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At least 685 records · Page 38Linked to original sources
PubMed2004

Maternal transfer of selenium in Alligator mississippiensis nesting downstream from a coal-burning power plant.

Selenium (Se) is embryotoxic in many oviparous vertebrates, but little is known about maternal transfer of Se and its impact in reptiles. Over a four-year period, we collected three clutches of eggs of the American alligator (Alligator mississippiensis) from a single nest at a site contaminated with Se and compared egg and hatchling Se concentrations and clutch viability from this nest to nests downstream from the contaminated site (two clutches from two nests) and at a reference site (two clutches from two nests). Eggs and hatchlings from the nest at the Se-contaminated site and downstream nests had elevated Se concentrations (2.1-7.8 ppm) and lower viability (30-54%) compared to reference nests (1.4-2.3 ppm and 67-74% viability), but Se concentrations did not exceed reproductive toxicity thresholds established for other oviparous vertebrates. Selenium concentrations were higher in chorioallantoic membranes of eggs from Se-contaminated sites, suggesting that this tissue may be useful as a nondestructive index of Se exposure for embryos of A. mississippiensis. Examination of these data suggests that further studies on uptake, accumulation, and reproductive success of crocodilian embryos exposed to excessive Se are warranted.

Alligators and Crocodiles↗
PubMed2001

Growth hormone induces cellular insulin resistance by uncoupling phosphatidylinositol 3-kinase and its downstream signals in 3T3-L1 adipocytes.

Growth hormone (GH) is well known to induce in vivo insulin resistance. However, the molecular mechanism of GH-induced cellular insulin resistance is largely unknown. In this study, we demonstrated that chronic GH treatment of differentiated 3T3-L1 adipocytes reduces insulin-stimulated 2-deoxyglucose (DOG) uptake and activation of Akt (also known as protein kinase B), both of which are downstream effects of phosphatidylinositol (PI) 3-kinase, despite enhanced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, association of IRS-1 with the p85 subunit of PI 3-kinase, and IRS-1-associated PI 3-kinase activity. In contrast, chronic GH treatment did not affect 2-DOG uptake and Akt activation induced by overexpression of a membrane-targeted form of the p110 subunit of PI 3-kinase (p110(CAAX)) or Akt activation stimulated by platelet-derived growth factor. Fractionation studies indicated that chronic GH treatment reduces insulin-stimulated translocation of Akt from the cytosol to the plasma membrane. Interestingly, chronic GH treatment increased insulin-stimulated association of IRS-1 with p85 and IRS-1-associated PI 3-kinase activity preferentially in the cytosol. These results indicate that cellular insulin resistance induced by chronic GH treatment in 3T3-L1 adipocytes is caused by uncoupling between activation of PI 3-kinase and its downstream signals, which is specific to the insulin-stimulated PI 3-kinase pathway. This effect of GH might result from the altered subcellular distribution of IRS-1-associated PI 3-kinase.

3T3 Cells↗
PubMed2002

Roles of ERBB family receptor tyrosine kinases, and downstream signaling pathways, in the control of cell growth and survival.

Within the last 20 years, multiple novel intracellular signal transduction pathways, downstream of plasma membrane receptors, have been discovered. These pathways have been linked to the regulation of diverse cellular events such as proliferation, senescence, differentiation and apoptosis. This review will focus upon the roles of signaling by the ErbB receptor tyrosine kinase family (ErbB1-4) in the survival of cells in response to cytotoxic stresses. In addition, plasma membrane-to-nucleus signaling pathways downstream of these receptors, such as mitogen activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K), in the control of cell survival will be discussed. Recent evidence suggests that signaling by the MAPK and PI3K pathways can both enhance proliferation as well as protect cells from apoptosis. We describe potential mechanisms by which modulation of pathway activities following inhibition of ErbB receptor function may alter the sensitivity of cells to toxic insults, leading to increased apoptosis and loss of clonogenic survival.

Animals↗
PubMed1997

Genes encoding proteins homologous to halobacterial Gvps N, J, K, F & L are located downstream of gvpC in the cyanobacterium Anabaena flos-aquae.

