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81.
Ethidium analogues with improved resolution in the dye-buoyant density procedure. 总被引:1,自引:0,他引:1 下载免费PDF全文
Analogues of ethidium chloride with large quaternary side chains have been synthesized and evaluated for improved resolution in the dye-buoyant density system for the separation of open and closed circular DNA. These compounds are similar to propidium which differs from ethidium by the replacement of an ethyl group by a methyldiethylaminopropyl group. The new analogues contain a triethylamino group attached to a methylene chain with 3, 5 or 7 carbons. With PM2 DNA the observed separation for propidium is 1.97 times that of ethidium and the new dyes show separations relative to ethidium of 2.27, 2.66 and 2.77. A correlation is established between the mass of the dye component and the observed separation which is retionalized on the basis of the four component thermodynamics describing this system. 相似文献
82.
Bertha C. Elias Takuya Suzuki Ankur Seth Francesco Giorgianni Gautam Kale Le Shen Jerrold R. Turner Anjaparavanda Naren Dominic M. Desiderio Radhakrishna Rao 《The Journal of biological chemistry》2009,284(3):1559-1569
Occludin is phosphorylated on tyrosine residues during the oxidative
stress-induced disruption of tight junction, and in vitro
phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the
present study mass spectrometric analyses of C-terminal domain of occludin
identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation
sites, which are located in a highly conserved sequence of occludin, YETDYTT;
Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion
of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and
the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin
also abolished the c-Src-mediated phosphorylation and regulation of ZO-1
binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding
even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A
mutant was localized at the plasma membrane and cell-cell contact sites in
Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at
the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant
occludin in Madin-Darby canine kidney cells was significantly delayed compared
with that of wild type occludin or its T398A/T402A mutant. Furthermore,
expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for
hydrogen peroxide-induced barrier disruption. This study reveals a unique
motif in the occludin sequence that is involved in the regulation of ZO-1
binding by reversible phosphorylation of specific Tyr residues.Epithelial tight junctions
(TJs)2 form a
selective barrier to the diffusion of toxins, allergens, and pathogens from
the external environment into the tissues in the gastrointestinal tract, lung,
liver, and kidney (1).
Disruption of TJs is associated with the gastrointestinal diseases such as
inflammatory bowel disease, celiac disease, infectious enterocolitis, and
colon cancer
(2–4)
as well as in diseases of lung and kidney
(5,
6). Numerous inflammatory
mediators such as tumor necrosis factor α, interferon γ, and
oxidative stress
(7–12)
are known to disrupt the epithelial TJs and the barrier function. Several
studies have indicated that hydrogen peroxide disrupts the TJs in intestinal
epithelium by a tyrosine kinase-dependent mechanism
(11,
12).Four types of integral proteins, occludin, claudins, junctional adhesion
molecules, and tricellulin are associated with TJs. Occludin, claudins, and
tricellulin are tetraspan proteins, and their extracellular domains interact
with homotypic domains of the adjacent cells
(1,
2,
13). The intracellular domains
of these proteins interact with a variety of soluble proteins such as ZO-1,
ZO-2, ZO-3, 7H6, cingulin, and symplekin
(14–23);
this protein complex interacts with the perijunctional actomyosin ring. The
interactions among TJ proteins are essential for the assembly and the
maintenance of TJs. Therefore, regulation of the interactions among TJ
proteins may regulate the TJ integrity. A significant body of evidence
indicates that numerous signaling molecules are associated with the TJs.
Protein kinases and protein phosphatases such as protein kinase Cζ
(PKCζ), PKCι/λ
(24), c-Src
(25), c-Yes
(26,
27), mitogen-activated protein
kinase (28), PP2A, and PP1
(29) interact with TJs,
indicating that TJs are dynamically regulated by intracellular signal
transduction involving protein phosphorylation. Additionally, other signaling
molecules such as calcium
(30), phosphatidylinositol
3-kinase (31), Rho
(32), and Rac
(33) are involved in the
regulation of TJs.Occludin, a ∼65-kDa protein, has been well characterized to be
assembled into the TJs. Although occludin knock-out mice showed the formation
of intact TJs in different epithelia
(34), numerous studies have
emphasized that it plays an important role in the regulation of TJ integrity.
Occludin spans the membrane four times to form two extracellular loops and one
intracellular loop, and the N-terminal and C-terminal domains hang into the
intracellular compartment
(35–37).
