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Biomedical subjects

G Kay

Publications and source records attributed to G Kay.

At least 37 records · Page 2Linked to original sources
PubMed1994

The lymphoblast beta-adrenergic receptor in bipolar depressed patients: effect of chronic incubation with lithium chloride.

We have recently reported a study of beta-adrenergic receptor binding characteristics in lymphoblast cell lines derived from patients with bipolar disorder (BD) and healthy, matched control subjects. In the present study we have investigated the effects of incubating cells from the same subjects with lithium chloride (1 mM) for 7 days prior to assay. There was no difference in beta-adrenergic receptor number between control and BD cell lines and incubation with lithium had no effect on receptor number in either group. Exposure of the cells to isoprenaline (1 nM) for 24 h immediately prior to assay caused significantly less down-regulation in BD cells (15 +/- 5%) than control cells (39 +/- 4%), as described previously. Incubation with lithium significantly increased the down-regulation response to isoprenaline in BD cells (39 +/- 6%) but not in control cells (30 +/- 7%). After lithium, the agonist-induced decrease in beta-AR number in BD cells was no longer significantly different from that in control cells. We conclude that lithium selectively enhanced the agonist down-regulation of beta-adrenergic receptors in cells derived from patients with bipolar disorder. The functional significance of this result and the potential biochemical mechanisms responsible for this effect are discussed.

Adult↗
PubMed1994

Transcranial Doppler sonography in headache-free migraineurs.

We used transcranial Doppler ultrasonography to determine whether intracranial blood velocities in 182 headache-free migraineurs (60 with aura, 122 without aura) differed from velocities in 38 nonheadache prone control subjects. During the headache-free period, migraineurs with and without aura had significantly elevated mean velocities in all intracranial arteries except the right internal carotid artery at the level of the siphon. Velocities in migraineurs with aura did not differ significantly from velocities in those without aura. Markedly increased velocities were noted in a subset of interictal migraineurs. Two explanations are possible: 1) Blood flow velocities may increase in response to a decrease in the cross sectional area of a vessel at or near the point of insonation. 2) Alterations at the level of the cerebral arteriole may affect regional cerebral blood flow, thereby changing blood flow velocities at the point of insonation. Transcranial Doppler sonography alone cannot sort out which process is responsible for the vascular response. This work may have therapeutic as well as diagnostic implications. When interictal flow velocities are markedly increased, it is possible that therapeutic agents with vasoconstrictor action might cause an excessive response. Further study is needed to clarify the sites of vasoreactivity as well as the magnitude of drug-induced vasoconstrictor response.

Adult↗
PubMed1993

The lymphoblast beta-adrenergic receptor in bipolar depressed patients: characterization and down-regulation.

beta-Adrenergic receptor binding and adenylyl cyclase activity were examined in lymphoblast cell lines established from 12 patients with bipolar disorder and 10 unrelated healthy control subjects. No significant differences were found in [125I]iodocyanopindolol binding affinity or capacity or in isoprenaline-stimulated cAMP response. Incubation of lymphoblasts with isoprenaline (1 nM) for 24 h prior to assay reduced both receptor number and adenylyl cyclase activity. The extent of receptor down-regulation was significantly less in cells of bipolar disorder patients (20 +/- 5%) compared to controls (40 +/- 4%). Desensitization of adenylyl cyclase, however, was reduced to a similar degree in bipolar (65 +/- 14%) and control (68 +/- 20%) subjects. We conclude that basal beta-adrenergic receptor characteristics are not altered in bipolar disorder but that agonist down-regulation of receptor number may be less efficient than in control cells. The functional implications of this effect are discussed.

Adenylyl Cyclases↗
PubMed1992

Lymphoid development in mice congenitally lacking T cell receptor alpha beta-expressing cells.

