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At least 829 records · Page 46Linked to original sources
PubMed2003

Functional proteomics of nonalcoholic steatohepatitis: mitochondrial proteins as targets of S-adenosylmethionine.

Recent work shows that S-adenosylmethionine (AdoMet) helps maintain normal liver function as chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. The mechanisms by which these nontraditional functions of AdoMet occur are unknown. Here, we use knockout mice deficient in hepatic AdoMet synthesis (MAT1A(-/-)) to study the proteome of the liver during the development of steatohepatitis. One hundred and seventeen protein spots, differentially expressed during the development of steatohepatitis, were selected and identified by peptide mass fingerprinting. Among them, 12 proteins were found to be affected from birth, when MAT1A(-/-) expression is switched on in WT mouse liver, to the rise of histological lesions, which occurs at approximately 8 months. Of the 12 proteins, 4 [prohibitin 1 (PHB1), cytochrome c oxidase I and II, and ATPase beta-subunit] have known roles in mitochondrial function. We show that the alteration in expression of PHB1 correlates with a loss of mitochondrial function. Experiments in isolated rat hepatocytes indicate that AdoMet regulates PHB1 content, thus suggesting ways by which steatohepatitis may be induced. Importantly, we found the expression of these mitochondrial proteins was abnormal in obob mice and obese patients who are at risk for nonalcoholic steatohepatitis.

Animals↗
PubMed2006

Coupling of STIM1 to store-operated Ca2+ entry through its constitutive and inducible movement in the endoplasmic reticulum.

Depletion of intracellular calcium (Ca(2+)) stores induces store-operated Ca(2+) (SOC) entry across the plasma membrane (PM). STIM1, a putative Ca(2+) sensor in the endoplasmic reticulum (ER), has been recently shown to be necessary for SOC channel activation. Here we show that STIM1 dynamically moves in tubulovesicular shape on the ER and its subcompartment in resting living cells, whereas, upon Ca(2+) store depletion, it is rapidly redistributed into discrete puncta that are located underneath, but not inserted into the PM. Normal constitutive movement of STIM1 is mediated through the coiled-coil and Ser/Thr-rich C-terminal domains in the cytoplasmic region of STIM1, whereas subsequent inducible puncta formation further requires the sterile alpha motif domain protruding into the ER lumen. Each of these three domains (coiled-coil, Ser/Thr-rich, and sterile alpha motif) was essential for activating SOC channels. Hence, our findings based on structure-function experiments suggest that constitutive dynamic movement of STIM1 in the ER and its subcompartment is obligatory for subsequent depletion-dependent redistribution of STIM1 into puncta underneath the PM and activation of SOC channels.

Animals↗
PubMed2007

In the regulation of cytochrome P450 genes, phenobarbital targets LKB1 for necessary activation of AMP-activated protein kinase.

Transcriptional activation of cytochrome P450 (CYP) genes and various drug metabolizing enzymes by the prototypical inducer phenobarbital (PB) and many other drugs and chemicals is an adaptive response of the organism to exposure to xenobiotics. The response to PB is mediated by the nuclear receptor constitutive androstane receptor (CAR), whereas the chicken xenobiotic receptor (CXR) has been characterized as the PB mediator in chicken hepatocytes. Our previous results suggested an involvement of AMP-activated protein kinase (AMPK) in the molecular mechanism of PB induction. Here, we show that the mechanism of AMPK activation is related to an effect of PB-type inducers on mitochondrial function with consequent formation of reactive oxygen species (ROS) and phosphorylation of AMPK by the upstream kinase LKB1. Gain- and loss-of-function experiments demonstrate that LKB1-activated AMPK is necessary in the mechanism of drug induction and that this is an evolutionary conserved pathway for detoxification of exogenous and endogenous chemicals. The activation of LKB1 adds a proximal target to the so far elusive sequence of events by which PB and other drugs induce the transcription of multiple genes.

Animals↗
PubMed2000

The in vitro and in vivo phosphotyrosine map of activated MuSK.

