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Biomedical subjects

K Page

Publications and source records attributed to K Page.

At least 37 records · Page 2Linked to original sources
PubMed2000

The effect of ceruloplasmin on iron release from placental (BeWo) cells; evidence for an endogenous Cu oxidase.

The mechanism of iron release from the placenta into the fetal circulation is not well understood. Ceruloplasmin, a plasma ferroxidase, has been implicated in iron efflux from a variety of cell types. The hypothesis is that circulating ceruloplasmin facilitates iron efflux by oxidizing the released Fe(II) to Fe(III) for incorporation into transferrin. We tested whether this mechanism mediates iron release from placental cells into the fetal circulation, using the BeWo cell line, a choriocarcinoma which can differentiate into a syncytium.(59)Fe release from undifferentiated or differentiated cells and from cells grown on porous filters was not stimulated by extracellular ceruloplasmin. Instead, we found that BeWo cells express an endogenous ferroxidase. The protein is membrane bound and cross-reacts with an anti-ceruloplasmin antibody, but has a different size; 100 and 140 kDa. Similar immunoreactivity was identified in first- and third-trimester human placentae. In BeWo cells, the protein has a perinuclear localization but does not entirely co-localize with markers for the endoplasmic reticulum or Golgi apparatus. We propose that this oxidase performs the same function as serum ceruloplasmin and is involved in iron release into the fetal circulation.

Ceruloplasmin↗
PubMed2000

Regulation of cyclin D(1) expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells.

We have shown in bovine tracheal myocytes that extracellular signal-regulated kinase (ERK) and Rac1 function as upstream activators of transcription from the cyclin D(1) promoter. We now examine the role of phosphatidylinositol (PI) 3-kinase in this process. PI 3-kinase activity was increased by platelet-derived growth factor (PDGF) and attenuated by the PI 3-kinase inhibitors wortmannin and LY294002. These inhibitors also decreased cyclin D(1) promoter activity, protein abundance, and DNA synthesis. Overexpression of the active catalytic subunit of PI 3-kinase (p110(PI) (3-K)CAAX) was sufficient to activate the cyclin D(1) promoter. Wortmannin and LY294002 failed to attenuate PDGF-induced ERK activation, and overexpression of p110(PI) (3-K)CAAX was insufficient to activate ERK. p110(PI) (3-K)CAAX-induced cyclin D(1) promoter activity was not blocked by PD98059, an inhibitor of mitogen-activated protein kinase/ERK kinase. We next examined whether PI 3-kinase and the 21-kD guanidine triphosphatase Rac1 regulate cyclin D(1) promoter activity by similar mechanisms. p110(PI) (3-K)CAAX-induced cyclin D(1) promoter activity was decreased by two inhibitors of Rac1-mediated signaling, catalase and diphenylene iodonium. Further, PDGF, PI 3-kinase, and Rac1 each activated the cyclin D(1) promoter at the cyclic adenosine monophosphate response element binding protein (CREB)/activating transcription factor (ATF)-2 binding site, as evidenced by expression of a CREB/ATF-2 reporter plasmid. Finally, PI 3-kinase and Rac1-induced CREB/ATF-2 transactivation were each inhibited by catalase. Together, these data suggest that in airway smooth muscle (ASM) cells, PI 3-kinase regulates transcription from the cyclin D(1) promoter and DNA synthesis in an ERK-independent manner. Further, PI 3-kinase and Rac1 regulate ASM cell cycle traversal via a common cis-regulatory element in the cyclin D(1) promoter.

Activating Transcription Factor 2↗
PubMed2000

Modification of the Koken nasal splint.

Koken nasal splints have been found effective postoperatively in treating cleft-lip nose deformity. In our experience, their retention has been poor, so reducing the molding effect. This paper presents a modification designed to overcome the problem of retention.

Cleft Lip↗
PubMed1999

Characterization of a Rac1 signaling pathway to cyclin D(1) expression in airway smooth muscle cells.

