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At least 541 records · Page 30Linked to original sources
PubMed2003

SPL8, an SBP-box gene that affects pollen sac development in Arabidopsis.

SQUAMOSA PROMOTER BINDING PROTEIN-box genes (SBP-box genes) encode plant-specific proteins that share a highly conserved DNA binding domain, the SBP domain. Although likely to represent transcription factors, little is known about their role in development. In Arabidopsis, SBP-box genes constitute a structurally heterogeneous family of 16 members known as SPL genes. For one of these genes, SPL8, we isolated three independent transposon-tagged mutants, all of which exhibited a strong reduction in fertility. Microscopic analysis revealed that this reduced fertility is attributable primarily to abnormally developed microsporangia, which exhibit premeiotic abortion of the sporocytes. In addition to its role in microsporogenesis, the SPL8 knockout also seems to affect megasporogenesis, trichome formation on sepals, and stamen filament elongation. The SPL8 mutants described help to uncover the roles of SBP-box genes in plant development.

Alleles↗
PubMed2003

Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis.

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.

Actin-Related Protein 2↗
PubMed2003

KIC, a novel Ca2+ binding protein with one EF-hand motif, interacts with a microtubule motor protein and regulates trichome morphogenesis.

Kinesin-like calmodulin binding protein (KCBP) is a microtubule motor protein involved in the regulation of cell division and trichome morphogenesis. Genetic studies have shown that KCBP is likely to interact with several other proteins. To identify KCBP-interacting proteins, we used the C-terminal region of KCBP in a yeast two-hybrid screen. This screening resulted in the isolation of a novel KCBP-interacting Ca2+ binding protein (KIC). KIC, with its single EF-hand motif, bound Ca2+ at a physiological concentration. Coprecipitation with bacterially expressed protein and native KCBP, gel-mobility shift studies, and ATPase assays with the KCBP motor confirmed that KIC interacts with KCBP in a Ca2+-dependent manner. Interestingly, although both Ca2+-KIC and Ca2+-calmodulin were able to interact with KCBP and inhibit its microtubule binding activity, the concentration of Ca2+ required to inhibit the microtubule-stimulated ATPase activity of KCBP by KIC was threefold less than that required for calmodulin. Two KIC-related Ca2+ binding proteins and a centrin from Arabidopsis, which contain one and four EF-hand motifs, respectively, bound Ca2+ but did not affect microtubule binding and microtubule-stimulated ATPase activities of KCBP, indicating the specificity of Ca2+ sensors in regulating their targets. Overexpression of KIC in Arabidopsis resulted in trichomes with reduced branch number resembling the zwichel/kcbp phenotype. These results suggest that KIC modulates the activity of KCBP in response to changes in cytosolic Ca2+ and regulates trichome morphogenesis.

Adenosine Triphosphatases↗
PubMed2003

The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development.

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.

Amino Acid Sequence↗
PubMed2004

Mutations in the pale aleurone color1 regulatory gene of the Zea mays anthocyanin pathway have distinct phenotypes relative to the functionally similar TRANSPARENT TESTA GLABRA1 gene in Arabidopsis thaliana.

The pale aleurone color1 (pac1) locus, required for anthocyanin pigment in the aleurone and scutellum of the Zea mays (maize) seed, was cloned using Mutator transposon tagging. pac1 encodes a WD40 repeat protein closely related to anthocyanin regulatory proteins ANTHOCYANIN11 (AN11) (Petunia hybrida [petunia]) and TRANSPARENT TESTA GLABRA1 (TTG1) (Arabidopsis thaliana). Introduction of a 35S-Pac1 transgene into A. thaliana complemented multiple ttg1 mutant phenotypes, including ones nonexistent in Z. mays. Hybridization of Z. mays genomic BAC clones with the pac1 sequence identified an additional related gene, mp1. PAC1 and MP1 deduced protein sequences were used as queries to build a phylogenetic tree of homologous WD40 repeat proteins, revealing an ancestral gene duplication leading to two clades in plants, the PAC1 clade and the MP1 clade. Subsequent duplications within each clade have led to additional WD40 repeat proteins in particular species, with all mutants defective in anthocyanin expression contained in the PAC1 clade. Substantial differences in pac1, an11, and ttg1 mutant phenotypes suggest the evolutionary divergence of regulatory mechanisms for several traits that cannot be ascribed solely to divergence of the dicot and monocot protein sequences.

Alleles↗
PubMed2005

Morphological and physiological characteristics of Saprolegnia spp. strains pathogenic to Atlantic salmon, Salmo salar L.

