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Biomedical subjects

A M Brown

Publications and source records attributed to A M Brown.

At least 127 records · Page 7Linked to original sources
PubMed1998

Successful fertilization after superovulation and laparoscopic intrauterine insemination of the brushtail possum, Trichosurus vulpecula, and tammar wallaby, Macropus eugenii.

Fertilization has been achieved in superovulated brushtail possums and tammar wallabies after laparoscopic intrauterine artificial insemination. Various superovulation protocols and insemination times were examined but a maximum of 2-5 eggs including 1-2 embryos per possum were recovered. The female possums were superovulated by treatment with 15 iu pregnant mares' serum gonadotrophin and then either GnRH (4 x 50 micrograms, at intervals of 90 min) or 4 mg LH, 3 days later. Inseminations were performed within 6 h before or 4-10 h after (pregnant mares' serum gonadotrophin-GnRH group only) the expected onset of ovulation using epididymal spermatozoa. Superovulation in wallabies was achieved by treatment with FSH (8 x 6 mg, at intervals of 12 h for 4 days) followed by 4 mg LH on day 5. Inseminations were performed 4-6 h before the expected onset of ovulation using ejaculated spermatozoa, which resulted in the recovery of 7-8 eggs including 3-4 embryos per female. All embryos recovered were from possums and wallabies examined 1-2 days after insemination and included fertilized eggs, two-cell and four-cell embryos. Motile spermatozoa were recovered from the oviducts and uteri but only immotile spermatozoa were found in the vaginal complex. Five to thirty per cent of spermatozoa recovered from the oviducts of possums examined 2-6 h after insemination had thumbtack morphology, which is thought to be correlated with capacitation. Although embryo yields per female were low, this study has established that intrauterine artificial insemination after superovulation is a feasible assisted breeding strategy for marsupials with implications for species conservation and population control.

Animals↗
PubMed1998

Blockade of HERG and Kv1.5 by ketoconazole.

Ketoconazole, a widely used fungicide in patients, has been associated with Q-T prolongation and torsade de pointes when co-administered with terfenadine (Seldane). Both compounds use the same cytochrome-P450 metabolic pathway, resulting in an increase in plasma concentration of terfenadine. We previously showed that terfenadine blocked HERG (Human Ether-a-Gogo Related Gene), an important component of the repolarizing cardiac delayed rectifier IK with concentration needed to obtain 50% of the block (IC50) in the therapeutic range (300 nM). Another target is Kv1.5 (delayed outward rectifier potassium current), an important component of human atrial ultrarapid delayed rectifier current. Whether Kv1.5 and HERG proteins are direct targets for ketoconazole has yet to be addressed. We heterologously expressed HERG and Kv1.5 in Xenopus oocytes and compared their sensitivities to ketoconazole. HERG and Kv1.5 currents were reduced comparably with apparent IC50 values of 49 microM and 107 microM, respectively, when measured using the two-microelectrode recording technique. The differences in the IC50 may help explain the preferential ventricular origin of the ketoconazole-associated arrhythmias during overdose. The mechanism of block was different between Kv1.5 and HERG. Cumulative application of terfenadine and ketoconazole at their respective IC50 concentrations resulted in current reductions that suggest an additive rather than a competitive type of block by the two drugs. We conclude that ketoconazole may potentiate the effects of terfenadine first by an indirect pharmacokinetic action to elevate plasma levels and second by a direct pharmacodynamic action on HERG currents. These potential dual actions on HERG currents suggest that precautions should be taken in long-term ketoconazole treatment, particularly for patients who have decreased liver function or are on a drug regimen requiring simultaneous medications that use cytochrome-P450 for breakdown, such as terfenadine or erythromycin, or Class III antiarrhythmic drugs.

Action Potentials↗
PubMed1998

Influence of concomitant disease on patterns of hospitalization in patients with heart failure discharged from Scottish hospitals in 1995.

AIMS: To determine the prevalence of common, serious, concomitant conditions complicating admissions with heart failure and how such conditions influence the length of hospital stay. METHODS AND RESULTS: Data from Scottish morbidity records (SMR1) were used to determine the rate of deaths and discharges for heart failure (ICD-9 428.0, 428.9), concomitant discharge diagnoses and length of stay in 1995. 27,477 SMR1 records listing heart failure as a diagnosis were identified with heart failure in the first position in 11,560 (42%) records. 63.3% of deaths or discharges results from emergency admission. 13.2% of admissions were associated with acute myocardial infarction, 7.3% with angina or chest pain, 11.8% with chronic airways obstruction, 8.3% with chronic or acute renal failure and 5.3% had had a stroke. Length of stay including those patients who died was 7.6 days when acute myocardial infarction was the principal diagnosis but 26.3 days when stroke was the principal diagnosis. CONCLUSION: A large proportion of deaths and discharges for heart failure are associated with conditions other than heart failure that may precipitate, contribute to or complicate admission. Treatment for heart failure that does not also seek to reduce the risk associated with common concomitant diseases may miss opportunities to reduce the overall risk of hospitalization.