We examined the importance of the Rho family GTPase Rac1 for cyclin D(1) promoter transcriptional activation in bovine tracheal myocytes. Overexpression of active Rac1 induced transcription from the cyclin D(1) promoter, whereas platelet-derived growth factor (PDGF)-induced transcription was inhibited by a dominant-negative allele of Rac1, suggesting that Rac1 functions as an upstream activator of cyclin D(1) in this system. Rac1 forms part of the NADPH oxidase complex that generates reactive oxygen species such as H(2)O(2). PDGF stimulated a substantial increase in intracellular reactive oxygen species, as measured by the fluorescence of dichlorofluorescein-loaded cells, and this was blocked by the glutathione peroxidase mimetic ebselen. Pretreatment with ebselen, catalase, and the flavoprotein inhibitor diphenylene iodonium each attenuated PDGF- and Rac1-mediated cyclin D(1) promoter activation, while having no effect on the induction of cyclin D(1) by mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase-1 (MEK1), the upstream activator of ERKs. Antioxidant treatment also inhibited PDGF-induced cyclin D(1) protein expression and DNA synthesis. Overexpression of an N-terminal fragment of p67(phox), a component of NADPH oxidase which interacts with Rac1, attenuated PDGF-induced cyclin D(1) promoter activity, whereas overexpression of the wild-type p67 did not. Finally, Rac1 was neither required nor sufficient for ERK activation. Taken together, these data suggest a model by which two distinct signaling pathways, the ERK and Rac1 pathways, positively regulate cyclin D(1) and smooth muscle growth.

Animals↗
PubMed1999

Restenosis following angioplasty in the swine coronary artery is inhibited by an orally active PDGF-receptor tyrosine kinase inhibitor, RPR101511A.

BACKGROUND: Platelet-derived growth factor (PDGF), a purported mediator of arterial response to injury, stimulates proliferation, chemotaxis, and matrix production by activation of its membrane receptor tyrosine kinase. Because these activities underlie restenosis, inhibition of the PDGF-receptor tyrosine kinase (PDGFr-TK) is postulated to decrease restenosis. METHODS AND RESULTS: RPR101511A is a novel compound which selectively and potently inhibits the cell-free and in situ PDGFr-TK and PDGFr-dependent proliferation and chemotaxis in vascular smooth muscle cells (VSMC). To evaluate the effect of RPR101511A (30 mg. kg-1. d-1 BID for 28 days following PTCA) on coronary restenosis, PTCA was performed in hypercholesterolemic minipigs whose left anterior descending (LAD) coronary artery had been injured by overdilation and denudation, yielding a previously existing lesion. Angiographically determined prePTCA minimal lumen diameters (MLD) were similar in vehicle and RPR101511A-treated pigs (1.98+/-0.09 versus 2.01+/-0.08 mm) and increased to the same extent in the 2 groups following successful PTCA (2.30+/-0.06 versus 2.52+/-0.13). At termination, there was an average 50% loss of gain in the vehicle-treated group but no loss of gain with RPR101511A (2.16+/-0. 05 versus 2.59+/-0.11, P<0.001). Morphometric analysis of the LAD showed that RPR101511A caused a significant decrease in total intimal/medial ratio (0.96+/-0.58 versus 0.67+/-0.09, P<0.05). CONCLUSIONS: RPR101511A, which acts by inhibition of the PDGFr-TK, completely prevented angiographic loss of gain following PTCA and significantly reduced histological intimal hyperplasia.

Administration, Oral↗
PubMed1999

Salmonella infection in a commercial line of ducks; experimental studies on virulence, intestinal colonization and immune protection.

Experimental infections of different salmonella serotypes were established in a commercial line of ducks to provide baseline information on which control measures might be based. The ducks were very resistant to systemic infection with Salmonella typhimurium, S. enteritidis and S. gallinarum within 36 h of hatching. This was associated with an inherent inability of the strains to multiply in the reticulo-endothelial system. The resistance was not associated with poor invasiveness or serum sensitivity. Individual strains of S. typhimurium, S. enteritidis, S. heidelberg and S. orion colonized the gut well and were excreted in the faeces for at least 6 weeks by ducks when they were infected orally within 2 days of hatching. The main sites of colonization were the caeca and, to a lesser extent, the crop. Viable counts of each inoculated strain in the caeca remained in excess of 10(6) c.f.u. 3 weeks after infection although the organisms had been cleared from the spleen by this time. Much less excretion occurred when the birds were infected at 3 weeks of age. When infected ducks, which had cleared themselves of infection, were challenged orally with the homologous strain expressing a different genetic marker, very low levels of excretion of the challenge strain were detected when compared with a control group. After infection low titres of circulating lipopolysaccharide-specific IgG antibodies were detected by an ELISA. Intestinal colonization of newly hatched ducks with an aroA strain of S. enteritidis resulted in extensive colonization which exerted an exclusion effect on the parent strain inoculated 24 h later.