In epithelium with intact TJs, occludin is highly phosphorylated on Ser and
Thr residues (38), whereas Tyr
phosphorylation is undetectable. However, the disruption of TJs in Caco-2 cell
monolayers by oxidative stress and acetaldehyde leads to Tyr phosphorylation
of occludin; the tyrosine kinase inhibitors attenuate the disruption of TJs
(39,
40). Furthermore, a previous
in vitro study demonstrated that Tyr phosphorylation of the
C-terminal domain of occludin leads to the loss of its interaction with ZO-1
and ZO-3 (25).In the present study we identified the Tyr residues in occludin that are
phosphorylated by c-Src and determined their role in regulated interaction
between occludin and ZO-1 and its assembly into the TJs. Results show that 1)
Tyr-379 and Tyr-383 in chicken occludin and Tyr-398 and Tyr-402 in human
occludin are the exclusive sites of phosphorylation by c-Src, and these Tyr
residues are located in a highly conserved sequence of occludin, YET-DYTT, 2)
deletion of YEDTYTT or point mutation of Tyr-398 and Tyr-402 in human occludin
attenuates the phosphorylation-dependent regulation of ZO-1 binding, 3)
Y398D/Y402D mutation of human occludin leads to loss of ZO-1 binding and
prevents its translocation to the plasma membrane and cell-cell contact sites
in Rat-1 cells, 4) Y398D/Y402D mutation of occludin delays its assembly into
the intercellular junctions during the calcium-induced assembly of TJs, and 5)
expression of Y398D/Y402D mutant occludin sensitizes cell monolayers for
hydrogen peroxide-induced disruption of barrier function. 相似文献
83.
S. A. Angioi D. Rau M. Rodriguez G. Logozzo F. Desiderio R. Papa G. Attene 《Molecular breeding : new strategies in plant improvement》2009,23(3):413-429
Studies of the level and the structure of the genetic diversity of local varieties of Phaseolus vulgaris are of fundamental importance, both for the management of genetic resources and to improve our understanding of the pathways
of dissemination and the evolution of this species in Europe. We have here characterized 73 local bean populations from Sardinia
(Italy) using seed traits and molecular markers (phaseolins, nuSSRs and cpSSRs). American landraces and commercial varieties
were also included for comparison. We see that: (a) the Sardinian material is distinct from the commercial varieties considered;
(b) the variation in the seed traits is high and it mostly occurs among populations (95%); (c) compared to the American sample
and the commercial varieties, the Sardinian collection has a low level of diversity; (d) the majority (>95%) of the Sardinian
individuals belong to the Andean gene pool; (e) the Sardinian material shows a strong genetic structure, both for cpSSRs and
nuSSRs; (f) the nuSSRs and cpSSRs concur in differentiating between gene pools, but a lack of congruence between nuclear and
chloroplast has been observed within gene pools; and (g) there are three putative hybrids between the Andean and Mesoamerican
gene pools. Despite the relatively low level of diversity, which is probably due to a strong founder effect, the Sardinian
landraces are worth being conserved and studied further because of their distinctiveness and because hybridization within
and between the gene pools could generate variation that will be useful for breeding.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
84.
De Rossi A Desiderio C 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2006,839(1-2):6-11
High sensitivity analysis of oxprenolol in spiked human urine has been performed by capillary zone electrophoresis (CZE) in ammonium formate buffer pH 2.5 using an uncoated capillary with 1cm length C18 on-capillary preconcentrator at the inlet side. The preconcentrator was fabricated in laboratory using the packing method and not encapped C18 5 microm particles as stationary phase material. The packed path was retained into the capillary by sintered stationary phase frits. Before running the CZE analysis, the oxprenolol was eluted from the preconcentrator by injecting a short plug of acetonitrile/water mixtures. With respect to classical CZE, the use of on-line preconcentrator widely increased the method sensitivity allowing the detection of the drug at 0.5 ng/mL (injected concentration). The method showed a linear response in the range of 1-150 ng/mL oxprenolol standard compound. The intra-day repeatability (n = 11) R.S.D. values for migration time, peak area and normalized peak area were 0.72%, 3.96% and 3.66%, respectively, while inter-day repeatability (n = 5 days) R.S.D. values were 2.74%, 9.41% and 9.83%, respectively. The method was successfully applied to the analysis of oxprenolol in extracted urine spiked at 250 pg/mL (oxprenolol LOQ concentration in urine). 相似文献
85.
Marina Ferrarini Nathalie Steimberg Maurilio Ponzoni Daniela Belloni Angiola Berenzi Stefania Girlanda Federico Caligaris-Cappio Giovanna Mazzoleni Elisabetta Ferrero 《PloS one》2013,8(8)
Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patients''sera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo.Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy. 相似文献
86.
