Majid Ghassemian - Profile on Academia.edu (original) (raw)

Papers by Majid Ghassemian

Research paper thumbnail of Corrigendum: Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

Corrigendum: Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

Nature, Jan 19, 2015

Research paper thumbnail of Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

Nature

Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; h... more Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4) exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pat...

Research paper thumbnail of Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss

Science (New York, N.Y.), Jan 9, 1998

Desiccation of plants during drought can be detrimental to agricultural production. The phytohorm... more Desiccation of plants during drought can be detrimental to agricultural production. The phytohormone abscisic acid (ABA) reduces water loss by triggering stomatal pore closure in leaves, a process requiring ion-channel modulation by cytoplasmic proteins. Deletion of the Arabidopsis farnesyltransferase gene ERA1 or application of farnesyltransferase inhibitors resulted in ABA hypersensitivity of guard cell anion-channel activation and of stomatal closing. ERA1 was expressed in guard cells. Double-mutant analyses of era1 with the ABA-insensitive mutants abi1 and abi2 showed that era1 suppresses the ABA-insensitive phenotypes. Moreover, era1 plants exhibited a reduction in transpirational water loss during drought treatment.

Research paper thumbnail of Proteomic Analysis of Highly Prevalent Amyloid A Amyloidosis Endemic to Endangered Island Foxes

Proteomic Analysis of Highly Prevalent Amyloid A Amyloidosis Endemic to Endangered Island Foxes

PLoS ONE, 2014

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated wi... more Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤ 0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.

Research paper thumbnail of Analysis of mRNA recognition by human thymidylate synthase

Research paper thumbnail of Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

Nature, Jan 11, 2014

Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; h... more Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4) exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pat...

Research paper thumbnail of Protein profiling of isolated uterine AA amyloidosis causing fetal death in goats

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 24, 2014

Pathologic amyloid accumulates in the CNS or in peripheral organs, yet the mechanism underlying t... more Pathologic amyloid accumulates in the CNS or in peripheral organs, yet the mechanism underlying the targeting of systemic amyloid deposits is unclear. Serum amyloid A (SAA) 1 and 2 are produced predominantly by the liver and form amyloid most commonly in the spleen, liver, and kidney. In contrast, SAA3 is produced primarily extrahepatically and has no causal link to amyloid formation. Here, we identified 8 amyloidosis cases with amyloid composed of SAA3 expanding the uterine wall of goats with near-term fetuses. Uterine amyloid accumulated in the endometrium, only at the site of placental attachment, compromising maternal-fetal gas and nutrient exchange and leading to fetal ischemia and death. No other organ contained amyloid. SAA3 mRNA levels in the uterine endometrium were as high as SAA2 in the liver, yet mass spectrometry of the insoluble uterine peptides identified SAA3 as the predominant protein, and not SAA1 or SAA2. These findings suggest that high local SAA3 production led ...

Research paper thumbnail of An integrated Arabidopsis annotation database for Affymetrix Genechip® data analysis, and tools for regulatory motif searches

Trends in Plant Science, 2001

Genome-scale sequencing projects have provided the essential information required for the constru... more Genome-scale sequencing projects have provided the essential information required for the construction of entire genome chips or microarrays for RNA expression studies. The Arabidopsis and rice genomes have been sequenced and whole-genome oligonucleotide arrays are being manufactured. These should soon become available to researchers. Expression studies using genomic-scale expression arrays are providing us with a vast quantity of information at a rapid pace. The rate-limiting step in this type of experiments is not the data generation step but rather the data analysis component of experiments. We report improvements that should facilitate the analysis of Affymetrix Genechip ® expression data.

Research paper thumbnail of Regulation of Abscisic Acid Signaling by the Ethylene Response Pathway in Arabidopsis

THE PLANT CELL ONLINE, 2000

Although abscisic acid (ABA) is involved in a variety of plant growth and developmental processes... more Although abscisic acid (ABA) is involved in a variety of plant growth and developmental processes, few genes that actually regulate the transduction of the ABA signal into a cellular response have been identified. In an attempt to determine negative regulators of ABA signaling, we identified mutants, designated enhanced response to ABA3 ( era3 ), that increased the sensitivity of the seed to ABA. Biochemical and molecular analyses demonstrated that era3 mutants overaccumulate ABA, suggesting that era3 is a negative regulator of ABA synthesis. Subsequent genetic analysis of era3 alleles, however, showed that these are new alleles at the ETHYLENE INSENSITIVE2 locus. Other mutants defective in their response to ethylene also showed altered ABA sensitivity; from these results, we conclude that ethylene appears to be a negative regulator of ABA action during germination. In contrast, the ethylene response pathway positively regulates some aspects of ABA action that involve root growth in the absence of ethylene. We discuss the response of plants to ethylene and ABA in the context of how these two hormones could influence the same growth responses.

