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At least 1,153 records · Page 64Linked to original sources
PubMed2006

Genetic modifications of seizure susceptibility and expression by altered excitability in Drosophila Na(+) and K(+) channel mutants.

A seizure-paralysis repertoire characteristic of Drosophila "bang-sensitive" mutants can be evoked electroconvulsively in tethered flies, in which behavioral episodes are associated with synchronized spike discharges in different body parts. Flight muscle DLMs (dorsal longitudinal muscles) display a stereotypic sequence of initial and delayed bouts of discharges (ID and DD), interposed with giant fiber (GF) pathway failure and followed by a refractory period. We examined how seizure susceptibility and discharge patterns are modified in various K(+) and Na(+) channel mutants. Decreased numbers of Na(+) channels in nap(ts) flies drastically reduced susceptibility to seizure induction, eliminated ID, and depressed DD spike generation. Mutations of different K(+) channels led to differential modifications of the various components in the repertoire. Altered transient K(+) currents in Sh(133) and Hk mutants promoted ID induction. However, only Sh(133) but not Hk mutations increased DD seizure and GF pathway failure durations. Surprisingly, modifications in sustained K(+) currents in eag and Shab mutants increased thresholds for DD induction and GF pathway failure. Nevertheless, both eag and Shab, like Sh(133), increased DD spike generation and recovery time from GF pathway failure. Interactions between channel mutations with the bang-sensitive mutation bss demonstrated the role of membrane excitability in stress-induced seizure-paralysis behavior. Seizure induction and discharges were suppressed by nap(ts) in bss nap double mutants, whereas Sh heightened seizure susceptibility in bss Sh(133) and bss Sh(M) double mutants. Our results suggest that individual seizure repertoire components reflect different neural network activities that could be differentially altered by mutations of specific ion channel subunits.

Animals↗
PubMed1995

Frequency of mutations of insulin receptor gene in Japanese patients with NIDDM.

To examine the prevalence of abnormalities in the insulin receptor structure gene in Japanese with non-insulin-dependent diabetes mellitus (NIDDM), a population of 51 patients with NIDDM was screened for mutations in this gene. Patient genomic DNAs of both alleles corresponding to 22 exons of the gene were amplified by polymerase chain reaction (PCR). The PCR products on pUC19 were sequenced. Three patients with heterozygous missense mutation Thr831-->Ala831 in exon 13 and one patient with heterozygous missense mutation Tyr1334-->Cys1334 in exon 22 of the beta-subunits were identified. Linkage analysis of one of the families plus statistical studies showed that the mutation Thr831-->Ala831 is possibly responsible for the onset of NIDDM. In COS cells transiently expressing both mutant receptor cDNAs and a cDNA of a M(r) 85,000 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), the mutation Tyr1334-->Cys1334 impaired binding of the receptor with the M(r) 85,000 subunit of PI 3-kinase, but linkage analysis of the family showed that the mutation did not cosegregate with NIDDM in the pedigree. Therefore, one missense mutation (Thr831-->Ala831) in the insulin receptor, as found in three patients, is possibly involved in the etiology of a subset of the 51 NIDDM patients.

Adult↗
PubMed2000

Combined pituitary hormone deficiency caused by a novel mutation of a highly conserved residue (F88S) in the homeodomain of PROP-1.

Mutations in the pituitary-specific paired-like homeodomain transcription factor, PROP-1, result in combined pituitary hormone deficiency. We studied a Brazilian girl, offspring of first cousins, who presented with short stature and deficiencies of GH, TSH, PRL, LH, and FSH. Her cortisol response to hypoglycemia was determined at age 4.9, 10.7, and 14.1 yr and remained normal. Magnetic resonance imaging at the age of 9 yr revealed an anterior pituitary lobe of diminished height (3 mm; normal, 4.5 +/- 0.6), but radiography revealed a sella turcica volume above the normal mean. Direct sequencing of the PROP-1 gene revealed homozygosity for a novel 263T>C transition that results in the replacement of a highly conserved phenylalanine by serine at codon 88 (F88S). F88 constitutes the hydrophobic core of the first helix of the homeodomain of PROP-1, and the substitution by the polar residue serine is expected to alter the secondary structure and impair binding of the mutated PROP-1 to DNA target sequences. The F88S mutation (which corresponds to murine F85S) was introduced into the murine Prop-1 complementary DNA and its consequences on DNA binding and trans-activation were assessed in vitro. In contrast to wild-type Prop-1, the F88S mutant showed no significant DNA binding to a PRDQ9 Prop-1 response element in gel shift assays. Transcriptional activation of a luciferase reporter gene containing a PRDQ9 site upstream of a simian virus 40 promoter was reduced to approximately 34% compared with that of wild-type Prop-1 in transiently transfected TSA-201 human embryonic kidney cells. The F88S mutation further expands the repertoire of mutations in PROP-1.

