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Biomedical subjects

K A Jones

Publications and source records attributed to K A Jones.

At least 91 records · Page 5Linked to original sources

D2, D3, and D4 dopamine receptors couple to G protein-regulated potassium channels in Xenopus oocytes.

Human D2, D3, D4 and dopamine receptors were individually coexpressed in Xenopus oocytes with a G protein-regulated inwardly rectifying potassium channel (GIRK1). At -100 mV in 96 mM potassium, dopamine (0.1-100 nM) evoked an inward current; the current showed inward rectification, reversed polarity at 0 mV, and was blocked by barium (50% inhibition by 10 microM). The concentrations of dopamine activating 50% of the maximal current (EC50) were not different (2-4 nM) for D2, D3, and D4 receptors, but the maximal current was 3-fold larger for D2 and D4 than for D3 receptors. Dopamine evoked reproducible inward currents at D2 and D4 receptors when applied repeatedly, but second responses could not be observed in oocytes expressing D3 receptors. 7-Hydroxy-N,N-di-n-propyl-2-aminotetralin mimicked the effect of dopamine (EC50 of approximately 2, approximately 3, and approximately 19 nM at D2, D3, and D4, respectively). (-) Sulpiride reversibly blocked the dopamine-induced current with IC50 values of 5, 300, and 2000 nM for D2, D3, and D4 receptors, respectively. Dopamine was ineffective in oocytes injected 2 hr previously with pertussis toxin. We concluded that all three D2-like dopamine receptors share the potential to activate inwardly rectifying potassium channels.

Animals↗

Use of nitric oxide-nucleophile adducts as biological sources of nitric oxide: effects on airway smooth muscle.

Nitric oxide (NO)-nucleophile adducts are compounds of the general structure [XN(O)NO]-, where X is a nucleophile residue, and they release NO spontaneously in aqueous solution. This study determined the effects of two NO-nucleophile adducts [diethylaminodiazen-1-ium-1,2-diolate (DEA-NO) and sperminediazen-1-ium-1,2-diolate (DEA-NO) and sperminediazen-1-ium-1,2-diolate (spermine-NO)] on isometric force and the cytosolic concentrations of cyclic GMP ([cGMP]1), cyclic AMP ([cAMP]i) and calcium in canine tracheal smooth muscle. The ratio of fura-2 emission fluorescence intensities with excitation at 340- and 380-nm wavelengths (F340/ F380) was used as an index of cytosolic [Ca+2] concentration. Both DEA-NO and spermine-NO caused a concentration-dependent and reversible decrease in force (EC50 of 0.13 +/- 0.02 microM and 4.1 +/- 0.3 microM, respectively) and F340/F380, a concentration-dependent increase in [cGMP]i and no change in [cAMP]i. There were no significant differences in the relationship between [cGMP]l and the percentage decrease in force or in the relationship between the percentage decrease in F340/ F380 and the percentage decrease in force between tissues relaxed with DEA-NO or spermine-NO. Oxyhemoglobin increased the EC50 for both DEA-NO (from 0.13 +/- 0.03 microM to 8.1 +/- 0.2 microM) and spermine-NO (from 3.9 +/- 0.3 microM to 81.6 +/- 6.4 microM) and completely scavenged NO released by 0.13 (EC50) and 1 (EC100)microM DEA-NO. These results suggest that both DEA-NO and spermine-NO generate NO extracellularly and relax airway smooth muscle in association with an increase in [cGMP]i and a decrease in cytosolic Ca+2 concentration. NO-nucleophile adducts may serve as vehicles for the controlled delivery of NO into biological systems and thus provide a useful tool by which the physiology of NO can be investigated.

Animals↗

Localization of the human RNA polymerase I transcription factor gene (UBTF) to the D17S183 locus on chromosome 17q21 and construction of a long-range restriction map of the region.

Human upstream binding factor (hUBF) is a sequence-specific DNA-binding protein that is essential for the activation of human 18s and 28s rRNA gene transcription. We have isolated and localized the gene (UBTF) encoding hUBF to the D17S183 locus on chromosome 17q21 by analyzing a cosmid from the region and carrying out Southern analysis on a previously constructed chromosome 17 somatic cell hybrid mapping panel using a probe from the hUBF cDNA. Confirmation of its location at this region was obtained from the results of pulsed-field gel electrophoresis analysis of genomic DNA using the hUBF cDNA and other probes from the region. These data also enabled the construction of a long-range restriction map of the region.