Di Carli M Zamboni A Pè ME Pezzotti M Lilley KS Benvenuto E Desiderio A 《Journal of proteome research》2011,10(2):429-446
The practice of postharvest withering is commonly used to correct quality traits and sugar concentration of high quality wines. To date, changes in the metabolome during the berry maturation process have been well documented; however, the biological events which occur at the protein level have yet to be fully investigated. To gain insight into the postharvest withering process, we studied the protein expression profiles of grape (Corvina variety) berry development focusing on withering utilizing a two-dimensional differential in gel electrophoresis (2D-DIGE) proteomics approach. Comparative analysis revealed changes in the abundance of numerous soluble proteins during the maturation and withering processes. On a total of 870 detected spots, 90 proteins were differentially expressed during berry ripening/withering and 72 were identified by MS/MS analysis. The majority of these proteins were related to stress and defense activity (30%), energy and primary metabolism (25%), cytoskeleton remodelling (7%), and secondary metabolism (5%). Moreover, this study demonstrates an active modulation of metabolic pathways throughout the slow dehydration process, including de novo protein synthesis in response to the stress condition and further evolution of physiological processes originated during ripening. These data represent an important insight into the withering process in terms of both Vitis germplasm characterization and knowledge which can assist quality improvement. 相似文献
87.
Castagnola M Inzitari R Fanali C Iavarone F Vitali A Desiderio C Vento G Tirone C Romagnoli C Cabras T Manconi B Sanna MT Boi R Pisano E Olianas A Pellegrini M Nemolato S Heizmann CW Faa G Messana I 《Molecular & cellular proteomics : MCP》2011,10(1):M110.003467
Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins β(4) and β(10), antileukoproteinase, histone H1c, and α and γ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures. 相似文献
88.
Desiderio C D'Angelo L Rossetti DV Iavarone F Giardina B Castagnola M Massimi L Tamburrini G Di Rocco C 《Proteomics》2012,12(13):2158-2166
Posterior cranial fossa is the most frequent location of pediatric brain tumors. Its diagnosis is currently performed by postsurgery histopathology and the identification of biomarkers in cerebrospinal fluid (CSF) could provide a less invasive tool. Patient CSF was collected during surgery before the tumor removal (PRE-CSF) and 6 days after the resection (POST-CSF) and analyzed by top down LC-MS proteomics for comparison. The PRE-CSFs generally exhibited a less complex LC-MS profile than the relative POST-CSFs suggesting a suppressive role of the tumor toward proteins and peptides production or release. Particularly, a panel of peptides, identified as alpha- and beta-hemoglobin chains fragments, were generally absent in the PRE-CSF and present in the POST ones independently from contaminant blood hemoglobin. Among them, the LVV- and VV-hemorphin-7 showed the most repeatable trend and with a few remarkable exceptions: their unusual absence in POST surgery CSF was in fact interestingly correlated to the presence of tumor in the patient despite surgery due to metastases or to subtotal resection. These results ascribed a relevant biological role to LVV- and VV-h7 peptides in the disease and a strong potential as biomarkers. Their analysis in POST surgery CSF could be used to predict patient prognosis. 相似文献
89.
Giorgia Carletti Andrea Carra Gianni Allegro Lorenzo Vietto Francesca Desiderio Paolo Bagnaresi Alberto Gianinetti Luigi Cattivelli Giampiero Valè Giuseppe Nervo 《PloS one》2016,11(3)
The genus Populus represents one of the most economically important groups of forest trees. It is composed by approximately 30 species used for wood and non-wood products, phytoremediation and biomass. Poplar is subjected to several biological and environmental threats although, compared to annual crops, we know far less about the genetic bases of biotic stress resistance. Woolly poplar aphid (Phloeomyzus passerinii) is considered a main pest of cultivated poplars in European and American countries. In this work we present two high density linkage maps in poplar obtained by a genotyping by sequencing (GBS) approach and the identification of QTLs involved in Ph. passerinii resistance. A total of 5,667 polymorphic markers (5,606 SNPs and 61 SSRs) identified on expressed sequences have been used to genotype 131 plants of an F1 population P ×canadensis obtained by an interspecific mate between Populus deltoides (resistant to woolly poplar aphid) and Populus nigra (susceptible to woolly poplar aphid). The two linkage maps, obtained following the two-way pseudo-testcross mapping strategy, have been used to investigate the genetic bases of woolly poplar aphid resistance. One major QTL and two QTLs with minor effects (mapped on LGV, LGXVI and LG XIX) explaining the 65.8% of the genetic variance observed in the progeny in response to Ph. passerinii attack were found. The high density coverage of functional markers allowed the identification of three genes belonging to disease resistance pathway as putative candidates for P. deltoides resistance to woolly poplar aphid. This work is the first report on genetic of woolly poplar aphid genetic resistance and the resistant loci associated markers identified represent a valuable tool in resistance poplar breeding programs. 相似文献
90.