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PubMed2007

Osteoactivin acts as downstream mediator of BMP-2 effects on osteoblast function.

Our laboratory previously showed that osteoactivin (OA) is a novel, osteoblast-related glycoprotein that plays a role in osteoblast differentiation and function. The purpose of this study was to examine the regulation of OA expression by BMP-2 and the role OA plays as a downstream mediator of BMP-2 effects in osteoblast function. Using primary osteoblast cultures, we tested different doses of BMP-2 on the regulation of OA expression during osteoblast development. To test whether Smad-1 signaling is responsible for BMP-2 regulation of OA expression, osteoblast cultures were transfected with Smad1 siRNA, treated with 50 ng/ml of BMP-2 and analyzed by Western blot. BMP-2 treatment increased OA mRNA and protein expression in a dose-dependent manner and this upregulation was blocked in Smad1 siRNA transfected cultures. We next examined whether the role of OA as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Osteoblast cultures were transfected with OA antisense oligonucleotides and treated with 50 ng/ml of BMP-2. Cultures transfected with OA antisense oligonucleotides and treated with BMP-2 showed a reduction of OA expression associated with a significant reduction in early and late differentiation markers induced by BMP-2. Therefore, OA acts, at least in part, as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Our findings suggest that BMP-2 regulates OA expression through the Smad1 signaling pathway. Our data also emphasize that OA protein acts as a downstream mediator of BMP-2 effects on osteoblast differentiation and function.

Animals↗
PubMed2003

A gene necessary for normal male courtship, yellow, acts downstream of fruitless in the Drosophila melanogaster larval brain.

The fruitless (fru) gene is a member of the Drosophila melanogaster somatic sex determination genetic pathway. Although it has been hypothesized that the primary function of fru is to regulate a genetic hierarchy specifying development of adult male courtship behavior, genes acting downstream of fru have not yet been identified. Here we demonstrate that the yellow (y) gene is genetically downstream of fru in the 3(rd)-instar larval brain. Yellow protein is present at elevated levels in neuroblasts, which also show expression of male-specific FRU proteins, compared to control neuroblasts without FRU. A location for y downstream of fru in a genetic pathway was experimentally demonstrated by analysis of fru mutants lacking transcription of zinc-finger DNA binding domains, and of animals with temporal, spatial, or sexual mis-expression of male-specific FRU. A subset of fru and y mutants is known to reduce levels of a specific behavioral component of the male courtship ritual, wing extension, and FRU and Yellow were detected in the general region of the brain whose maleness is necessary for development of that behavior. We therefore hypothesized that ectopic expression of Yellow in the 3(rd)-instar brain, in a y null background, would rescue low levels of wing extension and male competitive mating success, and this was found to be the case. Overall, these data suggest that y is a downstream member of the fru branch of the D. melanogaster sex determination hierarchy, where it plays a currently unknown role in the development of adult male wing extension during courtship.

Animals↗
PubMed1995

The region 3' to the major transcriptional start site of the MDR1 downstream promoter mediates activation by a subset of mutant P53 proteins.

We have examined the response of the human multidrug resistance gene-1 (MDR1) downstream promoter to various mutants of human p53 in a reporter assay system. Our findings indicate that mutant 175H inhibits reporter activity driven by the MDR1 downstream promoter (base pairs -189 to +133 relative to the major transcriptional initiation site) in a dose-dependent manner in cotransfection assays in the BHK and the Saos-2 cell lines. A 123 base-pair segment of DNA (-119 to +4 relative to the major transcriptional initiation site) and a 193 base-pair segment (-189 to +4) have been isolated from the MDR1 downstream promoter which, like the full promoter, are negatively controlled by mutant 175H. However, a 135 base-pair segment (-2 to +133) of the promoter is activated by mutant 175H as well as mutant 248Q, but not by mutants 213Q and 234H. Thus some mutants of p53 are able to activate transcription from the 3' region of the MDR1 downstream promoter, an activity that characterizes these p53 mutants as "gain of function" mutants.

