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Biomedical subjects

G Palmer

Publications and source records attributed to G Palmer.

At least 55 records · Page 3Linked to original sources
PubMed1999

Characterization of the human Glvr-1 phosphate transporter/retrovirus receptor gene and promoter region.

The cell surface receptor for gibbon ape leukemia virus (Glvr-1) belongs to the type III sodium-dependent phosphate transporter/retrovirus receptor gene family. Several observations have suggested an important role for Glvr-1 in regulated Pi handling in bone forming cells and prompted us to investigate further the molecular mechanisms regulating Glvr-1 gene expression. In addition, the regulation of Glvr-1 gene expression also has potential applications to gene therapy, since retroviral vectors carrying gibbon ape leukemia virus envelope proteins are used for gene delivery into different cell types. The aim of this study was thus to clone the human Glvr-1 gene in order to describe its structure and its promoter region. Our results indicate that the Glvr-1 gene consists of 11 exons and 10 introns spread over 18kb of genomic DNA. The translation initiation site is located within exon II and the translation stop codon within exon XI. Rapid amplification of cDNA ends (5'-RACE) suggests that, in human SaOS-2 osteoblast-like cells, transcription of Glvr-1 is initiated at multiple sites, mostly located between bp 32 and 50 of the published cDNA sequence, which was initially obtained from HL-60 cells. The 5'-flanking region of the gene is characterized by a very high GC content. Reporter gene assays demonstrate the presence of a functional promoter upstream of exon I and indicate that a GC-rich region, containing two potential SP1 binding sites, is required for high promoter activity in transiently transfected SaOS-2 cells. The description of the human Glvr-1 gene structure, as well as the analysis of some structural and functional characteristics of its promoter region, provide a basis for more detailed investigation of the molecular mechanisms controlling expression of the Glvr-1 gene in bone forming cells and in other cell types.

Base Sequence↗
PubMed1999

Preimplantation genetic diagnosis in 10 couples at risk for transmitting beta-thalassaemia major: clinical experience including the initiation of six singleton pregnancies.

Preimplantation genetic diagnosis (PGD) offers couples at risk for transmitting an inherited disorder the possibility to avoid the need to terminate affected pregnancies, since it allows the selection of unaffected IVF embryos for transfer. PGD for monogenic diseases is most commonly accomplished by blastomere biopsy from cleavage stage embryos, followed by polymerase chain reaction (PCR)-based DNA analysis. However, PCR-based DNA analysis of single cells is subject to several problems including sample contamination, total PCR failure, or failure of one allele to amplify--a phenomenon known as allelic drop-out (ADO). Furthermore, the molecular heterogeneity of many monogenic diseases requires a diagnostic strategy capable of detecting a spectrum of mutations and compound genotypes. With the above considerations we developed an accurate and reliable strategy for analysis of beta-globin gene mutations, applicable for PGD for the wide spectrum of beta-thalassaemia major genotypes in the Greek population. The strategy involves nested PCR followed by denaturing gradient gel electrophoresis (DGGE) analysis. DGGE is an advantageous method for mutation detection since it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it identifies the presence of normal alleles and in addition can monitor the occurrence of ADO. This report describes the application of the DGGE-based diagnostic strategy in 11 clinical IVF/PGD cycles, in 10 couples at risk for transmitting beta-thalassaemia major. The transfer of at least one embryo diagnosed as unaffected for beta-thalassaemia major in nine couples has resulted in the initiation of six pregnancies. Four pregnancies have so far been confirmed as unaffected for beta-thalassaemia major by first or second-trimester prenatal diagnosis, two of which have resulted in the birth of two healthy babies. Three singleton pregnancies are still on-going and one ectopic pregnancy was terminated.

Biopsy↗
PubMed1999

Sodium bicarbonate can be used as an ergogenic aid in high-intensity, competitive cycle ergometry of 1 h duration.