Cell Line↗

Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro.

Lymphoid enhancer-binding factor 1 (LEF-1) is a regulatory high mobility group (HMG) protein that activates the T cell receptor alpha (TCR alpha) enhancer in a context-restricted manner in T cells. In this paper we demonstrate that the distal region of the human immunodeficiency virus-1 (HIV-1) enhancer, which contains DNA-binding sites for LEF-1 and Ets-1, also provides a functional context for activation by LEF-1. First, we show that mutations in the LEF-1-binding site inhibit the activity of multimerized copies of the HIV-1 enhancer in Jurkat T cells, and that LEF-1/GAL4 can activate a GAL4-substituted HIV-1 enhancer 80- to 100-fold in vivo. Second, recombinant LEF-1 is shown to activate HIV-1 transcription on chromatin-assembled DNA in vitro. By using a nucleosome-assembly system derived from Drosophila embryos, we find that the packaging of DNA into chromatin in vitro strongly represses HIV-1 transcription and that repression can be counteracted efficiently by preincubation of the DNA with LEF-1 (or LEF-1 and Ets-1) supplemented with fractions containing the promoter-binding protein, Sp1. Addition of TFE-3, which binds to an E-box motif upstream of the LEF-1 and Ets-1 sites, further augments transcription in this system. Individually or collectively, none of the three enhancer-binding proteins (LEF-1, Ets-1, and TFE-3) could activate transcription in the absence of Sp1. A truncation mutant of LEF-1 (HMG-88), which contains the HMG box but lacks the trans-activation domain, did not activate transcription from nucleosomal DNA, indicating that bending of DNA by the HMG domain is not sufficient to activate transcription in vitro. We conclude that transcription activation by LEF-1 in vitro is a chromatin-dependent process that requires a functional trans-activation domain in addition to the HMG domain.

Animals↗

Physical mapping, cloning, and identification of genes within a 500-kb region containing BRCA1.

BRCA1 is a breast/ovarian cancer susceptibility gene on human chromosome 17q21. We describe a complete and detailed physical map of a 500-kb region of genomic DNA containing the BRCA1 gene and the partial cloning in phage P1 artificial chromosomes. Approximately 70 exons were isolated from this region, 11 of which were components of the BRCA1 gene. Analysis of the other exons revealed a rho-related G protein and the interferon-induced leucine-zipper protein IFP-35.

Amino Acid Sequence↗

Cloning of an SNF2/SWI2-related protein that binds specifically to the SPH motifs of the SV40 enhancer and to the HIV-1 promoter.

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

Adenosine Triphosphatases↗

Efficacy trials with tissue-culture grown, inactivated vaccines against chlamydial abortion in sheep.

Vaccines containing inactivated, semi-purified antigen of ovine abortifacient Chlamydia psittaci were prepared from tissue-culture grown harvests of two strains. These were evaluated for efficacy against experimentally reproduced enzootic abortion of ewes (EAE), the protection index (PI) of each vaccine being calculated from three variables. Trial 1 tested the adjuvants Marcol 52/Arlacel A, ISCOM matrix, Novasomes and Alhydrogel, each administered with a total antigen dose of 4 micrograms chlamydial protein (cp), against challenge with the two homologous vaccine strains. The first three formulations yielded PIs of 39-42%, while that of Alhydrogel was 23%. Marked granuloma and/or abscess formation occurred in the Marcol 52/Arlacel A group but in none of the others, and only this group seroconverted following vaccination. In Trial 2, ISCOM matrix containing 16 micrograms cp per dose was administered once or twice. Two challenges were used, one involving the two vaccine strains and the other these two strains plus five others, but no significant differences were seen between them. The double dose schedule, which was better than the single dose and also several Novasomes formulations tested concurrently, gave PIs of 64 and 77% against the two challenges (combined value 71.5%). Trial 3 studied duration of immunity over two lambing seasons using three different administration schedules of 16 micrograms cp in ISCOM matrix, namely single dose in year 1, single dose in year 2 (Group 1), double dose then booster (Group 2), and double dose without booster (Group 3). PIs against the homologous bivalent challenge were 79-80% for Groups 1 and 3 and 91% for Group 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Abortion, Veterinary↗

Halothane reduces force and intracellular Ca2+ in airway smooth muscle independently of cyclic nucleotides.