Animals↗
PubMed1995

The MDR1 downstream promoter contains sequence-specific binding sites for wild-type p53.

We have examined the interaction of the wild-type p53 protein with the downstream promoter of the human multidrug resistance gene-1 (MDR1). Our findings indicate that wild-type p53 inhibits reporter activity driven by the MDR1 downstream promoter (base pairs -189 to +133 relative to the major transcriptional initiation site) in a dose-dependent manner in cotransfection assays in the BHK and the Saos-2 cell lines. A 123 base-pair segment of DNA (-119 to +4 relative to the major transcriptional initiation site), a 193 base-pair segment (-189 to +4), and a 135 base-pair segment (-2 to +133) have been isolated from the MDR1 downstream promoter which, like the full promoter, are negatively controlled by wild-type p53. In addition, we show sequence-specific binding of wild-type p53 protein to the MDR1 downstream promoter. These in vitro results suggest that the presence of wild-type p53 negatively affects expression of the MDR1 gene product, p-glycoprotein, at the transcriptional level.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

DNA footprints of the two kinetically significant intermediates in formation of an RNA polymerase-promoter open complex: evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA.

Kinetic studies of formation and dissociation of open-promoter complexes (RPo) involving Esigma70 RNA polymerase (R) and the lambdaPR promoter (P) demonstrate the existence of two kinetically significant intermediates, designated I1 and I2, and facilitate the choice of conditions under which each accumulates. For such conditions, we report the results of equilibrium and transient DNase I and KMnO4 footprinting studies which characterize I1 and I2. At 0 degreesC, where extrapolation of equilibrium data indicates I1 is the dominant complex, DNA bases in the vicinity of the transcription start site (+1) do not react with KMnO4, indicating that this region is closed in I1. However, the DNA backbone in I1 is extensively protected from DNase I cleavage; the DNase I footprint extends approximately 30 bases downstream and at least approximately 40 bases upstream from the start site. I1 has a short lifetime (</=15 seconds), based on its sensitivity to competition with heparin. Shortly after a temperature downshift from 37 degreesC to 0 degreesC, in the time-range where we conclude that the dominant, transiently accumulated complex is I2, DNase I and KMnO4 footprinting reveal a complex with a closed-start site and an extended DNase I footprint like that of I1. However, unlike I1, I2 is insensitive to heparin competition and has a much longer dissociation lifetime at 0 degreesC. Based on footprinting, kinetic and thermodynamic studies, we conclude that in the short-lived intermediate I1 the promoter start site and downstream region are bound in a cleft defined by the open clamp-like jaws of Esigma70. We propose that binding of the start site and downstream DNA in this cleft triggers massive, relatively slow conformational changes which likely include RNA polymerase jaw closing with coupled folding. These proposed conformational changes occur prior to opening of the promoter start site region, and are responsible for the much longer lifetime of I2. Closing of the jaws of polymerase around the downstream region of promoter DNA appears to trigger opening of the start site region. From a quantitative analysis of the biphasic decay of KMnO4 reactivity of RPo at 0 degreesC, we obtain the equilibrium constant K3 for the conversion of I2 to RPo and the rate constant k-2 for the conversion of I2 to I1 (i.e. jaw opening). These quantitative results were previously unavailable at any temperature, and are necessary for the dissection of dissociation kinetic data at higher temperatures.

Bacteriophage lambda↗
PubMed1987

Three-dimensional visualization of axial velocity profiles downstream of six different mechanical aortic valve prostheses, measured with a hot-film anemometer in a steady state flow model.

Hot-film anemometry was used for in vitro steady-state measurements downstream of six mechanical aortic valve prostheses at flow rates 10, 20 and 30 l.min-1. Three-dimensional visualizations of velocity profiles at two downstream levels were made with the valves rotated 0 and 60 degrees in relation to the sinuses of valsalvae. The velocity fields downstream of the disc valves were generally skew with increasing velocity gradients and laminar shear stresses with increasing flow rates. Furthermore, increased skewness of the velocity profiles was noticed when the major orifices of the disc valves were towards the commissure than when approaching a sinus of valsalvae. The velocity profiles downstream of the ball valve were generally flat but with considerably more disturbed flow, consistent with the findings in turbulent flow.

