[Dyslipidemia, diabetes and cardiovascular disease].
Explore the source record for details and available documents.
Cardiovascular Diseases↗
Biomedical subjects
T J Jensen
Publications and source records attributed to T J Jensen.
Plant cell growth and differentiation may involve GAP regulation of Rac activity.
Two Rac GTPase cDNAs, LjRac1 and LjRac2, were identified in the legume Lotus japonicus. Two-hybrid screening with dominant-constitutive mutations in the two Rac GTPases target three plant cDNAs, LjRacGAP1, LjRacGAP2 and LjRacGAP3, that encode putative GTPase activating proteins of Rho-GTPase subfamily members. Employing Rac antiserum, purified recombinant LjRac GTPases and recombinant LjRacGAP1, for ligand overlay assays, in vitro GAP affinity assays and GTPase activation, we confirmed that eukaryote Rac/RacGAP interplay is conserved in plants. In this investigation we have developed some tools that can be used to characterize the role of enhanced LjRac2 expression in developing root nodules.
Amino Acid Sequence↗
A protein phosphatase 2C gene, LjNPP2C1, from Lotus japonicus induced during root nodule development.
Symbiotic interactions between legumes and compatible strains of rhizobia result in root nodule formation. This new plant organ provides the unique physiological environment required for symbiotic nitrogen fixation by the bacterial endosymbiont and assimilation of this nitrogen by the plant partner. We have isolated two related genes (LjNPP2C1 and LjPP2C2) from the model legume Lotus japonicus that encode protein phosphatase type 2C (PP2C). Expression of the LjNPP2C1 gene was found to be enhanced specifically in L. japonicus nodules, whereas the LjPP2C2 gene was expressed at a similar level in nodules and roots. A glutathione S-transferase-LjNPP2C1 fusion protein was shown to have Mg2+- or Mn2+-dependent and okadaic acid-insensitive PP2C activity in vitro. A chimeric construct containing the full-length LjNPP2C1 cDNA, under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter, was found to be able to complement a yeast PP2C-deficient mutant (pct1Delta). The transcript level of the LjNPP2C1 gene was found to increase significantly in mature nodules, and its highest expression level occurred after leghemoglobin (lb) gene induction, a molecular marker for late developmental events in nodule organogenesis. Expression of the LjNPP2C1 gene was found to be drastically altered in specific L. japonicus lines carrying monogenic-recessive mutations in symbiosis-related loci, suggesting that the product of the LjNPP2C1 gene may function at both early and late stages of nodule development.
Amino Acid Sequence↗
Removal of multiple arginine-framed trafficking signals overcomes misprocessing of delta F508 CFTR present in most patients with cystic fibrosis.
Many cystic fibrosis transmembrane conductance regulator (CFTR) mutants are recognized as aberrant by the quality control apparatus at the endoplasmic reticulum (ER) and are targeted for degradation. The mechanism whereby nascent chains are distinguished as either competent or incompetent for ER export has not been elucidated. Here we show that export-incompetent chains display multiple arginine-framed tripeptide sequences like the one recently identified in ATP-sensitive K+ channels. Replacement of arginine residues at positions R29, R516, R555, and R766 with lysine residues to inactivate four of these motifs simultaneously causes delta F508 CFTR, present in approximately 90% of CF patients, to escape ER quality control and function at the cell surface. Interference with recognition of these signals may be helpful in the management of CF.
Arginine↗
Modulation of norepinephrine release from sympathetic neurons of the rabbit aorta by prejunctional prostanoid receptors.