The aim of this study was to determine whether a dose of 300-mg x kg(-1) body mass of sodium bicarbonate would effect a high-intensity, 1-h maximal cycle ergometer effort. Ten male, well-trained [maximum oxygen consumption 67.3 (3.3) ml x kg(-1) x min(-1), mean (SD)] volunteer cyclists acted as subjects. Each undertook either a control (C), placebo (P), or experimental (E) ride in a random, double-blind fashion on a modified, air-braked cycle ergometer, attached to a personal computer to which the work and power data was downloaded at 10 Hz. Fingertip blood was sampled at 10-min intervals throughout the exercise. Blood was also sampled at 1, 3, 5, and 10 min post-exercise. Blood was analysed for lactate, partial pressure of Carbon dioxide and oxygen, pH and plasma bicarbonate (HCO-) concentration. Randomly chosen pairs of subjects were asked to complete as much work as possible during the 60-min exercise periods in an openly competitive situation. The sodium bicarbonate had the desired effect of increasing blood HCO3- prior to the start of the test. The subjects in E completed 950.9 (81.1) kJ of work, which was significantly more (F(2,27) = 5.28, P < 0.01) than during either the C [835.5 (100.2) kJ] or P [839.0 (88.6) kJ] trials. No differences were seen in peak power or in the power:mass ratio between these three groups. The results of this study suggest that sodium bicarbonate may be used to offset the fatigue process during high-intensity, aerobic cycling lasting 60 min.

Adult↗
PubMed1999

Effects of chronic bicarbonate ingestion on the performance of high-intensity work.

We have evaluated whether sodium bicarbonate, taken chronically (0.5 g x kg(-1) body mass) for a period of 5 days would improve the performance of eight subjects during 60 s of high-intensity exercise on an electrically braked cycle ergometer. The first test was performed prior to chronic supplementation (pre-ingestion) while the post-ingestion test took place 6 days later. A control test took place approximately 1 month after the cessation of all testing. Acid-base and metabolite data (n = 7) were measured from arterialised blood both pre- and post-exercise, as well as daily throughout the exercise period. The work completed by the subjects in the control and pre-ingestion test [21.1 (0.9) and 21.1 (0.9) MJ, respectively] was less than (P<0.05) that completed in the post-ingestion test [24.1 (0.9) MJ; F(2,21) = 3.4, P<0.05, power = 0.57]. Peak power was higher after the 5-day supplementation period (P<0.05). Ingestion of the sodium bicarbonate for a period of 5 days resulted in an increase in pH (F(5,36) = 12.5, P<0.0001, power = 1.0) over the 5-day period. The blood bicarbonate levels also rose during the trial (P<0.05) from a resting level of 22.8 (0.4) to 28.4 (1.1) mmol x l(-1) after 24 h of ingestion. In conclusion, the addition of sodium bicarbonate to a normal diet proved to be of ergogenic benefit in the performance of short-term, high-intensity work.

Acid-Base Equilibrium↗
PubMed1999

The effect of curing regime on the release of hydroxyethyl methacrylate (HEMA) from resin-modified glass-ionomer cements.

This study investigated the effects of degree of cure and specimen maturation on the release of HEMA from four resin-modified glass-ionomer cements. Disc specimens were light-cured for either the manufacturers' recommended time (MRT) for that material, or were under-cured (irradiated for 0.5 MRT), over-cured (1.5 MRT) or allowed to dark-cure (0 MRT). Specimens were matured in the mould at 37 degrees C until set or for 10 min, 40 min or 24 h prior to immersion in distilled water (at 37 degrees C) for 4 h. The HEMA release was determined by measuring the HEMA concentration in the storage water by HPLC. Vitremer, Fuji II LC and Fuji Lining LC set without light-curing (0 MRT) in less than 6 min and these specimens released low levels of HEMA (approximately 1, 7 and 4% of the included HEMA, respectively). Dark-cured Vitrebond took 15 min to set and the HEMA release was high (approximately 50% of the included HEMA) indicating that the material had only set by the acid-base reaction. HEMA release from light-cured Vitremer or Vitrebond was not affected by under- or over-curing but release from Fuji II LC was reduced by over-curing (1.5 MRT). Under-curing Fuji Lining LC caused a significant increase in HEMA release. Specimen maturation times of 10 min, or longer, had little effect on the measured HEMA release. All the materials released HEMA into their storage solutions. In order to minimise HEMA release, resin-modified glass-ionomers should always be cured for at least the manufacturers' recommended time at thicknesses no greater than the maximum recommended.