Animals↗
PubMed1999

Effect of maternal polyunsaturated fatty acid concentration on transport by the human placenta.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentas (n = 5) from normal pregnancies. The maternal side was perfused ex vivo for 90 min with a modified Krebs Ringer solution containing a physiological mixture of fatty acids - designed to mimic the composition of non-esterified fatty acids (NEFA) measured in the last trimester of pregnancy (n = 10) - and ratio of fatty acid to human albumin. The selectivity for alpha-linolenic acid (alphaLN) transfer to the fetal circulation was not significantly different from that observed when using the triglyceride (TG) composition (1.21 +/- 0.04), but significantly different for AA (1.43 +/- 0.12; p < 0.001) and docosahexaenoic acid (DHA; 2.02 +/- 0.09; p = 0.048). The absolute rate of transfer (nmol. ml-1) compared to that using the TG maternal perfusate composition was significantly different for LA (0.562 +/- 0.038; p = 0.50), alphaLN (0.130 +/- 0.009; p < 0.001), arachidonic acid (AA; 0.218 +/- 0.022; p = 0.001) and DHA (0.383 +/- 0.04; p < 0.001). Thus, placental selectivity for alphaLN and DHA appears to be relatively unresponsive to changes in the mixture of fatty acids in the maternal circulation but the selectivity for AA increased with the increase in the maternal AA concentration. For an 8-fold increase in the concentration of DHA in the maternal circulation there was a 13-fold increase in the transfer of DHA to the fetal circulation. For a 2-fold increase in the concentration of AA, transfer was increased 8-fold. For a 1.3-fold increase in the concentration of alphaLN, transfer was increased 2.1-fold. These results suggest that the maternal concentration of individual fatty acids, and hence the composition of the maternal diet, can have large effects on polyunsaturated/long-chain polyunsaturated fatty acids delivery to the fetus.

Adult↗
PubMed1999

Platelet-derived growth factor stimulation of mitogen-activated protein kinases and cyclin D1 promoter activity in cultured airway smooth-muscle cells. Role of Ras.

We hypothesized that in bovine tracheal myocytes, growth factor treatment induces transcription from the cyclin D1 promoter that is dependent on the activation of both Ras and extracellular signal-related kinase (ERK). We found that platelet-derived growth factor (PDGF) treatment induced substantial activation of ERK2 that was blocked by expression of a dominant-negative Ha-Ras. Further, expression of a constitutively active Ha-Ras induced substantial ERK2 activity, consistent with the notion that Ras is required and sufficient for ERK activation. PDGF treatment induced only modest activation of the Jun amino terminal kinase-1 (JNK1) and p38 mitogen-activated protein kinases (MAPKs). Active Ras induced similar responses, implying that complete activation of the JNK and p38 pathways requires additional or alternative upstream signaling intermediates besides Ras. In contrast, expression of a constitutively active Rac1, an alternative guanosine triphosphatase involved in intracellular signaling, produced a high level of JNK1 activation, suggesting that Rac1 is an important upstream activator of JNK in this system. Active Ras and MAPK/ ERK kinase-1 (MEK1) (the upstream activator of ERK) each induced cyclin D1 promoter activity, whereas active stress-activated protein kinase/ERK kinase-1 (SEK1), an upstream activator of JNK, did not. Finally, the synthetic MEK inhibitor PD98059 blocked Ras-induced cyclin D1 promoter activity. Together, these data suggest that in bovine tracheal myocytes: (1) activation of MAPK by PDGF is dependent on Ras; (2) active Ras is sufficient for ERK activation but is insufficient for maximal activation of JNK or p38; (3) activation of Rac1 is sufficient for maximal JNK activation; and (4) Ras, MEK, and ERK constitute a distinct pathway to cyclin D1 transcriptional activation.

Animals↗
PubMed1998

Transforming growth factor-beta1 in plasma and liver of children with liver disease.

