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At least 973 records · Page 54Linked to original sources
PubMed1998

Downstream defects in beta-adrenergic signaling and relation to myocyte contractility after cardioplegic arrest.

OBJECTIVE: Transient left ventricular dysfunction can occur after hypothermic, hyperkalemic cardioplegic arrest and is associated with decreased beta-adrenergic receptor responsiveness. Occupancy of the beta-adrenergic receptor activates adenylate cyclase, which phosphorylates the L-type Ca2+ channel-enhancing myocyte contractility. The goal of this study was to identify potential mechanisms that contribute to the defects in the beta-adrenergic receptor signaling cascade after cardioplegic arrest. METHODS: Isolated left ventricular porcine myocytes were assigned to one of two treatment groups: (1) cardioplegic arrest (24 mEq/L K+, 4 degrees C x 2 hours, then 5 minutes in 37 degrees C cell media; n = 130) or (2) normothermic control (cell media, 37 degrees C x 2 hours; n = 222). Myocyte contractility was assessed at baseline and after either beta-adrenergic receptor occupancy (25 nmol/L isoproterenol [INN: isoprenaline]), activation of adenylate cyclase (0.5 mumol forskolin), or direct activation of the L-type Ca(2+)-channel (10 nmol/L or 100 nmol/L (-)BayK 8644). RESULTS: Myocyte velocity of shortening (micron/sec) was increased with beta-adrenergic receptor occupancy or direct adenylate cyclase stimulation compared with baseline in the normothermic group (187.3 +/- 6.9, 181.7 +/- 10.2, and 73.9 +/- 2.9, respectively; p < 0.0001) and after cardioplegic arrest (128.6 +/- 8.9, 124.3 +/- 9.4, and 46.1 +/- 2.6, respectively; p < 0.0001). However, the response after cardioplegic arrest was significantly reduced compared with normothermic values under all conditions (p = 0.012). Direct activation of the L-type Ca(2+)-channel, which eliminates beta-adrenergic receptor-dependent events, increased myocyte contractility in the normothermic group (161.90 +/- 12.0, p < 0.0001) and after cardioplegic arrest (92.78 +/- 6.8, p < 0.0001), but the positive inotropic response appeared reduced compared with normothermic control values (p = 0.003). CONCLUSION: These findings suggest that contributory mechanisms for the reduced beta-adrenergic receptor-mediated response after hypothermic, hyperkalemic cardioplegic arrest lie downstream from these specific components of the transduction pathway and likely include defects in Ca2+ homeostasis, myofilament Ca2+ sensitivity, or both.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2003

Modulation of IR/PTP1B interaction and downstream signaling in insulin sensitive tissues of MSG-rats.

PTP1B has been shown to be a negative regulator of the insulin signal transduction in insulin resistant states. Herein we investigated IR/PTP1B interaction and downstream signaling in insulin sensitive tissues of 10 and 28-week-old MSG-insulin resistant rats which represent different stages of insulin resistance. Our results demonstrated that the increase in PTP1B expression and/or association with IR in MSG animals may contribute to the impaired insulin signaling mainly in liver and muscle. Although, adipose tissue of 10-week-old MSG rats showed higher PTP1B expression and IR/PTP1B interaction, they were not sufficient to impair all insulin signaling since IRS-2 phosphorylation and association with PI3-kinase and Akt serine phosphorylation were increased, which may contribute for the increased adiposity of these animals. In 28-week-old-MSG rats there was an increase in IR/PTP1B interaction and reduced insulin signaling in liver, muscle and adipocytes, and a more pronounced insulin resistance.

Adipose Tissue↗
PubMedDate not supplied

Concentrations of polychlorinated dibenzo-p-dioxins and furans in fish downstream from a Ky-5 manufacturing.

Concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) were studied in seven fish species, burbot (Lota lota), pike (Esox lucius), perch (Perca fluviatilis), pikeperch (Stizopedion lucioperca), bream (Abramis brama), salmon (Salmo salar) and Baltic herring (Clupea harengus membras) in River Kymijoki and its estuary polluted by Ky-5 manufacturing. The fish were caught at 14 localities along the river and its estuary. The selected species represent different trophic levels and/or inhabit different environments. The concentrations of PCDD/Fs were low, in most samples below 1 pg g(-1) ITEQ (NATO/CCMS 1988) fresh weight (fw) in muscle, except salmon and Baltic herring. These two species graze at the open sea and consequently accumulate contaminants at a large area in the Baltic Sea. The lipid content in salmon and Baltic herring was an order of magnitude higher than in other species. PCDD/Fs in fish muscle showed only slightly elevated levels in the Kymijoki area and its estuary as compared to the levels in the same species in Finnish freshwaters and sea areas. The concentration of the main impurities of the fungicide Ky-5 was higher in the Kymijoki River downstream the Ky-5 manufacturing place compared to the up-stream locations. The PCDD/F concentrations in fish liver and spawn were 10-100 times higher than the concentration in muscle, because of the much higher lipid concentrations of these organes. Consequently, the tolerable daily intake values could be as much as 100 times smaller (M. Korhonen, M. Verta, T. Vartiainen, Organohalog. Comp. 32 (1997) 305-310; P. Mikkelson, J. Paasivirta, H. Kiviranta, Organohalog. Comp. 39 (1998) 59-62).

Animals↗
PubMed1998

Pyrolysis-GC-MS and CuO-oxidation-HPLC in the characterization of HMMs from sediments and surface waters downstream of a pulp mill.

High molecular mass fractions of lignin and humic compounds in sediments and waters downstream of a pulp mill were characterized applying pyrolysis-gas chromatography-mass spectrometry. The results were compared to those obtained using reversed phase HPLC on the cupric oxide oxidation products. The chromatographic data of both pyrolysis and cupric oxide oxidation were also subjected to the principal component analysis (PCA). The sediment samples and fractions obtained by ultrafiltration of river water samples were freeze dried prior to characterization. The sediment samples were also extracted using 2 M sodium hydroxide solution. The extracts were ultrafiltrated, freeze dried and compared to the freeze dried original sediments using the procedures mentioned above. The amounts of HMMs obtained from the sediments ranged from 62 mg to 97 mg per one gram of sediment. Gel permeation chromatography was applied to samples obtained from sediments by extraction with tetrahydrofuran. The weight average molecular weights of these fractions were in the range of 1500-2300 amu.

Chromatography, High Pressure Liquid↗
PubMed2000

The water quality of the River Thames at a rural site downstream of Oxford

Water quality information is presented for the River Thames 34 km downstream of the market town of Oxford in Oxfordshire to provide an overview of the hydrochemical functioning of a major agriculturally impacted river entering the North Sea. The data, which cover the period from the spring of 1997 to the spring of 1999, relate to three types of data. These types are: (1) weekly spot sampling for determination of major, minor and trace elements, pH, alkalinity and herbicides; (2) tri-weekly spot sampling for pH, alkalinity and dissolved silicon; and (3) continuous measurements of pH and dissolved oxygen. Calcium and bicarbonate provide, respectively, the dominant cation and anion in solution and their compositions remain relatively constant through time, irrespective of flow levels. In contrast, many determinands show seasonally related fluctuations. Concentrations for most of the major anions, sodium, potassium as well as soluble reactive phosphorus and several soluble trace elements such as boron, antimony, arsenic and molybdenum decrease as flow increases. A reverse pattern is observed for nitrate, some herbicides and trace elements associated with particulate phases: concentrations increase with increasing flow. These patterns reflect the influence of: (1) a calcium carbonate rich groundwater system which provides the main stream flow component; (2) dilution of point source pollutant inputs associated with sewage and possibly light industry at high flows for several major, nutrient and trace elements; (3) enhanced nitrate and herbicide runoff from agricultural land at high flows; (4) enhanced microparticulate trace metal levels associated with increased suspended sediment loads at high flows; and (5) biological processes which affect pH, dissolved silicon, dissolved carbon dioxide and dissolved oxygen levels. An examination of soluble reactive phosphorus (SRP) and boron relationships reveals a reduction in concentrations for SRP associated possibly with phosphorus removal from a major sewage treatment works on an upstream tributary of the Thames, the Thame.

Journal Article↗
PubMed2001

The impact of a hydroelectric power plant on the sediment load in downstream water bodies, Svartisen, northern Norway.