Halothane relaxes airway smooth muscle in part by a direct effect on the smooth muscle cell. The purpose of this study was to investigate the possible role of cyclic nucleotides in this direct effect. Strips of canine tracheal smooth muscle in vitro were contracted with acetylcholine (ACh) and then exposed to 0.7-2.6% halothane. Isometric force and the intracellular concentrations of adenosine cyclic 3',5'-monophosphate ([cAMP]i) guanosine cyclic 3',5'-monophosphate ([cGMP]i), and free calcium ([Ca2+]i, using the fluorescent Ca(2+)-sensitive dye fura 2) were measured. ACh caused significant increases in force, [cAMP]i, [cGMP]i, and [Ca2+]i. Subsequent exposure of the strips to halothane caused an additional increase in [cAMP]i, decreases in force and [Ca2+]i, and no effect on [cGMP]i. The additional increase in [cAMP]i was similar to that produced by a concentration of isoproterenol (0.03 microM) that caused equipotent relaxation. Indomethacin abolished the increase in [cAMP]i produced by ACh and abolished the additional increase in [cAMP]i produced by halothane. In contrast, indomethacin had no effect on the decreases in force and [Ca2+]i. These findings suggest that in canine tracheal smooth muscle contracted with ACh 1) halothane increases [cAMP]i by a cyclooxygenase-dependent mechanism and 2) the increase in [cAMP]i produced by halothane is not responsible for the relaxation or the decrease in [Ca2+]i.

1-Methyl-3-isobutylxanthine↗

Molecular genetics of sporadic and familial breast cancer.

Molecular genetic analysis of breast cancers indicates that the mechanisms underlying tumorigenesis are complicated. Many oncogenes and tumour suppressor genes have been implicated, encoding proteins that are important at many levels of cell regulation, from cell surface molecules responding to external signals (eg ERBB2) to nuclear factors controlling gene transcription (eg TP53, MYC). Several correlations have been found between certain genetic events and clinical outcome and have therefore proved useful prognostic indicators. The mapping and cloning of genes important in familial breast cancers (eg BRCA1) have provided the essential tools for pinpointing the genes that may be critical in early stage breast cancer as well as for developing genetic tests for predicting carrier status in breast cancer families. Clarification of the molecular consequences of mutation in breast cancer associated genes is beginning to address the factors that drive a normal breast cell to change into a breast cancer cell. However, these studies are still in their infancy, and considerable research will be required to complete the picture.

Ataxia Telangiectasia↗

Functional properties of a cloned 5-hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors.

1. A comparative study of the whole-cell and single-channel properties of cloned and native mouse 5-hydroxytryptamine ionotropic receptors (5-HT3) was undertaken using mammalian cell lines expressing the cloned 5-HT3 receptor subunit A (5-HT3R-A), superior cervical ganglia (SCG) neurones and N1E-115 cells. 2. No pharmacological difference was found in the sensitivity to the agonists 5-HT and 2-methyl-5-HT, or to the antagonists d-tubocurare and 3-tropanyl-3,5-dichlorobenzoate (MDL-72222). 3. Current-voltage (I-V) relationships of whole-cell currents showed inward rectification in the three preparations. Rectification was stronger both in cells expressing the 5-HT3R-A subunit and in N1E-115 cells when compared with SCG neurones. 4. No clear openings could be resolved in 5-HT-activated currents in patches excised from cells expressing the 5-HT3R-A subunit or N1E-115 cells. Current fluctuation analysis of whole-cell and excised-patch records revealed a slope conductance of 0.4-0.6 pS in both preparations. Current-voltage relationships of these channels showed strong rectification that fully accounted for the whole-cell voltage dependence. 5. In contrast, single channels of about 10 pS were activated by 5-HT in patches excised from SCG neurones. The weak voltage dependence of their conductance did not account completely for the rectification of whole-cell currents. A lower unitary conductance (3.4 pS) was inferred from whole-cell noise analysis. 6. We conclude that the receptor expressed from the cloned cDNA is indistinguishable from the 5-HT3 receptor of N1E-115 cells, suggesting an identical structure for these two receptors. The higher conductance and different voltage dependence of the 5-HT3 receptor in SCG neurones might indicate the participation of an additional subunit in the structure of native ganglionic 5-HT3 receptors. Homo-oligomeric 5-HT3R-A channels may also be present as suggested by the lower conductance estimated by whole-cell noise analysis.

Animals↗

Single channel properties of the 5-HT3 subtype of serotonin receptor in primary cultures of rodent hippocampus.