Although several liver diseases of childhood, particularly biliary atresia (BA) and cystic fibrosis (CF) liver disease (CFLD) are characterized by hepatic fibrosis, the pathogenesis of this process is incompletely understood. The cytokine transforming growth factor-beta1 (TGF-beta1) has been implicated in hepatic fibrosis in experimental animals, in which both the hepatic expression and plasma concentration of this cytokine are increased. The objective of our study was to determine whether there are similar alterations of TGF-beta1 in patients with hepatic fibrosis secondary to either BA and/or CFLD. The study design was as follows. In study 1, plasma TGF-beta1 was assessed by ELISA in 9 children with BA undergoing liver transplantation, 11 patients with CFLD, and appropriate control subjects. In study 2, hepatic expression of TGF-beta1 protein (assessed immunohistochemically) and hepatic fibrosis were scored semiquantitatively, on a 1-3 scale, by blinded investigators, in archival liver biopsy specimens from 10 children with BA, 10 with CFLD, and from 10 older children with normal hepatic histology, as well as in 4 patients with liver diseases of various etiologies. Simultaneous plasma and liver TGF-beta1 studies were performed in 8 patients with liver disease. Results were as follows. Plasma TGF-beta1 values were inversely correlated with age in healthy subjects (r=-0.54, p < 0.0001). The plasma TGF-beta1 protein of children with BA was decreased (13+/-2 ng/mL) compared with values for healthy children (42+/-6 ng/mL, n=10, p < 0.005). Similarly, the plasma TGF-beta1 concentration in patients with CFLD was also decreased compared with values for children with CF and normal serum liver profiles (n=14) (2+/-1 ng/mL versus 12+/-1, p < 0.05). However, the plasma TGF-beta1 concentration was increased in two patients with other types of liver disease. The hepatic expression of TGF-beta1 was increased in the presence of hepatic fibrosis in all types of liver diseases studied. Forty-six percent of patients had both marked hepatic fibrosis and marked TGF-beta1 labeling; 86% of samples without fibrosis showed no TGF-beta1 labeling, p=0.007. In conclusion, these studies have established the association of hepatic TGF-beta1 protein and hepatic fibrosis in several common liver diseases of childhood. Our data also suggest that, in children, plasma TGF-beta1 does not appear to be a useful marker of hepatic expression of this cytokine.

Adult↗
PubMed1997

Cell adhesion to fibronectin regulates membrane lipid biosynthesis through 5'-AMP-activated protein kinase.

We have shown that attachment to a fibronectin substrate stimulates two pathways of lipid biosynthesis in cultured human fibroblasts. Detachment of these cells (mechanically, with trypsin, or by RGDS peptides) caused a significant decrease in their 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and in their incorporation of [3H]acetate into fatty acids. This inhibition was substantially reversed by the reattachment of cells to fibronectin substrates, but not to poly-L-lysine substrates or to fibronectin in solution. Inhibiting phosphoprotein phosphatase activity with okadaic acid blocked the recovery of both biosynthetic activities. Both 3-hydroxy-3-methylglutaryl-coenzyme A reductase and fatty acid biosynthesis are known to be inhibited by the action of 5'-AMP-activated protein kinase, which is activated by an increase in the level of AMP relative to ATP. For example, in our system, sodium azide and 2-deoxy-D-glucose increased the ratio of cellular AMP to ATP and caused a decrease in lipid biosynthesis. We then verified the prediction that detachment of cells from substrates also caused an increase in the AMP/ATP ratio. We therefore conclude that the attachment of cells to fibronectin promotes lipid biosynthesis, presumably in coordination with the cellular growth response evoked by attachment to the extracellular matrix.

AMP-Activated Protein Kinases↗
PubMed1997

An interleukin 5 mutant distinguishes between two functional responses in human eosinophils.

Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.

Cells, Cultured↗
PubMed1997

Long-chain polyunsaturated fatty acid transport across the perfused human placenta.

The role of the placenta in controlling the supply of fatty acids to the fetus was investigated in term placentae (n = 9) from normal pregnancies. The maternal side was perfused ex vivo for 90 min with a modified Krebs Ringer solution containing a physiological mixture of fatty acids and ratio of fatty acid to human albumin. There was no evidence of chain elongation and desaturation of the essential fatty acids. Relative to the value for oleic acid, the rate of transfer to the fetal circulation was: 1.30 +/- 0.02 (P < 0.001) for linoleic acid, 1.61 +/- 0.09 (P = 0.002) for alpha-linolenic acid, 0.67 +/- 0.10 (P = 0.033) for arachidonic acid and 2.10 +/- 0.16 (P = 0.003) for docosahexaenoic acid. For tissue accumulation the values were 1.47 +/- 0.39 (P < 0.001) for linoleic acid, 2.24 +/- 0.37 (P = 0.027) for alpha-linolenic acid, 9.84 +/- 1.03 (P = 0.001) for arachidonic acid, and 3.01 +/- 0.79 (P = 0.064) for docosahexaenoic acid. The order of selectivity for transfer from the maternal to the fetal circulation was docosahexaenoic > alpha-linolenic > linoleic > oleic > arachidonic acid. Such a mechanism would allow the preferential transfer of docosahexaenoic acid and the essential fatty acids to the fetal circulation, thereby protecting the polyunsaturated fatty acid supply to the fetus during a critical period of development.

Adult↗
PubMed1997

Developmental regulation of presenilin mRNA expression parallels notch expression.

