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PubMed2004

bFGF rescues 423-cells from serum starvation-induced apoptosis downstream of activated caspase-3.

Serum withdrawal rapidly induces apoptosis in rat 423-cells, while addition of bFGF results in cell survival. However, surviving cells initially display morphological changes characteristic for apoptotic cells and even process caspases. Active caspase-3 was detected at the single-cell level in those finally bFGF-rescued cells, while mitochondrial integrity was maintained. Generation of cleavage products of caspase targets was confirmed in surviving cells. Proteome analysis indicated multi-faceted survival activities of bFGF including upregulation of inhibitor-of-apoptosis and heat shock protein family members directly interfering with caspases. Our data suggest that the "point-of-no-return" in death-induced cells has to be moved downstream of activated caspase-3.

Animals↗

PubMed2005

Pinpointing phosphotyrosine-dependent interactions downstream of the collagen receptor DDR1.

Activation of the receptor tyrosine kinase DDR1 by collagen results in robust and sustained phosphorylation, however little is known about its downstream mediators. Using phosphopeptide mapping and site-directed mutagenesis, we here identified multiple tyrosine phosphorylation sites within DDR1. We found that Nck2 and Shp-2, two SH2 domain-containing proteins, bind to DDR1 in a collagen-dependent manner. The binding site of Shp-2 was mapped to tyrosine-740 of DDR1 within an ITIM-consensus sequence. Lastly, ablation of DDR1 in the mouse mammary gland resulted in delocalized expression of Nck2, suggesting that defects observed during alveologenesis are caused by the lack of the DDR1-Nck2 interaction.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Aspergillus oryzae produces compounds inhibiting cholesterol biosynthesis downstream of dihydrolanosterol.

The formation of cholesterol synthesis inhibiting molecules by five different strains of the koji mold Aspergillus oryzae was studied. After growing these strains on a complex liquid medium we found in crude organic phase extracts and specific fractions there from compounds inhibiting cholesterol synthesis in human hepatic T9A4 cells in vitro at enzyme sites downstream of dihydrolanosterol. This was evidenced by using different radioactively labeled precursors, namely acetate, mevalonate, 24,25-dihydro-[24,25-(3)H2]-lanosterol or [3-(3)H]-lathosterol.

Aspergillus oryzae↗

PubMed2003

Sp and GATA factors are critical for Apolipoprotein AI downstream enhancer activity in human HepG2 cells.

The factors that bind to the hepatic-specific human apolipoprotein AI (apoAI) 48-bp downstream enhancer (DSE) were identified and characterized by electrophoretic mobility shift assays. A significant homology was shown between the histone 4 (H4) promoters and the hepatic-specific human apoAI DSE at Sp1 and H4TF2 binding sites. Human HepG2 nuclear extracts were used to form four specific complexes with the DSE (referred to as apoAI DSE-1, -2, -3, and -4). The apoAI DSE-1 and -2 complexes showed similar binding specificity to the Sp/H4TF1 consensus site within the apoAI DSE. The apoAI DSE-1 complex was predominantly recognized by anti-Sp1 and Sp3 sera in gel shift assays, indicating that the DSE was recognized by multiple Sp family members. Nuclear extracts that were prepared from retinoic acid treated HepG2 cells showed increased levels of Sp factors in gel shift and Western blot assays. The apoAI DSE-2 complex was identified as H4TF1 and formed in the absence of magnesium chloride. The apoAI DSE-3 complex bound to a consensus GATA element within the DSE that was recognized by recombinant human GATA-6 as well. The apoAI DSE-3 complex was completely disrupted by a GATA-4 antibody in EMSA. GATA-4 and -6 were detected in nuclear extracts prepared from retinoic acid treated HepG2 cells using Western blot assays. The highest apoAI DSE-3 levels were observed with retinoic acid treated HepG2 cell nuclear extracts in EMSA. ApoAI DSE-4 is a multi-factor complex that includes an Sp/H4TF1 factor and either H4TF2 or apoAI DSE-3. Because apoAI DSE mutations revealed transcription defects in transient transfection assays, we conclude that the entire DSE sequence is required for full apoAI transcriptional activity in HepG2 cells.

Apolipoprotein A-I↗

PubMed2004

A unique ATG triplet downstream of gene start in archaea: implications for translation initiation and evolution.