Only two gas vesicle genes have been previously identified in the cyanobacteria, gvpA and gvpC, both of which encode structural gas vesicle proteins. Analysis of the nucleotide sequence immediately downstream of gvpC in the cyanobacterium Anabaena flos-aquae has revealed the presence of 4 ORFs (open reading frames) the products of which share significant homology with a number of the gene products derived from halobacterial gvp gene clusters. In halobacteria the gas vesicle gene clusters consist of 14 genes involved in gas vesicle synthesis and assembly. The product of Anabaena ORF 1, located immediately downstream of gvpC is homologous to halobacterial GvpNs. For the remaining ORFs the predicted gene products show homology to both GvpJ and GvpA for ORF 2, to GvpK and GvpA for ORF 3, and to both GvpF and GvpL for ORF 4.

Amino Acid Sequence↗
PubMed1987

Measured wall shear stress distribution pattern upstream and downstream of a unilateral stenosis by an electrochemical method.

An electrochemical surface shear stress measurement was applied to a model of unilateral arterial stenosis. The unilateral stenosis model was made up of a removable stenosis plug, in an electrochemical shear stress measurement test section with 100 cathodes. Three dimensional wall shear stress distribution was measured under steady flow field. At a relatively low Reynolds number, Re = 270, there was a characteristic high and low wall shear distribution pattern downstream of the unilateral stenosis. There were also remarkable high shear stress areas on the opposite wall up- and downstream, and both side walls of the stenosis upstream. It was clearly shown that detailed three dimensional structure of the flow field must be studied in order to correlate it to pathological findings.

Arterial Occlusive Diseases↗
PubMed2024

Heat Inactivation of Nipah Virus for Downstream Single-Cell RNA Sequencing Does Not Interfere with Sample Quality.

Single-cell RNA sequencing (scRNA-seq) technologies are instrumental to improving our understanding of virus-host interactions in cell culture infection studies and complex biological systems because they allow separating the transcriptional signatures of infected versus non-infected bystander cells. A drawback of using biosafety level (BSL) 4 pathogens is that protocols are typically developed without consideration of virus inactivation during the procedure. To ensure complete inactivation of virus-containing samples for downstream analyses, an adaptation of the workflow is needed. Focusing on a commercially available microfluidic partitioning scRNA-seq platform to prepare samples for scRNA-seq, we tested various chemical and physical components of the platform for their ability to inactivate Nipah virus (NiV), a BSL-4 pathogen that belongs to the group of nonsegmented negative-sense RNA viruses. The only step of the standard protocol that led to NiV inactivation was a 5 min incubation at 85 °C. To comply with the more stringent biosafety requirements for BSL-4-derived samples, we included an additional heat step after cDNA synthesis. This step alone was sufficient to inactivate NiV-containing samples, adding to the necessary inactivation redundancy. Importantly, the additional heat step did not affect sample quality or downstream scRNA-seq results.

Nipah Virus↗
PubMed2003

Variations of mitochondrial D-loop region plus downstream gene 1 2S rRNA-tRNA(phe) and gastric carcinomas.

AIM: To explore the instabilities, polymorphisms and other variations of mitochondrial D-loop region and downstream gene 12S rRNA-tRNA(phe) in gastric cancers, and to study their relationship with gastric cancer. METHODS: Three adjacent regions (D-loop, tRNA(phe) and 12S rRNA) were detected for instabilities, polymorphisms and other variations via PCR amplification followed by direct DNA sequencing in 22 matched gastric cancerous tissues and para-cancerous normal tissues. RESULTS: PolyC or (CA) (n) instabilities were detected in 13/22(59.1 %) gastric cancers and 9/22(40.9 %) in the control (P>0.05). There existed 2/12(16.7 %) and 6/10(60 %) alterations of 12S rRNA-tRNA(phe) in well differentiated gastric cancers and poorly differentiated ones, respectively (P<0.05). Some new variations were found, among which np 318 and np 321 C-T transitions in D-loop region were two of the five bases for H-strand replication primer. np 523 AC-deletion and np 527 C-T transition occurred at mtTF1 binding site (mtTFBS), which were associated with the transcription of downstream mitochondrial genome. Seven samples showed the np 16 182 polyC instabilities, five of which simultaneously showed np 16 189 T-C transitions. CONCLUSION: There is no statistic significance of instabilities and polymorphisms in mitochondrial D-loop region between gastric cancerous and para-cancerous normal tissues, which suggests that the instability might relate to heredity or be dependent on aging. There is a significant correlation between differentiation degree of gastric cancer and variant frequencies of 12S rRNA-tRNA(phe). The poorly differentiated gastric cancers are more prone to 12S rRNA-tRNA(phe) variations, or gastric cancers with 12S rRNA-tRNA(phe) variations are more likely to be poorly differentiated. np 16 189 T-C transition may be one of the important reasons for polyC instability in gastric cancer.