Chromatography, High Pressure Liquid↗
PubMed1999

In vivo expression of transcripts encoding the Glvr-1 phosphate transporter/retrovirus receptor during bone development.

In vitro observations suggest that inorganic phosphate (Pi) transport plays an important functional role in osteogenic cells and in their matrix vesicles for the initiation of matrix calcification. Recent studies have shown that the type III sodium-dependent Pi transporters, Glvr-1 and Glvr-2, are expressed in human osteoblast-like cells and have suggested a potential role for type III transporters in regulated Pi handling in osteogenic cells. To address the relevance of these findings in the context of bone formation in vivo and, in particular, in relation to matrix calcification, we investigated expression of the Glvr-1 transporter by in situ hybridization in developing embryonic murine metatarsals, using human Glvr-1 cDNA as a probe. In this model of endochondral ossification, expression of transcripts encoding Glvr-1 could be detected from day 17 of embryonic development. A hybridization signal for Glvr-1 was specifically observed in a subset of hypertrophic chondrocytes and could not be detected in osteoblasts. The expression of Glvr-1 mRNA was compared with that of transcripts encoding extracellular matrix proteins. Glvr-1 mRNA expression was confined to a population of early hypertrophic chondrocytes expressing type X collagen and to slightly more mature cells that express transcripts encoding osteopontin but lack type X collagen mRNA. No Glvr-1 transcripts were detected in fully differentiated hypertrophic chondrocytes. This pattern of Glvr-1 mRNA expression was maintained throughout embryonic development until after birth. In conclusion, the Glvr-1 phosphate transporter is selectively expressed in a subset of hypertrophic chondrocytes during endochondral bone formation, in a region where matrix mineralization proceeds. This observation represents the first in vivo evidence consistent with a potential role for this phosphate transporter in matrix calcification.

Animals↗
PubMed1999

Regulation of alkaline phosphatase activity by p38 MAP kinase in response to activation of Gi protein-coupled receptors by epinephrine in osteoblast-like cells.

The signaling mechanisms responsible for the regulation of alkaline phosphatase (ALP) activity by exogenous factors in osteoblast-like cells remain poorly understood. Among various agents, epinephrine was recently found to increase ALP activity in differentiating MC3T3-E1 cells by stimulating alpha1 adrenergic receptors coupled to Gi proteins. In the present study, we investigated the role of both ERK2 and p38 mitogen-activated protein (MAP) kinases in mediating this response in MC3T3-E1 cells. Our results indicate that both MAP kinases are transiently stimulated by epinephrine in differentiating cells via a pertussis toxin sensitive mechanism. The role of each MAP kinase pathway in mediating the stimulation of ALP activity by epinephrine was investigated using specific inhibitors. The MEK inhibitor PD98059, blocked ERK2 activity induced by epinephrine but had no effect on the stimulation of ALP activity. In contrast, low concentrations of SB203580, a specific inhibitor of the p38 MAP kinase, completely blunted this cellular response. However, this inhibitor had no influence on the stimulation of ALP activity induced by ascorbic acid. In conclusion, the results of this study suggest distinct roles for ERK and p38 MAP kinase pathways in regulating activity of MC3T3-E1 osteoblastic cells. The ERK pathway is likely involved in the control of cell proliferation whereas the p38 MAP kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors.