Research paper thumbnail of Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function

The Journal of Cell Biology, 2014

Research paper thumbnail of Tyrosine Phosphorylation of the G -Interacting Protein GIV Promotes Activation of Phosphoinositide 3-Kinase During Cell Migration

Science Signaling, 2011

GIV (Ga-interacting vesicle-associated protein; also known as Girdin) enhances Akt activation dow... more GIV (Ga-interacting vesicle-associated protein; also known as Girdin) enhances Akt activation downstream of multiple growth factor-and G protein (heterotrimeric guanosine 5′-triphosphate-binding protein)-coupled receptors to trigger cell migration and cancer invasion. We demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at tyrosine-1764 and tyrosine-1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the amino-and carboxyl-terminal Src homology 2 domains of p85a, a regulatory subunit of PI3K; stabilized receptor association with PI3K; and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85a increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIV-PI3K interaction a potential therapeutic target within the PI3K-Akt pathway.

Research paper thumbnail of A Protein Farnesyl Transferase Involved in Abscisic Acid Signal Transduction in Arabidopsis

Science, 1996

Although studies in plant and animal cell culture systems indicate farnesylation is required for ... more Although studies in plant and animal cell culture systems indicate farnesylation is required for normal cell cycle progression, how this lipid modi®cation of select proteins translates into whole-organism developmental decisions involving cell proliferation or dierentiation is largely unknown. The era1 mutant of the higher plant Arabidopsis thaliana (L.) Heynh. oers a unique opportunity to understand the role farnesylation may play in regulating various processes during the development of a multicellular organism. Loss of farnesylation aects many aspects of Arabidopsis growth and development. In particular, apical and axillary meristem development is altered and these phenotypes are contingent on the growth conditions. Abbreviations: ABA = abscisic acid; CL = continuous light; FTase = farnesyltransferase; SD = short day Correspondence to: P. McCourt;

Research paper thumbnail of Interactions of the NPXY microdomains of the low density lipoprotein receptor-related protein 1

PROTEOMICS, 2009

The LDL receptor-related protein 1 (LRP1) mediates internalization of a large number of proteins ... more The LDL receptor-related protein 1 (LRP1) mediates internalization of a large number of proteins and protein-lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1-CT) can be phosphorylated by activated protein-tyrosine kinases at two NPXY motifs in LRP1-CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull-down experiments from brain lysate revealed numerous proteins binding to LRP1-CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non-phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC-MS/MS, and confirmed by western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY 4507 (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine-phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY 4473 (membrane proximal) bound many fewer proteins and only to the phosphorylated form.

Research paper thumbnail of Phosphorylation of the Rab exchange factor Sec2p directs a switch in regulatory binding partners

Proceedings of the National Academy of Sciences, 2013

Sec2p is a guanine nucleotide exchange factor that promotes exocytosis by activating the Rab GTPa... more Sec2p is a guanine nucleotide exchange factor that promotes exocytosis by activating the Rab GTPase Sec4p. Sec2p is highly phosphorylated, and we have explored the role of phosphorylation in the regulation of its function. We have identified three phosphosites and demonstrate that phosphorylation regulates the interaction of Sec2p with its binding partners Ypt32p, Sec15p, and phosphatidyl-inositol-4-phosphate. In its nonphosphorylated form, Sec2p binds preferentially to the upstream Rab, Ypt32p-GTP, thus forming a Rab guanine nucleotide exchange factor cascade that leads to the activation of the downstream Rab, Sec4p. The nonphosphorylated form of Sec2p also binds to the Golgi-associated phosphatidyl-inositol-4-phosphate, which works in concert with Ypt32p-GTP to recruit Sec2p to Golgi-derived secretory vesicles. In contrast, the phosphorylated form of Sec2p binds preferentially to Sec15p, a downstream effector of Sec4p and a component of the exocyst tethering complex, thus forming a positivefeedback loop that prepares the secretory vesicle for fusion with the plasma membrane. Our results suggest that the phosphorylation state of Sec2p can direct a switch in its regulatory binding partners that facilitates maturation of the secretory vesicle and helps to promote the directionality of vesicular transport. membrane traffic | yeast | phospho-regulation | vesicle maturation

Research paper thumbnail of Cortactin as a Target for FAK in the Regulation of Focal Adhesion Dynamics

PLoS ONE, 2012

Background: Efficient cell movement requires the dynamic regulation of focal adhesion (FA) format... more Background: Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.