Searching for unique features of archaeal genome may shed light on the mechanism of gene regulation in primitive life forms. Statistical analysis of ATG frequency on the complete genome sequences of 16 archaea, 20 bacteria and 2 eukaryotes revealed that most of the archaeal genomes have a remarkably high ATG frequency at the position of nine nucleotide (nt) downstream of the translation initiation site (the first nucleotide of the translation initiation codon is designated as 0). To understand the role of this unique ATG in archaea, we further analyzed the ATG-initiated genes and non-ATG-initiated genes separately, and the results indicated that only the non-ATG-initiated genes contribute to the high ATG frequency at position +9. This led us to speculate that the in-frame ATG at +9 may serve as a remedial initiation site for archaea in case of initiation failure at the regular site. In addition, it seems that this phenomenon does not result from the harsh environment that archaea are usually viable according to the fact that no considerably high ATG frequency at +9 was observed in all the four thermophilic bacteria that also live in harsh environment. We proposed that the high ATG frequency at position +9 might reflect the decreased efficiency of the translation initiation machinery in archaea. Since archaea evolve very slowly, this unique characteristic of high ATG frequency at position +9 may present the primitive state of the Universal Ancestor.

Archaea↗

PubMed2005

The gonadotropin-regulated long-chain acyl CoA synthetase gene: a novel downstream Sp1/Sp3 binding element critical for transcriptional promoter activity.

The 79 kD gonadotropin-regulated testicular long chain acyl-CoA synthetase gene (GR-LACS) is a hormone-regulated member of the acyl-CoA synthetase family that is expressed abundantly in Leydig cells and to a lesser extent in germinal cells of the adult testis. GR-LACS possesses an ATP/AMP binding domain and the fatty acyl-CoA synthetase (FACS) signature motif. To gain insights into the transcriptional regulation of GR-LACS in gonadal cells, we determined the genomic organization of the gene, including the upstream flanking sequences. The mouse GR-LACS gene spans over at least 45 kb and the coding region is encoded by exons 1-14. All exon-intron junction sites correspond to the consensus splice sequence GT-AG. Exon 7 and 11 comprise the conserved ATP/AMP binding domain and the FACS signature motif, respectively. Primer extension and S1 nuclease analyses demonstrated four transcriptional start sites located at -266/-216 bp 5' to the ATG codon. The minimal promoter domain resides within -254/-217 bp 5' to ATG codon, and upstream sequences to -404 bp (-1035/-405 bp) contribute to the inhibition of transcription in the expressing mouse Leydig tumor cells. Removal of -217/-1 bp, containing a 23 nt GC rich sequence (-112/-90) with an Sp1/Sp3 binding element, within the 1st exon of this TATA-less promoter, significantly reduced GR-LACS gene transcription. Transcriptional activity was abolished by a 2 nt mutation of this element. Thus, functional analyses of this promoter domain indicate that transcription of GR-LACS gene requires an Sp1/Sp3 binding element downstream of the transcriptional start sites which is essential for basal promoter activity.

Animals↗

PubMed2006

Newly established in vitro system with fluorescent proteins shows that abnormal expression of downstream prion protein-like protein in mice is probably due to functional disconnection between splicing and 3' formation of prion protein pre-mRNA.

We and others previously showed that, in some lines of prion protein (PrP)-knockout mice, the downstream PrP-like protein (PrPLP/Dpl) was abnormally expressed in brains partly due to impaired cleavage/polyadenylation of the residual PrP promoter-driven pre-mRNA despite the presence of a poly(A) signal. In this study, we newly established an in vitro transient transfection system in which abnormal expression of PrPLP/Dpl can be visualized by expression of the green fluorescence protein, EGFP, in cultured cells. No EGFP was detected in cells transfected by a vector carrying a PrP genomic fragment including the region targeted in the knockout mice intact upstream of the PrPLP/Dpl gene. In contrast, deletion of the targeted region from the vector caused expression of EGFP. By employing this system with other vectors carrying various deletions or point mutations in the targeted region, we identified that disruption of the splicing elements in the PrP terminal intron caused the expression of EGFP. Recent lines of evidence indicate that terminal intron splicing and cleavage/polyadenylation of pre-mRNA are functionally linked to each other. Taken together, our newly established system shows that the abnormal expression of PrPLP/Dpl in PrP-knockout mice caused by the impaired cleavage/polyadenylation of the PrP promoter-driven pre-mRNA is due to the functional dissociation between the pre-mRNA machineries, in particular those of cleavage/polyadenylation and splicing. Our newly established in vitro system, in which the functional dissociation between the pre-mRNA machineries can be visualized by EGFP green fluorescence, may be useful for studies of the functional connection of pre-mRNA machineries.