Adult↗
PubMed1998

Use of cytosolic triacylglycerol hydrolysis products and of exogenous fatty acid for the synthesis of triacylglycerol secreted by cultured rat hepatocytes.

The fatty acyl moieties incorporated into triacylglycerol (TAG) secreted by rat hepatocytes are derived from diacylglycerol (DAG) that is synthesized de novo through the phosphatidate pathway or derived from endogenous, cytosolic TAG after hydrolysis to DAG and re-esterification. We have used a dual-labeling technique (overnight labeling of cell TAG with [3H]oleate, followed by 3 h incubation with [14C]oleate) to quantify the contributions of the two sources towards TAG secretion by cultured rat hepatocytes. A wide range of TAG secretion rates was achieved by short-term incubation of the cells under a variety of conditions. There was no correlation between the overall amount of exogenous 14C-labeled fatty acid metabolized and the rate of either [14C]- or [3H]TAG secretion. By contrast, there was a strong positive correlation between the fraction of newly synthesized [14C]TAG that was secreted (the fractional secretion rate, FSR) and the absolute rate of TAG secretion. This suggests that the partitioning of DAG between (re)synthesis of cytosolic TAG and synthesis of secreted TAG is an important locus for the control of the rate of TAG secretion. Comparison of the ratio: oxidation/TAG secretion for 3H- and 14C-labeled acyl moieties indicated that, for all conditions studied, approximately half the acyl moieties already esterified to the glyceroyl backbone within cytosolic TAG remain unavailable for oxidation when this pool of TAG is mobilized for the synthesis of secreted TAG. The data provide evidence that hydrolysis of cytosolic triacylglycerol (TAG) does not proceed fully to the constituent fatty acids and glycerol, but only to the level of diacylglycerol, followed by re-modelling of approximately half of its acyl chains, before re-esterification to form secretory TAG.

Animals↗
PubMed1997

Isolation of two novel WNT genes, WNT14 and WNT15, one of which (WNT15) is closely linked to WNT3 on human chromosome 17q21.

The Wnt gene family consists of at least 15 structurally related genes that encode secreted extracellular signaling factors. Wnt proteins function in a range of critical developmental processes in both vertebrates and invertebrates and are implicated in regulation of cell growth and differentiation in certain adult mammalian tissues, including the mammary gland. We have isolated a number of WNT sequences from human genomic DNA, two of which, designated WNT14 and WNT15, represent novel members of the Wnt gene family. We also isolated WNT sequences from human mammary cDNA and present evidence that WNT13 is expressed in human breast tissue, in addition to those previously described. WNT14 and WNT15 appear to have originated from an ancestral branch of the Wnt gene family that also includes the Wnt9 sequences found in jawless and cartilaginous fishes. A Wnt14 cDNA was also isolated from chicken and a partial Wnt15 sequence from mouse. We show that human WNT14 maps to chromosome 1 and that WNT15 maps distal to BRCA1 on chromosome 17q21, where it lies within 125 kb of another WNT family member, WNT3.

Amino Acid Sequence↗
PubMed1997

Multiple regulatory elements control transcription of the peripheral myelin protein zero gene.

The gene encoding protein zero (P0), the most abundant protein of peripheral nervous system myelin, is expressed uniquely in Schwann cells. Previous studies have demonstrated that much of the cell type specificity of this expression is due to transcriptional control elements in the 1.1-kilobase pair 5'-regulatory region of the gene. We have now analyzed this region and have identified a set of functional elements in the 500 base pairs proximal to the transcription start site. DNA sequence conservation within the 5' regions of the human, mouse, and rat P0 genes correlates closely with the results of promoter deletion analysis of the 1.1-kilobase pair region assayed in Schwann cell cultures and reveals a potent proximal region from position -350 to +45. Sites of protein/DNA interaction within the proximal 500 base pairs of the promoter were identified by footprinting assays. Functional transcriptional elements were identified within the protected regions in the proximal promoter by mutation and transient transfection analysis in P0-expressing cell lines. The core (or basal) P0 promoter is identified as two regulatory elements, a G/C-rich element that binds nuclear factor Sp1 and a CAAT box that binds NF-Y. These core elements are essential for the transcription observed from the transfected promoter in cultured Schwann cells.