The pharmacological properties and subtypes of prostanoid receptors involved in the prejunctional modulation of [(3)H]norepinephrine release from sympathetic neurons were studied using isolated rabbit aorta. Rings preincubated with [(3)H]norepinephrine were washed with physiological salt solution that contained cocaine plus corticosterone, uptake(1) and uptake(2) inhibitors, respectively, and rauwolscine to block prejunctional alpha(2)-adrenoceptors. Electrical field stimulation was used to evoke (3)H overflow. Prostaglandin (PG)E(2) (10(-9) to 3 x 10(-7) M) reduced the stimulation-evoked (3)H overflow; the pEC(50) value was 8.3, and E(max) value was 98%. This effect was also seen with PGE(1), PGD(2), PGF(2alpha), the EP(1)/EP(3) receptor agonist sulprostone, the EP(2)/EP(3) receptor agonist misoprostol, and the EP(1)/IP receptor agonist iloprost; the rank order (pEC(50)) was sulprostone (8.4) > PGE(2) (8.3) > misoprostol (8.1) > PGE(1) (7.9) > PGF(2alpha) (6.0) > PGD(2) (<5.0). This rank order suggests that these agents act on prejunctional prostaglandin receptors of the EP(3) subtype. The stable thromboxane A(2) analog U46619 (9,11-dideoxy-11alpha, 9alpha-epoxymethano-PGF(2alpha)) slightly reduced the stimulation-evoked (3)H overflow. The FP receptor agonist fluprostenol and the EP(2) receptor agonist butaprost had no effect. The EP receptor antagonist AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) did not alter the inhibitory effect of PGE(2) and sulprostone. AH6809 did not modulate the stimulation-evoked (3)H overflow. This suggests that prejunctional EP(1) receptors are not involved. The IP receptor agonist cicaprost reduced the (3)H overflow only at concentrations higher than 3 x 10(-5) M. We conclude that the postganglionic sympathetic neurons in rabbit aorta are endowed with prejunctional inhibitory EP(3) receptors. FP and IP receptors are not present, and the possible presence of inhibitory DP receptors requires further study.
Animals↗
Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.
Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.
ATP Binding Cassette Transporter, Subfamily B↗
Dibasic protein kinase A sites regulate bursting rate and nucleotide sensitivity of the cystic fibrosis transmembrane conductance regulator chloride channel.
1. The relationship between phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its gating by nucleotides was examined using the patch clamp technique by comparing strongly phosphorylated wild-type (WT) channels with weakly phosphorylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA). 2. The open probability (Po) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after correcting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP-PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated Po of WT was due to its higher bursting rate. 3. The ATP dependence of the 10SA mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between Po and [ATP] was noticeably sigmoid for 10SA channels (Hill coefficient, 1.8), consistent with positive co-operativity between two sites. Increasing ATP concentration to 10 mM caused the Po of both WT and 10SA channels to decline. 4. Wild-type and mutant CFTR channels became locked in open bursts when exposed to mixtures of ATP and the non-hydrolysable analogue AMP-PNP. The rate at which the low phosphorylation mutants became locked open was about half that of WT channels, consistent with Po being the principal determinant of locking rate in WT and mutant channels. 5. We conclude that phosphorylation at 'weak' PKA sites is sufficient to sustain the interactions between the ATP binding domains that mediate locking by AMP-PNP. Phosphorylation of the strong dibasic PKA sites controls the bursting rate and Po of WT channels by increasing the apparent affinity of CFTR for ATP.
Adenosine Triphosphate↗
Inhibition of the 1,25-dihydroxyvitamin D3-induced increase in vitamin D receptor (VDR) levels and binding of VDR-retinoid X receptor (RXR) to a direct repeat (DR)-3 type response element by an RXR-specific ligand in human keratinocyte cultures.
The biological active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), mediates most of its actions through the intracellular vitamin D receptor (VDR). VDR binds to vitamin D responsive elements (VDREs) in the promoter region of responsive genes and regulates transcription. Usually the VDREs consist of a direct repeat of two hexanucleotides spaced by three nucleotides (DR-3), to which VDR preferentially binds as a heterodimer with the retinoid X receptor (RXR). In the present study, we examined the effect of 1,25(OH)2D3 and a specific ligand for RXR, CD2809, on VDR and RXR levels in cultured human keratinocytes and on the binding of RXR-VDR to a DR-3 type response element. Incubation with 1,25(OH)2D3 increased VDR levels as determined by Western blotting, increased VDR-RXR binding to a DR-3 type response element as determined by the electromobility shift assay (EMSA), and induced the 25-OH-D3 24-hydroxylase (24-hydroxylase) gene, containing a DR-3 type response element. CD2809 caused a slight decrease in RXRalpha levels, but had no effect on VDR levels. Addition of both CD2809 and 1,25(OH)2D3 decreased VDR levels as well as the VDR-RXR binding levels to the DR-3 type response element, compared to 1,25(OH)2D3 alone. In conclusion, an RXR-specific ligand interferes with the 1,25(OH)2D3-induced stimulation of VDR levels and VDR-RXR binding to DNA in keratinocyte cultures. It is therefore possible that RXR-specific ligands may counteract certain biological actions of vitamin D3.