Mutations in the presenilin (PS) 1 and 2 genes are associated with autosomal dominant Alzheimer disease. PS1 shares striking homology with sel-12, a C. elegans gene implicated in the function of lin-12/Notch, a protein important in neurogenesis. We studied mRNA expression using RT-PCR and in situ hybridization techniques during neural development in mouse and rat. Strong expression of PSs and Notch1 was observed in embryos, especially in the ventricular zone, which decreased gradually as embryos developed. Very low levels of PSs and Notch were present in adulthood, their signals present primarily in the hippocampus and cerebellum. These observations show that, like Notch, PS1 and PS2 are strongly developmentally regulated, and support the plausibility of an interaction between PSs and Notch.

Animals↗
PubMed1996

In situ hybridization analysis of presenilin 1 mRNA in Alzheimer disease and in lesioned rat brain.

Presenilin-1 (PS-1) gene mutations are responsible for the majority of the early onset familial forms of Alzheimer disease (AD). Neither PS-1's anatomic distribution in brain nor expression in AD have been reported. Using in situ hybridization in the rat forebrain, we show that PS-1 mRNA expression is primarily in cortical and hippocampal neurons, with less expression in subcortical structures, in a regional pattern similar to APP695. Excitotoxic lesions lead to loss of PS-1 signal. A neuronal pattern of expression of PS-1 mRNA was also observed in the human hippocampal formation. AD and control levels did not differ. PS-1 is expressed in brain areas vulnerable to AD changes more so than in areas spared in AD; however, PS-1 expression is not sufficient to mark vulnerable regions. Collectively, these data suggest that the neuropathogenic process consequent to PS-1 mutations begins in neuronal cell populations.

Aged↗
PubMed1996

Transient protection of human T-cells from human immunodeficiency virus type 1 infection by transduction with adeno-associated viral vectors which express RNA decoys.

RNA decoys are oligonucleotides corresponding to the TAR and RRE sequences of HIV which inhibit the HIV-encoded regulatory proteins Tat and Rev, respectively. Adeno-associated viral vectors encoding RNA decoys stably transduced into the human T-cell line CEM-SS expressed transactivating region (TAR) and Rev-responsive element (RRE) RNA decoys from tRNA polIII promoters at high levels, without any apparent deleterious effects on cell growth or expression of CD4. DNA blot analysis indicated that RNA decoy-encoding vectors were not rearranged and were integrated into the genomic DNA of selected cell lines. Vector DNA with the appropriate TAR and RRE sequences was isolated from transduced cell lines after prolonged growth in culture, further confirming that the vector DNA was present in a stable form through multiple cell cycles. Cell lines expressing TAR and RRE decoys transiently inhibited HIV gene expression and replication by 70-99% as determined by measurement of intracellular and extracellular HIV p24 production. Adeno-associated vectors encoding RNA decoys may be useful for gene therapy of HIV infection.

Cell Line↗
PubMed1995

Voltage-dependent calcium channel beta-subunits in combination with alpha 1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by G alpha o in rat frontal cortex.

The dihydropyridine-sensitive calcium channel agonist (-)-BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by Go in rat frontal cortex membranes. An anti-calcium channel beta-subunit antiserum abolished the (-)-BayK 8644-stimulated hydrolysis of GTP by Go and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the beta-subunit binding domain of the calcium channel complex, also attenuated (-)-BayK 8644 activation of GTPase. This study suggests that the calcium channel beta-subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by Go. This may be a mechanism by which calcium channels can normally act to limit the duration of a G-protein modulatory signal.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Functional reconstitution of the NADPH-oxidase by adeno-associated virus gene transfer.

Chronic granulomatous disease (CGD) comprises a heterogeneous group of inherited conditions characterized biochemically by disordered function of a unique multicomponent enzyme system present in phagocytic cells, the NADPH-oxidase. Clinically, it is characterized by recurrent bacterial and fungal infections that are relatively resistant to treatment by conventional means. Curative bone marrow transplantation has been successfully achieved in a small number of cases, but the wider application of this procedure is limited by availability of suitable donor material. Somatic gene therapy would overcome this problem, and several groups have now shown correction of the biochemical defect in hematopoietic cells by retrovirus-mediated gene transfer. However, the failure of the current generation of retroviral vectors to efficiently transduce quiescent cells greatly restricts their potential for gene transfer to pluripotent hematopoietic stem cells. Given these limitations, we have constructed vectors based on adeno-associated virus and used these to transfer a functional copy of the p47phox gene to immortalized B cells derived from patients with p47phox-deficient autosomal recessive CGD. We show stable expression of protein and restoration of NADPH-oxidase function in these cells in the absence of selection. Adeno-associated virus vectors may overcome some of the limitations of retroviral gene delivery systems and may therefore be a useful vehicle for curative gene therapy of CGD and other primary immunodeficiencies.

B-Lymphocytes↗