Animals↗
PubMed1997

Interactions among inactivating and noninactivating Kvbeta subunits, and Kvalpha1.2, produce potassium currents with intermediate inactivation.

Experiments were carried out to determine whether coinjection of Kvalpha1.2 with inactivating and noninactivating Kvbeta subunits would produce currents with intermediate kinetics and channel complexes containing a mixture of these subunits. Upon coexpression with a saturating amount of Kvbeta1.2 and increasing levels of a noninactivating deletion mutant of Kvbeta1.2, we show that macroscopic Kvalpha1.2 currents have levels of fractional inactivation and inactivation time constants that are intermediate between those obtained with either the inactivating Kvbeta1.2 or the noninactivating Kvbeta1.2 mutant. We also find that coexpression of Kvalpha1.2 with saturating amounts of Kvbeta1.2 and the deletion mutant produces a population of single channels with properties intermediate to either the inactivating or noninactivating parental phenotype. Our data can best be explained by the presence of an intermediate population of heterooligomeric channels consisting of Kvalpha1.2 with different combinations of both types of subunits. Since Kvalpha1.2 subunits coexist in cells with inactivating and noninactivating Kvbeta subunits, our findings suggest that heterooligomeric assembly of these subunits occurs to increase the range of K+ current kinetics and expression levels.

Animals↗
PubMed1997

A mechanism for the proarrhythmic effects of cisapride (Propulsid): high affinity blockade of the human cardiac potassium channel HERG.

Cisapride (Propulsid) is a gastrointestinal prokinetic agent commonly used to treat nocturnal heartburn as well as a variety of other gastrointestinal disorders. The use of cisapride has been associated with acquired long QT syndrome and ventricular arrhythmias such as torsades de pointes which produces sudden cardiac death. These cardiotoxic effects can be due to blockade of one or more types of K+ channel currents in the human heart. For this reason we compared the effects of cisapride on two cloned human cardiac K+ channels, Kv1.5 and the human ether-a-go-go-related gene (HERG) stably transfected into mammalian cells. Using patch clamp electrophysiology, we found that cisapride was a potent inhibitor of HERG displaying an IC50 value of 44.5 nmol/l when tail currents at -40 mV were measured following a 2 s test depolarization to +20 mV. When HERG currents were measured at the end of prolonged (20 s) depolarizing steps to +20 mV, the apparent affinity of cisapride was increased and measured 6.70 nmol/l. The main effect of cisapride was to enhance the rate of HERG current decay thereby reducing current at the end of the voltage clamp pulse. Furthermore, the potency of cisapride for the HERG channel was similar to that observed for the class III antiarrhythmic agent dofetilide (IC50 = 15.3 nmol/l) and the nonsedating antihistamine terfenadine (IC50 = 56.0 nmol/l). In contrast to its effects on HERG, cisapride inhibited Kv1.5 channel currents weakly displaying an IC50 value of 21.2 micromol/l. It is concluded that cisapride displays specific, high affinity block of the human cardiac K+ channel HERG. It is likely that this interaction underlies the proarrhythmic effects of the drug observed under certain clinical settings.

Arrhythmias, Cardiac↗
PubMed1997

Separable Kvbeta subunit domains alter expression and gating of potassium channels.

Kvbeta subunits have been shown to affect kinetic properties of voltage-gated K+ channel Kv1alpha subunits and increase the number of cell surface dendrotoxin-binding sites when coexpressed with Kv1. 2. Here, we show that Kvbeta1.2 alters both current expression and gating of Kvalpha1 channels and that each effect is mediated by a distinct Kvbeta1.2 domain. The Kvbeta1.2 N terminus or Kvalpha1-blocking domain introduced steady state current block, an apparent negative shift in steady state activation, and a slowing of deactivation along with a dramatic reduction in single channel open probability. N-terminal deletions of Kvbeta1.2 no longer altered channel kinetics but promoted dramatic increases in Kv1.2 current. The conserved Kvbeta1 C terminus or Kvalpha1 expression domain alone was sufficient to increase the number of functional channels. The same effect was observed with the normally noninactivating subunit, Kvbeta2. By contrast, Kv1.5 currents were reduced when coexpressed with either the Kvbeta1 C terminus or Kvbeta2, indicating that the Kvalpha1 expression domain has Kvalpha1 isoform-specific effects. Our results demonstrate that Kvbeta subunits consist of two domains that are separable on the basis of both primary structure and functional modulation of voltage-gated K+ channels.

Animals↗
PubMed1997

Mapping the kidney potassium channel ROMK1. Glycosylation of the pore signature sequence and the COOH terminus.

