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A Gillespie

Publications and source records attributed to A Gillespie.

At least 19 recordsLinked to original sources
PubMed2002

Evaluation of Duddingtonia flagrans in reducing infective larvae of Haemonchus contortus in feces of sheep.

Consequences of nematode infections due to Haemonchus contortus are a serious constraint for the sheep industry worldwide. Development of anthelmintic resistance and increasing concern about the impact of anthelmintic use dictate the need of alternative control. Such an alternative is using the nematode trapping fungus Duddingtonia flagrans to reduce infective larvae levels on pasture. Two trials were conducted to determine the effect of D. flagrans in reducing infective larvae (predominantly H. contortus) in feces. The first trial determined the dose effect of D. flagrans in reducing infective larvae in feces. Eighteen ewes were dewormed to remove existing infections and randomly assigned to six treatment groups: 5 x 10(4), 1 x 10(5), 2.5 x 10(5), 5 x 10(5), 1 x 10(6) or no (control) spores of D. flagrans per kg of body weight mixed in their feed for 7 days. Fecal samples were collected daily from these and from infected donor ewes. Feces from individual-treated ewes were mixed with equal amounts of donor ewe feces, theoretically approximating oral dose spore concentrations of 2.5 x 10(4), 5 x 10(4), 1.25 x 10(5), 2.5 x 10(5), 5 x 10(5) and no spores, and were cultured. Across dosages and during the 7 days of fungus feeding, percent reduction of infective larvae ranged from 76.6 to 100.0%. The second trial determined the effect of D. flagrans at the dose of 10(5) spores per kg body weight on reducing infective larvae in feces from naturally infected lambs. Twenty lambs were randomly assigned to either treatment or control groups based on fecal egg count. Treatment lambs were fed spores mixed in feed for 7 days. Feces were collected daily and cultured. During the 7 days of fungus feeding, the percent reduction of infective larvae ranged from 82.8 to 99.7%. Results of these trials demonstrated that the nematode trapping fungus D. flagrans was highly effective in reducing infective larvae in sheep feces and should be considered as a biological control agent for integrated nematode control programs.

Administration, Oral↗
PubMed2001

Dual action of n-alcohols on neuronal nicotinic acetylcholine receptors.

Alcohol is known to modulate the activity of a variety of neuroreceptors and ion channels. Recently, neuronal nicotinic acetylcholine receptors (nnAChRs) have become a specific focus of study because not only are they potently modulated by alcohol but also they regulate the release of various transmitters, including gamma-aminobutyric acid (GABA) and dopamine, which play an important role in the behavioral effects of ethanol. Whereas the potency of normal alcohols (n-alcohols) to potentiate GABA(A) receptors and to inhibit N-methyl-D-aspartate receptors increases with carbon chain length, we have found that n-alcohols, depending on the carbon chain length, exert a dual action, potentiation and inhibition, on nnAChRs in primary cultured rat cortical neurons. The mechanism of dual action of n-alcohols on nnAChRs was further analyzed using human embryonic kidney cells expressing the alpha 4 beta 2 subunits. Shorter chain alcohols from methanol to n-propanol potentiated acetylcholine (ACh)-induced currents, whereas longer chain alcohols from n-pentanol to n-dodecanol inhibited the currents. n-Butanol either potentiated or inhibited the currents depending on the concentrations of ACh and butanol. The parameters for both potentiation (log EC(200)) and inhibition (log IC(50)) were linearly related to carbon number, albeit with different slopes. The slope for potentiation was -0.299, indicating a change in free energy change (Delta Delta G) of 405 cal/mol/methylene group, whereas the slope for inhibition was -0.584, indicating a Delta Delta G of 792 cal/mol. These results suggest that potentiating and inhibitory actions are exerted through two different binding sites. Ethanol decreased the potency of n-octanol to inhibit ACh currents, possibly resulting from an allosteric mechanism.

1-Octanol↗
PubMed2000

Neonatal mortality in an aquaporin-2 knock-in mouse model of recessive nephrogenic diabetes insipidus.

