite the relative increase in the number of the nucleus pulposus cells treated with the bone morphogenetic protein 2, the differences in metabolic and synthetic activity compared with control group was not significant. Conclusion. The bone morphogenetic protein 2 has an anabolic effect towards the intervertebral disc cells, particularly in the production of proteoglycans by the annulus fibrosis cells.

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Li, Zhonghui, Zijun Qiao, Wenling Zheng i Wenli Ma. "Network Cluster Analysis of Protein–Protein Interaction Network–Identified Biomarker for Type 2 Diabetes". Diabetes Technology & Therapeutics 17, nr 7 (lipiec 2015): 475–81. http://dx.doi.org/10.1089/dia.2014.0204.

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Cullen, C. Fiona, Peter Deák, David M. Glover i Hiroyuki Ohkura. "mini spindles". Journal of Cell Biology 146, nr 5 (6.09.1999): 1005–18. http://dx.doi.org/10.1083/jcb.146.5.1005.

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We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.

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TANG, Zhaohua, Norbert F. KÄUFER i Ren-Jang LIN. "Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation". Biochemical Journal 368, nr 2 (1.12.2002): 527–34. http://dx.doi.org/10.1042/bj20021133.

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The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1—Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast.

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Price, M. G., i R. H. Gomer. "Skelemin, a cytoskeletal M-disc periphery protein, contains motifs of adhesion/recognition and intermediate filament proteins." Journal of Biological Chemistry 268, nr 29 (październik 1993): 21800–21810. http://dx.doi.org/10.1016/s0021-9258(20)80613-9.

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Steele-Ogus, Melissa C., Ava M. Obenaus, Nathan J. Sniadecki i Alexander R. Paredez. "Disc and Actin Associated Protein 1 influences attachment in the intestinal parasite Giardia lamblia". PLOS Pathogens 18, nr 3 (25.03.2022): e1010433. http://dx.doi.org/10.1371/journal.ppat.1010433.

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The deep-branching eukaryote Giardia lamblia is an extracellular parasite that attaches to the host intestine via a microtubule-based structure called the ventral disc. Control of attachment is mediated in part by the movement of two regions of the ventral disc that either permit or exclude the passage of fluid under the disc. Several known disc-associated proteins (DAPs) contribute to disc structure and function, but no force-generating protein has been identified among them. We recently identified several Giardia actin (GlActin) interacting proteins at the ventral disc, which could potentially employ actin polymerization for force generation and disc conformational changes. One of these proteins, Disc and Actin Associated Protein 1 (DAAP1), is highly enriched at the two regions of the disc previously shown to be important for fluid flow during attachment. In this study, we investigate the role of both GlActin and DAAP1 in ventral disc morphology and function. We confirmed interaction between GlActin and DAAP1 through coimmunoprecipitation, and used immunofluorescence to localize both proteins throughout the cell cycle and during trophozoite attachment. Similar to other DAPs, the association of DAAP1 with the disc is stable, except during cell division when the disc disassembles. Depletion of GlActin by translation-blocking antisense morpholinos resulted in both impaired attachment and defects in the ventral disc, indicating that GlActin contributes to disc-mediated attachment. Depletion of DAAP1 through CRISPR interference resulted in intact discs but impaired attachment, gating, and flow under the disc. As attachment is essential for infection, elucidation of these and other molecular mediators is a promising area for development of new therapeutics against a ubiquitous parasite.

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Tang, Ngang Heok, Hirofumi Takada, Kuo-Shun Hsu i Takashi Toda. "The internal loop of fission yeast Ndc80 binds Alp7/TACC-Alp14/TOG and ensures proper chromosome attachment". Molecular Biology of the Cell 24, nr 8 (15.04.2013): 1122–33. http://dx.doi.org/10.1091/mbc.e12-11-0817.

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The Ndc80 outer kinetochore complex plays a critical role in kinetochore–microtubule attachment, yet our understanding of the mechanism by which this complex interacts with spindle microtubules for timely and accurate chromosome segregation remains limited. Here we address this issue using an ndc80 mutant (ndc80-NH12) from fission yeast that contains a point mutation within a ubiquitous internal loop. This mutant is normal for assembly of the Ndc80 complex and bipolar spindle formation yet defective in proper end-on attachment to the spindle microtubule, with chromosome alignment defects and missegregation happening later during mitosis. We find that ndc80-NH12 exhibits impaired localization of the microtubule-associated protein complex Alp7/transforming acidic coiled coil (TACC)-Alp14/tumor-overexpressed gene (TOG) to the mitotic kinetochore. Consistently, wild-type Ndc80 binds these two proteins, whereas the Ndc80-NH12 mutant protein displays a substantial reduction of interaction. Crucially, forced targeting of Alp7–Alp14 to the outer kinetochore rescues ndc80-NH12-mutant phenotypes. The loop was previously shown to bind Dis1/TOG, by which it ensures initial chromosome capture during early mitosis. Strikingly, ndc80-NH12 is normal in Dis1 localization. Genetic results indicate that the loop recruits Dis1/TOG and Alp7/TACC-Alp14/TOG independently. Our work therefore establishes that the Ndc80 loop plays sequential roles in spindle–kinetochore attachment by connecting the Ndc80 complex to Dis1/TOG and Alp7/TACC-Alp14/TOG.

