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At least 1,279 records · Page 71Linked to original sources
PubMed2007

FOXA1: Growth inhibitor and a favorable prognostic factor in human breast cancer.

The transcription factor Forkhead-box A1 (Foxa1), a member of the FOX class of transcription factors, has been implicated in the pathogenesis of lung, esophageal and prostate cancers. We have recently identified transcriptional activation of p27 by FOXA1. In this study, we analyzed the activities and expression pattern of FOXA1 in breast cancer. Forced expression of FOXA1 inhibited clonal growth of breast cancer cell lines, and FOXA1 levels inversely correlated with growth stimuli. In the estrogen receptor (ER)-positive MCF-7 cells, FOXA1 increased p27 promoter activity and inhibited the ER pathway activity. Analysis of FOXA1 expression in breast tissue arrays revealed significantly higher expression in pure ductal carcinomas in situ compared to invasive ductal carcinomas (IDC); and in IDC, high expression of FOXA1 was associated with favorable prognostic factors. Yet, FOXA1 expression was noted in a subset of the ER-negative tumors. Taken together, our findings suggest a growth inhibitory role for FOXA1, and identify it as a novel, potential prognostic factor in breast cancer.

Breast↗
PubMed1994

GDI1 encodes a GDP dissociation inhibitor that plays an essential role in the yeast secretory pathway.

GTP binding proteins of the Sec4/Ypt/rab family regulate distinct vesicular traffic events in eukaryotic cells. We have cloned GDI1, an essential homolog of bovine rab GDI (GDP dissociation inhibitor) from the yeast Saccharomyces cerevisiae. Analogous to the bovine protein, purified Gdi1p slows the dissociation of GDP from Sec4p and releases the GDP-bound form from yeast membranes. Depletion of Gdi1p in vivo leads to loss of the soluble pool of Sec4p and inhibition of protein transport at multiple stages of the secretory pathway. Complementation analysis indicates that GDI1 is allelic to sec19-1. These results establish that Gdi1p plays an essential function in membrane traffic and are consistent with a role for Gdi1p in the recycling of proteins of the Sec4/Ypt/rab family from their target membranes back to their vesicular pools.

Amino Acid Sequence↗
PubMed1994

A C-terminal domain conserved in precursor processing proteases is required for intramolecular N-terminal maturation of pro-Kex2 protease.

The Kex2 protease of the yeast Saccharomyces cerevisiae is the prototype of a family of eukaryotic subtilisin homologs thought to process prohormones and other precursors in the secretory pathway. Deletion analysis of Kex2 protease shows that a sequence of 154-159 residues carboxyl to the subtilisin domain is essential for the formation of active enzyme. Disruption of this region, termed the 'P-domain', blocks the normally rapid intra-molecular cleavage of the N-terminal pro-segment of pro-Kex2 protease in the endoplasmic reticulum (ER). The C-terminal boundary of the P-domain coincides closely with the endpoint of similarity between Kex2 protease and its mammalian homologues. The conservation of and functional requirement for the P-domain sharpens the distinction between a 'Kex2 family' of processing enzymes and degradative 'subtilases', and implies that the Kex2-related enzymes have in common entirely novel structural features that are important in the maturation of precursor polypeptide substrates. Failure to cleave the N-terminal pro-domain, due either to truncation of the P-domain or to mutation of the active site histidine or serine, results in stable, intracellular retention of pro-enzyme, apparently in the ER. Thus pro-Kex2 protease appears to contain an ER retention signal which is removed or destroyed by cleavage of the pro-domain.

Amino Acid Sequence↗
PubMed2004

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: II. Analysis of activating signal transduction pathways, transcriptional, post-transcriptional, and post-translational control.