Vertebrate T cells express either an alpha beta or gamma delta T cell receptor (TCR). The developmental relatedness of the two cell types is unresolved. alpha beta + T cells respond to specific pathogens by collaborating with immunoglobulin-producing B cells in distinct lymphoid organs such as the spleen and Peyer's patches. The precise influence of alpha beta + T cells on B cell development is poorly understood. To investigate the developmental effects of alpha beta + T cells on B cells and gamma delta + T cells, mice homozygous for a disrupted TCR alpha gene were generated. The homozygotes showed elimination of alpha beta + T cells and the loss of thymic medullae. Despite this, gamma delta + T cells developed in normal numbers, and there was an increase in splenic B cells.

Animals↗
PubMed1992

The synthesis, kinetic characterization and application of a novel biotinylated affinity label for cathepsin B.

In this study we report on the synthesis, kinetic characterization and application of a novel biotinylated and active-site-directed inactivator of cathepsin B. Thus the peptidyldiazomethane biotinyl-Phe-Ala-diazomethane has been synthesized by a combination of solid-phase and solution methodologies and has been shown to be a very efficient inactivator of bovine and human cathepsin B. The respective apparent second-order rate constants (k0bs./[I]) for the inactivation of the human and bovine enzymes by this reagent, namely approximately 5.4 x 10(4) M-1.min-1 and approximately 7.8 x 10(4) M-1.min-1, compare very favourably with those values determined for the urethane-protected analogue benzyloxycarbonyl-Phe-Ala-chloromethane first described by Green & Shaw [(1981) J. Biol. Chem. 256, 1923-1928], thus demonstrating that the presence of the biotin moiety at the P3 position is compatible with inhibitor effectiveness. The utilization of this reagent for the detection of cathepsin B in electrophoretic gels, using Western blotting and in combination with a streptavidin/alkaline phosphatase detection system, is also demonstrated. Given that the peptidyldiazomethanes exhibit a pronounced reactivity towards cysteine proteinases, we feel that the present label may well constitute the archetypal example of a wide range of reagents for the selective labelling of this class of proteinase, even in a complex biological milieu containing additional classes of proteinases.

Affinity Labels↗
PubMed1992

The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases.

The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.

Affinity Labels↗
PubMed1992

The application of a novel biotinylated affinity label for the detection of a cathepsin B-like precursor produced by breast-tumour cells in culture.

In this report we demonstrate how the recently developed biotinylated affinity label biotinyl-Phe-Ala-diazomethane (Bio-Phe-Ala-CHN2) [Cullen, McGinty, Walker, Nelson, Halliday, Bailie & Kay (1990) Biochem. Soc. Trans. 18, 315-316; Walker, Cullen, Kay, Halliday, McGinty & Nelson (1992) Biochem. J. 283, 449-453] can be used for the detection of a precursor form of a cathepsin B-like enzyme produced by breast-tumour cells in culture. Thus the cell lines MDA-MB-436, ZR-75-1 and T47-D produce a soluble protein that can be allowed to react with the biotinylated affinity label to yield an SDS-resistant complex; this can be revealed with a streptavidin/alkaline phosphatase label after PAGE and Western blotting. This protein (molecular mass 47 kDa) can also be detected by immunoblotting using sheep anti-(cathepsin B) antibodies in conjunction with a donkey anti-sheep IgG label. None of the cell lines studied produced any mature cathepsin B-like activity, as gauged by the lack of turnover of the fluorogenic substrate benzyloxycarbonyl-Arg-Arg-4-methylcoumarin-7-ylamide (Cbz-Arg-Arg-NH-Mec). However, treatment of medium samples with pepsin resulted in the generation of such activity. When the pepsin-catalysed activation step was analysed by SDS/PAGE, the protein of 47 kDa was completely converted into two species of very similar molecular masses of 30.5 kDa and 29 kDa. Both these proteins can incorporate the biotinylated probe and, in common with the 47 kD species, they can be detected with the streptavidin/alkaline phosphatase label and immunoblotting. We propose that the 47 kD form is the pepsin-activable proform of these lower-molecular-mass species. The release of the proform from the oestrogen-receptor (ER)-positive breast-tumour cell lines ZR-75-1 and T47-D is stimulated 5-10-fold when these cells are grown in medium containing epidermal growth factor (EGF) at a concentration of 10 ng/ml. In contrast, there is no modulation in the amount of proform released by the ER-negative cell line MDA-MB-436, over a range of EGF concentrations from 0 to 100 ng/ml.