Alkaline Phosphatase↗
PubMed1999

A widely applicable strategy for single cell genotyping of beta-thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) allows the selection of unaffected IVF embryos for transfer in couples that are at risk for transmitting genetic diseases. For monogenic diseases, polymerase chain reaction (PCR)-based diagnosis is usually performed on single blastomeres. In Greece, up to 10 per cent of the population are carriers for beta-thalassaemia and related haemoglobinopathies, and more than 20 pathological mutations in the beta-globin gene have been described. In this study we report a strategy which includes a first round of PCR, allowing subsequent nested PCR and DGGE analysis for at least 95 per cent of beta-thalassaemia major genotypes in the Greek population. The use of DGGE for beta-globin genotype analysis is advantageous: it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it detects the presence of normal alleles and monitors the occurrence of allelic drop-out (ADO) through the expectation that heterozygous samples have more than one electrophoretic band on DGGE analysis. The optimization, accuracy and reliability of the method was evaluated by genotyping 325 single blastomeres, 110 amniocytes and 55 lymphocytes. Results confirmed that PCR efficiency and occurrence of ADO are improved by higher denaturation temperatures in the first cycles of first-round PCR, influenced by the size of the fragment amplified in the first round of PCR and additionally by the quality and type of cells being genotyped. The proposed strategy was accurate and reliable, and thus for application to PGD should ensure the transfer of unaffected embryos. Furthermore it is widely applicable in most of the populations worldwide where beta-thalassaemia is common.

Blastomeres↗
PubMed1998

An improved sample packing device for rapid freeze-trap electron paramagnetic resonance spectroscopy kinetic measurements.

A new method has been developed for sample packing in rapid freeze-quench electron paramagnetic resonance spectroscopy (EPR) kinetic experiments. Sample particles freeze-quenched in chilled isopentane are filtered under pressure through a stainless steel funnel attached to an EPR tube fitted with a porous disk at its bottom. Isopentane exits through the porous disk and the sample particles can be transferred essentially quantitatively into the receiving EPR tube. This device provides a more predictable, reproducible, and time-saving method for sample packing, enables use of a wider range of flow velocity, and allows efficient use of valuable reactants.

Animals↗
PubMed1998

Diazene--a not so innocent ligand for the binuclear center of cytochrome c oxidase.

Diazene reacts rapidly with cytochrome c oxidase to reduce cytochrome a and CuA and to form a charge-transfer complex with ferric cytochrome a3; the diazene may serve to bridge the heme iron of this cytochrome and CuB. The complex is characterized by an intense, optically active absorbance located at 847 nm. A similar band had been observed previously upon reduction of cytochrome oxidase with hydrazine [Markossian, K. A., Paitian, N. A., and Nalbandyan, R. M. (1983) FEBS Lett. 156, 235-238], but it appears that this band is actually due to the diazene produced as a result of the oxidation of the hydrazine that occurs in this process. A similar diazene to iron charge-transfer band is found following the reaction of diazene with ferric horseradish peroxidase and with hemin chloride but not with met-myoglobin.

Animals↗
PubMed1998

Sequence and activity of parathyroid hormone/parathyroid hormone-related protein receptor promoter region in human osteoblast-like cells.

The parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor gene has been characterized in various species. The structure of its promoter and the regulation of its expression in human tissues have, however, not been clearly established yet. We characterized the region upstream of the PTH/PTHrP receptor gene and investigated its promoter activity in the human osteoblast-like SaOS-2 cells. In this region, three untranslated exons were localized, U1 and U2 by using a kidney cDNA, and U3 by homology with the mouse gene. In human osteoblast-like cells, a distal promoter (P1) was found to be inactive, as evaluated by luciferase reporter gene assays, in contrast to the situation found in human and mouse kidney tissue. A second promoter (P2), previously described in mouse and human kidney, was shown to be active in human osteoblast-like SaOS-2 cells. We found a hitherto uncharacterized promoter (P3), closely upstream of the ATG start codon. The activities of P2 and P3 were not additive. These results provide important information on the structure of the 5' flanking region of the human PTH/PTHrP gene receptor.