Seventeen strains of Saprolegnia spp. were examined for morphological and physiological characteristics, and seven were examined for their pathogenicity to Atlantic salmon, Salmo salar L. Two of the Saprolegnia strains tested caused 89 and 31% cumulative mortality in challenged salmonids and were significantly more pathogenic than the other strains tested. The positive control (Saprolegnia parasitica ATCC 90213) caused 18% mortality, but this was not significantly higher than non-pathogenic strains (0-3% cumulative mortality). All the pathogenic Saprolegnia strains and two non-pathogenic strains had secondary cysts with long, hooked hairs, a characteristic which is claimed to be typical of S. parasitica. This characteristic is apparently necessary, but does not in itself determine the ability to cause mortality in Atlantic salmon. However, all the pathogenic Saprolegnia strains in the present study showed a significantly higher initial growth rate of cysts in sterilized tap water than did non-pathogenic strains. The results of the present study suggest that initial growth rate of germinating cysts in pure water, together with the presence of long hooked hairs on the secondary cysts, may be indicators of pathogenicity of Saprolegnia strains to Atlantic salmon.

Animals↗
PubMed2007

A role of local signalling in the establishment and maintenance of the asymmetrical architecture of a neuron.

Significant progress has been made in the identification of intrinsic and extrinsic factors involved in the development of nervous system. It is remarkable that the establishment and maintenance of the asymmetrical architecture of a neuron is coordinated by a limited repertoire of signalling machineries. However, the details of signalling mechanisms responsible for creating specificity and diversity required for proper development of the nervous system remain largely to be investigated. An emerging body of evidence suggests that specificity and diversity can be achieved by differential regulation of signalling components at distinct subcellular localizations. Many aspects of neuronal polarization and morphogenesis are attributed to localized signalling. Further diversity and specificity of receptor signalling can be achieved by the regulation of molecules outside the cell. Recent evidence suggests that extracellular matrix molecules are essential extrinsic cues that function to foster the growth of neurons. Therefore, it is important to understand where the signalling machineries are activated and how they are combined with other factors in order to understand the molecular mechanism underlying neuronal development.

Animals↗
PubMed2001

Translocational inefficiency of intracellular proteins in senescence of human diploid fibroblasts.

In order to investigate signal transduction pathways and related changes of actin cytoskeleton organization in cellular senescence, H-ras double mutants--V12S35, V12G37, and V12C40--were constitutively expressed in human foreskin fibroblast (HDF). Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells, V12S35 and V12G37 expressers, revealed a failure to export actin fiber from nucleus to cytoplasm and also to form stress fibers. Perinuclear expression of Rac1 was prominent in the HDF cells and V12C40 expresser; however, in the V12S35 expresser, translocation of Rac1 from perinucleus to nucleus and strong expression of RhoA were obvious. In summary, the H-ras double mutant expressers induced premature senescence through the MEK pathway, accompanied by nuclear accumulation of actin and Rac1 proteins, cytoplasmic retention of p-Erk1/2, and marked induction of RhoA expression, suggesting the translocational inefficiency of the intracellular proteins in the senescent HDF cells.

3T3 Cells↗
PubMed2005

Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.

Microglial cells represent the immune system of the mammalian brain and therefore are critically involved in various injuries and diseases. Little is known about their role in the healthy brain and their immediate reaction to brain damage. By using in vivo two-photon imaging in neocortex, we found that microglial cells are highly active in their presumed resting state, continually surveying their microenvironment with extremely motile processes and protrusions. Furthermore, blood-brain barrier disruption provoked immediate and focal activation of microglia, switching their behavior from patroling to shielding of the injured site. Microglia thus are busy and vigilant housekeepers in the adult brain.

Animals↗
PubMed2006

Localizing NADPH oxidase-derived ROS.

Reactive oxygen species (ROS) function as signaling molecules to mediate various biological responses, including cell migration, growth, and gene expression. ROS are diffusible and short-lived molecules. Thus, localizing the ROS signal at the specific subcellular compartment is essential for activating redox signaling events after receptor activation. NADPH (nicotinamide adenine dinucleotide phosphate) oxidase is one of the major sources of ROS in vasculature; it consists of a catalytic subunit (Nox1, Nox2, Nox3, Nox4, or Nox5), p22phox, p47phox, p67phox, and the small guanosine triphosphatase Rac1. Targeting of NADPH oxidase to focal complexes in lamellipodia and membrane ruffles through the interaction of p47phox with the scaffold proteins TRAF4 and WAVE1 provides a mechanism for achieving localized ROS production, which is required for directed cell migration. ROS are believed to inactivate protein tyrosine phosphatases, which concentrate in specific subcellular compartments, thereby establishing a positive feedback system that activates redox signaling pathways to promote cell movement. Additionally, ROS production may be localized through interactions of NADPH oxidase with signaling platforms associated with lipid rafts and caveolae, as well as with endosomes. There is also evidence that NADPH oxidase is found in the nucleus, indicating its involvement in redox-responsive gene expression. This review focuses on targeting of NADPH oxidase to discrete subcellular compartments as a mechanism of localizing ROS and activation of downstream redox signaling events that mediate various cell functions.