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Shimanuki, M., N. Kinoshita, H. Ohkura, T. Yoshida, T. Toda i M. Yanagida. "Isolation and characterization of the fission yeast protein phosphatase gene ppe1+ involved in cell shape control and mitosis." Molecular Biology of the Cell 4, nr 3 (marzec 1993): 303–13. http://dx.doi.org/10.1091/mbc.4.3.303.

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We isolated a fission yeast putative protein serine/threonine phosphatase gene designated ppe1+ by hybridization. The predicted amino acid sequence is similar to those of the fission yeast ppa2 (53% identity) and dis2 (39%) phosphatases, and highly similar to those of the budding yeast SIT4 (72%), Drosophila PPV (68%) and rabbit PPX (61%) phosphatases. Antibodies against ppe1 protein identified a 37-kd polypeptide in fission yeast. A gene disruption (designated delta ppe1) caused cold-sensitive lethality and short, pear-shaped cells. These phenotypes were fully suppressed by a plasmid carrying ppe1+. Three classes of multicopy suppressor genes for delta ppe1 were identified as follows: 1) ppa1+ and ppa2+ encoding type 2A-like phosphatases, 2) mitotically essential dis3+ similar to the budding yeast SSD1/SRK1, a suppressor for sit4, and 3) pck1+ coding for a protein kinase C-like kinase. Consistently, the budding yeast SIT4 gene was also a multicopy suppressor for delta ppe1. Phosphatase ppe1 may play a role in cell morphogenesis and mitosis by either regulating or being regulated by these multicopy suppressor gene products. Consistent with this hypothesis, double mutants ppe1-ppa2 and ppe1-pck1 are lethal at the permissive temperature.

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Shi, Wei, Argyrios Stampas, Cynthia Zapata i Nicholas E. Baker. "The pineapple eye Gene Is Required for Survival of Drosophila Imaginal Disc Cells". Genetics 165, nr 4 (1.12.2003): 1869–79. http://dx.doi.org/10.1093/genetics/165.4.1869.

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Abstract Each ommatidium of the Drosophila eye is constructed by precisely 19 specified precursor cells, generated in part during a second mitotic wave of cell divisions that overlaps early stages of ommatidial cell specification. Homozygotes for the pineapple eye mutation lack sufficient precursor cells due to apoptosis during the period of fate specification. In addition development is delayed by apoptosis during earlier imaginal disc growth. Null alleles are recessive lethal and allelic to l(2)31Ek; heteroallelic combinations can show developmental delay, abnormal eye development, and reduced fertility. Mosaic clones autonomously show extensive cell death. The pineapple eye gene was identified and predicted to encode a novel 582-aminoacid protein. The protein contains a novel, cysteine-rich domain of 270 amino acids also found in predicted proteins of unknown function from other animals.

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Tessari, E. N. C., C. A. F. Oliveira, A. L. S. P. Cardoso, D. R. Ledoux i G. E. Rottinghaus. "EFEITOS DA AFLATOXINA B 1 E FUMONISINA B1 SOBRE OS NÍVEIS SÉRICOS DE ASPARTATO AMINO-TRANSFERASE E PROTEÍNA TOTAL DE FRANGOS DE CORTE". Arquivos do Instituto Biológico 72, nr 2 (kwiecień 2005): 187–91. http://dx.doi.org/10.1590/1808-1657v72p1872005.