Whole cell and outside-out patch clamp recordings were obtained from primary cultures of rat and mouse hippocampal neurons. Serotonin (5-HT) evoked short-latency fast inward currents in about 5% of neurons. These currents reversed near 0 mV, showed inward rectification, and were inhibited by the 5-HT3 receptor antagonists ICS 205-930, ondansetron and tubocurare. 5-HT activated single channel currents in 14 of 24 membrane patches obtained from neurons which showed 5-HT3-activated whole cell currents; mean amplitude of channel openings was 0.95 +/- 0.09 pA at -100 mV. Chord conductances measured at -80 and -160 mV were 8.3 and 10.5 pS, respectively. 5-HT-induced unitary currents were blocked reversibly by tubocurare (1 microM), ICS 205-930 (30 nM) and ondansetron (100 nM). Thus, single-channel properties of 5-HT3 receptors in hippocampal neurons are similar to those present in peripheral neurons and do not exhibit solely the sub-picosiemen conductance characteristic of the neuroblastoma and neuroblastoma-derived cloned 5-HT3 receptor.

Animals↗

Organization of the mouse 5-HT3 receptor gene and functional expression of two splice variants.

The structure of the mouse 5-HT3 receptor gene, 5-HT3R-A, is most similar to nicotinic acetylcholine receptor (nAChR) genes, in particular to the gene encoding the neuronal nAChR subunit alpha 7. These genes share among other things the location of three adjacent introns, suggesting that 5-HT3R-A and nAChR genes arose from a common precursor gene. The alternative use of two adjacent splice acceptor sites in intron 8 creates, in addition to the original 5-HT3R-A cDNA (5-HT3R-AL), a shorter isoform (5-HT3R-AS) which lacks six codons in the segment that translates into the major intracellular domain. This splice consensus sequence is not found in human genomic DNA. In mouse, we demonstrate by RNAse protection assay that 5-HT3R-AS mRNA is approximately 5 times more abundant than 5-HT3R-AL mRNA in both neuroblastoma cell lines and neuronal tissues. We used the Semliki Forest virus expression system for electrophysiological characterization of 5-HT3R-AS and 5-HT3R-AL in mammalian cells. No differences in electrophysiological characteristics, such as voltage dependence, desensitization kinetics, or unitary conductance were found between homomeric 5-HT3R-AS and 5-HT3R-AL receptors. Their properties are very similar to those of 5-HT3 receptors in mouse neuroblastoma cell lines.

Alternative Splicing↗

Cortical neurons containing calretinin are selectively resistant to calcium overload and excitotoxicity in vitro.

Calbindin and the more recently identified protein calretinin are structurally related calcium-binding proteins having a broad distribution in the brain. Recent evidence supports a neuroprotective role for calbindin in regulating calcium homeostasis during periods of heightened Ca2+ influx. It is not known if calretinin might have a similar function. We investigated if calretinin-containing neurons have a survival advantage in rat neocortical cultures treated with a calcium ionophore or excitatory amino acids. Neuronal cultures were challenged with the calcium ionophore A23187 at different concentrations to produce a broad range of cell death. Cell loss was quantified for both the calretinin immunopositive and the calretinin immunonegative populations of neurons. We found that 3 h after exposure to 2 microM A23187 there was a 48% loss of the calretinin immunonegative population of neurons whereas the calretinin immunopositive set of neurons was reduced by only 18%. Calretinin positive neurons were still relatively spared after treatment with 3 microM A23187. The ionophore had no cytotoxic effect when calcium ions were removed from the extracellular medium. We also studied glutamate excitotoxicity by treating the neuronal cultures with the excitatory amino acids glutamate, N-methyl-D-aspartate or kainate for 5 min and examining survival three hours later. We found again that calretinin-containing neurons were relatively spared after exposure to the excitatory amino acids; at doses of N-methyl-D-aspartate and kainate that produced a 32-40% loss of calretinin immunonegative neurons, only 2-10% of calretinin immunopositive neurons died. Similar results were obtained for glutamate. These results demonstrate that neurons containing calretinin are better able to survive disturbances in calcium homeostasis than cells not containing this calcium-binding protein. The fact that this effect was observed with ionophore treatment, as well as excitatory amino acids, suggests that neither the density nor distribution of glutamate receptors on the different cell types was a factor in determining selective vulnerability. We hypothesize that the neuroprotective effect of calretinin is due to the buffering capacities of the protein in a manner analogous to that suggested for calbindin.