Aortic Valve↗
PubMed1988

Nucleosome loss activates yeast downstream promoters in vivo.

Nucleosome depletion can be made to occur in yeast by addition of glucose to strains containing the histone H4 gene under GAL promoter control. This leads to the activation of downstream promoter elements (TATA box and initiation, I, region) of three different regulated yeast promoters fused to the E. coli lacZ gene. Nucleosome loss activates the PHO5 downstream element in the presence or absence of the upstream activator sequences (UAS) through which PHO5 induction is normally mediated. The cytochrome C (CYC1) and galactokinase (GAL1) promoters are normally repressed by glucose through their UAS elements. However, when these UAS are deleted, the remaining downstream promoters are also activated by glucose-mediated nucleosome loss. These data suggest that nucleosome loss increases transcription initiation and subsequent elongation in vivo. They also indicate that the proteins which recognize the downstream promoter are activated and functional, at least in part, even in the absence of the UAS complex.

Cytochrome c Group↗
PubMed1994

Sp1, USF, and GAL4 activate transcription independently of TFIID-downstream promoter interactions.

One way specific transcription factors are thought to activate transcription initiation is by facilitating interactions between the general transcription factor TFIID and DNA sequences downstream of the TATA element. Examples supporting this model include transcription activation from the core adenovirus E4 promoter by either the human gene-specific transcription factor ATF or the acidic-domain fusion protein GAL4-AH. In these cases, appearance of downstream promoter binding by TFIID correlated directly with transcription activation by these proteins. Previously we had shown that downstream promoter binding by TFIID involved recognition of the initiator DNA control element and that the extent of this binding correlated directly with initiator-dependent transcription activation in vitro. We now report our use of DNase I footprinting and in vitro transcription to investigate the effects of various stimulatory transcription factors on TFIID binding and transcription efficiency from different initiator-containing promoters. Transcription factors investigated included Sp1, USF, and several GAL4-acidic domain fusion proteins. We found that none of these transcription factors appreciably affected downstream promoter binding by TFIID, whether qualitatively or quantitatively. In fact, all of these transcription factors stimulated transcription in vitro regardless of the strength of the initiator element present. When both elements were present, transcription stimulation mediated by proximally bound transcription factors and by TFIID-initiator interaction seemed to be synergistic. Taken together, our data would suggest that transcription activation by these two means occurs through different steps within the transcriptional process.

Base Sequence↗
PubMed1994

Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-beta 1 gene.

Transcription of the human gene encoding transforming growth factor beta 1 (TGF-beta 1), which is a key regulator of cell growth and differentiation, is inducible by phorbol esters. DNA sequences resembling phorbol ester response elements (TREs) are present upstream and downstream of this gene. TREs are recognized by proteins from the AP-1 family of transcription factors. We examined a 16 basepair (bp) sequence downstream of the TGF-beta 1 gene that contains three putative TREs. This sequence had been shown to stimulate reporter gene expression from a downstream location in response to phorbol ester treatment. Electrophoretic mobility shift assays suggest that minor proteins from the related AP-1 and CREB families of transcription factors bind to the overlapping TREs within the 16 bp element. A site beginning at the end of this 16 bp element matches the consensus sequence of a DNA-binding protein called MDBP and was shown to bind to this protein. When the intact MDBP site was present in a reporter gene construct in addition to the TREs, the phorbol ester-induced stimulation of reporter gene expression was no longer observed. This suggests that MDBP can counteract the stimulation of transcription by AP-1/CREB-like proteins binding to this downstream enhancer element.

Base Sequence↗
PubMed2005

Solution structure of a common substrate mimetic of cyclooxygenase-downstream synthases bound to an engineered thromboxane A2 synthase using a high-resolution NMR technique.