Base Sequence↗
PubMed2000

The adaptor protein Gads (Grb2-related adaptor downstream of Shc) is implicated in coupling hemopoietic progenitor kinase-1 to the activated TCR.

The hemopoietic-specific Gads (Grb2-related adaptor downstream of Shc) adaptor protein possesses amino- and carboxyl-terminal Src homology 3 (SH3) domains flanking a central SH2 domain and a unique region rich in glutamine and proline residues. Gads functions to couple the activated TCR to distal signaling events through its interactions with the leukocyte-specific signaling proteins SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activated T cells). Expression library screening for additional Gads-interacting molecules identified the hemopoietic progenitor kinase-1 (HPK1), and we investigated the HPK1-Gads interaction within the DO11.10 murine T cell hybridoma system. Our results demonstrate that HPK1 inducibly associates with Gads and becomes tyrosine phosphorylated following TCR activation. HPK1 kinase activity is up-regulated in response to activation of the TCR and requires the presence of its proline-rich motifs. Mapping experiments have revealed that the carboxyl-terminal SH3 domain of Gads and the fourth proline-rich region of HPK1 are essential for their interaction. Deletion of the fourth proline-rich region of HPK1 or expression of a Gads SH2 mutant in T cells inhibits TCR-induced HPK1 tyrosine phosphorylation. Together, these data suggest that HPK1 is involved in signaling downstream from the TCR, and that SH2/SH3 domain-containing adaptor proteins, such as Gads, may function to recruit HPK1 to the activated TCR complex.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation.

Recombinant TCR ligands (RTLs) consisting of covalently linked alpha(1) and beta(1) domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP(72-89)) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP(72-89) peptide-specific CD4(+) T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP(72-89)-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP(72-89) peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3zeta p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3epsilon treatment produced stronger activation of these cellular events with additional activation of NF-kappaB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-kappaB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4(+) T cells.

Amino Acid Sequence↗
PubMed2005

Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses.

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.

Blotting, Northern↗
PubMed2005

Aberrant laminin beta3 isoforms downstream of EWS-ETS fusion genes in Ewing family tumors.

Ewing family tumors (EFTs) are associated with a chromosomal translocation resulting in a fusion of the amino-terminus of EWS with the DNA-binding domain of an ETS transcription factor. Although previous reports suggested that these chimeric proteins would act as aberrant transcription factors, their downstream targets have not been fully elucidated. To identify downstream targets of these EWS-ETS fusion proteins, we introduced EWS-ETS fusion constructs into a human fibrosarcoma cell line, HT-1080, by retroviral transduction. Here we report that the LAMB3 gene encoding the beta3 chain of basement membrane protein laminin-5 is induced to a significantly higher level in cells expressing EWS-ETSs than in cells expressing normal ETSs. Additionally through use of an antisense oligonucleotide for EWS-ERG in the W-ES EFT cell line, laminin beta3 protein was reduced coordinately with EWS-ERG fusion protein expression. Furthermore, we found small mRNAs were preferentially transcribed from the LAMB3 gene in EFT cell lines. Molecular cloning of the entire coding region shows that the alternative transcripts from different promoter(s) located within the intron 14, which encode small proteins, likely are major products of the LAMB3 gene in EFT cells. We show that the small isoforms conferred increased anchorage-independent proliferation to NIH3T3 cells. Together with previous studies showing that laminin-5 is involved in the invasive and malignant phenotype of several tumor types, our data suggest that the oncogenic effect of EWS-ETS may be mediated in part by upregulation of LAMB3 expression.

Animals↗
PubMed1998

Cloning, sequencing and characterisation of a downstream region of ksdDI operon of Arthrobacter simplex.

An 8-kb region downstream of the ketosteroid dehydrogenase (ksdD)-ketosteroid isomerase (ksdI) genes of Arthrobacter simplex was cloned. The nucleotide sequence of the first 3-kb segment downstream of ksdD-ksdI operon was determined. Three open reading frames (ORFs) preceded by Shine-Dalgarno (SD) sequences have been found. Homology search revealed that the putative product encoded by ORF3 has high level of similarity with the 3-oxosteroid dehydrogenases from A. simplex and P. testosteroni (90% identity in their putative active sites). The role of ORF3 product as a FAD-containing dehydrogenase in steroid degradation pathway is discussed.

Amino Acid Sequence↗
PubMed1999

A novel role for HEB downstream or parallel to the pre-TCR signaling pathway during alpha beta thymopoiesis.

