The effect of meconium exposure on the expression and differentiation of amniotic fluid mesenchymal stem cells.

BACKGROUND: The goal of this study was to determine if exposure to meconium would alter the phenotype of amniotic fluid mesenchymal stem cells (AF-MSCs) and the ability of these cells to be differentiated into distal airway type cells. METHODS: Meconium was collected, lyophilized and resuspended in PBS at 3 different concentrations (high, medium, and low). AF-MSCs were cultured in the presence of this meconium suspension for 8 hours and then analyzed for changes in gene expression. Additionally, AF-MSCs exposed to meconium were differentiated for 14 days using modified small airway growth medium (mSAGM) and gene expression was determined. As a spontaneous differentiation control, meconium exposed AF-MSCs were cultured in amniotic fluid stem cell medium (AF medium). RESULTS: After 8 hours of exposure in culture, AF-MSCs had increased expression of distal airway genes aquaporin 5 (AQP5) and surfactant protein c (SPC) when cultured in AF medium containing meconium. These gene expression levels were similar to that of AF-MSCs that were differentiated in mSAGM for 14 days. Furthermore, there was an up regulation of pluripotency genes NANOG and OCT4 in response to low meconium concentration for 8 hours. Following 14 days of culture in mSAGM, there was an upregulation of TTF1, SPC and AQP5 expression in the control, as well as in the low and medium meconium exposed groups indicating that these cells were still able to be differentiated. High meconium concentration did, however, appear to influence the level of distal airway gene expression after 14 days in mSAGM. After 14 days in AF medium, there was significant downregulation in pluripotency and mesenchymal markers as well as distal airway gene expression in all groups. CONCLUSION: The phenotype of AF-MSCs is modulated by meconium exposure; however, the cells were still able to differentiate into distal airway gene and protein expression. This result supports the hypothesis that progenitor cells exist in the amniotic fluid and the presence of meconium may affect their initial phenotype. However, these cells were still able to be differentiated to a distal lung phenotype.

Reactive oxygen species regulate properties of transformation in UROtsa cells exposed to monomethylarsonous acid by modulating MAPK signaling.

UROtsa cells exposed to 50 nM monomethylarsonous acid [MMA(III)] for 52 wk (MSC52) achieved hyperproliferation, anchorage independent growth, and enhanced tumorgenicity. MMA(III) has been shown to induce reactive oxygen species (ROS), which can lead to activation of signaling cascades causing stress-related proliferation of cells and even cellular transformation. Previous research established the acute activation of MAPK signaling cascade by ROS produced by MMA(III) as well as chronic up regulation of COX-2 and EGFR in MSC52 cells. To determine if ROS played a role in the chronic pathway perturbations by acting as secondary messengers, activation of Ras was determined in UROtsa cells [exposed to MMA(III) for 0-52 wk] and found to be increased through 52 wk most dramatically after 20 wk of exposure. Ras has been shown to cause an increase in O2(-) and be activated by increases in O2(-), making ROS important to study in the transformation process. COX-2 upregulation in MSC52 cells was confirmed by real time RT-PCR. By utilizing both antioxidants or specific COX inhibitors, it was shown that COX-2 upregulation was dependent on ROS, specifically, O2(-). In addition, because previous research established the importance of MAPK activation in phenotypic changes associated with transformation in MSC52 cells, it was hypothesized that ROS play a role in maintaining phenotypic characteristics of the malignant transformation of MSC52 cells. Several studies have demonstrated that cancer cells have lowered superoxide dismutase (MnSOD) activity and protein levels. Increasing levels of MnSOD have been shown to suppress the malignant phenotype of cells. SOD was added to MSC52 cells resulting in slower proliferation rates (doubling time=42h vs. 31h). ROS scavengers of OH also slowed proliferation rates of MSC52 cells. To further substantiate the importance of ROS in these properties of transformation in MSC52 cells, anchorage independent growth was assessed after the addition of antioxidants, both enzymatic and non-enzymatic. Scavengers of OH, and O2(-) blocked the colony formation of MSC52 cells. These data support the role for the involvement of ROS in properties of transformation of UROtsa cells exposed to MMA(III).

The role of reactive oxygen species in arsenite and monomethylarsonous acid-induced signal transduction in human bladder cells: acute studies.

Arsenicals are known to induce ROS, which can lead to DNA damage, oxidative stress, and carcinogenesis. A human urothelial cell line, UROtsa, was used to study the effects of arsenicals on the human bladder. Arsenite [As(III)] and monomethylarsonous acid [MMA(III)] induce oxidative stress in UROtsa cells after exposure to concentrations as low as 1 microM and 50 nM, respectively. Previous research has implicated ROS as signaling molecules in the MAPK signaling pathway. As(III) and MMA(III) have been shown to increase phosphorylation of key proteins in the MAPK signaling cascade downstream of ErbB2. Both Src phosphorylation (p-Src) and cyclooxygenase-2 (COX-2) are induced after exposure to 50 nM MMA(III) and 1 microM As(III). These data suggest that ROS production is a plausible mechanism for the signaling alterations seen in UROtsa cells after acute arsenical exposure. To determine importance of ROS in the MAPK cascade and its downstream induction of p-Src and COX-2, specific ROS antioxidants (both enzymatic and non-enzymatic) were used concomitantly with arsenicals. COX-2 protein and mRNA was shown to be much more influenced by altering the levels of ROS in cells, particularly after MMA(III) treatment. The antioxidant enzyme superoxide dismutase (SOD) effectively blocked both As(III)-and MMA(III)- associated COX-2 induction. The generation of ROS and subsequent altered signaling did lead to changes in protein levels of SOD, which were detected after treatment with either 1 microM As(III) or 50 nM MMA(III). These data suggest that the generation of ROS by arsenicals may be a mechanism leading to the altered cellular signaling seen after low-level arsenical exposure.

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.

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.

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.

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.

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.

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.

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.

[Microalbuminuria as predictor of atherosclerotic cardiovascular disease in IDDM]. / Mikroalbuminuri som praediktor af aterosklerotisk kardiovaskulaer sygdom ved 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.

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.

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.

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).

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.

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.

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.