ROMK1, also known as Kir 1.1, is an inwardly rectifying K+ channel and is the prototypical member of the large Kir gene family. The accepted model of Kir topology predicts intracellular NH2 and COOH termini, and two membrane-spanning segments, M1 and M2, connected by an intramembranous pore-forming segment, H5. The sequence of H5 is similar in voltage-dependent K+ channels and features a strictly conserved GY/FG in its mid-region, which has been proposed as the selectivity filter of the pore. We have been using N-glycosylation substitution mutants to map the extracellular topology of ROMK1 biochemically and have described several loci in H5 that were glycosylated. We now report glycosylation at loci Tyr144 and Phe146, which indicates that the signature GYG sequence (143-145) rather than being intramembranous is extracellular. The COOH terminus was predicted to begin at position 178, but contrary to the model, we observed that position 257 was glycosylated and surrounding positions at 199, 222, and 298 were unglycosylated. N-Glycosylation sequon substitution at the latter three positions abolished K+/Na+ selectivity. Our results suggest a major revision of the topology of ROMK1 with H5 and the pore signature sequence now completely extracellular. The COOH terminus appears to form two additional membrane-spanning segments and to contribute to the ion conduction pathway.

Amino Acid Sequence↗
PubMed1997

On the role of calcium in the regulatory volume decrease (RVD) response in Ehrlich mouse ascites tumor cells.

The putative role for Ca2+ entry and Ca2+ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P3 content, as measured in extracts from [2-3H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca2+-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca2+-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca2+ (1% only at 0.1-mM Ca2+ and in Ca2+-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca2+ upon the volume-induced K+ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca2+ chelator BAPTA when the BAPTA-loading is performed in K+ equilibrium medium; (vi) an inhibition of the swelling-induced K+ loss (about 50%) at 1-mM Ba2+, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 microM), reported inhibitors of the K+ loss induced by Ca2+-mobilizing agonists. Thus, Ca2+signaling by Ca2+ release or Ca2+ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells.

Animals↗
PubMed1997

Inhibition of chondrogenesis by Wnt gene expression in vivo and in vitro.

The Wnt family of secreted signaling proteins are implicated in regulating morphogenesis and tissue patterning in a wide variety of organ systems. Several Wnt genes are expressed in the developing limbs and head, implying roles in skeletal development. To explore these functions, we have used retroviral gene transfer to express Wnt-1 ectopically in the limb buds and craniofacial region of chick embryos. Infection of wing buds at stage 17 and tissues in the head at stage 10 resulted in skeletal abnormalities whose most consistent defects suggested a localized failure of cartilage formation. To test this hypothesis, we infected micromass cultures of prechondrogenic mesenchyme in vitro and found that expression of Wnt-1 caused a severe block in chondrogenesis. Wnt-7a, a gene endogenously expressed in the limb and facial ectoderm, had a similar inhibitory effect. Further analysis of this phenomenon in vitro showed that Wnt-1 and Wnt-7a had mitogenic effects only in early prechondrogenic mesenchyme, that cell aggregation and formation of the prechondrogenic blastema occurred normally, and that the block to differentiation was at the late-blastema/early-chondroblast stage. These results indicate that Wnt signals can have specific inhibitory effects on cytodifferentiation and suggest that one function of endogenous Wnt proteins in the limbs and face may be to influence skeletal morphology by localized inhibition of chondrogenesis.

Animals↗
PubMed1997

Simultaneous measurement of [3H]noradrenaline release and neurogenic contraction under identical conditions, to determine the prejunctional inhibitory effects of SKF 99101H and BRL 56905 in dog saphenous vein.