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RESUMO O objetivo do presente trabalho foi avaliar os efeitos da aflatoxina B 1 (AFB1) e da fumonisina B1 (FB1) sobre os níveis séricos da enzima aspartato amino-transferase (AST) e de proteínas totais de frangos de corte alimentados com ração contendo as toxinas isoladas e em associação, nos níveis de 0, 50 e 200 ?g de AFB1/kg e 0, 50 e 200 mg de FB1/kg. O delineamento experimental foi o inteiramente casualizado em esquema fatorial 3 x 3, com 9 tratamentos e 12 repetições, totalizando 108 aves. As aves foram alimentadas com as rações contaminadas a partir do 8 o dia até o 41o dia de idade. Aos 41 dias de idade, houve um aumento (p < 0,05) nos níveis de AST das aves que receberam dietas contendo AFB1 e FB1, com exceção do grupo que recebeu somente 50 ?g de FB1/kg. Este aumento foi maior quanto mais elevados foram os níveis de AFB1, sendo que um efeito tóxico aditivo foi observado nos tratamentos de associação com 50 mg de AFB1/ kg e 50 ou 200 mg de FB1/kg. Aos 28 dias de idade, observou-se uma redução (p < 0,05) na concentração de proteína dos grupos que receberam ração com 200 ?g de AFB1/kg, com ou sem FB1, porém esta redução não foi observada aos 41 dias de idade. Conclui-se que a intoxicação de frangos de corte com AFB1 e FB1, isoladas ou associadas, causa um aumento na concentração sérica de AST e uma redução nos níveis de proteínas totais após 20 dias de exposição contínua através da ração.

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Greenberg, Robert M., i Paul N. Adler. "Protein synthesis during imaginal disc pattern regulation". Journal of Experimental Zoology 242, nr 1 (kwiecień 1987): 17–25. http://dx.doi.org/10.1002/jez.1402420104.

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Turpin, JA, CA Schaeffer, SJ Terpening, L. Graham, M. Bu i WG Rice. "Reverse Transcription of Human Immunodeficiency Virus Type 1 is Blocked by Retroviral Zinc Finger Inhibitors". Antiviral Chemistry and Chemotherapy 8, nr 1 (luty 1997): 60–69. http://dx.doi.org/10.1177/095632029700800107.

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The Cys-Xaa2-Cys-Xaa4-His-Xaa4-Cys zinc fingers of retroviral nucleocapsid (NC) proteins are prime antiviral targets due to conservation of the Cys and His chelating residues and the absolute requirement of these fingers in both early and late phases of retroviral replication. Certain 2,2′-dithiobisbenzamides (DIBAs) chemically modify the Cys residues of the fingers, thereby inhibiting in vitro replication of human immunodeficiency virus type 1 (HIV-1). We examined the consequences of DIBA interaction with cell-free virions and their subsequent ability to initiate new rounds of infection. The DIBAs entered intact virions and chemically modified the p7NC proteins, resulting in extensive disulphide cross-linkage among zinc fingers of adjacent p7NC molecules. Likewise, treatment of Pr55gag-laden pseudovirions, used as a model of virion particles, with DIBAs resulted in Pr55gag cross-linkage. In contrast, monomeric p7NC protein did not form cross-linkages after DIBA treatment, indicating that the retroviral zinc finger proteins must exist in close proximity for cross-linkage to occur. Cross-linkage of p7NC in virions correlated with loss of infectivity and decreased proviral DNA synthesis during acute infection, even though DIBAs did not inhibit virus attachment to host cells or reverse transcriptase enzymatic activity. Thus, DIBA-type molecules impair the ability of HIV-1 virions to initiate reverse transcription through their action on the retroviral zinc finger, thereby blocking further rounds of replication.

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Toba, Gakuta, Jan Qui, Sandhya P. Koushika i Kalpana White. "Ectopic expression ofDrosophilaELAV and human HuD inDrosophilawing disc cells reveals functional distinctions and similarities". Journal of Cell Science 115, nr 11 (1.06.2002): 2413–21. http://dx.doi.org/10.1242/jcs.115.11.2413.

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Drosophila ELAV and human HuD are two neuronal RNA binding proteins that show remarkable sequence homology, yet differ in their respective documented roles in post-transcriptional regulation. ELAV regulates neural-specific alternative splicing of specific transcripts, and HuD stabilizes specific mRNAs that are otherwise unstable due to AU-rich elements(AREs) in their 3′ untranslated region (UTR). AREs are major determinants of transcript stability in mammalian cells. The role of each of these proteins was investigated and compared, by ectopically expressing them in Drosophila imaginal wing disc cells, which lack endogenous expression of either protein. The effect of the ectopic expression of ELAV and HuD was assessed on two sets of green fluorescent protein reporter transgenes,which were all driven with a broadly expressing promoter. Each set consisted of three reporter transgenes: (1) with an uninterrupted open reading frame(ORF); (2) with a constitutively spliced intron inserted into the ORF; and (3)with the intron nASI whose splicing is regulated in neurons by ELAV,inserted into the ORF. The two sets differed from each other only in their 3′UTR: Heat-shock-protein-70Ab (Hsp70Ab) trailer with ARE-like characteristics or Actin 5C (Act5C) trailer. Our results show that:(1) both ectopically expressed ELAV and HuD can enhance expression of transgenes with the Hsp70Ab 3′UTR, but not of transgenes with Act5C 3′UTR; (2) this enhancement is accompanied by an increase in mRNA level; (3) only ELAV can induce neural-specific splicing of nASI; and (4) although HuD is localized primarily to the cytoplasm,ELAV is localized to both the cytoplasm and the nucleus.