Amino Acid Sequence↗
PubMed2001

Upstream stimulating factor-1 (USF1) and USF2 bind to and activate the promoter of the adenomatous polyposis coli (APC) tumor suppressor gene.

The adenomatous polyposis coli (APC) gene product is involved in cell cycle arrest and apoptosis, and loss of function is associated with the development of colorectal carcinogenesis. Although it has been demonstrated that the APC gene is inducible, its transcriptional regulation has not been elucidated. Therefore, we characterized the promoter region of the APC gene and transcription factors required for basal expression. The APC gene has a TATA-less promoter and contains consensus binding sites for Octamer, AP2, Sp1, a CAAT-box, and three nucleotide sequences for E-box A, B, and M. The E-boxes are functional in several cancer cell lines and upstream stimulating factor-1 (USF1) and USF2 interact with these sites, with a preferred sequence-specificity for the B site. Analysis of activation of the cloned APC promoter by USF1 and USF2 in transient transfection assays in HCT-116 cells demonstrated that mutation of the E-box B site completely abolished the basal promoter activity. Further, the ectopic USF1 and USF2 expression in HCT-116 cells with deletion mutations of E-box A, B, and M sites showed that these E-boxes contribute to USF1- and USF2-mediated transcriptional activation of the APC promoter, with maximum promoter activity being associated with the E-box B site. Thus, USF1 and USF2 transcription factors are critical for APC gene expression.

Adenomatous Polyposis Coli Protein↗
PubMed1991

Nucleocytoplasmic shuttling of the progesterone receptor.

The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild-type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo-cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.

Animals↗
PubMed2002

Basic fibroblast growth factor decreases elastin gene transcription in aortic smooth muscle cells.

The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system by imparting elasticity to blood vessel wall. In this study, we examined the effect of basic fibroblast growth factor (bFGF) on the expression of elastin in aortic smooth muscle cells (SMC) to gain insight into events associated with cardiovascular diseases. The results show that bFGF treatment of SMC causes a significant decrease in elastin mRNA and secreted tropoelastin levels. Nuclear run-on analyses demonstrate that the downregulation is due to a decrease in the level of elastin gene transcription. Transient transfections of SMC with wild-type and mutated elastin gene promoter/chloramphenicol acetyl transferase (CAT) constructs show that a previously identified activator protein-1-cAMP response element (AP1/CRE) (-564 to -558-bp) within the elastin promoter mediates the bFGF-dependent downregulation of elastin gene transcription in SMC. Addition of bFGF to SMC activates the extracellular signal-regulated kinases 1/2 (ERK1/2) resulting in their translocation into the nucleus and subsequent induction of Fra-1. The addition of PD-98059, an inhibitor of ERK1/2 kinase, abrogates the bFGF-dependent decrease of elastin mRNA in SMC. The described inhibitory effect of bFGF on elastin gene expression in SMC may significantly contribute to the inefficient repair of elastin in early stages of vascular wall injury.

Animals↗
PubMed2000

Amino acid substitution within the S2 and S4 transmembrane segments in Shaker potassium channel modulates channel gating.

To investigate of the gating properties in the voltage-activated potassium channel, we have mutated a variety of S2 and S4 residues in the Shaker potassium protein. Results showed that the R365C and R368C, but not the E283C, R362C, R365S, R368S or the ShB-IR, were sensitive to micromolar concentrations of Cd(2+) ions. This indicates that R365 and R368 play a crucial role in the channel gating due to a conformational modulation of the channel structure. Doubly mutated channels of the E283C/R365E and E283C/R368E caused a transient increase in current amplitude, which reached a peak within a few seconds and then decreased toward initial levels, despite the continual presence of Cd(2+). Taken together, our results suggest that E283, R365, and R368 form a network of strong, local, and electrostatic interactions that relate closely to the mechanism of the channel gating.

Amino Acid Substitution↗
PubMed2001

Negative regulation of LPS-stimulated expression of inducible nitric oxide synthase by AP-1 in macrophage cell line J774A.1.