The muscle-specific receptor tyrosine kinase MuSK plays a crucial role in neuromuscular synapse formation. Activation of MuSK is induced by agrin leading to clustering of several proteins, including acetylcholine receptors, at synaptic sites. In a first step to elucidate the signal transduction cascade following MuSK activation and leading to clustering of synaptic proteins, we sought to identify the tyrosine residues that are phosphorylated in activated MuSK. We mapped the tyrosine residues that are phosphorylated in vitro and in vivo using methods that provide high sensitivity and do not require radioactive tracers. We expressed MuSK in insect cells by using a baculovirus expression vector and mapped the tyrosines that are phosphorylated in MuSK in an in vitro kinase assay using matrix-assisted laser desorption ionization MS to sequence tryptic peptides fractionated by HPLC. In addition, we isolated MuSK from Torpedo electric organ and used nanoelectrospray tandem mass spectrometry and parent ion scanning to identify the tyrosine residues that are phosphorylated in activated, endogenous MuSK in vivo. We found that six of the nineteen intracellular tyrosine residues in MuSK are phosphorylated in activated MuSK: the juxtamembrane tyrosine (Y553), the tyrosines within the activation loop (Y750, Y754, and Y755), a tyrosine near the beginning of the kinase domain (Y576), and a tyrosine (Y812) within the C-terminal lobe of the kinase domain. Our biochemical data are consistent with results from functional experiments and establish a good correlation between tyrosine residues that are phosphorylated in activated MuSK and tyrosines that are required for MuSK signaling.

Amino Acid Sequence↗
PubMed2001

Neuralized is essential for a subset of Notch pathway-dependent cell fate decisions during Drosophila eye development.

Neuralized (neur) is a neurogenic mutant of Drosophila in which many signaling events mediated by the Notch (N) receptor are disrupted. Here, we analyze the role of neur during eye development. Neur is required in a cell-autonomous fashion to restrict R8 and other photoreceptor fates and is involved in lateral inhibition of interommatidial bristles but is not required for induction of the cone cell fate. The latter contrasts with the absolute requirement for Suppressor of Hairless and the Enhancer of split-Complex for cone cell induction. Using gain-of-function experiments, we further demonstrate that ectopic wild-type and truncated Neur proteins can interfere with multiple N-controlled aspects of eye development, including both neur-dependent and neur-independent processes.

Animals↗
PubMed2003

Cytosol-endoplasmic reticulum interplay by Sec61alpha translocon in polyglutamine-mediated neurotoxicity in Drosophila.

Intracellular deposition of aggregated and ubiquitinated proteins is a prominent cytopathological feature of most neurodegenerative disorders frequently correlated with neural cell death. To elucidate mechanisms in neural cell death and degeneration, we characterized the Drosophila ortholog of Sec61alpha (DSec61alpha), a component of the translocon that is involved in both protein import and endoplasmic reticulum-associated degradation. Loss-of-function experiments for DSec61alpha revealed that the translocon contributes to expanded polyglutamine-mediated neuronal toxicity, likely resulting from proteasome inhibition and leading to accumulation of ubiquitinated proteins. Taken together, proteasome inhibition by expanded polyglutamine tracts may lead to the accumulation of toxic undegraded proteins normally transported by the Sec61alpha translocon.

Animals↗
PubMed2001

Expression of the REB transcriptional activator in rice grains improves the yield of recombinant proteins whose genes are controlled by a Reb-responsive promoter.

The gene encoding the rice transcription factor, REB (rice endosperm bZIP) was cloned from a bacterial artificial chromosome library of rice. The cloned 6,227-bp-long Reb gene is composed of six exons and five introns and is flanked by a 1.2-kb 5' promoter and a 1.2-kb 3' terminator region. The function of the Reb gene was explored by a transient assay by using a rice immature endosperm system. The effector constructs containing the native gene or fusion genes linking Reb to the rice actin (Act) or globulin (Glb) gene promoters and the reporter gene construct Glb-beta-glucuronidase (GUS) were used in this study. When these effector constructs were cotransferred with the reporter uidA gene encoding GUS under the control of the Glb promoter into immature rice endosperm cells, the Glb promoter was activated. The transient GUS expression was 2.0 to 2.5-fold higher with the effector construct than without. When the upstream activation sequence containing the GCCACGT(A/C)AG motifs of the Glb promoter was deleted, the activation by REB was abolished. On the other hand, a gain-of-function experiment showed that inserting the upstream activation sequence into the glutelin-1 (Gt1) promoter made it responsive to activation by REB. When cotransformed with Reb gene, mature transgenic rice grains containing the human lysozyme gene driven by the Glb promoter produced 3.7-fold more lysozyme. Accumulation of recombinant lysozyme in mature seed ranged from 30.57 to 279.61 microg.mg(-1) total soluble protein in individual transformants from 30 independent transformation events. Thus, our results show that REB is not only a transcriptional activator, it can also be used to increase the expression of recombinant protein in transgenic rice grains.

