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Biomedical subjects

G Palmer

Publications and source records attributed to G Palmer.

At least 37 records · Page 2Linked to original sources

Evaluation of the performance of diagnosis-related groups and similar casemix systems: methodological issues.

With the increasing recognition and application of casemix for managing and financing healthcare resources, the evaluation of alternative versions of systems such as diagnosis-related groups (DRGs) has been afforded high priority by governments and researchers in many countries. Outside the United States, an important issue has been the perceived need to produce local versions, and to establish whether or not these perform more effectively than the US-based classifications. A discussion of casemix evaluation criteria highlights the large number of measures that may be used, the rationale and assumptions underlying each measure, and the problems in interpreting the results. A review of recent evaluation studies from a number of countries indicates that considerable emphasis has been placed on the predictive validity criterion, as measured by the R2 statistic. However, the interpretation of the findings has been affected greatly by the methods used, especially the treatment and definition of outlier cases. Furthermore, the extent to which other evaluation criteria have been addressed has varied widely. In the absence of minimum evaluation standards, it is not possible to draw clear-cut conclusions about the superiority of one version of a casemix system over another, the need for a local adaptation, or the further development of an existing version. Without the evidence provided by properly designed studies, policy-makers and managers may place undue reliance on subjective judgments and the views of the most influential, but not necessarily best informed, healthcare interest groups.

Acute Disease↗

Study of the regulation by nutrients of the expression of genes involved in lipogenesis and obesity in humans and animals.

Dietary digestible carbohydrates are able to modulate lipogenesis, by modifying the expression of genes coding for key lipogenic enzymes, like fatty acid synthase. The overall objective of the Nutrigene project (FAIR-CT97-3011) was to study the efficiency of various carbohydrates to modulate the lipogenic capacity and relevant gene expression in rat and human species (control and obese subjects) and to understand the underlying molecular mechanisms involved in the regulation of lipogenic genes by carbohydrates. Key cellular mediators (namely SREBP-1c and 2, AMP activated protein kinase, cholesterol content) of the regulation of lipogenic gene expression by glucose and/or insulin were identified and constitute new putative targets in the development of plurimetabolic syndrome associated with obesity. In humans, hepatic lipogenesis and triglyceride synthesis, assessed in vivo by the use of stable isotopes, was promoted by a high-carbohydrate diet in non obese subjects, and in non alcoholic steatotic patients, but was not modified in the adipose tissue of obese subjects. Non digestible/fermentable carbohydrates, such as fructans, were shown to decrease hepatic lipogenesis in non obese rats, and to lessen hepatic steatosis and body weight in obese Zucker rats. If confirmed in obese humans, this would allow the development of functional food able to counteract the metabolic disturbances linked to obesity.

Adipose Tissue↗

Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1.

The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.

Animals↗

Structure of the murine Pit1 phosphate transporter/retrovirus receptor gene and functional characterization of its promoter region.

The Pit1 phosphate transporter (formerly also called Glvr-1) probably plays an important role in regulated phosphate handling in bone-forming cells. In this study, we describe the structure of the mouse Pit1 gene, as well as some functional characteristics of its promoter region in murine bone cells. Screening of a genomic library led to the isolation of two overlapping lambda clones containing 7kb of 5' flanking region, as well as the 10 exons of the mouse Pit1 gene corresponding to the published cDNA. The translation start site is located within exon I and the stop codon within exon X. The overall structure of the mouse gene is very similar to that of its human homolog, except for the presence of an additional 5' untranslated exon in human. The structure of the 5' untranslated region of the mouse gene was thus further investigated using rapid amplification of cDNA ends in murine ATDC5, MC3T3-E1 and Swiss 3T3 cells. The results indicate that, compared to the published cDNA, the mouse Pit1 gene contains in fact one additional 5' exon, which we named exon IA. Reporter gene assays demonstrate the presence of a functional TATA box containing promoter upstream of exon IA. This description of the murine Pit1 gene and of its promoter region paves the way to more detailed analyses concerning the regulation of Pit1 transcription in mouse cells. Furthermore, a comparison of mouse and human promoters will hopefully allow a better understanding of general mechanisms regulating Pit1 expression in different species.