CCAAT/enhancer binding protein delta (C/EBPdelta) plays a key role in mammary epithelial cell G0 growth arrest. C/EBPdelta gene expression is down-regulated in rodent mammary tumorigenesis and in human breast cancer, suggesting that "loss of function" alterations in C/EBPdelta gene expression are common in mammary gland malignancies. The goal of this study was to systematically investigate the mechanisms controlling C/EBPdelta gene expression in MCF-10A and MCF-12A human mammary epithelial cell lines. The results demonstrate that G0 growth arrest conditions (i.e., serum and growth factor withdrawal or Oncostatin M (OSM) treatment) result in the activation of JAK1, JAK2, and Tyk 2, members of the Janus kinase family of non-receptor tyrosine kinases, in MCF-10A and MCF-12A cells. Growth arrest or OSM treatment also specifically increases activated (phosphorylated) signal transduction and activators of transcription 3 (STAT3) levels, demonstrating that STAT3, not STAT1 or STAT5, is the downstream target of the activated Janus kinases in MCF-10A and MCF-12A cells. Whole cell lysates from G0 growth arrested (GA) and OSM-treated MCF-12A cells exhibit increased acute phase response element (APRE) binding compared to lysates from growing (GR) MCF-12A cells. Transient transfection using C/EBPdelta promoter-luciferase constructs demonstrated that the APRE (STAT3) consensus binding site is essential for growth arrest or OSM induction of the C/EBPdelta promoter. Mutation of the C/EBPdelta promoter STAT3 site or expression of a dominant negative STAT3 construct (STAT3delta) reduces C/EBPdelta promoter activity in response to growth arrest conditions. The human C/EBPdelta promoter also contains an Sp1 site at -61 bp (relative to the transcriptional start site) which is required for basal transcriptional activation. Mutation or deletion of the Sp1 site decreases promoter activity in response to growth arrest conditions. Treatment with the transcriptional inhibitor actinomycin D demonstrated that the C/EBPdelta mRNA exhibits a relatively short half-life (approximately 40 min). Similarly, treatment with the translational inhibitor anisomysin demonstrated that the C/EBPdelta protein half-life was also relatively short (approximately 160 min). These results indicate that the human C/EBPdelta gene is controlled at multiple levels, consistent with a role for C/EBPdelta in cell cycle control and/or cell fate determination.

Animals↗
PubMed2004

Effects of neonatal capsaicin deafferentation on neuromuscular adjustments, performance, and afferent activities from adult tibialis anterior muscle during exercise.

To investigate the role played by muscle afferents in the sensorimotor loops, we measured the effects of capsaicin injection in newborns on the mechano- and metabosensitive discharges and the running performance at adulthood. Female Sprague Dawley rats received a subcutaneous injection of either 50 mg/kg capsaicin or solvent (10% ethanol, 10% Tween 80 in 0.9% saline) during their second day of life. These two groups were compared with a control, untreated group. Four months later, treadmill running performance and muscle afferent (mechanosensitive and metabosensitive) activities from the tibialis anterior muscle were measured. The capsaicin-treated group demonstrated a reduced maximal exercise capacity (time to exhaustion) and a reduced response of muscle metabosensitive fibers (group III and IV nerve endings) to the examined stimuli (arterial KCl and lactic acid injections, electrically induced fatigue) compared with the sham-injected solvent and control groups. Group IV afferent responses were absent in the compound nerve action potentials evoked by peripheral nerve stimulation. The response to mechanosensitive fibers to tendon vibration was also affected in the capsaicin group compared with the control and sham-injected groups, which presented a bimodal response corresponding to the activation of muscle spindles and Golgi tendon organs. Finally, measurements of the force developed by the tibialis anterior muscle from the beginning to the end of a 3-min muscle stimulation revealed a more significant fall in the capsaicin group compared with the others. The present experiments reveal that the pharmacological alteration of muscular metabosensitive afferent resulted in drastic changes in the neuromuscular sensory encoding and in the central neural network that could accelerate failure of the task during fatigue.

Action Potentials↗
PubMed2005

Short latency afferent inhibition and facilitation in patients with writer's cramp.

Patients with writer's cramp (WC) show abnormalities of sensorimotor integration possibly contributing to their motor deficit. We studied sensorimotor integration by determining short-latency afferent inhibition (SAI) in 12 WC patients and 10 age-matched healthy controls. A conditioning electrical median nerve stimulus was followed 14 to 36 msec later by transcranial magnetic stimulation of the contralateral primary motor cortex, and motor evoked potentials (MEP) were recorded from the relaxed or contracting abductor pollicis brevis muscle (APB). SAI was normal in WC but during APB relaxation SAI was followed by abnormal MEP facilitation, which was absent during APB contraction and in the controls. These findings suggest that somatosensory short-latency inhibitory input into the primary motor cortex is normal in WC, whereas a later excitatory input, which very likely reflects the long-latency reflex II, is exaggerated.

