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61.
Paloma Acebo María Teresa Alda Manuel Espinosa Gloria del Solar 《FEMS microbiology letters》1996,140(1):85-91
Abstract Streptococcus pneumoniae genetic systems designed for isolation of plasmid mutants with copy-up phenotypes have been developed. The target plasmids have the pLS1 replicon, and two different strategies have been followed: (i) selection of clones exhibiting augmented resistance to antibiotics, or (ii) obligatory co-existence of incompatible plasmids. We have isolated 23 plasmid mutants exhibiting increased number of copies. All the mutations corresponded to four different alleles of the copG gene of plasmid pLS1. These strategies could be used with other plasmids. 相似文献
62.
Jos Antonio García-Espinoza Jos Francisco Muoz-Valle Mariel García-Chagolln Jorge Hernndez-Bello Claudia Azucena Palafox-Snchez Erika Fabiola Lpez-Villalobos Gabriela Athziri Snchez-Zuno Gloria Esther Martínez-Bonilla Sergio Cerpa-Cruz Francisco Josue Carrillo-Ballesteros Edith Oregon-Romero 《Current issues in molecular biology》2022,44(2):764
Background: Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease, which affects exocrine glands. T cell activation is a trigger mechanism in the immune response. Hyperreactivity of T cells and antibody production are features in pSS. ICOS can be critical in the pathogenesis of pSS. Methods: A total of 134 pSS patients and 134 control subjects (CS) were included. Genotyping was performed by PCR-RFLP. ICOS mRNA expression was quantified by real-time PCR, and CD4+ ICOS+ T cells were determined by flow cytometry. Results: The ICOS IVS1 + 173 T>C polymorphisms were not associated with susceptibility to pSS (p = 0.393, CI = 0.503–1.311). However, the c.1624 C>T polymorphism was associated with a reduction in the risk of development of pSS (p = 0.015, CI = 0.294–0.884). An increase in ICOS mRNA expression in patients was observed (3.7-fold). Furthermore, pSS patients showed an increase in membranal-ICOS expression (mICOS). High expression of mICOS (MFI) was associated with lymphocytic infiltration. Conclusions: The IVS1 + 173 polymorphism is not a genetic marker for the development of pSS, while c.1624 T allele was associated with a low risk. However, elevated mICOS expression in pSS patients with high lymphocytic infiltration was found. ICOS may have an important role in the immunopathogenesis of pSS and should be analyzed in T cell subsets in pSS patients as a possible disease marker. 相似文献
63.
Arabidopsis glt1-T mutant defines a role for NADH-GOGAT in the non-photorespiratory ammonium assimilatory pathway 总被引:1,自引:0,他引:1
Lancien M Martin M Hsieh MH Leustek T Goodman H Coruzzi GM 《The Plant journal : for cell and molecular biology》2002,29(3):347-358
The physiological role of the NADH-dependent glutamine-2-oxoglutarate aminotransferase (NADH-GOGAT) enzyme was addressed in Arabidopsis using gene expression analysis and by the characterization of a knock-out T-DNA insertion mutant (glt1-T) in the single NADH-GOGAT GLT1 gene. The NADH-GOGAT GLT1 mRNA is expressed at higher levels in roots than in leaves. This expression pattern contrasts with GLU1, the major gene encoding Fd-GOGAT, which is most highly expressed in leaves and is involved in photorespiration. These distinct organ-specific expression patterns suggested a non-redundant physiological role for the NADH-GOGAT and Fd-GOGAT gene products. To test the in vivo function of NADH-GOGAT, we conducted molecular and physiological analysis of the glt1-T mutant, which is null for NADH-GOGAT, as judged by mRNA level and enzyme activity. Metabolic analysis showed that the glt1-T mutant has a specific defect in growth and glutamate biosynthesis when photorespiration was repressed by 1% CO2. Under these conditions, the glt1-T mutant displayed a 20% decrease in growth and a dramatic 70% reduction in glutamate levels. Herein, we discuss the significance of NADH-GOGAT in non-photorespiratory ammonium assimilation and in glutamate synthesis required for plant development. 相似文献
64.
Laccase-like activity was detected in melanin-producing strains of Sinorhizobium meliloti mainly in cells at the stationary growth phase when copper was added to the medium. The laccase showed both syringaldazine and ABTS (2,2'-azino-bis-ethylbenzthiazoline-6-sulfonic acid) oxidase activities and was activated by the addition of 1.7 mM sodium dodecyl sulfate. Activity was totally inhibited by the addition of 1.0 mM EDTA, suggesting that the enzyme is a metal-dependent one. The enzyme was found to be cytosolic having an optimum pH of 5.0, an estimated molecular mass of 95 kDa and a K(m) of 4 microM for syringaldazine. Both laccase and tyrosinase activities were detected in melanin-producing S. meliloti strains. Plant growth-promoting (PGP) effect in rice by a laccase-producing S. meliloti strain when co-inoculated with Azospirillum brasilense Cd was observed. PGP effect by co-inoculation significantly increased plant yield compared to A. brasilense by itself. To the best of our knowledge this is the first report on laccase production in rhizobia and cooperation between Azospirillum and Sinorhizobium in rice. 相似文献
65.