Research paper thumbnail of Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite

Nature, 2007

From the standpoints of both basic research and biotechnology, there is considerable interest in ... more From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms 1 and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis 2 . Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding Nasutitermes species to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H 2 metabolism, CO 2 -reductive acetogenesis and N 2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-μl environment can be.

Research paper thumbnail of Automated Querying and Identification of Novel Peptides using MALDI Mass Spectrometric Imaging

Journal of Proteome Research, 2011

MSI is a molecular imaging technique that allows for the generation of topographic 2D maps for va... more MSI is a molecular imaging technique that allows for the generation of topographic 2D maps for various endogenous and some exogenous molecules without prior specification of the molecule. In this paper, we start with the premise that a region of interest (ROI) is given to us based on preselected morphological criteria. Given an ROI, we develop a pipeline, first to determine mass values with distinct expression signatures, localized to the ROI, and second to identify the peptides corresponding to these mass values. To identify spatially differentiated masses, we implement a statistic that allows us to estimate, for each spectral peak, the probability that it is over-or under-expressed within the ROI versus outside. To identify peptides corresponding to these masses, we apply LCÀMS/MS to fragment endogenous (nonprotease digested) peptides. A novel pipeline based on constructing sequence tags de novo from both original and decharged spectra and a subsequent database search is used to identify peptides. As the MSI signal and the identified peptide are only related by a single mass value, we isolate the corresponding transcript and perform a second validation via in situ hybridization of the transcript. We tested our approach, MSI-Query, on a number of ROIs in the medicinal leech, Hirudo medicinalis, including the central nervous system (CNS). The Hirudo CNS is capable of regenerating itself after injury, thus forming an important model system for neuropeptide identification. The pipeline helps identify a number of novel peptides. Specifically, we identify a gene that we name HmIF4, which is a member of the intermediate filament family involved in neural development and a second novel, uncharacterized peptide. A third peptide, derived from the histone H2B, is also identified, in agreement with the previously suggested role of histone H2B in axon targeting.

Research paper thumbnail of Genome organization in Arabidopsis thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism

Plant molecular biology, 2003

The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of na... more The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of natural products which serve numerous biochemical functions in plants: sterols (isoprenoids with a C30 backbone) are essential components of membranes; carotenoids (C40) and chlorophylls (which contain a C20 isoprenoid side-chain) act as photosynthetic pigments; plastoquinone, phylloquinone and ubiquinone (all of which contain long isoprenoid side-chains) participate in electron transport chains; gibberellins (C20), brassinosteroids (C30) and abscisic acid (C15) are phytohormones derived from isoprenoid intermediates; prenylation of proteins (with C15 or C20 isoprenoid moieties) may mediate subcellular targeting and regulation of activity; and several monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20) have been demonstrated to be involved in plant defense. Here we present a comprehensive analysis of genes coding for enzymes involved in the metabolism of isoprenoid-derived compou...

Research paper thumbnail of Abscisic acid-induced modulation of metabolic and redox control pathways in Arabidopsis thaliana