Adult↗
PubMed1994

Cloning of human and mouse brain cDNAs coding for S1, the second member of the mammalian elongation factor-1 alpha gene family: analysis of a possible evolutionary pathway.

We previously reported the cloning of a rat S1 cDNA whose deduced amino acid sequence shares high similarity (92%) with that of mammalian elongation factor-1 alpha (EF-1 alpha), a protein involved in the binding of aminoacyl-tRNA to the ribosome during peptide synthesis. We report here the isolation of a full-length cDNA from a mouse brain library and a partial-length cDNA from a human hippocampus library which share extensive sequence similarity to rat S1 cDNA. We show that, as with mammalian EF-1 alpha S, the predicted primary amino acid sequences of rat, mouse, and human S1 are almost identical, except for one conservative substitution. These results indicate that mouse and man contain a second member of the EF-1 alpha gene family, the S1 gene. They also suggest that our result obtained in rat may be extrapolated to mouse and man.

Amino Acid Sequence↗
PubMed2000

Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein.

Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis.

Amino Acid Sequence↗
PubMed2001

FMRFamide-related neuropeptides are agonists of the orphan G-protein-coupled receptor GPR54.

We have isolated and determined the coding sequences of human and mouse orthologs of the rat orphan G-protein-coupled receptor GPR54. Mouse and rat GPR54 are nearly 95% identical to each other, and both are approximately 85% identical to human GPR54 at the amino acid level. Screening of agonists for GPR54 identified several invertebrate neuropeptides of the RFamide and RWamide family that were able to activate GPR54 at microM range through the G(alpha)q pathway. Substitution analysis showed that the C-terminal optimal sequence of GPR54-activating peptides is Gly-Leu-Arg-Trp-NH2. Northern analysis of human GPR54 detected expression in several peripheral tissues and many regions of the central nervous system.

Amino Acid Sequence↗
PubMed1997

Polarity of constitutive and regulated von Willebrand factor secretion by transfected MDCK-II cells.

Von Willebrand factor (vWF), synthesized by endothelial cells, is both rapidly secreted by the constitutive pathway and stored in Weibel-Palade bodies. Secretion from these organelles occurs upon activation of the protein kinase C signal transduction pathway and yields highly multimerized vWF. Highly multimerized vWF acts as a more effective adhesive ligand than the lower molecular weight forms that are constitutively secreted. We employed the extensively characterized polar Madin-Darby Canine Kidney II (MDCK-II) epithelial cell line, stably transfected with full-length vWF cDNA or deletion mutants thereof, to gain insight in the polarity of vWF secretion by either one of the two pathways. Immunofluorescence analysis and metabolic labeling experiments revealed that multimeric "wild-type" vWF is stored in MDCK-II cells and released upon stimulation with phorbol esters. Furthermore, we show that 62.0 +/- 3.8% of constitutively secreted and 83.2 +/- 6.6% of the regulated secreted wild-type vWF is encountered at the apical side of the cell. The polarity of the constitutive secretion of deletion mutant vWFdelD'D3 is similar to that of constitutively secreted wild-type vWF, whereas deletion mutant vWFdelD1D2 displays no polar secretion (50.1 +/- 5.7% apical).

Animals↗
PubMed2001

Identification of Aspergillus nidulans genes essential for the accumulation of sterigmatocystin.