Affinity Labels↗
PubMed1992

Expression of growth factor mRNA in rabbit PVR model systems.

Proliferative vitreoretinopathy (PVR) involves the formation of intravitreal fibrocellular membranes which may lead to traction retinal detachment and blindness. The cellular component of epiretinal membranes originates from the proliferation and migration of cells within the eye. Several growth factors and other cytokines are plausible candidates for directing the processes leading to membrane formation. A reproducible animal model is needed for experimental studies of cytokine expression during PVR induction or treatment. We found that intravitreal injection of > 10(6) mixed mononuclear leukocytes or adherent monocytes along with a trans-scleral incision through the pars plana leads to the development of PVR-like disease in rabbit eyes. The severity of the disease was related to the number of monocytes injected. Typically, organized membranes extending from the incision toward the optic nerve formed within one week. Progression to extensive traction retinal detachment required 1 to 4 weeks. Injection of up to 5 x 10(6) lymphocytes or freeze-thaw killed monocytes was ineffective, and coinjecting 100 micrograms endotoxin with the monocytes did not result in enhanced disease. The histological appearance of the epiretinal membranes was similar to human PVR membranes. Macrophage, cytokeratin-positive (epithelial), and fibroblast-like cells were present. Northern blot analysis of RNA extracted from the rabbit membranes revealed the presence of mRNA for acidic fibroblast growth factor (aFGF). Acidic FGF mRNA was not expressed by the injected monocytes. A comparable level of aFGF mRNA and also mRNAs for basic FGF, platelet-derived growth factor-B, and transforming growth factor beta were found in epiretinal membranes induced by a scleral incision in association with cryopexy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

X-chromosome inactivation may explain the difference in viability of XO humans and mice.

Only about 1% of human XO conceptuses survive to birth and these usually have the characteristics of Turner's syndrome, with a complex and variable phenotype including short stature, gonadal dysgenesis and anatomical defects. Both the embryonic lethality and Turner's syndrome are thought to be due to monosomy for a gene or genes common to the X and Y chromosomes. These genes would be expected to be expressed in females from both active and inactive X chromosomes to ensure correct dosage of gene product. Two genes with these properties are ZFX and RPS4X, both of which have been proposed to play a role in Turner's syndrome. In contrast to humans, mice that are XO are viable with no prenatal lethality (P. Burgoyne, personal communication) and are anatomically normal and fertile. We have devised a system to analyse whether specific genes on the mouse X chromosome are inactivated, and demonstrate that both Zfx and Rps4X undergo normal X-inactivation in mice. Thus the relative viability of XO mice compared to XO humans may be explained by differences between the two species in the way that dosage compensation of specific genes is achieved.

Animals↗
PubMed1991

Molecular genetic analysis of the Ta25H deletion: evidence for additional deleted loci.

Seventeen linking clones sublocalized to the central region of the mouse X Chromosome (Chr) were screened against genomic DNA from male mice carrying the tabby-25H (Ta25H) deletion. Two of these linking clones, lambda EM131 and lambda EM169, were found to be deleted in Ta25H/Y animals. Genetic mapping through Mus musculus domesticus/Mus spretus interspecific backcross progeny, segregating for the original tabby (Ta) gene mutation, was utilized to order these markers and to define nearest flanking markers to the Ta25H deletion (lambda EM140 and lambda EM171). The size of the Ta25H deletion was thus estimated as up to 4.5 centiMorgans (cM). The order of markers, proximal to distal, was found to be lambda EM140/lambda EM131, mouse androgen receptor gene (Ar)/lambda EM169, Ta/lambda EM171. A putative CpG-rich island and a highly evolutionarily conserved DNA probe were isolated from the DXCrc169 locus which co-segregates with the Ta locus in this study.