Phytochemistry, 2008

Abscisic acid (ABA) has been implicated as a mediator in plant responses to various environmental... more Abscisic acid (ABA) has been implicated as a mediator in plant responses to various environmental stresses. To evaluate the transcriptional and metabolic events downstream of ABA perception, Arabidopsis thaliana seedlings were analyzed by transcript and metabolite profiling, and results were integrated, using the recently developed BioPathAt tool, in the context of the biochemical pathways affected by this treatment. Besides the up-regulation of pathways related to the biosynthesis of compatible solutes (raffinose family oligosaccharides and certain amino acids) as a response to ABA treatment, we also observed a down-regulation of numerous genes putatively localized to and possibly involved in the reorganization of cell walls, an association that had not been recognized previously. Metabolite profiling indicated that specific antioxidants, particularly a-tocopherol and L-ascorbic acid, were accumulated at higher levels in ABA-treated seedlings compared to appropriate controls. The transcription of genes involved in atocopherol biosynthesis were coordinately up-regulated and appeared to be integrated into a network of reactions controlling the levels of reactive oxygen species. Based upon the observed gene expression patterns, these redox control mechanisms might involve an ABA-mediated transition of mitochondrial respiration to the alternative, non-phosphorylating respiratory chain mode. The presented data herein provide indirect evidence for crosstalk between metabolic pathways and pathways regulating redox homeostasis as a response to ABA treatment, and allowed us to identify candidate genes for follow-up studies to dissect this interaction at the biochemical and molecular level. Our results also indicate an intricate relationship, at the transcriptional and possibly post-transcriptional levels, between ABA biosynthesis, the xanthophyll cycle, and ascorbic acid recycling.

Research paper thumbnail of Comprehensive post-genomic data analysis approaches integrating biochemical pathway maps

Phytochemistry, 2005

Post-genomic era research is focusing on studies to attribute functions to genes and their encode... more Post-genomic era research is focusing on studies to attribute functions to genes and their encoded proteins, and to describe the regulatory networks controlling metabolic, protein synthesis and signal transduction pathways. To facilitate the analysis of experiments using post-genomic technologies, new concepts for linking the vast amount of raw data to a biological context have to be developed. Visual representations of pathways help biologists to understand the complex relationships between components of metabolic networks, and provide an invaluable resource for the integration of transcriptomics, proteomics and metabolomics data sets. Besides providing an overview of currently available bioinformatic tools for plant scientists, we introduce BioPathAt, a newly developed visual interface that allows the knowledge-based analysis of genome-scale data by integrating biochemical pathway maps (BioPathAtMAPS module) with a manually scrutinized gene-function database (BioPathAtDB) for the model plant Arabidopsis thaliana. In addition, we discuss approaches for generating a biochemical pathway knowledge database for A. thaliana that includes, in addition to accurate annotation, condensed experimental information regarding in vitro and in vivo gene/protein function.

Research paper thumbnail of Corrigendum: Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

Corrigendum: Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

Nature, Jan 19, 2015

Research paper thumbnail of Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

Nature

Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; h... more Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4) exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pat...

Research paper thumbnail of Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss

Science (New York, N.Y.), Jan 9, 1998

Desiccation of plants during drought can be detrimental to agricultural production. The phytohorm... more Desiccation of plants during drought can be detrimental to agricultural production. The phytohormone abscisic acid (ABA) reduces water loss by triggering stomatal pore closure in leaves, a process requiring ion-channel modulation by cytoplasmic proteins. Deletion of the Arabidopsis farnesyltransferase gene ERA1 or application of farnesyltransferase inhibitors resulted in ABA hypersensitivity of guard cell anion-channel activation and of stomatal closing. ERA1 was expressed in guard cells. Double-mutant analyses of era1 with the ABA-insensitive mutants abi1 and abi2 showed that era1 suppresses the ABA-insensitive phenotypes. Moreover, era1 plants exhibited a reduction in transpirational water loss during drought treatment.

Research paper thumbnail of Proteomic Analysis of Highly Prevalent Amyloid A Amyloidosis Endemic to Endangered Island Foxes

Proteomic Analysis of Highly Prevalent Amyloid A Amyloidosis Endemic to Endangered Island Foxes

PLoS ONE, 2014

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated wi... more Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤ 0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.

Research paper thumbnail of Analysis of mRNA recognition by human thymidylate synthase

Research paper thumbnail of Carbonic anhydrases, EPF2 and a novel protease mediate CO2 control of stomatal development

Nature, Jan 11, 2014

Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; h... more Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4) exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pat...