Using a tissue bath system which allowed the simultaneous measurement of electrically-induced [3H]noradrenaline release and neurogenic contraction under identical conditions, we investigated the prejunctional inhibitory activity of the selective 5-HT(1D/1B) receptor agonists BRL 56905 ((+/-)-3-amino-6-carboxamido-1,2,3,4-tetrahydrocarbazole) and SKF 99101H (3-(2-dimethylaminoethyl)-4-chloro-5-propoxyindole hemifumarate), compared to sumatriptan and 5-HT. Transmural electrical stimulation (2 Hz) of dog saphenous vein induced consistent increases in [3H]noradrenaline release as well as reproducible contractile responses (<10% decrease over four stimulation periods). BRL 56905, SKF 99101H, sumatriptan and 5-HT (60 nM-6 microM) inhibited electrically-evoked [3H]noradrenaline release and neurogenic contractile responses in dog saphenous vein. However, despite being measured under identical conditions, the inhibition of [3H]noradrenaline release was consistently greater than the inhibition of neurogenic contraction induced by a particular concentration of agonist, suggesting that neurogenic contractile responses in dog saphenous vein result from the combined release of noradrenaline and other non-noradrenergic neurotransmitters. Under the present assay conditions, since the agonists produced only small (BRL 56905, sumatriptan and 5-HT) or marginal (SKF 99101H) contractile responses, it is unlikely that this is the cause of the discrepancy observed between inhibition of release and inhibition of contraction. The inhibitory effects of BRL 56905, sumatriptan and 5-HT were blocked by the 5-HT(1D/1B) receptor antagonist methiothepin, consistent with the involvement of canine ca-5-HT(1D/1B) receptors in inhibiting neurotransmitter release and subsequent smooth muscle contraction in dog saphenous vein. The present results show that the novel 5-HT(1D/1B) receptor agonists BRL 56905 and SKF 99101H are at least as potent as sumatriptan and 5-HT, at activating prejunctional inhibitory ca-5-HT(1D/1B) heteroreceptors on sympathetic axon terminals in dog saphenous vein. In addition, when measured simultaneously in the same tissue preparation, [3H]noradrenaline release was inhibited to a much greater extent than neurogenic contraction by any particular agonist.

Animals↗
PubMed1997

The effect of contralateral broad-band noise on acoustic distortion products from the human ear.

In the first part of the study, the effect of ipsilateral stimulus level on contralateral suppression of the 2f2-f2 distortion product by broad-band noise at 60 dB SPL RMS is examined. The levels of the primary stimuli were manipulated independently, giving f1 and f2 growth curves at four different f2 frequencies for four subjects. These typically bell-shaped, growth curves are shifted vertically to lower distortion levels and, in some cases, horizontally to higher primary stimulus levels. These results can be interpreted as an attenuation of both the primary stimuli and the distortion product and could be caused by simple acoustic attenuation produced by middle ear muscle activity, efferent activity or a combination of the two. In the second part of the study, the same contralateral stimulus was used while measuring both 3f1-2f2 and 2f1-f2 distortion products from the ipsilateral ear. The frequency separation of the primary tones was varied. This produced an approximately bandpass shape with the level peaking when the distortion frequency was approximately half an octave below f2, as previously described (Brown and Gaskill, 1990). This shape is thought to be linked with frequency selectivity in the cochlea. Contralateral broadband noise did not affect the tuning or the centre frequency of the bandpass shape or the mean group delay. It did reduce the size of the distortion peak and, in particular, it affected the peak-to-trough height of the 'fine structure' in the amplitude. Vector analysis revealed that the fine structure was due to a signal with substantial delay (probably from the distortion product 'place') which was summed with a larger, less delayed component (probably directly from the f2 'place'). The greater effect of contralateral stimulation on the more delayed component may reflect differences in efferent effect with complex (stimulus place), rather than simple (distortion product place) stimuli.

Acoustic Stimulation↗
PubMed1997

Selective aggregation of endogenous beta-amyloid peptide and soluble amyloid precursor protein in cerebrospinal fluid by zinc.

Zinc added to buffered solutions of synthetic beta-amyloid peptide (A beta) has been reported to induce accelerated formation of insoluble aggregates. This observation suggests that zinc may play a role in the formation of senile plaques, which contain A beta, in Alzheimer's disease. To test this hypothesis under conditions more representative of the brain, we investigated the ability of zinc to induce aggregation of A beta in freshly drawn canine CSF, which contains the same sequence as human A beta. Aggregates were separated from CSF by ultracentrifugation before and after incubation with zinc and assayed by quantitative western blotting and ELISA. We found that zinc induced the rapid aggregation of endogenous A beta in CSF, with an EC50 of 120-140 microM. The reaction was specific, because most (> or = 95%) CSF protein remained soluble under conditions where most A beta was insoluble, as assayed by scanning densitometry of Coomassie-stained gels. Staining of the precipitated material resulted in the visualization of punctate regions that were thioflavin positive or birefringent when stained with Congo red, suggesting the formation of amyloid-related structures. These results suggest that zinc could play a role in amyloid deposition, because there is overlap between the regions of the brain where zinc concentrations are highest and regions with the highest amyloid content. It is surprising that zinc induced the aggregation of endogenous soluble APP at lower concentrations than required for A beta (EC50 80 microM). The possibility that zinc-induced aggregation of APP may precede the deposition of A beta into plaques is discussed. Investigation of aggregation of A beta in CSF will aid in assessing the biological relevance of other agents that have been reported to accelerate amyloid formation.

Amyloid beta-Peptides↗