3' Untranslated Regions↗

PubMed2026

Identification of CD55 as a downstream factor of EP4 receptor signaling in colorectal cancer cells.

Prostaglandin E2 (PGE2) signaling through the E-type prostanoid 4 (EP4) receptor has been implicated in the pathophysiology of colorectal cancer (CRC). We herein identified decay-accelerating factor, also known as CD55, as a novel CRC-associated downstream factor of the EP4 receptor. The integration of transcriptomic profiling of PGE2-stimulated HCA-7 human colon cancer cells with analyses of cancer genomic databases predicted CD55 as a potential EP4 receptor-regulated target. Inhibitor-based experiments showed the induction of CD55 after a PGE2 stimulation required the EP4 receptor and Gi protein in HCA-7 cells, whereas protein kinase A signaling was dispensable. In combination with a toxicogenomic database analysis, p38 mitogen-activated protein kinase (MAPK) was identified as the predominant effector connecting the EP4 receptor to CD55 upregulation. A single-cell RNA-seq re-analysis of human CRC tissues revealed CD55 upregulation and p38 MAPK-related gene set enrichment in epithelial cells expressing the EP4 receptor, suggesting that this induction mechanism may operate in a subset of epithelial cells in clinical specimens. Collectively, these results delineate a PGE2/EP4 receptor/Gi protein/p38 MAPK signaling axis that induces CD55 expression in HCA-7 cells and epithelial tumor cells, provide new mechanistic clues for understanding the regulation of complement regulatory molecule CD55 expression by prostaglandin signaling.

Humans↗

PubMed2006

Role of the mitogen-activated protein kinase and phosphoinositide 3-kinase/AKT pathways downstream molecules, phosphorylated extracellular signal-regulated kinase, and phosphorylated AKT in colorectal cancer-a tissue microarray-based approach.

Tumor budding is defined as dedifferentiated cancer cells at the invasive margin of colorectal cancer (CRC) and correlates with a worse prognosis. The invasive margin and tumor budding are normally not present in a superficial diagnostic biopsy specimen. The aim of this study was to investigate the expression/overexpression of 2 downstream molecules of the mitogen-activated protein kinase and phosphoinositide 3-kinase/AKT pathways, phosphorylated AKT (pAKT) and phosphorylated extracellular signal-regulated kinase (pERK), in areas of CRC away from the invasive margin and determining if these variables were predictive of tumor budding or prognosis. A series of 1420 unselected, nonconsecutive CRC resections were subdivided into 3 groups: (1) DNA mismatch repair (MMR) proficient, (2) MLH1-negative, and (3) presumed HNPCC. Immunohistochemical analysis of pAKT and pERK expression (0% versus > 0%) and overexpression (increasing percentage of positivity) was performed using the tissue microarray technique. The results were correlated with clinicopathologic parameters. Fifty-seven samples of normal colon mucosa were included as a control group. Nuclear pERK expression (P = .008) was associated with presence of tumor budding in the MMR-proficient, but not in the MLH1-negative and presumed-HNPCC groups. In contrast, cytoplasmic pAKT overexpression was associated with early T stage (P = .04), early N stage (P = .02), and absence of tumor budding (P = .03) only in the MLH1-negative group. There was no association between pERK or pAKT and clinicopathologic parameters in the HNPCC group. Dysregulation of the mitogen-activated protein kinase pathway is likely to be implicated in the mechanism of tumor budding only in MMR-proficient CRC, whereas the phosphoinositide 3-kinase/AKT pathway is associated with early stage in MLH1-negative CRC.

Adenocarcinoma↗

PubMed2006

Increased resistance to beta-lactams in a Klebsiella pneumoniae strain: role of a deletion downstream of the Pribnow box in the blaSHV-1 promoter.

Two hundred and five isolates of Klebsiella pneumoniae were collected from Nantes University Hospital in 2002. A new 30bp deletion was detected downstream of the -10 box of the SHV-1 promoter in a clinical K. pneumoniae isolate with a high amoxicillin/clavulanic acid minimum inhibitory concentration. Reverse transcription polymerase chain reaction revealed increased transcription of bla(SHV-1) mRNA. All conjugation mating assays failed. This new promoter was cloned upstream of the cat gene of the reporter plasmid pKK232-8 and compared with previously described bla(SHV-1) promoters. The deletion induced a 15-fold increase in promoter strength compared with the usual weak promoter. This study reports a new genetic event that increases bla(SHV-1) chromosomal gene expression, which may be of clinical relevance when associated with porin deficiency.