Research paper thumbnail of Protein profiling of isolated uterine AA amyloidosis causing fetal death in goats

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 24, 2014

Pathologic amyloid accumulates in the CNS or in peripheral organs, yet the mechanism underlying t... more Pathologic amyloid accumulates in the CNS or in peripheral organs, yet the mechanism underlying the targeting of systemic amyloid deposits is unclear. Serum amyloid A (SAA) 1 and 2 are produced predominantly by the liver and form amyloid most commonly in the spleen, liver, and kidney. In contrast, SAA3 is produced primarily extrahepatically and has no causal link to amyloid formation. Here, we identified 8 amyloidosis cases with amyloid composed of SAA3 expanding the uterine wall of goats with near-term fetuses. Uterine amyloid accumulated in the endometrium, only at the site of placental attachment, compromising maternal-fetal gas and nutrient exchange and leading to fetal ischemia and death. No other organ contained amyloid. SAA3 mRNA levels in the uterine endometrium were as high as SAA2 in the liver, yet mass spectrometry of the insoluble uterine peptides identified SAA3 as the predominant protein, and not SAA1 or SAA2. These findings suggest that high local SAA3 production led ...

Research paper thumbnail of An integrated Arabidopsis annotation database for Affymetrix Genechip® data analysis, and tools for regulatory motif searches

Trends in Plant Science, 2001

Genome-scale sequencing projects have provided the essential information required for the constru... more Genome-scale sequencing projects have provided the essential information required for the construction of entire genome chips or microarrays for RNA expression studies. The Arabidopsis and rice genomes have been sequenced and whole-genome oligonucleotide arrays are being manufactured. These should soon become available to researchers. Expression studies using genomic-scale expression arrays are providing us with a vast quantity of information at a rapid pace. The rate-limiting step in this type of experiments is not the data generation step but rather the data analysis component of experiments. We report improvements that should facilitate the analysis of Affymetrix Genechip ® expression data.

Research paper thumbnail of Regulation of Abscisic Acid Signaling by the Ethylene Response Pathway in Arabidopsis

THE PLANT CELL ONLINE, 2000

Although abscisic acid (ABA) is involved in a variety of plant growth and developmental processes... more Although abscisic acid (ABA) is involved in a variety of plant growth and developmental processes, few genes that actually regulate the transduction of the ABA signal into a cellular response have been identified. In an attempt to determine negative regulators of ABA signaling, we identified mutants, designated enhanced response to ABA3 ( era3 ), that increased the sensitivity of the seed to ABA. Biochemical and molecular analyses demonstrated that era3 mutants overaccumulate ABA, suggesting that era3 is a negative regulator of ABA synthesis. Subsequent genetic analysis of era3 alleles, however, showed that these are new alleles at the ETHYLENE INSENSITIVE2 locus. Other mutants defective in their response to ethylene also showed altered ABA sensitivity; from these results, we conclude that ethylene appears to be a negative regulator of ABA action during germination. In contrast, the ethylene response pathway positively regulates some aspects of ABA action that involve root growth in the absence of ethylene. We discuss the response of plants to ethylene and ABA in the context of how these two hormones could influence the same growth responses.

Research paper thumbnail of Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function

The Journal of Cell Biology, 2014

Research paper thumbnail of Tyrosine Phosphorylation of the G -Interacting Protein GIV Promotes Activation of Phosphoinositide 3-Kinase During Cell Migration

Science Signaling, 2011

GIV (Ga-interacting vesicle-associated protein; also known as Girdin) enhances Akt activation dow... more GIV (Ga-interacting vesicle-associated protein; also known as Girdin) enhances Akt activation downstream of multiple growth factor-and G protein (heterotrimeric guanosine 5′-triphosphate-binding protein)-coupled receptors to trigger cell migration and cancer invasion. We demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at tyrosine-1764 and tyrosine-1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the amino-and carboxyl-terminal Src homology 2 domains of p85a, a regulatory subunit of PI3K; stabilized receptor association with PI3K; and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85a increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIV-PI3K interaction a potential therapeutic target within the PI3K-Akt pathway.

Research paper thumbnail of A Protein Farnesyl Transferase Involved in Abscisic Acid Signal Transduction in Arabidopsis

Science, 1996

Although studies in plant and animal cell culture systems indicate farnesylation is required for ... more Although studies in plant and animal cell culture systems indicate farnesylation is required for normal cell cycle progression, how this lipid modi®cation of select proteins translates into whole-organism developmental decisions involving cell proliferation or dierentiation is largely unknown. The era1 mutant of the higher plant Arabidopsis thaliana (L.) Heynh. oers a unique opportunity to understand the role farnesylation may play in regulating various processes during the development of a multicellular organism. Loss of farnesylation aects many aspects of Arabidopsis growth and development. In particular, apical and axillary meristem development is altered and these phenotypes are contingent on the growth conditions. Abbreviations: ABA = abscisic acid; CL = continuous light; FTase = farnesyltransferase; SD = short day Correspondence to: P. McCourt;