Hereditary non-X-linked nephrogenic diabetes insipidus (NDI) is caused by mutations in the aquaporin-2 (AQP2) water channel. In transfected cells, the human disease-causing mutant AQP2-T126M is retained at the endoplasmic reticulum (ER) where it is functional and targetable to the plasma membrane with chemical chaperones. A mouse knock-in model of NDI was generated by targeted gene replacement using a Cre-loxP strategy. Along with T126M, mutations H122S, N124S, and A125T were introduced to preserve the consensus sequence for N-linked glycosylation found in human AQP2. Breeding of heterozygous mice yielded the expected Mendelian distribution with 26 homozygous mutant offspring of 99 live births. The mutant mice appeared normal at 2-3 days after birth but failed to thrive and generally died by day 6 if not given supplemental fluid. Urine/serum analysis showed a urinary concentrating defect with serum hyperosmolality and low urine osmolality that was not increased by a V2 vasopressin agonist. Northern blot analysis showed up-regulated AQP2-T126M transcripts of identical size to wild-type AQP2. Immunoblots showed complex glycosylation of wild-type AQP2 but mainly endoglycosidase H-sensitive core glycosylation of AQP2-T126M indicating ER-retention. Biochemical analysis revealed that the AQP2-T126M protein was resistant to detergent solubilization. Kidneys from mutant mice showed collecting duct dilatation, papillary atrophy, and unexpectedly, some plasma membrane AQP2 staining. The severe phenotype of the AQP2 mutant mice compared with that of mice lacking kidney water channels AQP1, AQP3, and AQP4 indicates a critical role for AQP2 in neonatal renal function in mice. Our results establish a mouse model of human autosomal NDI and provide the first in vivo biochemical data on a disease-causing AQP2 mutant.

Animals↗
PubMed2000

Localization of nicotinic receptor subunit mRNAs in monkey brain by in situ hybridization.

Nicotinic receptors are implicated in memory, learning, locomotor activity, and addiction. Identification of the specific receptor subtypes that mediate these behaviors is essential for understanding their role in central nervous system (CNS) function. Although expression of nicotinic receptor transcript has been studied in rodent brain, their localization in the monkey CNS, which may be a better model for the human brain, is not yet known. We therefore investigated the distribution of alpha4, alpha6, alpha7, beta2, beta3, and beta4 receptors subunit mRNAs in the monkey brain by using in situ hybridization. alpha4 and alpha7 mRNAs were very widely expressed, with a substantial degree of overlap in their distribution, except for the reticular nucleus of the thalamus in which alpha7 mRNA was much more prominently expressed. beta2 and beta4 mRNA were also widely distributed, although beta4 was more prominently localized in thalamic nuclei than beta2. The distribution of alpha6 and beta3 mRNA was very distinct from that of the other transcripts, being restricted to catecholaminergic nuclei, the cerebellum, and a few other areas. Although there were similarities in distribution of the nicotinic receptor subunit mRNAs in monkey and rodent brain, there were prominent differences in areas such as the caudate, putamen, locus coeruleus, medial habenula, and cerebellum. In fact, the distribution of alpha4 and alpha7 mRNAs in the monkey caudate and putamen was more similar to that reported in the human than rodent brain. These findings have implications for the development of drug therapies for neurological disorders, such as Alzheimer's and Parkinson's disease, in which nicotinic receptors are decreased.

Animals↗
PubMed2000

Nephrogenic diabetes insipidus in mice lacking aquaporin-3 water channels.