The fungus Aspergillus nidulans (Emericella nidulans) was used as a genetic model for the identification of genes required for efficient accumulation of sterigmatocystin (ST). The required gene for sterigmatocystin expression was stc, which is an intermediate penultimate product in the aflatoxin biosynthetic pathway. Genetic analysis included studies of the sexual and parasexual cycles. The allelic segregation rates and recombination frequencies between linked and nonlinked genetic markers were determined by the crossing of the strains UT448 (stc) to UT196 (stc(+)) and UT448 (stc) to UT184 (stc). Low ST accumulation (4.0 ppm) in the UT196 strain and in 7.4% of the meiotic segregants allowed us to map the stc locus at chromosome I, 3.4% distant from riboA1. The diploid UT448 (stc)//UT184 (stc), prepared from nonproducing strains, was analyzed based on the parasexual cycle, and 28% of the haploid segregants accumulated the ST toxin. The results suggest that UT448 carries the stc mutant (or an inactivated) allele and that UT184, although carrying the stc(+) allele, is reactivated only by the R2(+) factor, which is located at chromosome VIII of UT448. In such a configuration, the diploid accumulates large amounts of sterigmatocystin (40 ppm). Another regulator factor (R1), located at the meth-w (II) chromosomic interval, was identified in the UT448 strain. At DNA level in chromosome I, the R1 product acts and blocks the stcZ(+) gene transcription. In a different genotypic configuration, the R1 product interacts with the R2 product (of chromosome VIII), allowing the stcZ(+) gene expression. Furthermore, the diploid UT448 (stc)//UT196 (stc(+)) accumulated the ST toxin at high level (40 ppm), indicating similar interaction between mentioned factors and the stc gene. Obtained data suggest that R1 (II) regulates the stcZ(+) transcription, by interacting with chromosome I (at the DNA level) and that R2 (VIII) controls R1 activity at the cytoplasm level. Based on these results, we propose a regulation model for the sterigmatocystin production.

Aflatoxins↗
PubMed2001

Cloning of two thyrotropin-releasing hormone receptor subtypes from a lower vertebrate (Catostomus commersoni): functional expression, gene structure, and evolution.

A PCR approach was used to clone thyrotropin-releasing hormone receptors (TRH-R) from the brain and anterior pituitary of the teleost Catostomus commersoni (cc), the white sucker. Two distinct TRH-R, designated ccTRH-R1 and ccTRH-R2, were identified. ccTRH-R1 was similar to mammalian TRH-R of the subtype 1, whereas ccTRH-R2 exhibited the highest identity (61% at the amino acid level) with the recently discovered rat TRH-R2. It is postulated that ccTRH-R2 and rat TRH-R2 are members of the same TRH-R subfamily 2. Functional expression of ccTRH receptors in human embryonic kidney cells and in Xenopus laevis oocytes demonstrated that both ccTRH receptors were fully functional in both systems. Oocytes expressing either receptor responded to the application of TRH by an induction of membrane chloride currents, indicating that ccTRH-R of both subtypes are coupled to the inositol phosphate/calcium pathway. The analysis of genomic clones revealed, for the first time, both similarities and differences in the structure of TRH-R subtype genes. Both ccTRH-R genes contained an intron within the coding region at the beginning of transmembrane domain (TM) 6. The position of this intron is highly conserved, as it was found at an identical position in the human TRH-R1 gene. The ccTRH-R2 gene contained an additional intron at the end of TM 3 that was not found in any of the TRH-R1 genes identified so far. The analysis of the gene structure of ccTRH-R and the amino acid sequence comparisons of mammalian and teleost TRH-R of both subtypes suggest that TRH receptors have been highly conserved during the course of vertebrate evolution. A common ancestral TRH receptor gene that could be found much earlier in evolution, possibly in invertebrates, might be the origin of ccTRH-R genes.

Amino Acid Sequence↗
PubMed1997

Following co-operative formation of secondary and tertiary structure in a single protein module.

We have prepared a family of peptide fragments of the 64 amino acid protein chymotrypsin inhibitor (CI2), corresponding to progressive elongation from the N terminus, in order to elucidate the basis of conformational preferences in single-domain proteins and to obtain insights into their conformational pathway. Structural analysis of the fragment comprising the first 50 residues, CI2(1-50), indicates that it is mainly disordered, with patches of hydrophobic residues exposed to the solvent. Structural characterisation of the fragment CI2(1-63) which lacks only the C-terminal glycine, Gly64, shows native-like structure in all regions of the fragment. The study provides insights into the contribution of specific residues to the stability and co-operativity of the intact protein. We define a phiNMR value, derived from chemical shift analysis, which describes the build-up of structure at the level of individual residues (protons). All the macroscopic probes used to study the growth of structure in CI2 on elongation of the chain (circular dichroism, fluorescence and gel filtration) are in agreement with the residue-by-residue description by NMR. It is seen that secondary and tertiary structure build up in parallel in the fragments and show similar structures to those developed in the transition state for folding of the intact protein.

Amino Acid Sequence↗
PubMed2001

A synaptic mechanism underlying the behavioral abnormalities induced by manganese intoxication.