Research paper thumbnail of Interactions of the NPXY microdomains of the low density lipoprotein receptor-related protein 1

PROTEOMICS, 2009

The LDL receptor-related protein 1 (LRP1) mediates internalization of a large number of proteins ... more The LDL receptor-related protein 1 (LRP1) mediates internalization of a large number of proteins and protein-lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1-CT) can be phosphorylated by activated protein-tyrosine kinases at two NPXY motifs in LRP1-CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull-down experiments from brain lysate revealed numerous proteins binding to LRP1-CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non-phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC-MS/MS, and confirmed by western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY 4507 (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine-phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY 4473 (membrane proximal) bound many fewer proteins and only to the phosphorylated form.

Research paper thumbnail of Phosphorylation of the Rab exchange factor Sec2p directs a switch in regulatory binding partners

Proceedings of the National Academy of Sciences, 2013

Sec2p is a guanine nucleotide exchange factor that promotes exocytosis by activating the Rab GTPa... more Sec2p is a guanine nucleotide exchange factor that promotes exocytosis by activating the Rab GTPase Sec4p. Sec2p is highly phosphorylated, and we have explored the role of phosphorylation in the regulation of its function. We have identified three phosphosites and demonstrate that phosphorylation regulates the interaction of Sec2p with its binding partners Ypt32p, Sec15p, and phosphatidyl-inositol-4-phosphate. In its nonphosphorylated form, Sec2p binds preferentially to the upstream Rab, Ypt32p-GTP, thus forming a Rab guanine nucleotide exchange factor cascade that leads to the activation of the downstream Rab, Sec4p. The nonphosphorylated form of Sec2p also binds to the Golgi-associated phosphatidyl-inositol-4-phosphate, which works in concert with Ypt32p-GTP to recruit Sec2p to Golgi-derived secretory vesicles. In contrast, the phosphorylated form of Sec2p binds preferentially to Sec15p, a downstream effector of Sec4p and a component of the exocyst tethering complex, thus forming a positivefeedback loop that prepares the secretory vesicle for fusion with the plasma membrane. Our results suggest that the phosphorylation state of Sec2p can direct a switch in its regulatory binding partners that facilitates maturation of the secretory vesicle and helps to promote the directionality of vesicular transport. membrane traffic | yeast | phospho-regulation | vesicle maturation

Research paper thumbnail of Cortactin as a Target for FAK in the Regulation of Focal Adhesion Dynamics

PLoS ONE, 2012

Background: Efficient cell movement requires the dynamic regulation of focal adhesion (FA) format... more Background: Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.

Research paper thumbnail of Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite

Nature, 2007

From the standpoints of both basic research and biotechnology, there is considerable interest in ... more From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms 1 and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis 2 . Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding Nasutitermes species to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H 2 metabolism, CO 2 -reductive acetogenesis and N 2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-μl environment can be.

Research paper thumbnail of Automated Querying and Identification of Novel Peptides using MALDI Mass Spectrometric Imaging

Journal of Proteome Research, 2011

MSI is a molecular imaging technique that allows for the generation of topographic 2D maps for va... more MSI is a molecular imaging technique that allows for the generation of topographic 2D maps for various endogenous and some exogenous molecules without prior specification of the molecule. In this paper, we start with the premise that a region of interest (ROI) is given to us based on preselected morphological criteria. Given an ROI, we develop a pipeline, first to determine mass values with distinct expression signatures, localized to the ROI, and second to identify the peptides corresponding to these mass values. To identify spatially differentiated masses, we implement a statistic that allows us to estimate, for each spectral peak, the probability that it is over-or under-expressed within the ROI versus outside. To identify peptides corresponding to these masses, we apply LCÀMS/MS to fragment endogenous (nonprotease digested) peptides. A novel pipeline based on constructing sequence tags de novo from both original and decharged spectra and a subsequent database search is used to identify peptides. As the MSI signal and the identified peptide are only related by a single mass value, we isolate the corresponding transcript and perform a second validation via in situ hybridization of the transcript. We tested our approach, MSI-Query, on a number of ROIs in the medicinal leech, Hirudo medicinalis, including the central nervous system (CNS). The Hirudo CNS is capable of regenerating itself after injury, thus forming an important model system for neuropeptide identification. The pipeline helps identify a number of novel peptides. Specifically, we identify a gene that we name HmIF4, which is a member of the intermediate filament family involved in neural development and a second novel, uncharacterized peptide. A third peptide, derived from the histone H2B, is also identified, in agreement with the previously suggested role of histone H2B in axon targeting.

