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Biomedical subjects

A Graham

Publications and source records attributed to A Graham.

At least 163 records · Page 9Linked to original sources
PubMed1994

Isolation and pharmacological characterization of a hamster urinary bladder neurokinin A receptor cDNA.

Functional cDNA clones for hamster neurokinin-2 receptor (NK-2R) were isolated from hamster urinary bladder using a polymerase chain reaction-based methodology. The hamster NK-2R consists of 384 amino acids with a relative molecular weight of 43,418. Hamster NK-2R shares significant amino acid sequence homology with other tachykinin receptors, particularly with rat, bovine, and human NK-2R (94.3, 84.4, and 86.5%, respectively). To examine the pharmacology of cloned hamster NK-2R, we transfected mouse erythroleukemia cells with this receptor, prepared high speed membranes, and studied the receptor properties utilizing the ligand [4,5-3H-Leu9]NKA in a receptor-binding assay. For pharmacological comparison, we also transfected the human NK-2R into mouse erythroleukemia cells. [3H]NKA bound to hamster NK-2R receptor in a protein-dependent, high affinity (Kd1 = 4.14 +/- 0.31 nM), saturable (Bmax1 = 679 +/- 26 fmol/mg of protein), and highly specific manner (89 +/- 2%). A smaller population (10% density) of lower affinity receptors (Kd2 = 150 +/- 92 nM), was also observed in competition experiments. [3H]NKA bound to the human receptor with significantly higher affinity and overall greater receptor density (Kd1 = 0.37 +/- 0.11 nM, Bmax1 = 234 +/- 175 fmol/mg of protein; Kd2 = 9.0 +/- 2 nM, Bmax2 = 1989 + 990 fmol/mg of protein). [3H]NKA binding to both hamster and human receptors was enhanced greatly by divalent cations, whereas GTP analogs weakly inhibited binding to hamster receptor, but potently inhibited binding to the human receptor. Competition experiments with agonists demonstrated binding to high and low affinity states of NK-2 receptors, with identical order of potency in hamster or human NK-2R; NKA > [Nle10]NKA(4-10) > [beta-Ala8]NKA(4-10) >> substance P >>> Senktide. However, remarkable differences were observed in studies with selective NK-2 antagonists (hamster, SR48,968 > L659,877 > R396 >> MEN10,376 versus human, SR48,968 > MEN10,376 > L659,877 > R396). The rank order of antagonist affinity is consistent with the observations of NK-2 receptor pharmacology in the native tissues.

Amino Acid Sequence↗
PubMed1993

Inhibition of glutathione synthesis increases the toxicity of oxidized low-density lipoprotein to human monocytes and macrophages.

Macrophages are thought to play an important role in the pathogenesis of atherosclerosis by mediating the oxidation of low-density lipoprotein (LDL). However, it is known that these cells show elevated glutathione levels after exposure to oxidized LDL. Here we demonstrate that this increase in the level of intracellular glutathione is due to synthesis de novo stimulated by oxidized LDL. Furthermore, inhibition of glutathione synthesis renders oxidized LDL cytotoxic to both monocytes and macrophages at a concentration well tolerated by untreated cells. The stimulation of cholesterol esterification in macrophages by low, non-toxic, concentrations of oxidized LDL is enhanced under conditions where glutathione synthesis is inhibited. These results suggest that the glutathione status of macrophages in the artery wall could be important in both controlling foam-cell formation and the detoxification of oxidized LDL.

Antimetabolites↗
PubMed1993

Peroxynitrite modification of low-density lipoprotein leads to recognition by the macrophage scavenger receptor.

Peroxynitrite is an oxidant which could be formed in the vasculature by the reaction of superoxide with nitric oxide. It is capable of modifying amino acid residues and of initiating lipid peroxidation. In the present study we have shown that peroxynitrite converts low density lipoprotein to a form recognized by the macrophage scavenger receptor and that this process is associated with modification of the protein and lipid, and with the oxidation of alpha-tocopherol to alpha-tocopherol quinone.

Cell Line↗
PubMed1993

Human bradykinin B2 receptor: nucleotide sequence analysis and assignment to chromosome 14.

Functional cDNA clones for human bradykinin B2 receptor were isolated from uterus RNA by a polymerase chain reaction (PCR)-based method and by screening a human cosmid library with rat bradykinin B2 receptor probe. We isolated several overlapping clones from the cosmid library, each of which encodes the entire protein coding sequence. The human bradykinin B2 receptor gene codes for a 364-amino-acid protein with a molecular mass of 41,442 Da that is highly homologous to rat bradykinin B2 receptor cDNA (81%). The entire human cDNA sequence was cloned into an expression vector and mRNA was synthesised by in vitro transcription. Applications of bradykinin caused membrane current responses in Xenopus oocytes injected with the in vitro-synthesized mRNA. Preincubation with the potent B2 antagonist, HOE140, prevented this response. The genomic clone is intronless, and we have identified an upstream promoter region and a downstream polyadenylation signal. The human bradykinin B2 receptor gene has been mapped to chromosome 14 using PCR to specifically amplify DNA from somatic cell hybrids.