Understanding the docking mechanism of the common substrate, prostaglandin H(2) (PGH(2)), into the active sites of different cyclooxygenase(COX)-downstream synthases is a key step toward uncovering the molecular basis of the isomerization of PGH(2) to different prostanoids. A high-resolution NMR spectroscopy was used to determine the conformational changes and solution 3D structure of U44069, a PGH(2) analogue, bound to one of the COX-downstream synthases-an engineered thromboxane A(2) synthase (TXAS). The dynamic binding was clearly observed by (1)D NMR titration. The detailed conformational change and 3D structure of U44069 bound to the TXAS were demonstrated by 2D (1)H NMR experiments using transferred NOEs. Through the assignments for the 2D (1)H NMR spectra, TOCSY, DQF-COSY, NOESY, and the structural calculations based on the NOE constraints, they demonstrated that the widely open conformation with a triangle shape of the free U44069 changed to a compact structure with an oval shape when bound to the TXAS. The putative substrate-binding pocket of the TXAS model fits the conformation of the TXAS-bound U44069 appropriately, but could not fit the free form of U44069. It was the first to provide structural information for the dynamic docking of the PGH(2) mimic of the TXAS in solution, and to imply that PGH(2) undergoes conformational changes when bound to different COX-downstream synthases, which may play important roles in the isomerization of PGH(2) to different prostanoids. The NMR technique can be used as a powerful tool to determine the conformations of PGH(2) bound to other COX-downstream synthases.

Binding Sites↗
PubMed2004

17-Beta estradiol and 4-nonylphenol delay smolt development and downstream migration in Atlantic salmon, Salmo salar.

The effect of 17-beta estradiol (E2) and 4-nonylphenol (4-NP) on smoltification and downstream migration of Atlantic salmon was studied in an integrated laboratory and field study. In a stock of hatchery-raised 1-year-old salmon, smoltification progressed from February until late May as judged by increased gill Na+, K+ -ATPase activity and 24 h sea water (SW)-tolerance. Starting late March, three groups of 150 fish were each given 6 serial injections over 20 days of 2 microg/g body weight E2, 120 microg/g 4-NP dissolved in peanut oil or peanut oil (4 microl/g) as control. After the last injection, all fish were individually tagged (Passive Integrated Transponder tags) and a non-lethal gill biopsy was taken. Two days later (8 April), 100 fish per group were transported to the field site and released into a small stream. Smolt migration was registered by measuring arrival time at a trap downstream of the release site. Serum vitellogenin levels increased several-fold in both male and female E2- and 4-NP-treated fish. Overall, E2- and 4-NP-treatment impaired smolting as judged by elevated condition factor, reduced gill Na+, K+ -ATPase activity and alpha-subunit Na+, K+ -ATPase mRNA level, reduced muscle water content and increased mortality following 24 h SW-challenge. After release, control fish initiated downstream migration immediately, with 50% of the total number of migrants appearing in the trap within 10 days. E2- and 4-NP-treated fish appeared in the trap with a delay in comparison to controls of 6 and 8 days, respectively. After the smolt run, no fish were registered by electro-fishing upstream of the trap. The total number of fish reaching the trap and thus post-release survival was in the order control (81%), E2 (53%), 4-NP (12%). Representatives from all treatment groups held under simulated natural conditions in the laboratory survived 100% through the migration period, suggesting that a combination of behavioural and in-stream factors (predation by herons) may contribute to the differential mortality. The study indicates that short-term exposure to natural and environmental estrogens may impair smolt development and survival and delay subsequent downstream migration in Atlantic salmon.

Analysis of Variance↗
PubMed2004

Endocrine disruption and health effects of caged mussels, Elliptio complanata, placed downstream from a primary-treated municipal effluent plume for 1 year.