Actins↗
PubMed2003

A comprehensive analysis of hydrogen peroxide-induced gene expression in tobacco.

Hydrogen peroxide plays a central role in launching the defense response during stress in plants. To establish a molecular profile provoked by a sustained increase in hydrogen peroxide levels, catalase-deficient tobacco plants (CAT1AS) were exposed to high light (HL) intensities over a detailed time course. The expression kinetics of >14000 genes were monitored by using transcript profiling technology based on cDNA-amplified fragment length polymorphism. Clustering and sequence analysis of 713 differentially expressed transcript fragments revealed a transcriptional response that mimicked that reported during both biotic and abiotic stresses, including the up-regulation of genes involved in the hypersensitive response, vesicular transport, posttranscriptional processes, biosynthesis of ethylene and jasmonic acid, proteolysis, mitochondrial metabolism, and cell death, and was accompanied by a very rapid up-regulation of several signal transduction components. Expression profiling corroborated by functional experiments showed that HL induced photoinhibition in CAT1AS plants and that a short-term HL exposure of CAT1AS plants triggered an increased tolerance against a subsequent severe oxidative stress.

Cell Death↗
PubMed2002

Coupling of c-Src to large conductance voltage- and Ca2+-activated K+ channels as a new mechanism of agonist-induced vasoconstriction.

The voltage-dependent and Ca(2+)-activated K(+) channel (MaxiK, BK) and the cellular proto-oncogene pp60(c-Src) (c-Src) are abundant proteins in vascular smooth muscle. The role of MaxiK channels as a vasorelaxing force is well established, but their role in vasoconstriction is unclear. Because Src participates in regulating vasoconstriction, we investigated whether c-Src inhibits MaxiK as a mechanism for agonist-induced vasoconstriction. Functional experiments in human and rat show that inhibitors of Src (Lavendustin A, PP2) but not inactive compounds (Lavendustin B, PP3) induce a pronounced relaxation of coronary or aortic smooth muscle precontracted with 5-hydroxytriptamine, phenylephrine, or Angiotensin II. Iberiotoxin, a MaxiK blocker, antagonizes the relaxation induced by Lavendustin A or PP2, indicating that c-Src inhibits the Iberiotoxin-sensitive component, likely MaxiK channels. In agreement, coronary muscle MaxiK currents were enhanced by Lavendustin A. To investigate the molecular mechanism of c-Src action on MaxiK channels, we transiently expressed its alpha subunit, hSlo, with or without c-Src in HEK293T cells. The voltage sensitivity of hSlo was right-shifted by approximately 16 mV. hSlo inhibition by c-Src is due to channel direct phosphorylation because: (i) excised patches exposed to protein tyrosine phosphatase (CD45) resulted in a partial reversal of the inhibitory effect by approximately 10 mV, and (ii) immunoprecipitated hSlo channels were recognized by an anti-phosphotyrosine Ab. Furthermore, coexpression of hSlo and c-Src demonstrate a striking colocalization in HEK293T cells. We propose that MaxiK channels via direct c-Src-dependent phosphorylation play a significant role supporting vasoconstriction after activation of G protein-coupled receptors by vasoactive substances and neurotransmitters.

Angiotensin II↗
PubMed1991

The C-terminal region of the Vibrio fischeri LuxR protein contains an inducer-independent lux gene activating domain.