Animals↗

A computer-controlled, closed-loop infusion system for infusing muscle relaxants: its use during motor-evoked potential monitoring.

A microcomputer-controlled closed-loop infusion system (MCCLIS) has been developed that provides stable intraoperative levels of partial neuromuscular blockade. Complete neuromuscular blockade interferes with intraoperative motor-evoked potential (MEP) monitoring used for patients undergoing surgical procedures that place them at risk for spinal cord ischemia. Nine patients were studied during which the MCCLIS maintained stable levels of partial neuromuscular blockade and allowed transcranial magnetic motor-evoked potential (TcM-MEP) monitoring during thoracoabdominal aortic aneurysmectomy. The use of TcM-MEP for monitoring intraoperative spinal cord function was balanced against surgical considerations for muscle relaxation with 80% to 90% neuromuscular blockade fulfilling each requirement. Intraoperative adjustment of partial neuromuscular blockade to facilitate TcM-MEP monitoring was also possible with the MCCLIS. The MCCLIS should allow for further investigation into the sensitivity, specificity, and predictability of TcM-MEP monitoring for any patient at risk for intraoperative spinal cord ischemia including those undergoing thoracoabdominal aortic aneurysmectomy.

Action Potentials↗

Changes in cytosolic cGMP and calcium in airway smooth muscle relaxed by 3-morpholinosydnonimine.

Nitrovasodilators relax airway smooth muscle by both guanosine 3',5'-cyclic monophosphate (cGMP)-dependent and cGMP-independent mechanisms and by mechanisms that reduce cytosolic calcium concentration ([Ca2+]i). This study was conducted to determine the relative importance of these mechanisms in relaxation of canine tracheal smooth muscle (CTSM) induced by 3-morpholinosydnonimine (SIN-1). We measured 1) the effect of SIN-1 on force, [cGMP]i, and [Ca2+]i, and 2) the ability of methylene blue (MB) to antagonize SIN-1-induced relaxation and cGMP accumulation. The ratio of fura 2 emission fluorescence intensities due to excitation at 340- and 380-nm wavelengths (F340/F380) was used as an index of [Ca2+]i. In strips contracted with 0.3 microM acetylcholine (ACh, n = 8) or 24 mM KCl (n = 8), SIN-1 (1-100 microM) caused a concentration-dependent decrease in force which was correlated with a concentration-dependent increase in [cGMP]i. MB (10 microM) proportionally attenuated both relaxation and cGMP accumulation. In fura 2-loaded strips contracted with 0.3 microM ACh (n = 7) or 30 mM KCl (n = 7), reductions in force induced by SIN-1 (1-100 microM) were accompanied by decreases in F340/F380. These findings suggest that in CTSM contracted with ACh or KCl, SIN-1 causes relaxation which appears to be mediated by cGMP-dependent mechanisms that reduce [Ca2+]i.

Animals↗

Effects of halothane on the relationship between cytosolic calcium and force in airway smooth muscle.

The mechanism of the direct relaxing effect of halothane on airway smooth muscle may involve a decrease in 1) cytosolic calcium concentration ([Ca2+]i) and/or 2) the force produced for a given [Ca2+]i (i.e., the "sensitivity" of the myofibrillar contractile system to Ca2+). This study was conducted to test the hypothesis that halothane reduces the sensitivity of the myofibrillar contractile system to Ca2+ during muscarinic receptor stimulation of canine tracheal smooth muscle. Isolated smooth muscle strips were mounted in a photometric superfusion system, stretched to their optimal length for force development, and loaded with the fluorescent Ca2+ indicator, fura 2, for simultaneous recording of fura 2 fluorescence and isometric force. Emission fluorescence intensities due to excitation at 340 (F340)- and 380 (F380)-nm wavelengths were measured and F340/F380 was used as an index of [Ca2+]i. After superfusion with Ca(2+)-free physiological salt solution (PSS) containing 1 or 100 microM acetylcholine (ACh), two consecutive cumulative concentration-response curves to CaCl2 (0.01-2.4 mM) were generated for each strip; one curve was generated in the presence of halothane. In strips stimulated with 1 (n = 6) or 100 (n = 6) microM ACh, the cumulative addition of CaCl2 to the Ca(2+)-free PSS caused concentration-dependent increases in both F340/F380 and force. In strips stimulated with 1 microM ACh, 2.4 +/- 0.3% halothane proportionally attenuated increases in both F340/F380 and force.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