3T3 Cells↗

Mitogen-activated protein kinase signaling in bovine articular chondrocytes in response to fluid flow does not require calcium mobilization.

In the present study, the role of mitogen-activated protein kinases (MAPKs) in chondrocyte mechanotransduction was investigated. We hypothesized that MAPKs participate in fluid flow-induced chondrocyte mechanotransduction. To test our hypothesis, we studied cultured chondrocytes subjected to a well-defined mechanical stimulus generated with a laminar flow chamber. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) were activated 1.6-3-fold after 5-15 min of fluid flow exposure corresponding to a chamber wall shear stress of 1.6 Pa. Activation of ERK1/2 was observed in the presence of both 10% FBS and 0.1% BSA, suggesting that the flow effects do not require serum agonists. Treatment with thapsigargin or EGTA had no significant effect on the ERK1/2 activation response to flow, suggesting that Ca2+ mobilization is not required for this response. To assess downstream effects of the activated MAPKs on transcription, flow studies were performed using chondrocytes transfected with a chimeric luciferase construct containing 2.4 kb of the promoter region along with exon 1 of the human aggrecan gene. Two-hour exposure of transfected chondrocytes to fluid flow significantly decreased aggrecan promoter activity by 40%. This response was blocked by treatment of chondrocytes with the MEK-1 inhibitor PD98059. These findings demonstrate that, under the conditions of the present study, fluid flow-induced signals activate the MEK-1/ERK signaling pathway in articular chondrocytes, leading to down-regulation of expression of the aggrecan gene.

Aggrecans↗

Separation and identification of the regioisomers of verdoheme by reversed-phase ion-pair high-performance liquid chromatography, and characterization of their complexes with heme oxygenase.

We report an HPLC method for separating the four regioisomers of verdoheme formed in the coupled oxidation of hemin with oxygen and ascorbate in aqueous pyridine. The reversed-phase ion-pair system uses hexafluoroacetone and pyridine as ion-pair agents. The regiochemistry of the separated isomers was established both by HPLC of the corresponding biliverdin IX derivatives and by 1H NMR of each isomer. Optical spectra of the pyridine verdohemochrome isomers were similar to each other, but showed differences in the absorption maxima in the red region, which appear at 680, 663, 648 and 660 nm for the alpha, beta, gamma, and delta-isomers, respectively. Each of the four isomers was incorporated anaerobically into heme oxygenase-1, yielding the corresponding verdoheme-enzyme complex. The ferrous forms had absorption maxima at 690, 667, 655, and 663 nm, and their CO-bound forms had maxima at 638, 624, 616, and 626 nm for alpha, beta, gamma, and delta-isomer, respectively. Addition of ferricyanide to the alpha-verdoheme-heme oxygenase complex brought about a ferric low-spin heme-like signal, which is identical with the ferric alpha-verdoheme complexed with the heme oxygenase that was observed in the heme oxygenase reaction.

Ascorbic Acid↗

A novel in vitro culture system for analysis of functional role of phosphate transport in endochondral ossification.

In vivo expression of the type III sodium-dependent phosphate transporter (NaPiT) Glvr-1 during endochondral ossification, suggests a functional role for inorganic phosphate (Pi) transport in cartilage calcification. For further analysis of this relationship, an in vitro model of endochondral ossification is required. In this context, we investigated the characteristics of Pi transport in the new chondrogenic cell line ATDC5 in relation to extracellular matrix (ECM) formation and mineralization. Pi uptake in ATDC-5 cells and in isolated matrix vesicles (MVs) is mediated by an Na-dependent Pi transporter with a pH dependency characteristic of a type III Pi carrier (lower activity at alkaline pH). Northern blot analysis indicated that ATDC-5 cells express Glvr-1 transcripts during the various stages of their maturation with a maximal level during the proliferating stage. In isolated MVs, Pi transport activity was maximal at day 21, concomitant with the beginning of type X collagen messenger RNA expression. These events preceded the initiation of matrix mineralization, which was apparent at day 25, and then gradually increased until day 47. This temporal relationship between maximal Pi transport activity in MVs and the expression of a marker of mineralizing chondrocytes is compatible with the possible involvement of Pi transport in the ECM calcification observed in ATDC-5 cell cultures. In conclusion, these observations suggest that ATCD-5 cells in culture represent a promising model for the analysis of a functional role of Pi transport in the initial events of endochondral ossification.