Aquaporin-3 (AQP3) is a water channel expressed at the basolateral plasma membrane of kidney collecting-duct epithelial cells. The mouse AQP3 cDNA was isolated and encodes a 292-amino acid water/glycerol-transporting glycoprotein expressed in kidney, large airways, eye, urinary bladder, skin, and gastrointestinal tract. The mouse AQP3 gene was analyzed, and AQP3 null mice were generated by targeted gene disruption. The growth and phenotype of AQP3 null mice were grossly normal except for polyuria. AQP3 deletion had little effect on AQP1 or AQP4 protein expression but decreased AQP2 protein expression particularly in renal cortex. Fluid consumption in AQP3 null mice was more than 10-fold greater than that in wild-type litter mates, and urine osmolality (<275 milliosmol) was much lower than in wild-type mice (>1,200 milliosmol). After 1-desamino-8-d-arginine-vasopressin administration or water deprivation, the AQP3 null mice were able to concentrate their urine partially to approximately 30% of that in wild-type mice. Osmotic water permeability of cortical collecting-duct basolateral membrane, measured by a spatial filtering optics method, was >3-fold reduced by AQP3 deletion. To test the hypothesis that the residual concentrating ability of AQP3 null mice was due to the inner medullary collecting-duct water channel AQP4, AQP3/AQP4 double-knockout mice were generated. The double-knockout mice had greater impairment of urinary-concentrating ability than did the AQP3 single-knockout mice. Our findings establish a form of nephrogenic diabetes insipidus produced by impaired water permeability in collecting-duct basolateral membrane. Basolateral membrane aquaporins may thus provide blood-accessible targets for drug discovery of aquaretic inhibitors.

Amino Acid Sequence↗
PubMed2000

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK293 cells.

HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.

Blotting, Northern↗
PubMed2000

Differential alterations in nicotinic receptor alpha6 and beta3 subunit messenger RNAs in monkey substantia nigra after nigrostriatal degeneration.

Our previous studies showed that alpha4, alpha6, alpha7, beta2, beta3 and beta4 nicotinic acetylcholine receptor messenger RNAs are present in monkey substantia nigra, with a particularly intense and localized labelling of the alpha6 and beta3 subunit messenger RNAs to this brain region. Because loss of nigrostriatal neurons is a central feature of Parkinson's disease and evidence suggests that nicotinic agonists potentiate antiparkinsonian effects of L-dopa, experiments were done to determine whether nicotinic receptor subunit messenger RNAs and binding sites were altered in the basal ganglia after nigrostriatal degeneration. Squirrel monkeys (Saimiri sciureus) were rendered parkinsonian by systemic injection of the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine. Behavioral studies showed that this treatment decreased baseline motor activity to 36+/-11% of control. One month after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, caudate and putamen dopamine levels were reduced to 51+/-15% and 43+/-6% of control, respectively, while the number of tyrosine hydroxylase-positive neurons in the substantia nigra was 75+/-6% of control. Despite the reduction in nigral cell number after nigrostriatal degeneration, there were no changes in alpha4, alpha7, beta2 and beta4 messenger RNA levels in the substantia nigra. In contrast, alpha6 mRNA levels were significantly increased (143+/-10%) and the beta3 transcript decreased (62+/-6%) in the substantia nigra after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Declines were also observed in [125I]epibatidine binding in both the caudate-putamen and substantia nigra, with no change in alpha7 receptor binding. These results may suggest a dissociation in the regulation of receptor messenger RNA and binding sites, and/or that there are differential alterations in the different receptor subtypes measured using [125I]epibatidine. The changes in the two nicotinic receptor subunit messenger RNAs, alpha6 and beta3, which exhibit a selective localization to the substantia nigra, may indicate that nicotinic receptors containing these subunits are altered after nigrostriatal degeneration.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1999

Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels.

Aquaporin-5 (AQP5) is a water-selective transporting protein expressed in epithelial cells of serous acini in salivary gland. We generated AQP5 null mice by targeted gene disruption. The genotype distribution from intercross of founder AQP5 heterozygous mice was 70:69:29 wild-type:heterozygote:knockout, indicating impaired prenatal survival of the null mice. The knockout mice had grossly normal appearance, but grew approximately 20% slower than litter-matched wild-type mice when placed on solid food after weaning. Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wild-type mice, the saliva from knockout mice was hypertonic (420 mosM) and dramatically more viscous. Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Water channels AQP1 and AQP4 have also been localized to salivary gland; however, pilocarpine stimulation studies showed no defect in the volume or composition of saliva in AQP1 and AQP4 knockout mice. These results implicate a key role for AQP5 in saliva fluid secretion and provide direct evidence that high epithelial cell membrane water permeability is required for active, near-isosmolar fluid transport.