The Vibrio fischeri luminescence genes are activated by an autoinducer and the 250-amino acid residue LuxR protein. To develop a general view of LuxR structure and function, a set of luxR 5'-deletion mutations was generated. Ten luxR mutant plasmids encoding active LuxR proteins with deletions ranging from residues 2-5 (delta 2-5) to residues 2-182 (delta 2-182) were studied. The degree of transcriptional activation of luminescence genes by the truncated LuxR proteins ranged from 0.01% to greater than 200% of the wild-type level. LuxR proteins with small deletions (up to delta 2-20) were active and remained autoinducer-dependent, LuxR proteins with deletions between residues 2-58 and 2-138 showed low activity and were not affected by autoinducer, and LuxR proteins with large deletions such as the delta 2-162 protein were highly active and autoinducer-independent. However, proteins with deletions equal to or greater than delta 2-20 were unable to autoregulate luxR. Our data indicate there is a C-terminal LuxR domain capable of functioning as a transcriptional activator. We suggest that an N-terminal region of LuxR starting between residues 20 and 58 and extending to the region of residues 138-162 masks the activator function of the C-terminal domain. Residues prior to position 20 are needed for autoregulatory function. Experiments showing that wild-type luxR is dominant over luxR genes coding the delta 2-58 through delta 2-138 proteins indicate the N-terminal arm masks lux DNA binding.

Bacterial Proteins↗
PubMed1993

Chimeric-transgenic mice represent a powerful tool for studying how the proliferation and differentiation programs of intestinal epithelial cell lineages are regulated.

An in vivo system has been developed for examining the effects of wild-type or mutant proteins on cell fate determination in the mouse intestinal epithelium or on the proliferation and differentiation programs of its component epithelial lineages. This system takes advantage of the fact that at the conclusion of gut morphogenesis, each intestinal crypt is composed of a monoclonal population of cells descended from a single active multipotent stem cell, each villus is supplied by several monoclonal crypts, and the four principal cell types of the intestinal epithelium differentiate during a rapid, geographically well-organized migration along the crypt-to-villus axis. Embryonic stem (ES) cells (129/Sv origin) are initially transfected with recombinant DNAs consisting of a reporter of interest linked to transcriptional regulatory elements that control the cell lineage-specific, differentiation-dependent, and axial patterns of expression of fatty acid binding protein genes in the gut. Stably transfected ES cells are subsequently introduced into host C57BL/6 blastocysts to generate chimeric-transgenic mice. At the borders of ES cell-derived and host blastocyst-derived epithelium, intestinal villi are found that are supplied by both ES cell- and host blastocyst-derived crypts. These villi can be rapidly identified in fixed whole-mount preparations of intestine using the alpha-L-fucose-specific Ulex europaeus agglutinin type I (UEA-I) lectin. They appear striped because UEA-I recognizes a cell-surface carbohydrate polymorphism between the inbred strains used to generate the chimeric animals. The strength of this system derives from the fact that two gut epithelial populations can be compared and contrasted that occupy virtually identical positions along the crypt-to-villus and duodenal-to-colonic axes within the same animal and differ only by the presence or absence of a single gene product. The band of blastocyst-derived epithelium in these striped, polyclonal villi can be used as an internal control to assess the biological effect of the transfected gene product produced in the adjacent stripe of ES-derived cells. The system can be used for either gain-of-function or loss-of-function experiments.

Animals↗
PubMed1993

Cell type-preferred expression of maize cab-m1: repression in bundle sheath cells and enhancement in mesophyll cells.

Different sets of genes for photosynthesis are expressed in mesophyll cells (MCs) and bundle sheath cells (BSCs)--the two adjacent but morphologically and functionally distinct types of photosynthetic cells in leaves of maize and other C4 plants. For example, light-harvesting chlorophyll a/b-binding proteins of photosystem II, which are encoded by a family of cab genes, are 3- to 4-fold more abundant in maize MCs than in BSCs. Each maize cab gene is different from the others in its relative expression in MCs vs. BSCs and in its degree of photoresponsiveness. The gene cab-m1 is positively photoregulated and is highly preferentially expressed in MCs. A 159-bp sequence in the 5' flanking region of this gene (-1026 to -868 relative to the translation start site) is required for MC-preferred expression of a reporter gene in greening maize leaves. Deletion as well as gain-of-function experiments have now shown that all of the sequence information required for MC-preferred expression resides within this mesophyll-specifying region and that cab-m1 is preferentially expressed in MCs because of the presence of two types of sequence elements: one is required for suppressing expression in BSCs and the other for promoting expression in MCs. One of the four cis-acting regions mapped within the mesophyll-specifying region resembles the AT-1 box of some plant gene negative regulatory elements. Various combinations of such MC-specific enhancing and BSC-specific repressing regions could make maize cab gene family members different from one another in their relative expression in MCs vs. BSCs.