TCR gene rearrangement and expression are central to the development of clonal T lymphocytes. The pre-TCR complex provides the first signal instructing differentiation and proliferation events during the transition from CD4-CD8-TCR- double negative (DN) stage to CD4+CD8+ double positive (DP) stage. How the pre-TCR signal leads to downstream gene expression is not known. HeLa E-box binding protein (HEB), a basic helix-loop-helix transcription factor, is abundantly detected in thymocytes and is thought to regulate E-box sites present in many T cell-specific gene enhancers, including TCR-alpha, TCR-beta, and CD4. Targeted disruption of HEB results in a 5- to 10-fold reduction in thymic cellularity that can be accounted for by a developmental block at the DN to DP stage transition. Specifically, a dramatic increase in the CD4low/-CD8+CD5lowHSA+TCRlow/- immature single positive population and a concomitant decrease in the subsequent DP population are observed. Adoptive transfer test shows that this defect is cell-autonomous and restricted to the alpha beta T cell lineage. Introduction of an alpha beta TCR transgene into the HEBko/ko background is not sufficient to rescue the developmental delay. In vivo CD3 cross-linking analysis of thymocytes indicates that TCR signaling pathway in the HEBko/ko mice appears intact. These findings suggest an essential function of HEB in early T cell development, downstream or parallel to the pre-TCR signaling pathway.

Adoptive Transfer↗
PubMed1999

Defective apoptotic signal transduction pathway downstream of caspase-3 in human B-lymphoma cells: A novel mechanism of nuclear apoptosis resistance.

Mitochondria play a central role in controlling apoptosis, and activation of the caspase cascade appears to be crucial event during the apoptotic process. Human B lymphoma Raji cells are resistant to nuclear apoptosis induced by various stimuli. Using this cell line, we have asked whether reduction of the mitochondrial transmembrane potential and activation of caspase-3 are sufficient to induce DNA fragmentation during the apoptotic process. After stimulation with cell-permeable C2-ceramide or mitochondrial permeability transition (PT) inducers, not only apoptosis-sensitive cell lines (HL-60, Jurkat, and Daudi cells), but also Raji cells showed reduction of the mitochondrial transmembrane potential (triangle uppsim), activation of caspase-3, and loss of clonogenic potential. However, Raji cells did not show detectable levels of nuclear apoptosis (DNA degradation). In a cell-free system, cell lysates from tetra-butylhydroperoxide (t-BHP)-treated HL-60 cells induced DNA degradation of Raji nuclei, whereas cell lysates from t-BHP-treated Raji cells failed to induce DNA degradation in either apoptosis-sensitive cell lines or apoptosis-resistant Raji cells. Cleavage of DFF-45, which is a downstream target molecule for caspase-3, was observed in Raji cells as well as in apoptosis-sensitive Daudi cells. These results indicate that there is a defective apoptotic pathway in the cytoplasm downstream of caspase-3 in Raji cells.

Alkylating Agents↗
PubMed1999

Direct coupling of expanded bed adsorption with a downstream purification step.

In the course of developing a cost-effective, scaleable process for the purification of a recombinant protein from Chinese hamster ovary (CHO) suspension cell culture, we investigated direct capture of this molecule using expanded bed adsorption (EBA). EBA combines clarification, purification, and concentration of the product into a single step. The unclarified bioreactor material was directly applied to a STREAMLINE 25 column containing an affinity STREAMLINE adsorbent. This work focused on simplifying the EBA operations and minimizing the overall processing time by running the EBA column unidirectionally, eluting in the expanded bed mode, and coupling the EBA column directly with ion exchange or hydrophobic interaction chromatography. Unidirectional EBA was clearly a simpler unit operation and did not require the use of specialized equipment. The increase in the elution pool volume was insignificant, especially when the EBA column was eluted directly onto the downstream column. Scale-down was simple and could be automated. Coupling of unidirectional EBA with a downstream purification step reduced processing time, equipment requirements and cost.