DNA, Bacterial↗

PubMed2004

The polymorphism of the locus control region lying downstream the human IgH locus is restricted to hs1,2 but not to hs3 and hs4 enhancers.

In human, three transcriptional enhancers called hs1,2, hs3 and hs4 were identified downstream the 3' Ig heavy (IgH) locus. We previously reported by PCR and Southern blotting the existence of various allelic forms for the hs1,2 enhancer, one allele being associated with a higher efficiency of switching to IgA in IgA nephropathy (IgAN) patients. Since it is strongly suggested in the mouse that the whole 3' regulatory region is broadly involved in the regulation of class switch recombination (CSR), we wondered if the reported hs1,2 polymorphism was the sole difference possibly accounting for the varying ability to produce non-IgM antibodies in the human population. In this study, we report the absence of additional polymorphism of the hs3 and hs4 enhancers either by using a PCR method or by Southern blotting. DNA sequence analysis confirmed the existence of an invariant core sequence for human hs3 and hs4 enhancers, featuring multiple nuclear factor potential binding sites. In conclusion, human hs3 and hs4 enhancers are not polymorphic, a result that markedly contrasts with the hs1,2 enhancer for which the generation of multiple alleles in both rodents and humans has likely been favored by its central position within a large palindromic region.

Base Sequence↗

PubMed2005

Influence of a downstream narrowing on the flow profile in a tube.

The distance over which the upstream flow conditions in a tube are disturbed by a stenosis downstream, i.e. the outlet length, was investigated for Reynolds numbers in the range 210-2900. Two methods were used, the Navier-Stokes equations were solved with a computer and a physical model was constructed and maximal velocities were measured with an ultrasound Doppler system. The computer model showed that Re number does not influence the outlet length, varying the stenosis area from 25% to 90% has an effect. However, the outlet length remained small, below 70% of the diameter of the tube. The physical model confirmed for a 75% stenosis that the outlet length is small, this method set the limit at not more than 1.2 times the tube diameter.

Algorithms↗

PubMed2005

Improved heterologous gene expression in Escherichia coli by optimization of the AT-content of codons immediately downstream of the initiation codon.

Escherichia coli is the most frequently used host for heterologous gene expression. This study focuses on the effect of AT-rich codons immediately downstream of the initiation codon of the target gene. The third to sixth codons of ndx3, a Nudix hydrolase gene from Thermus thermophilus HB8, were engineered by introducing several silent mutations. As a result, the expression level of ndx3 increased in proportion to the AT-content in the third to sixth codons. This result suggests that incorporation of AT-rich codons can be utilized as a general strategy for improving the expression efficiency of a recombinant protein.

AT Rich Sequence↗

PubMed2006

Downstream processing of triple layered rotavirus like particles.

Rotavirus like particles (RLPs) constitute a potential vaccine for the prevention of rotavirus disease, responsible for the death of more than half a million children each year. Increasing demands for pre-clinical trials material require the development of reproducible, scaleable and cost-effective purification strategies as alternatives to the traditional laboratory scale CsCl density gradient ultracentrifugation methods commonly used for the purification of these complex particles. Self-assembled virus like particles (VLPs) composed by VP2, VP6 and VP7 rotavirus proteins (VLPs 2/6/7) were produced in 5l scale using the insect cells/baculovirus expression system. A purification process using depth filtration, ultrafiltration and size exclusion chromatography as stepwise unit operations was developed. Removal of non-assembled rotavirus proteins, concurrently formed particles (RLP 2/6), particle aggregates and products of particle degradation due to shear was achieved. Particle stability during storage was studied and assessed using size exclusion chromatography as an analytical tool. Formulations containing either glycerol (10% v/v) or trehalose (0.5 M) were able to maintain 75% of intact triple layered VLPs, at 4 degrees C, up to 4 months. The overall recovery yield was 37% with removal of 95% of host cell proteins and 99% of the host cell DNA, constituting a promising strategy for the downstream processing of other VLPs.

Animals↗

PubMed2003

Boater preferences for beach characteristics downstream from Glen Canyon Dam, Arizona.