Characterization of an apoplastic basic thaumatin-like protein from recalcitrant chestnut seeds 总被引:5,自引:0,他引:5
Gloria Garcia-Casado Carmen Collada Isabel Allona Alvaro Soto Rosa Casado Emilio Rodriguez-Cerezo Luis Gomez Cipriano Aragoncillo 《Physiologia plantarum》2000,110(2):172-180
Mature chestnut seeds, with one of the highest moisture contents described to date, accumulate certain defensive proteins at unusually elevated levels. In this work a major 23-kDa thaumatin-like protein, termed CsTL1, has been purified from mature chestnut ( Castanea sativa ) cotyledons. Amino acid sequencing and characterization of its full-length cDNA indicate that CsTL1 is synthesized as a preprotein with a signal peptide 22 amino acids in length. The mature protein contains 16 conserved cysteine residues presumably involved in disulfide bonding and has a high isoelectric point (ca. 9). Unlike most basic pathogenesis-related (PR) proteins, mature CsTL1 is localized to the extracellular matrix, as revealed by immunoelectron microscopy studies of cotyledonary cells. The isolated protein has in vitro antifungal activity against Trichoderma viride and Fusarium oxysporum and shows strong synergistic effects with CsCh1, the most abundant chestnut cotyledon endochitinase. Moreover, both CsTL1 and CsCh1 appear to be regulated in the same manner during seed development and germination. These observations, along with the recent finding of endoglucanase activity for some TL proteins, support the notion that CsTL1 and CsCh1 are part of a complex seed defensive system against microbial growth. Another possibility is that these, and probably other seed PR proteins, have antifreeze activity. Both functions would be particularly relevant for chestnut seeds given their remarkable moisture content at maturity. 相似文献
66.
Mónica M. S. de Cano M. Cr de Mulé Gloria Z. de Caire Delia R. de Halperin 《Journal of applied phycology》1990,2(1):79-81
Phenolic compounds were determined in methanolic extract from the algal mass of aNostoc muscorum culture. Bioassays with two human pathogens,Candida albicans andStaphylococcus aureus indicated that algal phenolic compounds evoked significant growth inhibition for both species (89.1% and 88.2%, respectively). It is suggested that this strong inhibitory effect is of potential medicinal value. 相似文献
67.
Gloria Salazar Stephanie Zlatic Branch Craige Andrew A. Peden Jan Pohl Victor Faundez 《The Journal of biological chemistry》2009,284(3):1790-1802
The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting.
Disease is triggered by defects in any of 15 mouse gene products, which are
part of five distinct cytosolic molecular complexes: AP-3, homotypic fusion
and vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecular
associations of these complexes, we used in vivo cross-linking
followed by purification of cross-linked AP-3 complexes and mass spectrometric
identification of associated proteins. AP-3 was co-isolated with BLOC-1,
BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits;
clathrin; and phosphatidylinositol-4-kinase type II α (PI4KIIα).
We previously reported that this membrane-anchored enzyme is a regulator of
AP-3 recruitment to membranes and a cargo of AP-3 (Craige, B.,
Salazar, G., and Faundez, V. (2008) Mol. Biol.
Cell
19,1415
-1426). Using cells deficient
in different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1,
but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIα inclusion into
AP-3 complexes. BLOC-1, PI4KIIα, and AP-3 belong to a tripartite
complex, and down-regulation of either PI4KIIα, BLOC-1, or AP-3
complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1
complex modulates the association of PI4KIIα with AP-3. These results
suggest that AP-3 and BLOC-1 act, either in concert or sequentially, to
specify sorting of PI4KIIα along the endocytic route.Membranous organelles along the exocytic and endocytic pathways are each
defined by unique lipid and protein composition. Vesicle carriers communicate
and maintain the composition of these organelles
(2). Consequently defining the
machineries that specify vesicle formation, composition, and delivery are
central to understanding membrane protein traffic. Generally vesicle
biogenesis uses multiprotein cytosolic machineries to select membrane
components for inclusion in nascent vesicles
(2,
3). Heterotetrameric adaptor
complexes (AP-1 to AP-4) are critical to generate vesicles of specific
composition from the different organelles constituting the exocytic and
endocytic routes
(2-4).The best understood vesicle formation machinery in mammalian cells is the
one organized around the adaptor complex AP-2
(5). This complex generates
vesicles from the plasma membrane using clathrin. Our present detailed
understanding of AP-2 vesicle biogenesis mechanisms and interactions emerged
from a combination of organellar and in vitro binding proteomics
analyses together with the study of binary interactions in cell-free systems
(5-9).