When the Svartisen hydroelectric power plant was put into operation, extensive sediment pollution was observed in the downstream fjord area. This paper discusses the impact of the power plant and the contribution from various sources of sediment. Computation of the sediment load was based on samples collected one to four times per day. Grain size distribution analyses of suspended sediments were carried out and used as input in a routing model to study the movement of sediments through the system. Suspended sediment delivered to the fjord before the power station was constructed was measured as 8360 metric tons as an annual mean for a 12-year period. During the years 1995-1996 when the power plant was operating, the total suspended load through the power station was measured as 32609 and 30254 metric tons, respectively. Grain size distribution analyses indicate a major change in the composition of the sediments from 9% clay before the power plant was operative to 50-60% clay afterwards. This change, together with the increase in sediment load, is believed to be one of the main causes of the drastic reduction in secchi depths in the fjord. The effect of the suspended sediment load on the fjord water turbidity was evaluated by co-plotting secchi depth and power station water discharge. Measurements during 1995 and 1996 showed that at the innermost of these locations the water failed to attain the minimum requirement of 2 m secchi depth. In later years secchi depths were above the specified level. In 1997 and 1998 the conditions improved. At the more distal locality, the conditions were acceptable with only a few exceptions. A routing model was applied to data acquired at a location 2 km from the power station in order to calculate the contributions from various sediment sources. This model indicated that the contribution from reservoir bed erosion dominated in 1994 but decreased significantly in 1995. Future operation of the power station will mostly take place with a high water level in the reservoir and is likely to result in acceptable water quality in the fjord. However, during periods of low drawdown, sediment pollution may again become a problem.

Environmental Monitoring↗
PubMed1999

Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila.

The SH2/SH3 adaptor protein Dock has been proposed to transduce signals from guidance receptors to the actin cytoskeleton in Drosophila photoreceptor (R cell) growth cones. Here, we demonstrate that Drosophila p21-activated kinase (Pak) is required in a Dock pathway regulating R cell axon guidance and targeting. Dock and Pak colocalize to R cell axons and growth cones, physically interact, and their loss-of-function phenotypes are indistinguishable. Normal patterns of R cell connectivity require Pak's kinase activity and binding sites for both Dock and Cdc42/Rac. A membrane-tethered form of Pak (Pak(myr) acts as a dominant gain-of-function protein. Retinal expression of Pak(myr) rescues the R cell connectivity phenotype in dock mutants. These data establish Pak as a critical regulator of axon guidance and a downstream effector of Dock in vivo.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor.

Drosophila Roundabout (Robo) is the founding member of a conserved family of repulsive axon guidance receptors that respond to secreted Slit proteins. Little is known about the signaling mechanisms which function downstream of Robo to mediate repulsion. Here, we present genetic and biochemical evidence that the Abelson (Abl) tyrosine kinase and its substrate Enabled (Ena) play direct and opposing roles in Robo signal transduction. Genetic interactions support a model in which Abl functions to antagonize Robo signaling, while Ena is required in part for Robo's repulsive output. Both Abl and Ena can directly bind to Robo's cytoplasmic domain. A mutant form of Robo that interferes with Ena binding is partially impaired in Robo function, while a mutation in a conserved cytoplasmic tyrosine that can be phosphorylated by Abl generates a hyperactive Robo receptor.

Animals↗
PubMed1999

Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation.

Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.

ADP-Ribosylation Factor 1↗
PubMed2003

Linker scanning mutagenesis of the Plasmodium gallinaceum sexual stage specific gene pgs28 reveals a novel downstream cis-control element.

Protozoan parasites undergo complex life cycles that depend on regulated gene expression. However, limited studies on gene regulation in these parasites have repeatedly shown characteristics different from other eukaryotes. Within the Apicomplexa family, little is known about the mechanism of gene expression and regulation in Plasmodium spp. We have been investigating the cis-elements that control basal expression of a sexual stage specific gene in Plasmodium gallinaceum. Previously, we identified by 5' deletion analysis of a reporter construct that the 333bp upstream of the translational start site of pgs28 is sufficient for basal expression, and that the sequence between -333 and 316bp is necessary for such expression. In this report, we identified by linker scanning mutagenesis an 8-bp sequence that is essential for pgs28 transgene expression. This sequence is a target of sequence-specific nuclear factors. Primer extension studies demonstrate that, interestingly, the endogenous pgs28 transcript has two 5' ends, at -65 and +1. We suggest that this 8-bp sequence, CAGACAGC that is situated at +24 to +31 (with respect to the proximal start site), is a novel downstream promoter element in P. gallinaceum that appears to function independently of a TATA box or an Inr element.

Animals↗
PubMed1998

Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA.

