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immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma 总被引:1,自引:0,他引:1
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L Skriver LI Larsson V Kielberg LS Nielsen PB Andresen P Kristensen K Dano 《The Journal of cell biology》1984,99(2):753-758
The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA. 相似文献
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Yuri Ypez Mariana Marcano-Ruiz Rafael S Bezerra Bibiana Fam Joo PB Ximenez Wilson A Silva Jr Maria Ctira Bortolini 《Genetics and molecular biology》2022,45(1)
Our goal was to describe in more detail the evolutionary history of Gamma and two derived lineages (P.1.1 and P.1.2), which are part of the arms race that SARS-CoV-2 wages with its host. A total of 4,977 sequences of the Gamma strain of SARS-CoV-2 from Brazil were analyzed. We detected 194 sites under positive selection in 12 genes/ORFs: Spike, N, M, E, ORF1a, ORF1b, ORF3, ORF6, ORF7a, ORF7b, ORF8, and ORF10. Some diagnostic sites for Gamma lacked a signature of positive selection in our study, but these were not fixed, apparently escaping the action of purifying selection. Our network analyses revealed branches leading to expanding haplotypes with sites under selection only detected when P.1.1 and P.1.2 were considered. The P.1.2 exclusive haplotype H_5 originated from a non-synonymous mutational step (H3509Y) in H_1 of ORF1a. The selected allele, 3509Y, represents an adaptive novelty involving ORF1a of P.1. Finally, we discuss how phenomena such as epistasis and antagonistic pleiotropy could limit the emergence of new alleles (and combinations thereof) in SARS-COV-2 lineages, maintaining infectivity in humans, while providing rapid response capabilities to face the arms race triggered by host immuneresponses. 相似文献
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Integration of thylakoid proteins by the chloroplast signal recognition particle (cpSRP) posttranslational transport pathway requires the cpSRP, an SRP receptor homologue (cpFtsY), and the membrane protein ALB3. Similarly, Escherichia coli uses an SRP and FtsY to cotranslationally target membrane proteins to the SecYEG translocase, which contains an ALB3 homologue, YidC. In neither system are the interactions between soluble and membrane components well understood. We show that complexes containing cpSRP, cpFtsY, and ALB3 can be precipitated using affinity tags on cpSRP or cpFtsY. Stabilization of this complex with GMP-PNP specifically blocks subsequent integration of substrate (light harvesting chl a/b-binding protein [LHCP]), indicating that the complex occupies functional ALB3 translocation sites. Surprisingly, neither substrate nor cpSRP43, a component of cpSRP, was necessary to form a complex with ALB3. Complexes also contained cpSecY, but its removal did not inhibit ALB3 function. Furthermore, antibody bound to ALB3 prevented ALB3 association with cpSRP and cpFtsY and inhibited LHCP integration suggesting that a complex containing cpSRP, cpFtsY, and ALB3 must form for proper LHCP integration. 相似文献
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Black AP Bhayani H Ryder CA Pugh MT Gardner-Medwin JM Southwood TR 《Arthritis research & therapy》2003,5(5):R277-R284
The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients
with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to
a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients
with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients:
a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested;
and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial
fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients
without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association
between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence
recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses
to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells
in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association
between the acute phase response and the T cell population recruited to an inflammatory site. 相似文献
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Narayana PB Fazolini André LS Cruz Miriam BF Werneck Jo?o PB Viola Clarissa M Maya-Monteiro Patrícia T Bozza 《Cell cycle (Georgetown, Tex.)》2015,14(16):2667-2676
Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma. 相似文献
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Recent advances in technology have allowed for the identification of complex protein mixtures in a rapid fashion. This report highlights the use of 2D gel electrophoresis, mass spectrometry, and database analysis to determine contaminating species of the Escherichia coli genome that are present during immobilized metal affinity chromatography (IMAC), highlighting Co(2+) as the affinity ligand. Four proteins (triosephosphate isomerase, alpha galactosidase, Hsp90, and glucosamine 6-phosphate synthase) constitute the majority of E. coli proteins that bind and potentially may coelute during chromatography. Results are discussed within the context of changes that when implemented could lead to an increase in IMAC efficiency, not by altering column conditions, but rather by changing the nature of the nuisance proteins that principally reduce column capacity and extend processing times. Such a study illustrates the use of proteome data to aid in bioprocess design. 相似文献
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Naomi J. Marty Dakshinamurthy Rajalingam Alicia D. Kight Nathaniel E. Lewis Daniel Fologea Thallapuranam Krishnaswamy Suresh Kumar Ralph L. Henry Robyn L. Goforth 《The Journal of biological chemistry》2009,284(22):14891-14903
The chloroplast signal recognition particle (cpSRP) and its receptor
(cpFtsY) function in thylakoid biogenesis to target integral membrane proteins
to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions
between thylakoid membranes and the soluble stroma. Based on sequence