Freshwater mussels, Elliptio complanata, were caged in special benthic pens and were immersed at one upstream (Ups) site and two downstream sites (8 and 11 km) of a primary-treated municipal effluent plume for 1 year. The levels of metallothionein-like proteins (MT), lipid peroxidation, protein-free DNA strands and glutathione S-transferase (GST) activity were assayed in digestive gland, gill and gonad tissues to evaluate biological effects and damage. The levels of monoamines (serotonin and dopamine) in nerve ganglia, ATP-dependent transport activity and monoamine oxidase (MAO) activity were also investigated in the homogenates, synaptosomes and mitochondria, respectively. Results showed that significant amounts of sediment accumulated in cages and 82% of mussels survived the yearlong exposure period at the downstream sites. MT-like proteins were induced in all tissues with the following response intensity: gill (3-fold), digestive gland (1.4-fold) and gonad tissues (1.3-fold). Lipid peroxidation decreased (2.5-fold) in digestive gland but increased in gill (1.6-fold) and in gonad tissues (1.5-fold). GST activity was readily increased in digestive gland (2.5-fold), suggesting the presence of organic contaminants in the plume. Levels of protein-free DNA strands did not vary significantly in digestive gland and gill tissues but were significantly reduced in gonad tissues (2.5-fold) relative to the upstream site. In visceral nerve ganglia, both serotonin and ATP-dependent serotonin transport decreased 1.7-fold with a 4-fold increase of 5-hydroxyindole acetate (5-HIAA, a serotonin metabolite) level relative to the upstream site. However, MAO activity was somewhat reduced at downstream sites (0.7- to 0.9-fold of the activity at the upstream site). Dopamine levels were found to be decreased (1.5-fold), but dopamine ATP-dependent transport activity was increased 1.8-fold, suggesting reduced dopaminergic activity. These results indicate that estrogenic chemicals are likely at play, and the increased dopamine and decreased serotonin ATP-dependent transport suggest that the municipal plume was serotonergic for mussels located at the downstream sites. Mussels exposed for 1 year display a complex but characteristic pattern of responses that could lead to harmful health effects including neuroendocrine disruption of reproduction.

Animals↗
PubMed2006

Constitutive phosphorylation of ATM in lymphoblastoid cell lines from patients with ICF syndrome without downstream kinase activity.

Double strand DNA breaks in the genome lead to the activation of the ataxia-telangiectasia mutated (ATM) kinase in a process that requires ATM autophosphorylation at serine-1981. ATM autophosphorylation only occurs if ATM is previously acetylated by Tip60. The activated ATM kinase phosphorylates proteins involved in arresting the cell cycle, including p53, and in repairing the DNA breaks. Chloroquine treatment and other manipulations that produce chromatin defects in the absence of detectable double strand breaks also trigger ATM phosphorylation and the phosphorylation of p53 in primary human fibroblasts, while other downstream substrates of ATM that are involved in the repair of DNA double strand breaks remain unphosphorylated. This raises the issue of whether ATM is constitutively activated in patients with genetic diseases that display chromatin defects. We examined lymphoblastoid cell lines (LCLs) generated from patients with different types of chromatin disorders: Immunodeficiency, Centromeric instability, Facial anomalies (ICF) syndrome, Coffin Lowry syndrome, Rubinstein Taybi syndrome and Fascioscapulohumeral Muscular Dystrophy. We show that ATM is phosphorylated on serine-1981 in LCLs derived from ICF patients but not from the other syndromes. The phosphorylated ATM in ICF cells did not phosphorylate the downstream targets NBS1, SMC1 and H2AX, all of which require the presence of double strand breaks. We demonstrate that ICF cells respond normally to ionizing radiation, ruling out the possibility that genetic deficiency in ICF cells renders activated ATM incapable of phosphorylating its downstream substrates. Surprisingly, p53 was also not phosphorylated in ICF cells or in chloroquine-treated wild type LCLs. In this regard the response to chromatin-altering agents differs between primary fibroblasts and LCLs. Our findings indicate that although phosphorylation at serine-1981 is essential in the activation of the ATM kinase, serine-1981 phosphorylation is insufficient to render ATM an active kinase towards downstream substrates, including p53.