Giardia lamblia, a parasitic protozoan, has been regarded as one of the most conserved eukaryotes evolved from the prokaryotes. One of its unique features appears to be the unusually short 5'-untranslated regions (UTR) (1-6 nucleotides (nts)) and the apparent absence of 5'-cap structures from its mRNAs. Transfection of the Giardia trophozoites with luciferase-encoding chimeric transcripts, flanked by the 5'- and 3'-ends of giardiavirus (GLV) (+)-strand RNA, indicated that the translational efficiency was enhanced by 5000-fold when the 5'-viral sequence extended 264 nts into the capsid coding region and fused with the luciferase open reading frame (ORF). A 13-nt downstream box (DB) was identified within this region which complements a 15-nt sequence between nts # 1382 and 1396 near the 3'-end of the Giardia 16S-like ribosomal RNA (the anti-DB). Deletion or scrambling of this DB in the mRNA leads to a significant loss of the translational efficiency in Giardia. A Shine-Dalgarno (SD)-like element was also identified at 9-14 nts upstream from the initiation codon in the viral (+)-strand RNA, but alteration of its sequence led to no change in translation. Using the sequence complementary to ribosomal anti-DB to probe the Giardia mRNAs available in the databases, each mRNA was found to contain a putative DB with an average length from 8 to 13 nts. It is thus possible that initiation of translation in Giardia may involve a DB in the coding region of mRNA that may bind to a putative anti-DB in the small ribosomal RNA through base pairing. This mechanism of ribosome recruitment, which finds a potential parallel in Escherichia coli, could illustrate a relatively close distance between Giardia and prokaryotes in terms of translation initiation, and may provide a model for studying the evolution of translation machinery.

5' Untranslated Regions↗
PubMed2001

Identification of downstream target genes of latent membrane protein 1 in nasopharyngeal carcinoma cells by suppression subtractive hybridization.

Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China and is closely associated with infection of Epstein-Barr virus (EBV). The EBV encoded latent membrane protein 1 (LMP1) is frequently detected in NPC and may play a role in its pathogenesis. Previous studies have shown that LMP1 transformed rodent fibroblasts and altered growth properties in B cells and epithelial cells. However, the pathological role of LMP1 in NPC cells is still poorly understood. In order to investigate the downstream target genes of LMP1 in NPC cells, suppression subtractive hybridization was used to clone and identify the genes differentially expressed in a LMP1 expressing NPC cell line, CNE-2. Two subtractive cDNA libraries were constructed: one enriched for the genes upregulated by LMP1 and one was for the genes downregulated by LMP1. A total of 192 clones were screened by reverse Northern blotting. Fourteen of them were confirmed to be overexpressed while eight of them were suppressed. The upregulation of integrin alpha6, laminin 5gamma2, TAP1 and downregulation of p54nrb, RACK1 and p66Shc were further confirmed in three sets of LMP1 expressing NPC cell lines. The expression profiles of differentially expressed genes identified in this study suggest a role of LMP1 in promotion of cell survival and facilitation of tumor invasion.

DNA, Complementary↗
PubMed1998

Characterization of the region downstream of the pilus biogenesis gene pilC1 in Neisseria gonorrhoeae.

The nucleotide sequence of a 3 kb region downstream of pilC1 in Neisseria gonorrhoeae MS11 was analyzed. This region contains two open reading frames, ORF1 and ORF2, and several repetitive DNA elements. ORF1 encodes an outer membrane protein that shows homology to orf98 of Pediococcus acidilactici. PCR with primers specific for ORF1 revealed that the gene is present in all gonococcal strains tested. The other open reading frame, ORF2, is highly homologous to the putative integral membrane protein HI1680 of Haemophilus influenzae.

Amino Acid Sequence↗
PubMed2000

Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications.

Interest in producing large quantities of supercoiled plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNA vaccines. Owing to the commercial interest in these approaches, the development of production and purification strategies for gene-therapy vectors has been performed in pharmaceutical companies within a confidential environment. Consequently, the information on large-scale plasmid purification is scarce and usually not available to the scientific community. This article reviews downstream operations for the large-scale purification of plasmid DNA, describing their principles and the strategy used to attain a final product that meets specifications.

3' Untranslated Regions↗
PubMed1997

Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite.