Animals↗
PubMed2003

Decolorization of malachite green and crystal violet by waterborne pathogenic mycobacteria.

Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium marinum, and Mycobacterium chelonae tolerate high concentrations of the dyes malachite green and crystal violet. Cells of strains of those species decolorized (reduced) both malachite green and crystal violet. Because decolorized malachite green lacked antimicrobial activity, the resistance of these mycobacteria could be due, in part, to their ability to decolorize the dyes. Small amounts of malachite green and its reduced, decolorized product were detected in the lipid fraction of M. avium strain A5 cells grown in the presence of malachite green, suggesting that a minor component of resistance could be due to sequestering the dyes in the extensive mycobacterial cell surface lipid. The membrane fraction of M. avium strain A5 had at least a fivefold-higher specific decolorization rate than did the crude extract, suggesting that the decolorization activity is membrane associated. The malachite green-decolorizing activity of the membrane fraction of M. avium strain A5 was abolished by either boiling or proteinase exposure, suggesting that the decolorizing activity was due to a protein. Decolorization activity of membrane fractions was stimulated by ferrous ion and inhibited by dinitrophenol and metyrapone.

Anti-Infective Agents, Local↗
PubMed2003

Alpha-pheromone-induced "shmooing" and gene regulation require white-opaque switching during Candida albicans mating.

A 14-mer alpha-pheromone peptide of Candida albicans was chemically synthesized and used to analyze the role of white-opaque switching in the mating process. The alpha-pheromone peptide blocked cell multiplication and induced "shmooing" in a/a cells expressing the opaque-phase phenotype but not in a/a cells expressing the white-phase phenotype. The alpha-pheromone peptide induced these effects at 25 degrees C but not at 37 degrees C. An analysis of mating-associated gene expression revealed several categories of gene regulation, including (i) MTL-homozygous-specific, pheromone stimulated, switching-independent (CAG1 and STE4); (ii) mating type-specific, pheromone-induced, switching-independent (STE2); and (iii) pheromone-induced, switching-dependent (FIG1, KAR4, and HWP1). An analysis of switching-regulated genes revealed an additional category of opaque-phase-specific genes that are downregulated by alpha-pheromone only in a/a cells (OP4, SAP1, and SAP3). These results demonstrate that alpha-pheromone causes shmooing, the initial step in the mating process, only in a/a cells expressing the opaque phenotype and only at temperatures below that in the human host. These results further demonstrate that although some mating-associated genes are stimulated by the alpha-pheromone peptide in both white- and opaque-phase cells, others are stimulated only in opaque-phase cells, revealing a category of gene regulation unique to C. albicans in which alpha-pheromone induction requires the white-opaque transition. These results demonstrate that in C. albicans, the mating process and associated gene regulation must be examined within the context of white-opaque switching.

ATP-Binding Cassette Transporters↗
PubMed2004

Release of periplasmic proteins of Brucella suis upon acidic shock involves the outer membrane protein Omp25.

The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B. suis released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens. The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In B. suis, this protein contained 9 repeats, while 12 were present in the B. melitensis orthologue. B. suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product. However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.

Acetates↗
PubMed2004

Protein kinase Calpha activates c-Src and induces podosome formation via AFAP-110.