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Xie, Liang-yu, Sheng-nan Cao, Zi-teng Li, Dan-dan Wang i Bin Shi. "Effects of Fluid Shear Stress on Human Intervertebral Disc Nucleus Pulposus Cells Based on Label-Free Quantitative Proteomics". Disease Markers 2022 (14.09.2022): 1–9. http://dx.doi.org/10.1155/2022/3860898.

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Objective. To explore the possible mechanism of fluid shear stress on human nucleus pulposus cells based on label-free proteomics technology. Methods. The human nucleus pulposus cell line was purchased and subcultured in vitro. The Flexcell STR-4000 multiflow field cell fluid shear stress loading culture system was used to apply continuous laminar fluid shear stress (12 dyne/cm2, 45 mins) to the monolayer adherent cells. Those without mechanical loading were used as the control group, and those subjected to fluid shear loading were used as the experimental group. Differential protein expression was identified using mass spectrometry identification technology, and bioinformatics analysis was performed using Gene Ontology GO (Gene Ontology) and Kyoto Encyclopedia of Genes and Genomes KEGG (Kyoto Encyclopedia of Genes and Genomes). Results. The proteomics results of the experimental group and the control group showed that the total number of mass spectra was 638653, the number of matched mass spectra was 170110, the total number of identified peptides was 32050, the specific peptide was 30564, and the total number of identified proteins was 4745. Comparing the two groups, 47 proteins were significantly differentially expressed, namely, 25 upregulated proteins and 22 downregulated proteins. Bioinformatics analysis showed that significantly different proteins were mainly manifested in cellular process, biological regulation, metabolic process, binding, catalytic activity, cellular components (cell part), organelle part (organelle part), and other molecular biological functions. Conclusion. Using proteomics technology to screen human nucleus pulposus cells after fluid shear stress loading, the differential protein expression provides a basis for further exploration of the mechanism of mechanical factors on nucleus pulposus.

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Enke, Cathrin, Nadine Zekert, Daniel Veith, Carolin Schaaf, Sven Konzack i Reinhard Fischer. "Aspergillus nidulans Dis1/XMAP215 Protein AlpA Localizes to Spindle Pole Bodies and Microtubule Plus Ends and Contributes to Growth Directionality". Eukaryotic Cell 6, nr 3 (19.01.2007): 555–62. http://dx.doi.org/10.1128/ec.00266-06.

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ABSTRACTThe dynamics of cytoplasmic microtubules (MTs) is largely controlled by a protein complex at the MT plus end. InSchizosaccharomyces pombeand in filamentous fungi, MT plus end-associated proteins also determine growth directionality. We have characterized the Dis1/XMAP215 family protein AlpA fromAspergillus nidulansand show that it determines MT dynamics as well as hyphal morphology. Green fluorescent protein-tagged AlpA localized to MT-organizing centers (centrosomes) and to MT plus ends. The latter accumulation occurred independently of conventional kinesin or the Kip2-familiy kinesin KipA.alpAdeletion strains were viable and only slightly temperature sensitive. Mitosis, nuclear migration, and nuclear positioning were not affected, but hyphae grew in curves rather than straight, which appeared to be an effect of reduced MT growth and dynamics.

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Ishida, Takuma, Nobuyoshi Akimitsu, Tamami Kashioka, Masakazu Hatano, Toshio Kubota, Yasuyuki Ogata, Kazuhisa Sekimizu i Tsutomu Katayama. "DiaA, a Novel DnaA-binding Protein, Ensures the Timely Initiation ofEscherichia coliChromosome Replication". Journal of Biological Chemistry 279, nr 44 (23.08.2004): 45546–55. http://dx.doi.org/10.1074/jbc.m402762200.

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The DnaA protein is the initiator ofEscherichia colichromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitivednaAmutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. AdiaA::Tn5mutation suppresses the cold-sensitive growth of an overinitiation typednaAmutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in thediaAmutant cells as well as wild-type cells with pBR322 expressing thediaAgene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in anin vitrosystem especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication.