Amino Acid Sequence↗
PubMed1993

Tin protoporphyrin prolongs the biochemical remission produced by heme arginate in acute hepatic porphyria.

BACKGROUND: In acute porphyria, repletion of intrahepatic heme, with exogenously administered heme, suppresses the overproduction of delta-aminolaevulinic acid (ALA) and porphobilinogen (PBG). The effect of reducing heme breakdown has been assessed by administering tin protoporphyrin, a competitive inhibitor of heme oxygenase. METHODS: The effect of tin protoporphyrin, 1 mumol/kg, and heme arginate, 3 mg/kg, individually and combined was compared with placebo in patients with an acute porphyric crisis. The treatments were given by intravenous infusion on three successive mornings. Thirty-four attacks were studied in 8 patients (9 placebo, 10 heme arginate alone, 4 tin protoporphyrin alone, and 11 combination treatments). RESULTS: Placebo and tin protoporphyrin alone had little effect on ALA and PBG excretion. Following heme arginate alone or combined with tin protoporphyrin, there was a marked and similar suppression of both ALA and PBG excretion (P < 0.005 for each, compared with pretreatment values). However, on the 5th day after discontinuing treatment, the excretion of ALA and PBG were both lower following combination therapy than following heme arginate alone (P < 0.005 and P < 0.01, respectively). CONCLUSIONS: These findings suggest that inhibition of heme oxygenase by tin protoporphyrin prolongs the biochemical remission induced by heme arginate in the porphyric crisis.

Acute Disease↗
PubMed1993

Opioid regulation of parental behavior in juvenile rats.

When exposed to young rats for a period of days, juvenile rats will respond with full parental behavior (FPB: retrieval and grouping of, and crouching over, pups). Because the parental behavior of juveniles is so robust, and because opiates have been shown to be involved in the regulation of parental behavior in adult animals, we examined morphine's ability to disrupt the display of parental behavior in the juvenile animal. In Experiment 1, 25-day-old males and females were administered one of three injection regimens of morphine (MOR), saline (SAL), or naloxone (NAL)--[MOR (5.0 mg/kg) + SAL; MOR + NAL (0.5 mg/kg); or SAL + SAL]--and 1 h later were exposed to three 1- to 6-day-old neonates. Behavior was scored over a 60-min period and animals were considered parental if they responded with FPB for 2 consecutive testing days. Whereas the SAL + SAL and MOR + NAL were not different, MOR + SAL virtually failed to respond to young over a 10-day period. Experiment 2 exposed juveniles to young for a period of days until they displayed 2 consecutive days of FPB. Next, separate groups of juveniles were treated with the same regimen as above. Again, SAL + SAL and MOR + NAL responded rapidly to young, whereas MOR + SAL did not. These data suggest that the display of parental behavior, both its onset (Experiment 1) and maintenance (Experiment 2), appears to be regulated by opiates.

Animals↗
PubMed1993

'Correctable undetected visual acuity deficit' in patients aged 65 and over attending an accident and emergency department.

The rate of occurrence of correctable undetected visual acuity deficit (CUVAD) in a population of patients aged 65 and over was investigated, using a pinhole screening method, to compare the sociodemographic and optical eye care habits of patients with or without a functionally significant CUVAD. Of 136 patients 46 (34%) were found to have a functionally significant CUVAD in one or both eyes which was not significantly associated with optician or general practitioner contact, age, sex, social class, living situation, or number of medications. Half the patients with significant CUVAD had not attended for 2 years mainly because of financial considerations. Three quarters attended of their own volition; only one in seven were prompted by opticians' postal invitations. It was concluded that a significant degree of CUVAD could be detected using a simple procedure which can be carried out by general practitioners as part of their general elderly health screen.

Aged↗
PubMed1993

Even-numbered rhombomeres control the apoptotic elimination of neural crest cells from odd-numbered rhombomeres in the chick hindbrain.

Neural crest cells originate at three discontinuous levels along the rostrocaudal axis of the chick rhombencephalon, centred on rhombomeres 1 and 2, 4 and 6, respectively. These are separated by the odd-numbered rhombomeres r3 and r5 which are depleted of migratory neural crest cells. Here we show elevated levels of apoptosis in the dorsal midline of r3 and r5, immediately following the formation of these rhombomeres at the developmental stage (10-12) when neural crest cells would be expected to emerge at these neuraxial levels. These regions are also marked by their expression of members of the msx family of homeobox genes with msx-2 expression preceding apoptosis in a precisely colocalised pattern. In vitro and in ovo experiments have revealed that r3 and r5 are depleted of neural crest cells by an interaction within the neural epithelium: if isolated or distanced from their normal juxtaposition with even-numbered rhombomeres, both r3 and r5 produce migrating neural crest cells. When r3 or r5 are unconstrained in this way, allowing production of crest, msx-2 expression is concomitantly down regulated. This suggests a correlation between msx-2 and the programming of apoptosis in this system. The hindbrain neural crest is thus produced in discrete streams by mechanisms intrinsic to the neural epithelium. The crest cells that enter the underlying branchial region are organised into streams before they encounter the mesodermal environment lateral to the neural tube. This contrasts sharply with the situation in the trunk where neural crest production is uninterrupted along the neuraxis and the segmental accumulation of neurogenic crest cells is subsequently founded on an alternation of permissive and non-permissive qualities of the local mesodermal environment.