Calcitriol↗
[Microalbuminuria as predictor of atherosclerotic cardiovascular disease in IDDM].
The aim of this follow-up study was to assess whether slightly elevated urinary albumin excretion, i.e., microalbuminuria, precedes development of atherosclerotic vascular disease in IDDM. Out of 259 IDDM-patients 30 developed vascular disease during 2,457 person-years. Microalbuminuria was significantly predictive of vascular disease (hazard ratio (95% confidence interval) 1.06 (1.02-1.18) per 5 mg/24 hours increase in urinary albumin excretion; p = 0.002). The predictive effect was independent of age, sex, blood pressure, tobacco smoking, serum concentrations of total-cholesterol, HDL-cholesterol, sialic acid, and von Willebrand factor, and of haemoglobin A1c, insulin dose, diabetes duration, and diabetic nephropathy (hazard ratio (95% confidence interval) 1.04 (1.01-1.08) per 5 mg/24 hours increase in urinary albumin excretion; p = 0.03). It is concluded that slightly elevated urinary albumin excretion is an independent predictor of atherosclerotic vascular disease in insulin-dependent diabetes mellitus.
Adult↗
Identification of new protein species among 33 different small GTP-binding proteins encoded by cDNAs from Lotus japonicus, and expression of corresponding mRNAs in developing root nodules.
In this study, 266 cDNA clones were isolated from a cDNA library made from mRNA of three-week-old root nodules of Lotus japonicus, employing a degenerate oligonucleotide probe that corresponds to a conserved region of small GTP-binding (SMG) proteins. The clones were sorted into groups by cross hybridization and 3' sequencing, and 33 contigs were sequenced in an orderly fashion. Twenty-seven complete and six incomplete protein structures were deduced, which represent three subfamilies of the superfamily of signal transducing GTP-binding proteins. The 33 proteins are divided into nine subclasses, of which seven belong to the Ypt/Rab subfamily, one subclass represents the Rho/Rac subfamily, and one subclass represents the Ran subfamily of small GTP-binding proteins. The protein sequences were compared with related proteins from other plants, from mammals and other species, and discussed with respect to structure and function in different cellular processes. It is apparent that the number of genes encoding SMG proteins in plants must be quite large, since the large number of subclasses found in other eukaryotes is not fully represented in our analysis. Transcription patterns through root nodule development were analysed for 27 of the 33 cDNAs. Differential expression patterns may reflect whether the coded gene product is of importance for organ development. Most mRNAs appear to be constitutively expressed; however, a few unique mRNAs representing the subclasses Rab1, Rab2, Rab5, Rab7 and Rac show elevated levels in root nodules, and certain Rab7, Rab8 and Rab11 species are enriched in aerial parts of the plant. This suggests that most small GTPases have household functions, whereas a few may be required for specialized activities that are important for specialized cells.
Amino Acid Sequence↗
Prejunctional modulation by prostaglandin E2 of noradrenaline release from sympathetic neurones in rabbit aorta.
The modulating effect of prostaglandin E2 (PGE2) on the electrically-evoked 3H-overflow from rabbit isolated aorta preloaded with 3H-noradrenaline was examined. PGE2 (3 x 10(-9)-3 x 10(-7) M) inhibited the stimulation-evoked 3H-overflow (maximum inhibition: 81%; pIC50: 8.1). The inhibition was reversible and inversely related to stimulation frequency (1-30 Hz). Cocaine (3 x 10(-5) M) and corticosterone (4 x 10(-5) M) did not alter the inhibitory effect of PGE2 (3 x 10(-9)-10(-7) M). Rauwolscine (10(-6) M) enhanced the reduction caused by PGE2 (3 x 10(-9)-10(-7) M). Rauwolscine (10(-6) M) alone enhanced the 3H-overflow by 360%. Indomethacin (3 x 10(-6) M) and suprofen (4 x 10(-5) M) did not alter the PGE2 (3 x 10(-9)-10(-7) M)-induced reduction of the 3-H-overflow. Indomethacin (3 x 10(-6) M) and suprofen (4 x 10(-5) M) alone had no effect. We conclude that in the rabbit aorta (1) PGE2 modulates noradrenaline release from sympathetic neurones through a prejunctional inhibitory receptor mechanism; (2) that there is an interaction between alpha 2-adrenoceptors and EP-receptors; (3) that uptake inhibition does not affect the effect of PGE2; and (4) that the influence of endogenous prostaglandins on the noradrenaline release can be excluded.