The level of NOS II mRNA was markedly increased during 24 h lipopolysaccharide (LPS) stimulation, but showed no further increase thereafter. On the other hand, the level of NOS II mRNA in J774A.1 cells transfected with an expression vector containing the rat csk cDNA (J.Csk) was significantly increased during 3 h LPS stimulation, but rather decreased thereafter. Although no significant difference was observed in the activation of NF-kappaB by LPS among parental J774A.1, J774A.1 transfected with promoterless vector (J.pBK), and J.Csk cells, activity of c-Jun N-terminal kinase (JNK) and nuclear translocation of nuclear factor activator protein-1 (AP-1) were markedly upregulated in the J.Csk cells. Then luciferase reporter vectors containing NOS II promoter with mutations in two AP-1-like sites (U site, -1126 approximately -1120; L site, -524 approximately -518) were transiently transfected in J774A.1 cells. The promoter activity following LPS stimulation for 24 h was significantly increased by mutation at the L site, but not by mutation at the U site, suggesting that NOS II expression is negatively regulated, at least in part, through the AP-1-like L site in response to LPS.

Animals↗
PubMed2002

Mitogen-induced, FAK-dependent tyrosine phosphorylation of the SSeCKS scaffolding protein.

The ability of mitogens to rapidly induce tyrosine phosphorylation of cellular proteins has been taken as evidence of participation in subsequent signaling pathways. SSeCKS, a major protein kinase C (PKC) substrate with protein scaffolding and tumor suppressive properties, becomes tyrosine phosphorylated in NIH3T3 and rodent embryo fibroblasts after short-term treatment with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or fetal calf serum in the presence of pervanadate, but not by treatment with insulin or insulin-like growth factor-1. The relative phosphotyrosine level on SSeCKS was higher in actively dividing cells than in confluent cultures. Tyrosine phosphorylation of SSeCKS was apparent in cells deficient in Src, Fyn, Yes, or Abl tyrosine kinases or in NIH3T3 cells expressing a temperature-sensitive v-Src allele, but not in FAK-deficient embryo fibroblasts. Purified FAK or Src enzyme failed to directly phosphorylate SSeCKS in vitro. EGF failed to induce SSeCKS tyrosine phosphorylation in FAK-/- fibroblasts, indicating that the EGF receptor is probably not the direct kinase of SSeCKS. Phosphorylation under these conditions was rescued by the transient reexpression of wt-FAK but not FAK mutated at Y397, a major autophosphorylation and SH2-based docking site. Adhesion of FAK+/+ cells to fibronectin failed to significantly induce SSeCKS tyrosine phosphorylation although FAK was activated, suggesting that SSeCKS phosphorylation is mediated through a growth factor receptor-FAK rather than an integrin-FAK pathway. Moreover, PDGF could induce SSeCKS tyrosine phosphorylation in the absence of FAK activation, suggesting a role for FAK SH2-based docking rather than kinase activity. Immunofluorescence analysis showed that in FAK-/- cells, SSeCKS costains along F-actin stress fibers, in contrast to FAK+/+ cells, where most SSeCKS stains at the cell edge and along a cortical cytoskeletal matrix. This correlated with increased coprecipitation of SSeCKS with biotin-phalloidin-bound F-actin from FAK-/- compared to FAK+/+ cell lysates. Similarly, bacterially expressed, unphosphorylated SSeCKS cosedimented with F-actin in ultracentrifugation assays. These data suggest that mitogen-induced, FAK-dependent tyrosine phosphorylation of SSeCKS modulates its binding to the actin-based cytoskeleton, suggesting a role for SSeCKS in mitogen-induced cytoskeletal reorganization.

3T3 Cells↗
PubMed2005

Effect of hydro-osmotic pressure on polycystin-2 channel function in the human syncytiotrophoblast.