Animals↗
PubMed1993

No added benefit from nebulized amiloride in patients with cystic fibrosis.

In cystic fibrosis (CF) airway epithelial sodium absorption is increased 2-3 fold. Since sodium absorption is inhibited by the sodium channel blocker amiloride, our aim was to assess its therapeutic benefit in cystic fibrosis. A randomized, double-blind, placebo-controlled, cross-over trial of nebulized amiloride was performed in 23 patients with cystic fibrosis. Amiloride or placebo was administered four times daily for two six month periods. Existing treatment was continued, and any infective exacerbations treated in the usual way. Fourteen patients completed the study. No significant changes occurred in forced expiratory volume in one second, forced vital capacity, oxygen saturation, body weight, sputum volume, culture and rheology, serum urea, and electrolytes, white cell count and erythrocyte sedimentation rate during either treatment period. The frequency of infective exacerbations was also not different in either treatment period. We were thus unable to confirm the benefit shown in the only other clinical trial of nebulized amiloride in cystic fibrosis and conclude that, in the presence of established treatment for cystic fibrosis lung disease, nebulized amiloride offers no additional clinical benefit.

Administration, Intranasal↗
PubMed1992

Analysis of the murine Hox-2.7 gene: conserved alternative transcripts with differential distributions in the nervous system and the potential for shared regulatory regions.

In this study we have investigated the organization and regulation of the mouse Hox-2.7 gene. There are several alternative transcripts some of which are conserved between mouse and humans. By Northern and in situ analysis we are able to identify at least three types of transcripts which are different in size and splicing pattern and have distinctly different boundaries of expression in the nervous system. One subset of the endogenous transcripts has a boundary of expression that corresponds to the adjacent Hox-2.8 gene instead of Hox-2.7. In another type of transcript there is an alternative reading frame which predicts a protein that has homology to an enzyme ATPase and suggests that a non-homeobox containing gene may be located in the Hox-2 cluster. A Hox-2.7-lacZ transgene is expressed in a similar pattern to the endogenous gene in that spatially-restricted domains of expression are seen in the branchial arches, neural tube, paraxial mesoderm (somites), cranial ganglia, neural crest and gut. However, the anterior boundaries of transgene expression only correspond to the subset of Hox-2.7 transcripts which map to the Hox-2.8 boundary. The proximity of a Hox-2.7 promoter to regions which regulate the adjacent Hox-2.6 gene and the expression of transgenic and endogenous transcripts in a Hox-2.8 pattern, suggest that regulatory elements may be shared by neighbouring genes to establish the complete expression pattern.

Amino Acid Sequence↗
PubMed1992

A human aldehyde dehydrogenase (aldose reductase) pseudogene: nucleotide sequence analysis and assignment to chromosome 3.

Four cosmid clones containing putative pseudogenes for human aldehyde dehydrogenase (Aldose reductase) were isolated from libraries made to two individuals. These clones show different patterns on digestion with restriction endonucleases and probably represent distinct and separate loci. The DNA sequence of one of the putative pseudogenes (cosmid AR.F) was determined, and comparisons demonstrate 89.7% homology with the cDNA sequence of the functional aldose reductase gene. This pseudogene sequence contains no intronic sequences, whereas the functional aldose reductase has nine introns. In addition, the homology disappears in region 5' to the transcription start site for the cDNA, implying that regulatory elements such as the promoter are missing from this pseudogene. The pseudogene defined by cosmid AR.F has been mapped to chromosome 3 by polymerase chain reaction using amplimers specific for this pseudogene to amplify DNA from somatic cell hybrids.

Aldehyde Reductase↗
PubMed1992

Domains of cellular retinoic acid-binding protein I (CRABP I) expression in the hindbrain and neural crest of the mouse embryo.

We describe here the distribution of cellular retinoic acid-binding protein I (CRABP I) in the head of the early mouse embryo from day 8 to day 13 of gestation, using both in situ hybridisation to localise mRNA and immunocytochemistry to localise protein. The distribution of mRNA and protein was found to be identical. CRABP I first appeared in part of the presumptive hindbrain of the presomite embryo and then became localised to rhombomeres 2, 4, 5 and 6. The only other area of expression in the cephalic neuroepithelium was in a part of the midbrain roof. The neural crest and its mesenchymal derivatives, the branchial arches, expressed CRABP I and crest could be seen streaming from the neuroepithelium of individual rhombomeres into particular branchial arches. This suggested a fate map could be constructed describing the rhombomeric origin of branchial arch mesenchyme. Later in development, axons throughout the hindbrain expressed CRABP I. The results are considered in terms of the role of retinoic acid in the specification of neuronal phenotype in the hindbrain and in axon outgrowth.

Amino Acid Sequence↗