Biological Transport↗

Effects of differing heat and humidity on the performance and recovery from multiple high intensity, intermittent exercise bouts.

The aim of this study was to determine the effects of different conditions of heat and humidity on two multiple bouts of high intensity cycling with 60 min recovery between each bout. Eight males (age: 25.5+/-1.8 yr, height: 179.0+/-3.7 cm; weight: 72.3+/-4.0 kg; VO2peak: 51.5+/-2.4 ml x kg(-1) x min(-1), Peak Aerobic Power: 366+/-13 W) volunteered for this study. After undertaking VO2peak testing, all participated randomly, in three consecutive 30 s Wingate tests in three different environmental conditions being: Normal (22 degrees C/30% RH), Wet (30 degrees C/85% RH), and Hot (40 degrees C/40% RH). Subjects were then monitored for the 60 min post-exercise period after which time they repeated the Wingate tests and were again monitored for 60 min. Blood samples were taken pre, immediately post exercise, and at 1, 5, 10, 15, 30, 45, and 60 min into each of the recovery periods and analysed for lactate, pH, and hematocrit. Heart rate was monitored continuously throughout exercise (5 s average) and recovery (60 s average). Weight was measured pre exercise and at 15, 30, 45, and 60 min post-exercise. Urine samples were collected at the same time and analysed for osmolality. The results of the experiment indicated that environmental conditions had no effect on the performance of either series 1 or 2 Wingate tests. Neither were there any changes in weight throughout the three conditions or across the condition. Post exercise pH levels were lower than pre exercise values (p < 0.0001) and the reverse was true for blood lactate levels (p < 0.0003). We conclude that anaerobic exercise is not unduly affected by hot or humid conditions when subjects can re-hydrate according to decreases in body weight.

Adult↗

The performance of Australian DRGs.

This paper reports the results of an evaluation study of the Australian National Diagnosis Related Groups (AN-DRGs). The evaluation was based on statistical rather than clinical criteria with the principal goal being to provide information for the future development of the classification system. As well as comparing versions 1.0 to 3.0 of AN-DRGs, the project included a comparison of these systems with the most recent versions of the DRG systems from the United States. Taking all the evaluation criteria together, Version 3.0 of AN-DRGs performed best of all the systems except for the All Patient Refined (APR)-DRGs with its much larger number of groups. However, the differences between all the classifications were slight. Data of higher quality are needed if further refinements of the AN-DRGs are to produce substantial improvements in performance.

Australia↗

Transforming growth factor-beta stimulates inorganic phosphate transport and expression of the type III phosphate transporter Glvr-1 in chondrogenic ATDC5 cells.

Members of the transforming growth factor (TGF)-beta family are important regulators of skeletal development. In this study, we investigated the effect of TGF-beta1 on inorganic phosphate (Pi) transport and on expression of the type III Pi carriers Glvr-1 and Ram-1 in murine ATDC5 chondrocytes. TGF-beta1 induced a selective, dose- and time-dependent increase in sodium-dependent Pi transport in ATDC5 cells. This response was dependent on RNA and protein synthesis and reflected a change in the maximal rate of the transport system, suggesting that TGF-beta1 induces the synthesis of new Pi carriers and their insertion into the plasma membrane. Consistently, Northern blotting analysis showed a dose-dependent increase in Glvr-1 messenger RNA expression in response to TGF-beta1, which preceded the maximal stimulation of Pi transport by several hours. Glvr-1 thus likely mediates at least part of the increase in Pi uptake induced by TGF-beta1. Ram-1 messenger RNA expression was not affected by TGF-beta1. TGF-beta1 activated the Smad signaling pathway and the mitogen-activated protein kinases ERK and p38 in ATDC5 cells. Unlike the regulation of Pi transport by receptor tyrosine kinase agonists in osteoblasts, the effect of TGF-beta1 on Pi uptake in ATDC5 cells did not involve protein kinase C or mitogen-activated protein kinases, suggesting that a specific, possibly Smad-dependent, signal mediates this response. In conclusion, TGF-beta1 stimulates Pi transport and Glvr-1 expression in chondrocytes, suggesting that, like proliferation, differentiation, and matrix synthesis, Pi handling is subject to regulation by TGF-beta3 family members in bone-forming cells.