Release flow decisions are increasingly being influenced by an array of social values, including those related to river-based recreation. A substantial portion of past recreation research on downstream impacts of dams has focused on variability of instream flows. This study complements past research by assessing user preferences for beach characteristics affected by long-term impacts of flow regimes. Based upon a study of three recreational user groups (private trip leaders, commercial passengers, and river guides) of the Colorado River in Grand Canyon, preferences for beach size, presence of shade on beach, and presence of vegetation on beach are examined. Results indicate that large size beaches with shade from trees are setting characteristics with highly reliable and strong user preferences. The multinomial regression models developed for each user group indicate that 80% of all respondents would choose beach campsites 800 m(2); results were the same regardless of respondents' past boating experience, boat type (i.e. oar or motorized), or group size. In addition, size of beach was consistently reported to be a trip feature of moderate importance to respondents' river trip. Implications of this research are related to future prospects for controlled floods (i.e. spike flows) released from Glen Canyon Dam.

Arizona↗

PubMed2004

Identifying target genes regulated downstream of Cdx2 by microarray analysis.

The caudal-related homeobox transcription factor (Cdx2) plays an important role in intestinal development, differentiation, and homeostasis. However, only a limited number of Cdx2-regulated target genes have been elucidated. To delineate the molecular mechanism regulated downstream of Cdx2, we aimed to define Cdx2-regulated genes. We engineered a rat intestinal epithelial cell line, IEC-6, with minimal endogenous Cdx2 expression to express exogenous Cdx2. The gene expression patterns for Cdx2-inducing cells and control cells were examined using oligonucleotide arrays. In the present study, differential expression of 23 genes was confirmed by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis using gene-specific primers. Increased expression of genes was involved in the Notch signaling pathway, xenobiotic metabolism, enzymes associated with tumor suppression, RNA binding protein, receptors, signal transduction, and transcription factors. The wide-ranging collection of such inducing genes suggests to the functions of Cdx2 in cell fate decision and maintenance of intestinal epithelia.

Animals↗

PubMed2005

Akt regulates thrombin-induced HSP27 phosphorylation in aortic smooth muscle cells: function at a point downstream from p38 MAP kinase.

We previously reported that p38 MAP kinase takes part in thrombin-induced HSP27 phosphorylation in aortic smooth muscle A10 cells. In the present study, we investigated whether Akt is involved in the phosphorylation of HSP27 and the role of adenylyl cyclase-cAMP system. Thrombin time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) and Akt in aortic smooth muscle A10 cells. SB203580, a p38 MAP kinase inhibitor, significantly suppressed the thrombin-induced phosphorylation of Akt and the Akt inhibitor suppressed the phosphorylation of HSP27. Furthermore, the thrombin-induced phosphorylation of HSP27, p38 MAP kinase and Akt were decreased by dibutyryl-cAMP (DBcAMP). These results strongly suggest that Akt functions the thrombin-induced phosphorylation of HSP27 at a point downstream from p38 MAP kinase in aortic smooth muscle cells and the adenylyl cyclase-cAMP system is upstream regulator of the HSP27 phosphorylation in these cells.

Adenylyl Cyclases↗

PubMed2026

Global downstream BMP15 pathway analysis in human ovarian granulosa cells reveals novel genetic variations associated with primary ovarian insufficiency.

OBJECTIVES: Primary ovarian insufficiency (POI) is a fertility disorder with a well-established genetic component, but many cases still remain idiopathic. Approximately 1.5-12% of patients with POI can carry a variant in the BMP15 gene, depending on the population and the diagnostic criteria. We hypothesize that genetic variations within pathways downstream of BMP15 activity in ovarian granulosa cells (GCs) may contribute to unexplained cases of POI. The main goal of this study is to identify novel variants associated with POI in genes induced by BMP15 in GCs. STUDY DESIGN: Primary cultures of human GCs were stimulated with recombinant human BMP15. Microarray analysis profiled the BMP15-induced transcriptome in GCs. Validation was achieved by qPCR and immunoblot. Further, target exome sequencing of the differentially expressed genes was performed on 64 women with early POI onset in search of novel variants. MAIN OUTCOME MEASURES: Transcriptome profiling of human GCs stimulated with BMP15 and target exome sequencing in women with early onset of POI. RESULTS: Transcriptome analysis revealed significant upregulation of 19 genes (p < 0.05). Ontology analysis of these genes converged towards two main pathways: TGF-beta signaling and regulation of stem cell pluripotency. Target exome sequencing identified six novel rare variants in five BMP15-induced genes (SAMD11, SMAD6, ID1, USP35, GPCR137C) in 9 of the 64 women with early POI (14%). CONCLUSIONS: BMP15 action in human ovarian GCs defines TGF-beta signaling and pluripotency fate in ovarian follicles. In addition, this study uncovers new potential candidate genes for the pathogenesis of POI.

Humans↗