We report that the actin filament-associated protein AFAP-110 is required to mediate protein kinase Calpha (PKCalpha) activation of the nonreceptor tyrosine kinase c-Src and the subsequent formation of podosomes. Immunofluorescence analysis demonstrated that activation of PKCalpha by phorbol 12-myristate 13-acetate (PMA), or ectopic expression of constitutively activated PKCalpha, directs AFAP-110 to colocalize with and bind to the c-Src SH3 domain, resulting in activation of the tyrosine kinase. Activation of c-Src then directs the formation of podosomes, which contain cortactin, AFAP-110, actin, and c-Src. In a cell line (CaOV3) that has very little or no detectable AFAP-110, PMA treatment was unable to activate c-Src or effect podosome formation. Ectopic expression of AFAP-110 in CaOV3 cells rescued PKCalpha-mediated activation of c-Src and elevated tyrosine phosphorylation levels and subsequent formation of podosomes. Neither expression of activated PKCalpha nor treatment with PMA was able to induce these changes in CAOV3 cells expressing mutant forms of AFAP-110 that are unable to bind to, or colocalize with, c-Src. We hypothesize that one major function of AFAP-110 is to relay signals from PKCalpha that direct the activation of c-Src and the formation of podosomes.

Actins↗
PubMed2005

Role of phosphoinositide 3-kinase regulatory isoforms in development and actin rearrangement.

Class Ia phosphoinositide 3-kinases (PI3Ks) are heterodimers of p110 catalytic and p85 regulatory subunits that mediate a variety of cellular responses to growth and differentiation factors. Although embryonic development is not impaired in mice lacking all isoforms of the p85alpha gene (p85alpha-/- p55alpha-/- p50alpha-/-) or in mice lacking the p85beta gene (p85beta-/-) (D. A. Fruman, F. Mauvais-Jarvis, D. A. Pollard, C. M. Yballe, D. Brazil, R. T. Bronson, C. R. Kahn, and L. C. Cantley, Nat Genet. 26:379-382, 2000; K. Ueki, C. M. Yballe, S. M. Brachmann, D. Vicent, J. M. Watt, C. R. Kahn, and L. C. Cantley, Proc. Natl. Acad. Sci. USA 99:419-424, 2002), we show here that loss of both genes results in lethality at embryonic day 12.5 (E12.5). The phenotypes of these embryos, including subepidermal blebs flanking the neural tube at E8 and bleeding into the blebs during the turning process, are similar to defects observed in platelet-derived growth factor receptor alpha null (PDGFRalpha-/-) mice (P. Soriano, Development 124:2691-2700, 1997), suggesting that PI3K is an essential mediator of PDGFRalpha signaling at this developmental stage. p85alpha-/- p55alpha+/+ p50alpha+/+ p85beta-/- mice had similar but less severe defects, indicating that p85alpha and p85beta have a critical and redundant function in development. Mouse embryo fibroblasts deficient in all p85alpha and p85beta gene products (p85alpha-/- p55alpha-/- p50alpha-/- p85beta-/-) are defective in PDGF-induced membrane ruffling. Overexpression of the Rac-specific GDP-GTP exchange factor Vav2 or reintroduction of p85alpha or p85beta rescues the membrane ruffling defect. Surprisingly, reintroduction of p50alpha also restored PDGF-dependent membrane ruffling. These results indicate that class Ia PI3K is critical for PDGF-dependent actin rearrangement but that the SH3 domain and the Rho/Rac/Cdc42-interacting domain of p85, which lacks p50alpha, are not required for this response.

Actins↗
PubMed2002

Cholinergic and nitrergic interneurones in the myenteric plexus of the human colon.

BACKGROUND: Myenteric interneurones are involved in the reflexes that control the motility of the human colon. AIMS: The distribution of choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity in myenteric interneurones was investigated in this study. METHODS: DiI (1,1'- didodecyl 3,3,3',3'-indocarbocyanine perchlorate) was applied to the myenteric plexus of the human colon followed by organotypic culture. Retrogradely labelled neurones, with projections longer than motor neurones (>10 mm), were studied to exclude motor neurone populations. ChAT and NOS immunoreactivity was then determined in the interneurones. RESULTS: We found that 90% of interneurones projecting orally contained ChAT and none contained NOS. Ninety five per cent of descending interneurones were labelled with ChAT and/or NOS antisera; 46% contained NOS immunoreactivity alone, 20% contained ChAT immunoreactivity alone, and 29% contained both ChAT and NOS. Anally directed interneurones had significantly longer projections than orally projecting interneurones. CONCLUSIONS: Nearly all interneurones contain either NOS or ChAT immunoreactivity. Orally projecting interneurones are of two types: 90% contain ChAT alone and the remainder contain immunoreactivity for neither ChAT nor NOS. There are three main types of anally projecting interneurones: the largest, which contains NOS but not ChAT, and the two smaller classes which contain ChAT and NOS, and CHAT alone.

Aged↗
PubMed2004

Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance.

Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of "hemifusion" is observed and quantified. In this state, the outer leaflets of both vesicles' bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.

Cell Surface Extensions↗