Animals↗
PubMed1998

Effects of various imidazole ligands on heme conformation in endothelial nitric oxide synthase.

We have evaluated the influence of a series of substituted imidazoles on the heme structure of endothelial nitric oxide synthase (eNOS). Optical, MCD, and EPR spectra reveal widely differing effects on heme spin state and geometry. 1-Substituted imidazoles always yield low-spin heme complexes, but the size of the 2- and 4-substituent influences their structural effects on the heme. Methyl substituents lead to low-spin complexes while the bulky phenyl group yields high-spin complexes. The only exception to this behavior is provided by 2-aminoimidazole. Although this compound has three functional groups which can serve as an axial ligand to the heme, its binding to eNOS leads to a pure high-spin complex. This result can only be interpreted as due to a direct binding of 2-aminoimidazole to the guanidine binding subdomain of L-arginine. MCD spectra also imply that an O-ligand is present in the low-spin resting eNOS, while EPR data reveal the presence of two low-spin heme complexes in resting eNOS and its imidazole complexes. EPR also distinguishes four different high-spin forms of eNOS generated by different imidazole analogues. This series of ligands promises to be useful in probing the subtle structural difference among the active sites of three NOS isozymes and in developing selective inhibitors to these important enzymes.

Circular Dichroism↗
PubMed1998

Structural characterization of arachidonyl radicals formed by prostaglandin H synthase-2 and prostaglandin H synthase-1 reconstituted with mangano protoporphyrin IX.

A tyrosyl radical generated in the peroxidase cycle of prostaglandin H synthase-1 (PGHS-1) can serve as the initial oxidant for arachidonic acid (AA) in the cyclooxygenase reaction. Peroxides also induce radical formation in prostaglandin H synthase-2 (PGHS-2) and in PGHS-1 reconstituted with mangano protoporphyrin IX (MnPGHS-1), but the EPR spectra of these radicals are distinct from the initial tyrosyl radical in PGHS-1. We have examined the ability of the radicals in PGHS-2 and MnPGHS-1 to oxidize AA, using single-turnover EPR studies. One wide singlet tyrosyl radical with an overall EPR line width of 29-31 gauss (G) was generated by reaction of PGHS-2 with ethyl hydroperoxide. Anaerobic addition of AA to PGHS-2 immediately after formation of this radical led to its disappearance and emergence of an AA radical (AA.) with a 7-line EPR, substantiated by experiments using octadeuterated AA. Subsequent addition of oxygen resulted in regeneration of the tyrosyl radical. In contrast, the peroxide-generated radical (a 21G narrow singlet) in a Y371F PGHS-2 mutant lacking cyclooxygenase activity failed to react with AA. The peroxide-generated radical in MnPGHS-1 exhibited a line width of 36-38G, but was also able to convert AA to an AA. with an EPR spectrum similar to that found with PGHS-2. These results indicate that the peroxide-generated radicals in PGHS-2 and MnPGHS-1 can each serve as immediate oxidants of AA to form the same carbon-centered fatty acid radical that subsequently reacts with oxygen to form a hydroperoxide. The EPR data for the AA-derived radical formed by PGHS-2 and MnPGHS-1 could be accounted for by a planar pentadienyl radical with two strongly interacting beta-protons at C10 of AA. These results support a functional role for peroxide-generated radicals in cyclooxygenase catalysis by both PGHS isoforms and provide important structural characterization of the carbon-centered AA..

Arachidonic Acid↗
PubMed1998

Hydrogen peroxide is not released following reaction of cyanide with several catalytically important derivatives of cytochrome c oxidase.