Base Sequence↗
PubMed1994

Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter.

Promoters whose temporal activity can be directly manipulated in transgenic animals provide a tool for the study of gene functions in vivo. We have evaluated a tetracycline-responsive binary system for its ability to temporally control gene expression in transgenic mice. In this system, a tetracycline-controlled trans-activator protein (tTA), composed of the repressor of the tetracycline-resistance operon (tet from Escherichia coli transposon Tn10) and the activating domain of viral protein VP16 of herpes simplex virus, induces transcription from a minimal promoter (PhCMV*-1; see below) fused to seven tet operator sequences in the absence of tetracycline but not in its presence. Transgenic mice were generated that carried either a luciferase or a beta-galactosidase reporter gene under the control of PhCMV*-1 or a transgene containing the tTA coding sequence under the control of the human cytomegalovirus immediate early gene 1 (hCMV IE1) promoter/enhancer. Whereas little luciferase or beta-galactosidase activity was observed in tissues of mice carrying only the reporter genes, the presence of tTA in double-transgenic mice induced expression of the reporter genes up to several thousand-fold. This induction was abrogated to basal levels upon administration of tetracycline. These findings can be used, for example, to design dominant gain-of-function experiments in which temporal control of transgene expression is required.

Animals↗
PubMed1994

Paneth cell differentiation in the developing intestine of normal and transgenic mice.

Paneth cells represent one of the four major epithelial lineages in the mouse small intestine. It is the only lineage that migrates downward from the stem-cell zone located in the lower portion of the crypt of Lieberkühn to the crypt base. Mature Paneth cells release growth factors, digestive enzymes, and antimicrobial peptides from their apical secretory granules. Some of these factors may affect the crypt stem cell, its transit-cell descendants, differentiating villus-associated epithelial lineages, and/or the gut microflora. We used single and multilabel immunocytochemical methods to study Paneth cell differentiation during and after completion of gut morphogenesis in normal, gnotobiotic, and transgenic mice as well as in intestinal isografts. This lineage emerges coincident with cytodifferentiation of the fetal small intestinal endoderm, formation of crypts from an intervillus epithelium, and establishment of a stem-cell hierarchy. The initial differentiation program involves sequential expression of cryptdins, a phospholipase A2 (enhancing factor), and lysozyme. A dramatic increase in Paneth cell number per crypt occurs during postnatal days 14-28, when crypts proliferate by fission. Accumulation of fucosylated and sialylated glycoconjugates during this period represents the final evolution of the lineage's differentiation program. Establishment of this lineage is not dependent upon instructive interactions from the microflora. Transgenic mice containing nucleotides -6500 to +34 of the Paneth cell-specific mouse cryptdin 2 gene linked to the human growth hormone gene beginning at its nucleotide +3 inappropriately express human growth hormone in a large population of proliferating and nonproliferating cells in the intervillus epithelium up to postnatal day 5. Transgene expression subsequently becomes restricted to the Paneth cell lineage in the developing crypt. Cryptdin 2 nucleotides -6500 to +34 should be a useful marker of crypt morphogenesis and a valuable tool for conducting gain-of-function or loss-of-function experiments in Paneth cells.

Animals↗
PubMed1997

Epidermal muscle attachment site-specific target gene expression and interference with myotube guidance in response to ectopic stripe expression in the developing Drosophila epidermis.