In the present study we have characterized a rat model of manganese (Mn) intoxication leading to behavioral disinhibition in the absence of major motor alterations. These behavioral changes were associated with significantly increased brain Mn levels but were uncoupled to anatomical lesions of the striatum or to morphological and cytochemical changes of the nigrostriatal dopaminergic pathway. The analysis of this model at cellular level showed an enhanced dopaminergic inhibitory control of the corticostriatal excitatory transmission via presynaptic D2-like dopamine (DA) receptors in slices obtained from Mn-treated rats. Conversely, the use of agonists acting on presynaptic purinergic, muscarinic, and glutamatergic metabotropic receptors revealed a normal sensitivity. Moreover, membrane responses recorded from single dopaminergic neurons following activation of D2 DA autoreceptors were also unchanged following Mn intoxication. Thus, our findings indicate a selective involvement of the D2-like DA receptors located on glutamatergic corticostriatal terminals in this pathological condition and suggest that the behavioral symptoms described in the "early" clinical phase of manganism may be caused by an abnormal dopaminergic inhibitory control on corticostriatal inputs. The identification of the synaptic mechanism underlying the "early" phase of Mn intoxication might have a critical importance to understand the causes of the progression of this pathological condition towards an "established" phase characterized by motor abnormalities and anatomical lesions of the basal ganglia.

Animals↗
PubMed2002

Analysis of the infectious entry pathway of human papillomavirus type 33 pseudovirions.

Human papillomavirus type 33 (HPV-33) pseudovirus infection is a slow process dependent on the initial interaction with cell-surface heparan sulfate (T. Giroglou, L. Florin, F. Schafer, R. E. Streeck, and M. Sapp, 2001a, J. Virol. 75, 1565-1570). We have now further dissected the initial steps of pseudovirus uptake using removal of cell-surface proteoglycans and selective inhibition of entry pathways. Treatment of cells with heparinase I, but not with phosphoinositol-specific phospholipase C (PIPLC), prevented binding of papillomavirus-like particles and infection with HPV-33 pseudovirions, indicating that GPI-linked proteoglycans (glypicans) are not required for productive infection. The slow entry of pseudovirions was inhibited by cytochalasin D and nocodazole in a concentration-dependent manner, suggesting actin polymerization and intact microtubuli be required. Inhibitors of the caveolae-mediated uptake did not significantly affect pseudoinfection. Interestingly, pseudoinfection was blocked by selective inhibitors of endosomal acidification up to 12 h postinfection. Together, our results suggest that binding of HPV pseudovirions to heparan sulfate proteoglycans, most likely syndecans, is followed by delayed internalization via the endosomal pathway.

Actins↗
PubMed1995

Relative phase destabilization during interlimb coordination: the disruptive role of kinesthetic afferences induced by passive movement.

The disruption of three patterns of two-limb coordination, involving cyclical flexion-extension movements performed in the same or in different directions, was investigated through application of passive movement to a third limb by the experimenter. The three patterns referred to the homologous, homolateral, and heterolateral (diagonal) limb combinations which were performed in the sagittal plane. The passive movement involved a spatiotemporal trajectory that differed from the movements controlled actively. Even though subjects were instructed to completely ignore the passive limb movement, the findings of experiment 1 demonstrated a moderate to severe destabilization of the two-limb patterns, as revealed by analyses of power spectra, relative phase, cycle duration, and amplitude. This disruption was more pronounced in the homolateral and heterolateral than in the homologous effector combinations, suggesting stronger coupling between homologous than nonhomologous limb pairs. Moreover, passive mobilization affected antiphase (nonisodirectional) movements more than inphase (isodirectional) movements, pointing to the differential stability of these patterns. Experiment 2 focused on homolateral coordination and demonstrated that withdrawal of visual information did not alter the effects induced by passive movement. It was therefore hypothesized that the generation of extra kinesthetic afferences through passive limb motion was primarily responsible for the detriment in interlimb coordination, possibly conflicting with the sensory information accompanying active movement production. In addition, it was demonstrated that the active limbs were more affected by their homologous passive counterpart than by their nonhomologous counterpart, favoring the notion of "specific" interference. The findings are discussed in view of the potential role of kinesthetic afferences in human interlimb coordination, more specifically the preservance of relative phasing through a kinesthetic feedback loop.

Adolescent↗