Research paper thumbnail of Genome organization in Arabidopsis thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism

Plant molecular biology, 2003

The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of na... more The isoprenoid biosynthetic pathway provides intermediates for the synthesis of a multitude of natural products which serve numerous biochemical functions in plants: sterols (isoprenoids with a C30 backbone) are essential components of membranes; carotenoids (C40) and chlorophylls (which contain a C20 isoprenoid side-chain) act as photosynthetic pigments; plastoquinone, phylloquinone and ubiquinone (all of which contain long isoprenoid side-chains) participate in electron transport chains; gibberellins (C20), brassinosteroids (C30) and abscisic acid (C15) are phytohormones derived from isoprenoid intermediates; prenylation of proteins (with C15 or C20 isoprenoid moieties) may mediate subcellular targeting and regulation of activity; and several monoterpenes (C10), sesquiterpenes (C15) and diterpenes (C20) have been demonstrated to be involved in plant defense. Here we present a comprehensive analysis of genes coding for enzymes involved in the metabolism of isoprenoid-derived compou...

Research paper thumbnail of Abscisic acid-induced modulation of metabolic and redox control pathways in Arabidopsis thaliana

Phytochemistry, 2008

Abscisic acid (ABA) has been implicated as a mediator in plant responses to various environmental... more Abscisic acid (ABA) has been implicated as a mediator in plant responses to various environmental stresses. To evaluate the transcriptional and metabolic events downstream of ABA perception, Arabidopsis thaliana seedlings were analyzed by transcript and metabolite profiling, and results were integrated, using the recently developed BioPathAt tool, in the context of the biochemical pathways affected by this treatment. Besides the up-regulation of pathways related to the biosynthesis of compatible solutes (raffinose family oligosaccharides and certain amino acids) as a response to ABA treatment, we also observed a down-regulation of numerous genes putatively localized to and possibly involved in the reorganization of cell walls, an association that had not been recognized previously. Metabolite profiling indicated that specific antioxidants, particularly a-tocopherol and L-ascorbic acid, were accumulated at higher levels in ABA-treated seedlings compared to appropriate controls. The transcription of genes involved in atocopherol biosynthesis were coordinately up-regulated and appeared to be integrated into a network of reactions controlling the levels of reactive oxygen species. Based upon the observed gene expression patterns, these redox control mechanisms might involve an ABA-mediated transition of mitochondrial respiration to the alternative, non-phosphorylating respiratory chain mode. The presented data herein provide indirect evidence for crosstalk between metabolic pathways and pathways regulating redox homeostasis as a response to ABA treatment, and allowed us to identify candidate genes for follow-up studies to dissect this interaction at the biochemical and molecular level. Our results also indicate an intricate relationship, at the transcriptional and possibly post-transcriptional levels, between ABA biosynthesis, the xanthophyll cycle, and ascorbic acid recycling.

Research paper thumbnail of Comprehensive post-genomic data analysis approaches integrating biochemical pathway maps

Phytochemistry, 2005

Post-genomic era research is focusing on studies to attribute functions to genes and their encode... more Post-genomic era research is focusing on studies to attribute functions to genes and their encoded proteins, and to describe the regulatory networks controlling metabolic, protein synthesis and signal transduction pathways. To facilitate the analysis of experiments using post-genomic technologies, new concepts for linking the vast amount of raw data to a biological context have to be developed. Visual representations of pathways help biologists to understand the complex relationships between components of metabolic networks, and provide an invaluable resource for the integration of transcriptomics, proteomics and metabolomics data sets. Besides providing an overview of currently available bioinformatic tools for plant scientists, we introduce BioPathAt, a newly developed visual interface that allows the knowledge-based analysis of genome-scale data by integrating biochemical pathway maps (BioPathAtMAPS module) with a manually scrutinized gene-function database (BioPathAtDB) for the model plant Arabidopsis thaliana. In addition, we discuss approaches for generating a biochemical pathway knowledge database for A. thaliana that includes, in addition to accurate annotation, condensed experimental information regarding in vitro and in vivo gene/protein function.