The egr-type zinc-finger transcription factor encoded by the Drosophila gene stripe (sr) is expressed in a subset of epidermal cells to which muscles attach during late stages of embryogenesis. We report loss-of-function and gain-of-function experiments indicating that sr activity provides ectodermal cells with properties required for the establishment of a normal muscle pattern during embryogenesis and for the differentiation of tendon-like epidermal muscle attachment sites (EMA). Our results show that sr encodes a transcriptional activator which acts as an autoregulated developmental switch gene. sr activity controls the expression of EMA-specific target genes in cells of ectodermal but not of mesodermal origin. sr-expressing ectodermal cells generate long-range signals that interfere with the spatial orientation of the elongating myotubes.

Animals↗
PubMed2001

Reproduction-related behaviors of Swiss-Webster female mice living in a cold environment.

Based on a molecular neuroendocrine theory about cold environments, thyroid hormone levels, and liganded thyroid hormone receptor interference with estrogen receptor function, experiments were designed to test female mouse reproductive behaviors in the cold. Because natural seasonal temperature declines would usually be associated with decreased photoperiods and reduced food supplies, we combined cold temperatures with short days and metabolic challenge. The simplest hypothesis was that lordosis quotients would be significantly reduced as a result of cold temperatures. That hypothesis was denied. Instead, female approaches to the stud male declined. Because cold temperatures also led to significant reductions of activity in locomotor wheels, a straightforward reduction of activity could explain the female's behavior during mating tests. We suggest that cold temperatures accompanied by reduced photoperiod and reduced metabolic fuel can reduce overall activity in female mice, thus indirectly blocking untimely reproductive behaviors.

Adaptation, Physiological↗
PubMed1996

Transcriptional regulation of the SIS/PDGF-B gene in human osteosarcoma cells by the Sp family of transcription factors.

Expression of PDGF-B, the gene encoding the platelet-derived growth factor B chain, has been implicated as a participant in an autocrine growth loop in the human osteosarcoma cell line U2-OS. In previous work, we identified a primary site in the PDGF-B promoter, the SIS proximal element (SPE), which is critical for transcription of the PDGF-B gene in U2-OS cells. We also identified Sp1 as one of the SPE-binding proteins in U2-OS nuclear extracts. In the present work, we have identified another SPE-binding protein to be Sp3. Gel mobility shift assays showed that both Sp1 and Sp3 require the CACCC motif within the SPE for binding. In vitro transcription assays showed that Sp1 or/and Sp3 is necessary for transcription of the PDGF-B gene. Cotransfection experiments functionally demonstrated that Sp1 and Sp3 can independently or additively activate the PDGF-B promoter through the SPE as well as a synthetic promoter. However, the CACCC motif within the SPE is not the only site within the minimal PDGF-B promoter through which Sp1/Sp3 acts; additional nested deletion analyses showed that multiple cis-acting elements within the minimal promoter are required for full level transcription of the PDGF-B gene in U2-OS cells.

Base Sequence↗
PubMed1996

Comparison of binding and block produced by alternatively spliced Kvbeta1 subunits.

Voltage-gated K+ (Kv) channels consist of alpha subunits complexed with cytoplasmic Kvbeta subunits. Kvbeta1 subunits enhance the inactivation of currents expressed by the Kv1 alpha subunit subfamily. Binding has been demonstrated between the C terminus of Kvbeta1.1 and a conserved segment of the N terminus of Kv1.4, Kv1.5, and Shaker alpha subunits. Here we have examined the interaction and functional properties of two alternatively spliced human Kvbeta subunits, 1.2 and 1.3, with Kvalpha subunits 1.1, 1.2, 1.4, and 1.5. In the yeast two-hybrid assay, we found that both Kvbeta subunits interact specifically through their conserved C-terminal domains with the N termini of each Kvalpha subunit. In functional experiments, we found differences in modulation of Kv1alpha subunit currents that we attribute to the unique N-terminal domains of the two Kvbeta subunits. Both Kvbeta subunits act as open channel blockers at physiological membrane potentials, but hKvbeta1.2 is a more potent blocker than hKvbeta1.3 of Kv1.1, Kv1.2, Kv1.4, and Kv1. 5. Moreover, hKvbeta1.2 is sensitive to redox conditions, whereas hKvbeta1.3 is not. We suggest that different Kvbeta subunits extend the range over which distinct Kv1alpha subunits are modulated and may provide a variable mechanism for adjusting K+ currents in response to alterations in cellular conditions.

Alternative Splicing↗