Animals↗
PubMed1991

Effects of chronic alpha 2-adrenoceptor blockade on platelet and lymphocyte adrenoceptor binding in normal volunteers.

Platelet and lymphocyte adrenoceptor binding was measured in 12 healthy male volunteers before and after 22 days treatment with the alpha 2-adrenoceptor antagonist idazoxan 40 mg tds. Platelet alpha 2-adrenoceptor number assessed by the agonist 3H-UK 14304 [correction of UK 14303] was significantly increased following idazoxan, with a smaller increase in antagonist binding (3H-rauwolscine). Lymphocyte beta-adrenoceptor number was unaltered by idazoxan, although the variance within the sample was significantly increased. Plasma MHPG levels were significantly reduced by chronic idazoxan. These data indicate upregulation of the platelet alpha 2-adrenoceptor in response to chronic blockade and suggest that this may reflect a similar change in presynaptic alpha 2-adrenoceptors which regulate norepinephrine release.

Adrenergic alpha-Antagonists↗
PubMed1991

Determination of a molecular map position for Hyp using a new interspecific backcross produced by in vitro fertilization.

We have established a Mus spretus/Mus musculus domesticus interspecific backcross segregating for two X-linked mutant genes, Ta and Hyp, using in vitro fertilization. The haplotype of the recombinant X chromosome of each of 241 backcross progeny has been established using the X-linked anchor loci Otc, Hprt, Dmd, Pgk-1, and Amg and the additional probes DXSmh43 and Cbx-rs1. The Hyp locus (putative homologue of the human disease gene hypophosphatemic rickets, HYP) has been incorporated into the molecular genetic map of the X chromosome. We show that the most likely gene order in the distal portion of the mouse X chromosome is Pgk-1-DXSmh43-Hyp-Cbx-rs1-Amg, from proximal to distal. The distance in centimorgans (mean +/- SE) between DXSmh43 and Hyp was 2.52 +/- 1.4 and that between Hyp and Cbx-rs1 was 1.98 +/- 1.39. Thus closely linked flanking markers for the Hyp locus that will facilitate the molecular characterization of the gene itself have been defined.

Alkaline Phosphatase↗
PubMed1991

High-density molecular map of the central span of the mouse X chromosome.

A total of 17 linking clones previously sublocalized to the central span of the mouse X chromosome have been ordered by detailed analysis through interspecific Mus spretus/Mus musculus domesticus backcross progeny. These probes have been positioned with respect to existing DNA markers utilizing a new interspecific backcross segregating for the Tabby (Ta) locus. The density of clones within this 11.5-cM interval is now, on average, one clone every 1000 kb. This high-density map provides probes in the vicinity of a number of important genetic loci in this region which include the X-inactivation center, the Ta locus, and the mottled (Mo) locus, and therefore provides a molecular framework for identification of the genes encoded at these loci.

Animals↗
PubMed1991

Haemodynamic and electrophysiological effects of amlodipine, a new long-acting calcium antagonist.

Patients with stable angina pectoris received an intravenous bolus injection of 10 mg amlodipine followed by a second 10-mg bolus, 30 min later, if no haemodynamic compromise occurred. Systolic and diastolic blood pressures were significantly reduced and cardiac output increased. Left ventricular function was unaltered. Amlodipine did not affect the electrophysiological parameters studied; sinus node recovery time and the pacing rate to Wenckebach heart block did not significantly change from baseline. Amlodipine is a powerful arterial vasodilator with no negative inotropic effects. Cardiac electrophysiological function is not affected by amlodipine.

Adult↗