alignments, a membrane-binding motif identified in Escherichia coli
FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is
responsible for the membrane-binding function of cpFtsY has yet to be shown
experimentally. Our studies show that a small N-terminal region in cpFtsY
stabilizes a membrane interaction critical to cpFtsY function in
cpSRP-dependent protein targeting. This membrane-binding motif is both
necessary and sufficient to direct cpFtsY and fused passenger proteins to
thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may
be functionally replaced by the corresponding region from E. coli,
confirming that the membrane-binding motif is conserved among organellar and
prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding
correlates with liposome-induced GTP hydrolysis stimulation. Mutations that
debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP
hydrolysis, suggesting that negative regulation is provided by the intact
membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD
structural studies of the N-terminal region and the analogous region in the
E. coli SRP receptor revealed a conformational change in secondary
structure that takes place upon lipid binding. These studies suggest that the
cpFtsY membrane-binding motif plays a critical role in the intramolecular
communication that regulates cpSRP receptor functions at the membrane.Proper compartmentalization of proteins relies on the ability of protein
localization pathways to transport proteins efficiently from their sites of
synthesis to their sites of function. The signal recognition particle
(SRP)2 and its
receptor function in every kingdom of life to target proteins to the
endoplasmic reticulum (eukaryotes), cytoplasmic membrane (prokaryotes), and
thylakoid membrane (chloroplasts)
(1). The targeting function of
SRP relies on a conserved 54-kDa SRP subunit (SRP54; Ffh in Escherichia
coli and cpSRP54 in chloroplasts) as well as a conserved SRP receptor
(SRα; FtsY in E. coli and cpFtsY in chloroplasts). For
cytosolic SRPs (SRP54 and Ffh), interactions with a substrate signal sequence
and an SRP RNA moiety are prerequisite for interaction with the SRP receptor
(SRα and FtsY) (2). GTP
binding and hydrolysis by both SRP54 and SRα coordinate substrate
release from SRP to the translocon and release of SRP from SRα. In
chloroplasts, cpFtsY functions along with a unique SRP (cpSRP) to
post-translationally target nuclear encoded proteins to thylakoid membranes
(3). Light-harvesting
chlorophyll a/b-binding proteins (LHCPs) imported into the
chloroplast stroma are bound by cpSRP to form a soluble targeting complex,
which directs the LHCP substrate to the thylakoid membrane translocon Alb3
(Albino3) in a GTP- and cpFtsY-dependent manner
(14,
36). Although many general
steps of SRP protein targeting seem largely conserved across evolutionary
boundaries, the nature and dynamics of the receptor appear to have
diverged.In eukaryotic systems, SRα is peripherally bound to the membrane
through association with the integral membrane subunit SRβ. In contrast,
no chloroplast or bacterial homolog of SRβ has been identified. cpFtsY
and E. coli FtsY (EcFtsY) are found partitioned between the membrane
and the stroma or cytosol, respectively. The membrane-binding capacity of
EcFtsY serves to stimulate GTPase activity and appears critical in that only
membrane-associated EcFtsY supports the release of nascent chains from SRP to
the translocon (4,
5). However, the partitioning
activity is not strictly required because EcFtsY tethered to the membrane is
functional in vivo
(37). Given the conserved
nature of partitioning among bacterial and chloroplast SRP receptors,
partitioning may play an, as of yet, unidentified role in protein targeting by
SRP. Nevertheless, differences in lipid composition between bacterial and
thylakoid membranes make it interesting to speculate that there are
mechanistic differences in membrane partitioning.Like many prokaryotic FtsY homologs (e.g. Thermus aquaticus),
cpFtsY lacks the N-terminal acidic domain (A domain) implicated in EcFtsY
membrane binding (6). Although
the highly conserved FtsY GTPase domain (NG domain) of EcFtsY
(EcFtsYNG) fails to support protein targeting, the addition of the
last A domain residue, Phe-196 of a conserved double-Phe motif
(EcFtsYNG+1), restores protein targeting in vivo
(7). In vitro studies
also show that EcFtsYNG+1 retains the capacity to bind membranes
and support integration of SRP-dependent substrates, although at significantly
reduced levels compared with full-length EcFtsY
(8). A resolved structure of
EcFtsYNG+1 suggests that the amphipathic nature of the region
containing Phe-196 plays a critical role in membrane association
(9). Furthermore, it has been
demonstrated that liposomes stimulate GTP hydrolysis rates of SRP with
EcFtsYNG+1, but not with EcFtsYNG, supporting the idea
that the A domain in its entirety is not strictly required.For cpFtsY, the necessity and functional role(s) of partitioning between a
thylakoid-bound and a soluble phase, as well as the role of N-terminal
residues in these functions, remain unknown. In addition, both the
conformational state of membrane-bound cpFtsY and EcFtsY and the mechanism
responsible for controlling membrane partitioning and altered GTPase activity
remain unclear. Because of the gain of function exhibited by
EcFtsYNG+1 and the conserved nature of the surrounding motif
(9), it seems likely that this
conserved region is necessary to support membrane binding and corresponding
functions not only in EcFtsY but also in FtsY homologs.To examine the functional role of the N-terminal region of cpFtsY, we have
utilized deletion and point mutants in assays that reconstitute cpFtsY
activities, including the cpSRP-dependent integration of LHCP. Together, our
data indicate that the conserved lipid-binding motif identified in bacterial
FtsY homologs is present in cpFtsY and is both necessary and sufficient for
thylakoid binding and critical for LHCP targeting. 相似文献