We have looked for the production of hydrogen peroxide following reaction of oxidized cytochrome c oxidase and two oxy derivatives (compounds P and F) with cyanide. In each case the final product was the cyanide adduct of cytochrome c oxidase. In no case release of hydrogen peroxide was detected, as gauged by the scopoletin plus horse radish peroxidase assay. The simplest conclusion is that none of these forms of the enzyme contains intact hydrogen peroxide.

Cyanides↗
PubMed1998

Catecholamines stimulate the proliferation and alkaline phosphatase activity of MC3T3-E1 osteoblast-like cells.

A number of factors have been shown to influence osteoblastic proliferation, including fluoride. Recent observations suggest that heterotrimeric G proteins are probably involved in the mitogenic response induced by this agent, further suggesting a role of guanosine 5'-triphosphate (GTP)-binding protein-coupled receptors (GPCR) in the regulation of osteoblastic cell growth. We therefore explored what mitogenic factors known to activate GPCR can influence the replication of mouse osteoblast-like MC3T3-E1 cells. Among several candidates, epinephrine was found to be a potent mitogen for these cells, and its effect on the growth and differentiation of these cells was further investigated. Deoxyribonucleic acid (DNA) synthesis was dose dependently enhanced by this catecholamine in the concentration range of 1 nmol/L-10 micromol/L. Stimulation of DNA synthesis by catecholamines was in the order of epinephrine > norepinephrine >> isoproterenol, indicating that alpha adrenergic receptors mediated this cellular response. Further analysis with specific adrenergic receptor agonists and antagonists suggested that the mitogenic response induced by epinephrine in MC3T3-E1 cells is mediated by alpha1 adrenergic receptors. In addition to its effect on cell replication, epinephrine also enhanced alkaline phosphatase (ALP) activity in these cells but had little effect on collagen synthesis and osteocalcin production. As for the mitogenic response, the change in ALP activity was found to be mediated by alpha1 adrenergic receptors. Both effects of epinephrine on cell replication and ALP activity were markedly reduced by pretreatment of the cells with pertussis toxin (PTX), suggesting a role of Gi proteins. These effects were also completely blocked by pretreatment of the cells with 50 micromol/L genistein, a nonselective inhibitor of tyrosine kinase. In conclusion, the results indicate that epinephrine enhances replication and ALP activity of MC3T3-E1 osteoblast-like cells via alpha1 adrenergic receptors coupled to Gi proteins. The signaling mechanism probably involves a tyrosine phosphorylation mechanism. These observations suggest that PTX-sensitive G proteins are potent mediators of cell proliferation and ALP activity of osteoblast-like cells in response to factors acting through G protein-coupled receptors.

3T3 Cells↗
PubMed1998

Regulation of Drosophila Ca2+/calmodulin-dependent protein kinase II by autophosphorylation analyzed by site-directed mutagenesis.

In this study we demonstrate that Drosophila calcium/calmodulin-dependent protein kinase II (CaMKII) is capable of complex regulation by autophosphorylation of the three threonines within its regulatory domain. Specifically, we show that autophosphorylation of threonine-287 in Drosophila CaMKII is equivalent to phosphorylation of threonine-286 in rat alpha CaMKII both in its ability to confer calcium independence on the enzyme and in the mechanistic details of how it becomes phosphorylated. Autophosphorylation of this residue occurs only within the holoenzyme structure and requires calmodulin (CaM) to be bound to the substrate subunit. Phosphorylation of threonine-306 and threonine-307 in the CaM binding domain of the Drosophila kinase occurs only in the absence of CaM, and this phosphorylation is capable of inhibiting further CaM binding. Additionally, our findings suggest that phosphorylation of threonine-306 and threonine-307 does not mimic bound CaM to alleviate the requirement for CaM binding to the substrate subunit for intermolecular threonine-287 phosphorylation. These results demonstrate that the mechanism of regulatory autophosphorylation of this kinase predates the split between invertebrates and vertebrates.

Amino Acid Sequence↗