Animals↗

Positive and negative control of the expression of parathyroid hormone (PTH)/PTH-related protein receptor via proximal promoter P3 in human osteoblast-like cells.

Three promoters, P1, P2, and P3, regulate the expression of the receptor for the human PTH/PTH -related protein. The P3 promoter, proximal to the gene, seems to be turned on in many tissues and to be the most active of the three in the human adult kidney. P3 is also active in human osteoblastic SaOS-2 cells. Its structure to function relationship is, however, still poorly understood. To address this issue we assayed, in transiently transfected SaOS-2 cells, the expression of reporter gene constructs containing truncated P3 promoter fragments and substitution mutants. We thus localized cis-acting elements essential for P3 promoter activity and identified two key Sp1 binding sites. We also found in the 5'-untranslated exon U4, transcribed from promoter P3, an element that inhibits the expression of the receptor and is not promoter specific. This study provides new insights into PTH receptor expression in human osteoblast-like cells.

5' Untranslated Regions↗

Stimulation of sodium-dependent phosphate transport and signaling mechanisms induced by basic fibroblast growth factor in MC3T3-E1 osteoblast-like cells.

Physiological and pathological observations indicate that basic fibroblast growth factor (bFGF) is an important regulator of osteoblastic cell differentiation and in particular of cranial ossification. Experimental evidence suggests that inorganic phosphate (Pi) transport could be an important function of bone matrix calcification. In the present study, we address the influence of bFGF on Pi transport activity in MC3T3-E1 osteoblast-like cells derived from mouse calvaria. The results indicate that bFGF is a potent and selective stimulator of sodium-dependent Pi transport in these cells. The change in Pi transport activity induced by bFGF depends on transcription and translation and corresponds to a change in the maximum velocity of the Pi transport system (Vmax). These observations suggest that enhanced Pi transport activity in response to bFGF may result from insertion of newly synthesized Pi transporters into the plasma membrane. A selective inhibitor of fibroblast growth factor receptor (FGFR) tyrosine kinase, SU5402, blunted the stimulation of Pi transport induced by bFGF. It also prevented the increase in protein tyrosine phosphorylation induced by bFGF, including phosphorylation of FGFR-1, FGFR-2, phospholipase C-gamma (PLC-gamma), and Shc as well as the recruitment of the Grb2/Sos signaling complex. In addition, bFGF-induced the activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and p38, effects that were prevented by SU5402. Both the protein kinase C (PKC) inhibitor calphostin C and PKC down-regulation suppressed the stimulatory effect of bFGF on Pi transport. Selective inhibitors of ERK and p38 MAP kinases slightly reduced this cellular response with a significant effect observed with the highest concentration of the p38 MAP kinase inhibitor. In conclusion, the results of this study indicate that bFGF selectively stimulates Pi transport in calvaria-derived osteoblastic cells. The main signaling mechanism responsible for this effect involves tyrosine phosphorylation of PLC-gamma and activation of PKC, with a possible contribution of the p38 MAP kinase pathway.

3T3 Cells↗

Evidence for the involvement of two pathways in activation of extracellular signal-regulated kinase (Erk) and cell proliferation by Gi and Gq protein-coupled receptors in osteoblast-like cells.