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Pohl, Pedro Henrique Isoldi, Thais Cuperman, Thomas Lozito, Takashi Yurube, Rocky Tuan, James Kang, Nam Vo i Luciano Miller Reis Rodrigues. "Expression of matrix factors in the process of neovascularization of intervertebral disc". Coluna/Columna 14, nr 2 (czerwiec 2015): 77–81. http://dx.doi.org/10.1590/s1808-185120151402132735.

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<sec><title>OBJECTIVE:</title> To investigate the effects of proteins products of endothelial cells (ECs) on the annulus fibrosus (AF) cell metabolism in an in vitro culture.</sec><sec><title>METHODS:</title>Human AF cells were expanded in monolayer cultures and treated with proteins from the medium of cell line HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). After 72h of treatment RNA was isolated from AF cells for analysis of gene expression and the culture medium was collected for protein expression analysis.</sec><sec><title>RESULTS:</title> The qRT-PCR analysis demonstrated increased gene expression of matrix metalloproteinases (MMPs) in AF cells treated with protein products of endothelial cells compared with cells from control group of AF cells: MMP-1 243.10 times (p<0.05), MMP-2 1.37 time (p<0.05), MMP-3 39.83 times (p<0.05) and MMP-13 5.70 times (p<0.05). In contrast, tissue inhibitors of metalloproteinases (TIMPs) were suppressed; TIMP-2 (0.55 time) (p<0.05) and TIMP-3 (0.60 time) (p<0.05) in the exposed groups. The expression of aggrecan gene (0.83 time) (p<0.05), an important extracellular matrix component, was also reduced. MMP-1 and MMP-3 detection was performed, confirming the results of PCR by Western Blot technique.</sec><sec><title>CONCLUSIONS:</title> In this study, we observed that the proteins produced by ECs induced the MMPs expression and suppressed the TIMPs as well as the aggrecan in primary cells of the human intervertebral disc, targeting the development of potential treatments for intervertebral disc degeneration and associated discogenic pain.</sec>

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Charroux, Bernard, Corinne Angelats, Laurent Fasano, Stephen Kerridge i Christine Vola. "The Levels of the bancal Product, aDrosophila Homologue of Vertebrate hnRNP K Protein, Affect Cell Proliferation and Apoptosis in Imaginal Disc Cells". Molecular and Cellular Biology 19, nr 11 (1.11.1999): 7846–56. http://dx.doi.org/10.1128/mcb.19.11.7846.

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ABSTRACT We have characterized the Drosophila bancal gene, which encodes a Drosophila homologue of the vertebrate hnRNP K protein. The bancal gene is essential for the correct size of adult appendages. Reduction of appendage size in bancalmutant flies appears to be due mainly to a reduction in the number of cell divisions in the imaginal discs. Transgenes expressingDrosophila or human hnRNP K are able to rescue weakbancal phenotype, showing the functional similarity of these proteins in vivo. High levels of either human orDrosophila hnRNP K protein in imaginal discs induces programmed cell death. Expression of the antiapoptotic P35 protein suppresses this phenotype in the eye, suggesting that apoptosis is the major cellular defect caused by overexpression of K protein. Finally, the human K protein acts as a negative regulator of bancalgene expression. We propose that negative autoregulation limits the level of Bancal protein produced in vivo.

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Benichou, Axel, Abraham Aserin i Nissim Garti. "Protein-Polysaccharide Interactions for Stabilization of Food Emulsions". Journal of Dispersion Science and Technology 23, nr 1&3 (2002): 93–123. http://dx.doi.org/10.1081/dis-120003307.

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YIN, HONGWEI, XIAOYONG XIAO, XIAOQING WEN i TIANSHOU ZHOU. "STABILITY OF REGULATORY PROTEIN GRADIENTS INDUCED BY MORPHOGEN DPP IN DROSOPHILA WING DISC". International Journal of Bifurcation and Chaos 23, nr 08 (sierpień 2013): 1350138. http://dx.doi.org/10.1142/s0218127413501381.

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In the development of Drosophila wing disc, morphogen Dpp, which is a signaling molecule from a local region and disperses into anterior and posterior compartments, builds up a gradient with precise pattern information. Experiments have demonstrated that the key genes (brk, dad, omb and sal) and phosphorylated protein (pMad), which are activated by Dpp signaling molecules and form the gradients of the corresponding proteins of these genes, direct and control the spatial pattern of the wing disc. However, the regulatory network of these genes are in complex and nonlinear interaction with upstream regulators and downstream targets. In this paper, the mathematical model is built according to the regulatory relationships of these key genes. The stabilities of the gradients of these corresponding proteins are investigated. Furthermore, numerical simulations show that these gradients are robust with respect to some major reaction rates in this regulatory network.

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