Downstream processing by biospecific chromatography of maltodextrin phosphorylase from Escherichia coli, overexpressed in E. coli, was substantially improved by a novel approach using ceramic hydroxyapatite. Wild-type and a less active mutant enzyme were purified from crude bacterial cell extracts in one efficient separation step that yielded phosphorylase in purity > 95% in at least 90% recoveries. At pH 6.9 and 25 degrees C, wild-type and mutant phosphorylases eluted from the hydroxyapatite column at a phosphate concentration of 0.4 M whereas calcium ions failed to displace the enzymes. The dynamic capacity for phosphorylase binding in the presence of bulk proteins was approximately 3 mg enzyme ml-1 matrix. The interaction of E. coli phosphorylase with hydroxyapatite seems to be mediated by surface amino groups, so that the bound enzyme retained almost full catalytic activity. Compared to the soluble enzyme, immobilization onto hydroxyapatite resulted in a more than 30-fold stabilization of wild-type phosphorylase against thermal and proteolytic inactivation and thus could improve the operational stability of phosphorylase during conversion of polysaccharide to glucose 1-phosphate.

Chromatography↗
PubMed2000

Sequences upstream and downstream of two xylem-specific pine genes influence their expression.

The identification of regulatory elements conferring high levels of expression in differentiating pine xylem will be valuable for genetic engineering of wood properties and will contribute to our understanding of gene regulation in this important group of forest trees. We examined the roles of both upstream and downstream elements in regulating the expression of two genes with preferential expression in developing xylem of loblolly pine. Gene constructs containing a PtX3H6, PtX14A9, or CaMV 35S promoter, the uidA gene encoding beta-glucuronidase, and a PtX3H6, PtX14A9, or NOS terminator were used to transform tobacco and hybrid poplar. When combined with the NOS terminator, neither pine promoter conferred xylem-specific expression in tobacco. When combined with the PtX3H6 promoter, an element at the 3' end of PtX3H6 reduced GUS expression resulting in preferential expression in vascular tissues. This silencing effect was not observed when the pine terminator was tested in conjunction with the CaMV 35S promoter. The PtX14A9 terminator did not increase tissue specificity. In leaves of transgenic poplar, both pine promoters conferred preferential GUS expression in veins when combined with the NOS terminator. The PtX3H6 terminator greatly decreased expression in leaves and stems when combined with the PtX3H6 promoter but only slightly altered expression when combined with the CaMV 35S promoter. An element at the 3' end of PtX14A9 increased GUS expression in veins when used in conjunction with either the PtX14A9 or CaMV35S promoter.

Journal Article↗
PubMed2001

Downstream of Otx2, or how to get a head.

Otx2 is a member of a highly conserved family of homeodomain-containing transcription factors that function in early brain development. Recent studies have identified a significant number of target genes downstream of Otx2, allowing us to address the question of how it fulfils its diverse developmental roles. Interestingly, many of these target genes are not transcription factors or signaling molecules, and they probably have no direct affect on gene expression. Furthermore, there is evidence that Otx2 coordinates the activity of unrelated genes that have overlapping functions, and that it does so directly without intermediate transcriptional or signaling activity.

Animals↗
PubMed2001

Transcription of the Escherichia coli dcw cluster: evidence for distal upstream transcripts being involved in the expression of the downstream ftsZ gene.

Escherichia coli strains VIP596 and VIP597 have been constructed to compare the amount of transcription of the ftsZ gene derived from proximal promoters in the ddlB-ftsZ region with that originating in the upstream regions of the dcw cluster. Both strains have in common a beta-galactosidase reporter fusion located at the ddlB locus, but differ in that VIP597 has a transcription terminator Omega interposon located downstream from lacZ. In addition, these strains have the ddlB, ftsQ, ftsA and ftsZ genes under the control of the IPTG-inducible promoter P(tac), allowing to control artificially ftsZ expression for normal cell division to take place. When beta-galactosidase activity was measured in VIP596 and VIP597 and compared to the levels measured in strain VIP407, in which the lacZ reporter fusion is located in the ftsZ gene, they were found to account for nearly 66% of the total transcription entering into ftsZ. This result indicates that the reduction in ftsZ transcription observed when the promoters in the ddlB-ftsA region are disconnected from the upstream sequences of the dcw cluster (as observed by Flärdh et al., Mol. Microbiol. 30 (1998) 305-316) in strain VIP490) is the direct consequence of the interruption in the transcription originated upstream and not due to the effect of such sequences on the promoters proximal to ftsZ.

Bacterial Proteins↗