The mechanisms by which Gi and Gq protein- coupled receptors mediate mitogenic signaling in osteoblast-like cells are unknown and were investigated in MC3T3-E1 cells using specific receptor agonists such as lysophosphatidic acid (LPA) and prostaglandin F2alpha (PGF2alpha). In contrast to their implication in epidermal growth factor (EGF) receptor tyrosine kinase signaling, the adaptor protein Shc, the Grb2/Sos complex, and the small G protein Ras were not involved in the activation of Erk induced by either LPA or PGF2alpha in MC3T3-E1 cells, suggesting that activation of Erk by Gi and Gq protein-coupled receptors is Ras independent in these cells. Using specific kinase inhibitors and kinetic analyses, we provide evidence for two distinct components in the activation of Erk by Gi and Gq protein-coupled receptors in MC3T3-E1 cells including an Src-like kinase-dependent pathway and a protein kinase C (PKC)-dependent mechanism. Functional analyses suggested that these two components are required for optimal DNA synthesis in response to LPA and PGF2alpha. These results suggest the implication of two pathways in the stimulation of Erk and cell replication by growth factors acting through Gi and Gq protein-coupled receptors in bone-forming cells.

Adaptor Proteins, Signal Transducing↗

Mass spectrometric determination of dioxygen bond splitting in the "peroxy" intermediate of cytochrome c oxidase.

The "peroxy" intermediate (P form) of bovine cytochrome c oxidase was prepared by reaction of the two-electron reduced mixed-valence CO complex with (18)O(2) after photolytic removal of CO. The water present in the reaction mixture was recovered and analyzed for (18)O enrichment by mass spectrometry. It was found that approximately one oxygen atom ((18)O) per one equivalent of the P form was present in the bulk water. The data show that the oxygen-oxygen dioxygen bond is already broken in the P intermediate and that one oxygen atom can be readily released or exchanged with the oxygen of the solvent water.

Animals↗

Rapid kinetics of tyrosyl radical formation and heme redox state changes in prostaglandin H synthase-1 and -2.

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34-35 G) was formed by 4 ms, followed by transition to a wide singlet (33-34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25-26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.

Animals↗

Ferric alpha-hydroxyheme bound to heme oxygenase can be converted to verdoheme by dioxygen in the absence of added reducing equivalents.

Whether or not reducing equivalents are indispensable for the conversion of ferric alpha-hydroxyheme bound to heme oxygenase-1 to verdoheme remains controversial (Matera, K. M., Takahashi, S., Fujii, H., Zhou, H., Ishikawa, K., Yoshimura, T., Rousseau, D. L., Yoshida, T., and Ikeda-Saito, M. (1996) J. Biol. Chem. 271, 6618-6624; Liu, Y., Moënne-Loccoz, P., Loehr, T. M., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 6906-6917). To resolve this controversy, we have prepared a ferric alpha-hydroxyheme-heme oxygenase-1 complex and titrated the complex with O2 under strictly anaerobic conditions. The formation of verdoheme was monitored by optical and electron spin resonance spectroscopies. Electron spin resonance spectra of the complex showed that alpha-hydroxyheme exists as a mixture of resonance structures composed of the iron(III) porphyrin and the iron(II) porphyrin pi neutral radical. Upon addition of CO the latter species becomes dominant. The results obtained from these titration experiments indicate that alpha-hydroxyheme can be converted to verdoheme by an approximately equimolar amount of O2 without any requirement for exogenous electrons. The verdoheme formed from alpha-hydroxyheme was shown to be in the ferrous oxidation state by the addition of CO or potassium ferricyanide to the resultant verdoheme-heme oxygenase-1 complex.

Electron Spin Resonance Spectroscopy↗

Redox state of peroxy and ferryl intermediates in cytochrome c oxidase catalysis.

The redox states of the "peroxy" (P) and "ferryl" (F) intermediates formed during reoxidation of reduced bovine cytochrome c oxidase have been probed by reduction with both ferrocytochrome c and acetylpyridine NADH under anaerobic conditions using optical spectroscopy. The reduction of the P and F forms revealed that both are in very similar redox states. One-electron reduction of either the P or F form yields an optical spectrum primarily due to oxidized enzyme implying that the heme iron of cytochrome a3 is in the ferryl state in both forms. The F and P forms were found to be 1 and less than 1.3 oxidizing equiv, respectively, above the oxidized enzyme. The slightly higher oxidation state in the P form is interpreted as being due to an optically undetectable redox center presumably located in the binuclear cavity.

Catalysis↗