Polycystin-2 (PC2), one of the gene products whose mutations cause autosomal dominant polycystic kidney disease is a transient receptor potential (TRP)-type (TRPP2) Ca(2+)-permeable, non-selective cation channel. PC2 is localized in the plasma membrane, the primary cilium, and other cellular organelles of renal epithelial and other cells. Recent studies indicate that PC2 is involved in signal transduction events associated with the transient increase in cytosolic Ca(2+). Proof of evidence now hinges on involvement of the PC2 channel in the transduction of environmental signals. PC2 is abundantly expressed in the apical membrane of human syncytiotrophoblast (hST), a highly intricate epithelial tissue, which is essential for the maternal-fetal transfer of solutes, including ions. Physical forces such as hydrostatic (H) and osmotic (Pi) pressure play important roles in placenta homeostasis. In this study, we provide new information on PC2 channel regulation in the hST by these environmental factors, and propose a model as to how they may trigger the activation of PC2. Using apical hST vesicles reconstituted in a lipid bilayer system, we found that a change in either H or Pi modified PC2 channel activity. This stimulatory effect was no longer observed in hST vesicles pre-treated with the actin cytoskeleton disrupter cytochalasin D. As shown by immunofluorescence analysis PC2 co-localized with actin filaments in the vicinity of the plasma membrane. This co-localization was disrupted by cytochalasin D. Taken together, our findings indicate that physical forces exerted on cells regulate PC2 channel activity by a sensory mechanism involving the actin cytoskeleton.

Actins↗
PubMed1992

Homeotic transformations in the mouse induced by overexpression of a human Hox3.3 transgene.

A permanent transgenic mouse line was generated carrying 40 copies of the human Hox3.3 gene. The resulting mice express large amounts of Hox3.3 protein in posterior regions of the embryo where this homeodomain protein is normally not expressed. The transgene causes homeotic transformations of the skeleton, in particular the appearance of an extra pair of ribs in the lumbar region, transformation of the shape of posterior ribs into that of more anterior ones, and the joining of an additional pair of ribs to the sternum. The phenotype of this line resembles that obtained by the targeted loss-of-function mutation of Hox3.1 (Le Mouellic et al., 1992). In transient assays, the human Hox3.3 transgene leads to the formation of additional ribs in more posterior vertebrae as well.

Animals↗
PubMed2005

Structural requirements for O2 sensing by the human tandem-P domain channel, hTREK1.

TREK1 is a member of the tandem-P domain K+ channel family which is expressed almost exclusively in the nervous system. It is modulated by a number of important factors including arachidonic acid and cell swelling. Since both factors are associated with brain ischemia, it has been suggested that activation of TREK1 may confer neuroprotection. However, it has been reported that the stably expressed human homologue of TREK1 is inhibited by hypoxia, calling into question its neuroprotective role in ischemia. Here, using transient transfection of HEK 293 cells with several hTREK1 mutations and whole-cell patch-clamp, we show that: hypoxic inhibition: (a) requires the C-terminal domain of the channel; (b) does not involve redox modulation of the C-terminal domain cysteine residues C365 and C399; and (c) is critically dependent on the glutamate residue at position 306. These data suggest strongly that neuroprotection is unlikely to be provided by this channel in low O2 environments and continue to cast a shadow of doubt over the precise role that TREK may have during hypoxic episodes.

Cell Line↗
PubMed2006

Aaptamine, a spongean alkaloid, activates p21 promoter in a p53-independent manner.

Aaptamine, a benzonaphthyridine alkaloid was isolated from a marine sponge on the guidance of a bioassay using the transfected human osteosarcoma MG63 cells (MG63luc(+)). Aaptamine activated p21 promoter stably transfected in MG63 cells dose-dependently at the concentrations of 20-50microM. Expression of p21 and its mRNA in the wild-type MG63 cells also increased by aaptamine-treatment. Furthermore, the cell cycle of MG63 cells was arrested at the G2/M phase within 48h by the aaptamine-treatment. To analyze a responsive element of p21 promoter in the up-regulation of p21 by aaptamine, MG63 cells were transiently transfected with a series of the deleted or mutated promoter segments, and induction of luciferase with aaptamine treatment was examined by using these corresponding transfected cells. The activation of p21 promoter by aaptamine was led through acting Sp1 sites between -82 and -50bp in a p53-independent manner.

Animals↗
PubMed2001

Biological consequences of cyclobutane pyrimidine dimers.

In the skin many molecules may absorb ultraviolet (UV) radiation upon exposure. In particular, cellular DNA strongly absorbs shorter wavelength solar UV radiation, resulting in various types of DNA damage. Among the DNA photoproducts produced the cyclobutane pyrimidine dimers (CPDs) are predominant. Although these lesions are efficiently repaired in the skin, this CPD formation results in various acute effects (erythema, inflammatory responses), transient effects (suppression of immune function), and chronic effects (mutation induction and skin cancer). The relationships between the presence of CPD in skin cells and the subsequent biological consequences are the subject of the present review.

Animals↗
PubMed1996

Regulation of human C-reactive protein gene expression by two synergistic IL-6 responsive elements.