Abnormalities, Multiple↗
PubMed2004

Protein kinase Cdelta functions downstream of Ca2+ mobilization in FcepsilonRI signaling to degranulation in mast cells.

BACKGROUND: Mobilization of Ca 2+ plays an important role in the degranulation of mast cells. Although events upstream of Ca 2+ mobilization in the regulation of degranulation are relatively well characterized, the downstream mediators of Ca 2+ remain largely unknown. OBJECTIVE: We sought to characterize the downstream signaling mechanism by which Ca 2+ mobilization mediates degranulation in antigen-stimulated mast cells. METHODS: The effect of various inhibitors was examined in the antigen-induced or Ca 2+ ionophore A23187-induced degranulation process, and the effect of inhibitors on histamine release was tested in the mouse model of asthma. RESULTS: The delta isoform of protein kinase C (PKC) functions downstream of Ca 2+ in the signaling pathway from FcepsilonRI to degranulation in RBL-2H3 mast cells. Stimulation of cells with either antigen or the Ca 2+ ionophore A23187 induced a rapid translocation of PKC-delta from the cytosol to the cellular membranes, and either treatment with the PKC-delta-specific inhibitor rottlerin or infection with an adenovirus encoding a dominant negative mutant of PKC-delta markedly inhibited degranulation induced with antigen or A23187. Furthermore, both the translocation of PKC-delta and degranulation induced by A23187 were inhibited by prevention of the accumulation of reactive oxygen species normally elicited by the ionophore. Finally, intraperitoneal injection of rottlerin prevented the increase in the concentration of histamine in bronchoalveolar lavage fluid induced by means of antigen challenge in a mouse model of allergic asthma. conclusion: PKC-delta plays an essential downstream mediatory role in the degranulation elicited by Ca 2+ mobilization, and reactive oxygen species mediate the activation of PKC-delta by Ca 2+ in the regulation of degranulation.

Animals↗
PubMed2006

Downstream processing of monoclonal antibodies--application of platform approaches.

This paper presents an overview of large-scale downstream processing of monoclonal antibodies and Fc fusion proteins (mAbs). This therapeutic modality has become increasingly important with the recent approval of several drugs from this product class for a range of critical illnesses. Taking advantage of the biochemical similarities in this product class, several templated purification schemes have emerged in the literature. In our experience, significant biochemical differences and the variety of challenges to downstream purification make the use of a completely generic downstream process impractical. Here, we describe the key elements of a flexible, generic downstream process platform for mAbs that we have adopted at Amgen. This platform consists of a well-defined sequence of unit operations with most operating parameters being pre-defined and a small subset of parameters requiring development effort. The platform hinges on the successful use of Protein A chromatography as a highly selective capture step for the process. Key elements of each type of unit operation are discussed along with data from 14 mAbs that have undergone process development. Aspects that can be readily templated as well as those that require focused development effort are identified for each unit operation. A brief description of process characterization and validation activities for these molecules is also provided. Finally, future directions in mAb processing are summarized.

Antibodies, Monoclonal↗
PubMed2006

Cisplatin mediates selective downstream hydrolytic cleavage of Met-(Gly)(n)-His segments (n=1,2) in methionine- and histidine-containing peptides: the role of ammine loss trans to the initial Pt-S(Met) anchor in facilitating amide hydrolysis.