Adsorption↗
PubMed2000

A member of Forkhead family transcription factor, FKHRL1, is one of the downstream molecules of phosphatidylinositol 3-kinase-Akt activation pathway in erythropoietin signal transduction.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is important for the regulation of a number of cellular responses. Serine/threonine kinase Akt (protein kinase B; PKB) is downstream of PI3K and activated by growth factors. This study found that erythropoietin (EPO) induced tyrosine phosphorylation of Akt in a time- and dose-dependent manner in EPO-dependent human leukemia cell line UT-7/EPO. In vitro kinase assay using histone H2B and glucose synthase kinase as substrates demonstrated that Akt was actually activated by EPO. EPO-induced phosphorylation of Akt was completely blocked by a PI3K-specific inhibitor, LY294002, at 10 micromol/L, indicating that activation of Akt by EPO is dependent on PI3K activity. In addition, overexpression of the constitutively active form of Akt on UT-7/EPO cells partially blocked apoptosis induced by withdrawal of EPO from the culture medium. This finding suggested that the PI3K-Akt activation pathway plays some role in the antiapoptotic effect of EPO. EPO induced phosphorylation of a member of the trancription factor Forkhead family, FKHRL1, at threonine 32 and serine 253 in a dose- and time-dependent manner in UT-7/EPO cells. Moreover, results showed that Akt kinase activated by EPO directly phosphorylated FKHRL1 protein and that FKHRL1 phosphorylation was completely dependent on PI3K activity as is the case for Akt. In conjunction with the evidence that FKHRL1 is expressed in normal human erythroid progenitor cells and erythroblasts, the results suggest that FKHRL1 plays an important role in erythropoiesis as one of the downstream target molecules of PI3K-Akt.

DNA-Binding Proteins↗
PubMed2000

[Study on a downstream signal molecule of human CASK/LIN-2].

OBJECTIVE: To elucidate the function of human CASK/LIN-2, a novel member of membrane-associated guanylate kinase homologs family (MAGUK), by using the yeast two-hybrid system(LexA) to screen the protein interacting with the guanylate kinase-like domain(GK) of hCASK and identify possible protein that might involve downstream signal transduction of hCASK. METHODS: PCR strategy was used to amplify GK domain cDNA fragment of hCASK. The DNA was subcloned into pLexA to construct bait vector pLexA-GK. The pLexA-GK was transformed into yeast host strain EGY48; through testing the bait protein and a repression assay, it was demonstrated that the bait protein did not have transcription activation for leu and lacZ reporter genes, however it could enter nucleus, bind LexA operator. Human fetus brain cDNA library plasmids were transformed into EGY48 containing pLexA-GK and p8op-lacZ, and screened on plates of Gal/Raf his- trp- ura- leu- and Gal/Raf his- trp- ura-X-gal. The clones with galactose dependent leu+ and lacZ+ were isolated; their library plasmids were rescued by means of transforming E.coli KC8. These library plasmids were transformed into the yeast again to re-screen. The remaining positive clones were analyzed by PCR and restriction endonuclease. Finally two kinds of target DNA fragments were obtained. RESULTS: The specificity testing indicated that the two target proteins could specially bind GK domain of hCASK. After DNA sequencing and BLASTn analysis on NCBI, it was shown that the two target DNA fragments, 732 and 683 bp, had high sequence similarity with human inhibitors of differentiation 1(Id1) mRNA, with identities of 97%(630/645) and 98%(656/666), respectively. CONCLUSION: GK domain of hCASK could specially interact with Id1 in yeast. Id1 might be a downstream signal molecule of hCASK, which might involve in regulation of cell differentiation.

Base Sequence↗
PubMed2001

An evolutionarily conserved transmembrane protein that is a novel downstream target of neurotrophin and ephrin receptors.

Appropriate development of nervous system connectivity involves a variety of processes, including neuronal life-and-death decisions, differentiation, axon guidance and migration, and synaptogenesis. Although these activities likely require specialized signaling events, few substrates unique to these neurotrophic functions have been identified. Here we describe the cloning of ankyrin repeat-rich membrane spanning (ARMS), which encodes a novel downstream target of neurotrophin and ephrin receptor tyrosine kinases, Trk and Eph, respectively. The amino acid sequence of ARMS is highly conserved from nematode to human, suggesting an evolutionarily conserved role for this protein. The ARMS protein consists of 1715 amino acids containing four putative transmembrane domains, multiple ankyrin repeats, a sterile alpha motif domain, and a potential PDZ-binding motif. In the rat, ARMS is specifically expressed in the developing nervous system and in highly plastic areas of the adult brain, regions enriched in Trks and Eph receptors. ARMS can physically associate with TrkA and p75 neurotrophin receptors. Moreover, endogenous ARMS protein is tyrosine phosphorylated after neurotrophin treatment of pheochromocytoma 12 cells and primary hippocampal neurons or ephrin B treatment of NG108-15 cells, demonstrating that ARMS is a downstream target for both neurotrophin and ephrin receptors.

Amino Acid Motifs↗