In contrast, the vesicle biogenesis pathways controlled by AP-3 are far less
understood. AP-3 functions to produce vesicles that traffic selected membrane
proteins from endosomes to lysosomes, lysosome-related organelles, or synaptic
vesicles
(10-13).
AP-3 is one of the protein complexes affected in the Hermansky-Pudlak syndrome
(HPS;3 Online
Mendelian Inheritance in Man (OMIM) 203300). So far, mutations in any of 15
mouse or eight human genes trigger a common syndrome. This syndrome
encompasses defects that include pigment dilution, platelet dysfunction,
pulmonary fibrosis, and occasionally neurological phenotypes
(14,
15). All forms of HPS show
defective vesicular biogenesis or trafficking that affects lysosomes,
lysosome-related organelles (for example melanosomes and platelet dense
granules), and, in some of them, synaptic vesicles
(11-13).
Most of the 15 HPS loci encode polypeptides that assemble into five distinct
molecular complexes: the adaptor complex AP-3, HOPS, and the BLOC complexes 1,
2, and 3 (14). Recently binary
interactions between AP-3 and BLOC-1 or BLOC-1 and BLOC-2 suggested that
arrangements of these complexes could regulate membrane protein targeting
(16). Despite the abundance of
genetic deficiencies leading to HPS and genetic evidence that HPS complexes
may act on the same pathway in defined cell types
(17), we have only a partial
picture of protein interactions organizing these complexes and how they might
control membrane protein targeting.In this study, we took advantage of cell-permeant and reversible
cross-linking of HPS complexes followed by their immunoaffinity purification
to identify novel molecular interactions. Cross-linked AP-3 co-purified with
BLOC-1, BLOC-2, HOPS, clathrin, and the membrane protein PI4KIIα. We
previously identified PI4KIIα as a cargo and regulator of AP-3
recruitment to endosomes (1,
18). Using mutant cells
deficient in either individual HPS complexes or a combination of them, we
found that BLOC-1 facilitates the interaction of AP-3 and PI4KIIα. Our
studies demonstrate that subunits of four of the five HPS complexes co-isolate
with AP-3. Moreover BLOC-1, PI4KIIα, and AP-3 form a tripartite complex
as demonstrated by sequential co-immunoprecipitations as well as by similar
LAMP1 distribution phenotypes induced by down-regulation of components of this
tripartite complex. Our findings indicate that BLOC-1 complex modulates the
recognition of PI4KIIα by AP-3. These data suggest that AP-3, either in
concert or sequentially with BLOC-1, participates in the sorting of common
membrane proteins along the endocytic route. 相似文献
68.
SCARFACE encodes an ARF-GAP that is required for normal auxin efflux and vein patterning in Arabidopsis 下载免费PDF全文
Sieburth LE Muday GK King EJ Benton G Kim S Metcalf KE Meyers L Seamen E Van Norman JM 《The Plant cell》2006,18(6):1396-1411
To identify molecular mechanisms controlling vein patterns, we analyzed scarface (sfc) mutants. sfc cotyledon and leaf veins are largely fragmented, unlike the interconnected networks in wild-type plants. SFC encodes an ADP ribosylation factor GTPase activating protein (ARF-GAP), a class with well-established roles in vesicle trafficking regulation. Quadruple mutants of SCF and three homologs (ARF-GAP DOMAIN1, 2, and 4) showed a modestly enhanced vascular phenotype. Genetic interactions between sfc and pinoid and between sfc and gnom suggest a possible function for SFC in trafficking of auxin efflux regulators. Genetic analyses also revealed interaction with cotyledon vascular pattern2, suggesting that lipid-based signals may underlie some SFC ARF-GAP functions. To assess possible roles for SFC in auxin transport, we analyzed sfc roots, which showed exaggerated responses to exogenous auxin and higher auxin transport capacity. To determine whether PIN1 intracellular trafficking was affected, we analyzed PIN1:green fluorescent protein (GFP) dynamics using confocal microscopy in sfc roots. We found normal PIN1:GFP localization at the apical membrane of root cells, but treatment with brefeldin A resulted in PIN1 accumulating in smaller and more numerous compartments than in the wild type. These data suggest that SFC is required for normal intracellular transport of PIN1 from the plasma membrane to the endosome. 相似文献
69.
70.
While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits [small ribosomal subunits (SSUs)] and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized the function of Escherichia coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase-deficient form of this protein. Our data reveal cold sensitivity and altered ribosomal profiles are associated with a DeltaksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis-prompting discussion of KsgA as a possible antimicrobial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family. 相似文献