The pH- and time-dependent reactions of the antitumor drug cisplatin, cis-[PtCl(2)(NH(3))(2)], with the methionine- and histidine-containing pentapeptides Ac-Met-Gly-His-Gly-Gly-OH, Ac-Met-Gly-Gly-His-Gly-OH and Ac-Gly-Met-Gly-His-Gly-OH (Gly=glycyl, Met=L-methionyl, His=L-histidyl) at 313K have been investigated by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance. Cisplatin mediates a rapid "downstream" hydrolytic cleavage of the Met-Gly amide bond in weakly acid solution (pH < or =5) for all three peptides, leading to release of H-Gly-His-Gly-Gly-OH, H-Gly-Gly-His-Gly-OH and H-Gly-His-Gly-OH, respectively, and formation of kappa(2)S,N(M) chelate complexes of the methionine-containing residuals Ac-Met-OH or Ac-Gly-Met-OH. An alternative reaction pathway affords tridentate kappa(3)S,N(M),N(imidazole) macrochelates of the original pentapeptide following ammine loss. The downstream cleavage pathway is competitive with the likewise cisplatin-mediated upstream cleavage of the Ac-Gly linkage in the pentapeptide Ac-Gly-Met-Gly-His-Gly-OH. This leads to formation of both the kappa(3)S,N(M),N(G1) complex of H-Gly-Met-Gly-His-Gly-OH due to upstream cleavage and the analogous tridentate complex for H-Gly-Met-OH due to initial downstream loss of H-Gly-His-Gly-OH followed by upstream loss of acetic acid. As downstream cleavage is not observed for Ac-(Gly)(2)-Met-(Gly)(2)-OH under similar conditions, it may be concluded that rapid histidine imidazole substitution of the ammine ligand in trans-position to an anchoring methionine S atom must assist hydrolytic cleavage of the Met-Gly amide bond.

Amides↗
PubMed2006

The influences of stenosis on the downstream flow pattern in curved arteries.

The influence of stenosis on the pulsatile blood flow pattern in curved arteries with stenosis at inner wall was investigated by computer simulations. Numerical calculations were performed with various values of physiological parameters to examine the effect of a stenosis on the hemodynamic characteristics such as secondary flow, flow separation, wall shear stress (WSS) and pressure drop. The results demonstrated that when the severity of a stenosis at the inner wall of a curved artery reaches a certain level, the flow pattern in the downstream of the artery shows a dramatic change compared to that of a curved artery with no stenosis. According to previous studies, a flow separation occurs at the inner wall of the bend in a curved artery. The present work reports an analysis of such a flow separation area at the inner wall of the post stenosis region in curved arteries with a stenosis. In addition, another area of flow separation with low and oscillating WSS and blood pressure at the outer wall in a downstream tube was also found and investigated. The observed characteristic change of the flow downstream may suggest a formation of a new plaque at the outer wall downstream.

Animals↗
PubMed2004

Sequences downstream of the bHLH domain of the Xenopus hairy-related transcription factor-1 act as an extended dimerization domain that contributes to the selection of the partners.

XHRT1 is a member of the HRT/Hey protein subfamily that are known as Notch effectors. XHRT1 is expressed in the developing floor plate and encodes a basic helix-loop-helix (bHLH) transcription repressor. Here, we show that XHRT1 misexpression in the neural plate inhibits differentiation of neural precursor cells and thus may be important for floor plate cells to prevent them from adopting a neuronal fate. Deletion analysis indicated that inhibition of differentiation by XHRT1 requires the DNA-binding bHLH motif and either the Orange domain or the C-terminal region. XHRT1 could efficiently homodimerize and heterodimerize with hairy proteins. Among those hairy genes, Xhairy2b shows extensive overlap of expression with XHRT1 in floor plate precursors and may be a biologically relevant XHRT1 partner. Dimerization is mediated through both the bHLH and downstream sequences, the Orange domain being particularly important for the efficiency of the interaction. Using chimeric constructs between XHRT1 and the ESR9 bHLH-O protein that does not interact with Xhairy1 and Xhairy2b, we found that both the bHLH domain and downstream sequences of XHRT1 were required for heterodimerization with Xhairy2b, while only the XHRT1 sequences downstream of the Orange domain are required for the interaction with Xhairy1. Together, these results suggest that XHRT1 plays a role in floor plate cell development and highlight the importance of the Orange and downstream sequences in dimerization and in the selection of the bHLH partners.

Amino Acid Sequence↗