To study the mechanism of interleukin-6 (IL-6) induction of human C-reactive protein (CRP) gene expression, we have utilized a human hepatoma (PLC/PRF/5) cell culture system to analyze the trans-acting factors which bind to the 300 bp 5'-flanking region of human CRP gene. In vitro gel mobility shift analyses and methylation interference assays demonstrated that NFIL-6 alpha interacted with two IL-6 responsive elements, and HNF-1 alpha and HNF-3/Octamer-like factors interacted with the downstream IL-6 responsive element in the human CRP promoter. In vivo functional analysis by transient transfection of plasmid constructs containing site-specific mutations in one or two IL-6 responsive elements in the CRP promoter fused to a reporter gene, chloramphenicol acetyl transferase (CAT), demonstrated that the binding of NFIL-6 alpha to two IL-6 responsive elements resulted in synergistic induction of the gene. When HNF-1 alpha or HNF-3/Octamer-like factors were independently bound to their corresponding sites, they had either a positive or negative effect, respectively, on IL-6 inducible transcriptional activity.

Base Sequence↗
PubMed2006

Early intermediate in human prion protein folding as evidenced by ultrarapid mixing experiments.

An important step toward understanding the mechanism of the PrP(C)-to-PrP(Sc) conversion is to elucidate the folding pathway(s) of the prion protein. On the basis of stopped-flow measurements, we recently proposed that the prion protein folds via a transient intermediate formed on the submillisecond time scale, and mutations linked to familial diseases result in a pronounced increase in the population of this intermediate. Here, we have extended these studies to continuous-flow measurements using a capillary mixing system with a time resolution of approximately 100 micros. This allowed us to directly observe two distinct phases in folding of the recombinant human prion protein 90-231, providing unambiguous evidence for rapid accumulation of an early intermediate (with a time constant of approximately 50 micros), followed by a rate-limiting folding step (with a time constant of approximately 700 micros). The present study also clearly demonstrates that the population of the intermediate is significantly increased at mildly acidic pH and in the presence of urea. A similar three-state folding behavior was observed for the Gerstmann-Straussler-Scheinker disease-associated F198S mutant, in which case the population of an intermediate was greatly increased as compared to that of the wild-type protein. Overall, the present data strongly suggest that this partially structured intermediate may be a direct monomeric precursor of the misfolded PrP(Sc) oligomer.

Humans↗
PubMed2001

Involvement of multiple cis-elements in the regulation of GA responsive promoters: Definition of a new cis-element in the Amy32b gene promoter of barley (Hordeum vulgare).

The Amy32b gene is a member of the low-pI alpha-amylase gene family of barley, whose expression is tightly regulated by hormones in the aleurone layer. Four cis-elements are known to be important for the GA induction of this gene: GARE, amylase box, pyrimidine box, and O2S. These sequences are located between -101 and -149 relative to the transcription start site. In the present work, we have created a series of Amy32b promoter-GUS reporter constructs introducing mutations in the -79 to -93 region. Using a transient expression system, we have functionally defined an additional region (-81 to -89) essential for the GA activation of the Amy32b promoter. This region is highly conserved among barley, wheat, and wild oat low-pI alpha-amylase promoters. Interestingly, in contrast with the variability in the relative distances among other cis-elements, this region maintains a nearly constant distance to GARE, which suggests that the function of these elements might be coupled. The involvement of this and other sequences in the transactivation of Amy32b by a transcription factor, GAMyb, has also been studied. Our results indicate that the only indispensable element for the GAMyb transactivation of the alpha-amylase promoter is GARE. The present work brings new evidence to the proposed model that considers the GAMyb-GARE interaction as a critical point for the GA induction of alpha-amylase genes, but also strengthens the notion that multiple sequences are required for full regulation of alpha-amylase promoters.

Journal Article↗
PubMedDate not supplied

Inducible repair of oxidative DNA damage in Escherichia coli.

Hydrogen peroxide is lethal to many cell types, including the bacterium Escherichia coli. Peroxides yield transient radical species that can damage DNA and cause mutations. Such partially reduced oxygen species are occasionally released during cellular respiration and are generated by lethal and mutagenic ionizing radiation. Because cells live in an environment where the threat of oxidative DNA damage is continual, cellular mechanisms may have evolved to avoid and repair this damage. Enzymes are known which evidently perform these functions. We report here that resistance to hydrogen peroxide toxicity can be induced in E. coli, that this novel induction is specific and occurs, in part, at the level of DNA repair.

DNA Repair↗