Adrenergic alpha-2 Receptor Antagonists↗
[Pharmacological prevention of late complications of diabetes].
Explore the source record for details and available documents.
Aldehyde Reductase↗
Immunological properties of meningococcal lipopolysaccharide from serogroups A, B & C.
The aim of the study was to measure and compare the oxidative burst, chemotaxis and cytokine production of human white blood cells, stimulated with meningococcal lipopolysaccharides (LPS) extracted from three different serogroups (A, B and C) of Neisseria meningitidis, and to evaluate whether convalescent sera from patients with meningococcal disease could modify cell stimulation of LPS. All three preparations of LPS from groups A, B and C were tested using the Limulus amoebocyte lysate assay (LAL), and the KDO concentrations of the LPS extracts were measured. Equivalent amounts of biologically active LPS, judged by LAL, and LPS with the same KDO concentration were assayed. IL-1alpha, IL-1beta, IL-6 and TNF-alpha production was stimulated by all three LPS preparations. All three preparations stimulated oxidative burst in monocytes (MNC). Only group A LPS stimulated neutrophil chemotaxis, while none of the three LPS stimulated superoxide production. Pooled convalescent sera from five patients with meningococcal disease suppressed the activity of neutrophils stimulated with LPS from groups B and C (p<0.05, Mann-Whitney U-test).
Cells, Cultured↗
Multiple proteolytic systems, including the proteasome, contribute to CFTR processing.
The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Function and dysfunction of the CFTR chloride channel.
Explore the source record for details and available documents.
Adenosine Triphosphate↗
Phosphatase inhibitors activate normal and defective CFTR chloride channels.
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by phosphorylation and dephosphorylation at multiple sites. Although activation by protein kinases has been studied in some detail, the dephosphorylation step has received little attention. This report examines the mechanisms responsible for the dephosphorylation and spontaneous deactivation ("rundown") of CFTR chloride channels excised from transfected Chinese hamster ovary (CHO) and human airway epithelial cells. We report that the alkaline phosphatase inhibitors bromotetramisole, 3-isobutyl-1-methylxanthine, theophylline, and vanadate slow the rundown of CFTR channel activity in excised membrane patches and reduce dephosphorylation of CFTR protein in isolated membranes. It was also found that in unstimulated cells, CFTR channels can be activated by exposure to phosphatase inhibitors alone. Most importantly, exposure of mammalian cells to phosphatase inhibitors alone activates CFTR channels that have disease-causing mutations, provided the mutant channels are present in the plasma membrane (R117H, G551D, and delta F508 after cooling). These results suggest that CFTR dephosphorylation is dynamic and that membrane-associated phosphatase activity may be a potential therapeutic target for the treatment of cystic fibrosis.
1-Methyl-3-isobutylxanthine↗
Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion.
Technical difficulties in obtaining three-dimensional structures of intrinsic membrane proteins continues to limit understanding of their function. However, considerable insight can be gained from their two-dimensional topological arrangement in the lipid bilayer. Efficient molecular genetic approaches are available to discern the topology of prokaryotic but not of eukaryotic membrane proteins. The absolute asymmetry of the sidedness of their N-glycosylation was employed here to develop such a method using the cystic fibrosis transmembrane conductance regulator (CFTR). Insertion by in vitro mutagenesis of N-glycosylation consensus sequences (NXS/T) in predicted cytoplasmic and extracytoplasmic loops between hydrophobic sequences capable of traversing the membrane established the membrane topology of CFTR. This provides the first experimental evaluation of the original topological model of CFTR based solely on hydropathy algorithms and a method which may be generally applicable for the in vivo evaluation of the topology of other mammalian membrane proteins.
1-Methyl-3-isobutylxanthine↗
Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.
Amino Acid Sequence↗