Amino Acid Sequence↗
PubMed1999

Evaluation of a three-year school-based intervention to increase adolescent sun protection.

The efficacy of a school-based intervention was evaluated using a randomized controlled trial in Australia. In consecutive grades (8, 9, and 10), students in the intervention group received components of a program that addressed issues related to the need to protect yourself from the sun, behavioral strategies related to using sun-protective measures, personal and social images of having a tan, the use of sun-safe clothing, and how to change their schools through forms of structural change. Pre- and postintervention measures among junior high school students showed greatest improvement in the intervention group's knowledge scores and minimal changes in sun protection behavior from Grade 8 to Grade 9, which were not maintained through Grade 10. Results of the study highlight some limitations of school-based interventions for changing sun protection behaviors.

Adolescent↗
PubMed1998

Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels.

Water channel aquaporin-1 (AQP1) is strongly expressed in kidney in proximal tubule and descending limb of Henle epithelia and in vasa recta endothelia. The grossly normal phenotype in human subjects deficient in AQP1 (Colton null blood group) and in AQP4 knockout mice has suggested that aquaporins (other than the vasopressin-regulated water channel AQP2) may not be important in mammalian physiology. We have generated transgenic mice lacking detectable AQP1 by targeted gene disruption. In kidney proximal tubule membrane vesicles from knockout mice, osmotic water permeability was reduced 8-fold compared with vesicles from wild-type mice. Although the knockout mice were grossly normal in terms of survival, physical appearance, and organ morphology, they became severely dehydrated and lethargic after water deprivation for 36 h. Body weight decreased by 35 +/- 2%, serum osmolality increased to >500 mOsm, and urinary osmolality (657 +/- 59 mOsm) did not change from that before water deprivation. In contrast, wild-type and heterozygous mice remained active after water deprivation, body weight decreased by 20-22%, serum osmolality remained normal (310-330 mOsm), and urine osmolality rose to >2500 mOsm. Urine [Na+] in water-deprived knockout mice was <10 mM, and urine osmolality was not increased by the V2 agonist DDAVP. The results suggest that AQP1 knockout mice are unable to create a hypertonic medullary interstitium by countercurrent multiplication. AQP1 is thus required for the formation of a concentrated urine by the kidney.

Animals↗
PubMed1998

Regulation of nicotinic receptor subtypes following chronic nicotinic agonist exposure in M10 and SH-SY5Y neuroblastoma cells.

The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick alpha4beta2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the Ki values for binding inhibition and EC50 values for agonist-induced alpha4beta2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing alpha3, alpha5, beta2, and beta4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.

Animals↗
PubMed1998

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed1998

Phase II trial of topotecan administered as 72-hour continuous infusion in children with refractory solid tumors: a collaborative Pediatric Branch, National Cancer Institute, and Children's Cancer Group Study.

The antitumor activity of topotecan administered as a 72-h continuous i.v. infusion was evaluated in children with refractory neuroblastoma and sarcomas of soft tissue and bone. We also attempted to increase the dose intensity of topotecan by including an intrapatient dose escalation in the trial design. Ninety-three children (85 eligible and evaluable for response) with recurrent or refractory neuroblastoma, osteosarcoma, Ewing's sarcoma/peripheral neuroectodermal tumor, rhabdomyosarcoma, or other soft-tissue sarcomas received topotecan administered as a 72-h i.v. infusion every 21 days. The initial dose was 1.0 mg/m2/day, with subsequent intrapatient dose escalation to 1.3 mg/m2/day for those patients who did not experience dose-limiting toxicity after their first cycle of topotecan. There was one complete response in a patient with neuroblastoma (n = 26) and one partial response in a patient with Ewing's sarcoma/peripheral neuroectodermal tumor (n = 25). No complete or partial responses were observed in 17 patients with osteosarcoma, 15 patients with rhabdomyosarcoma, or 2 patients with other soft-tissue sarcomas; however, 8 patients had prolonged (15-48 weeks) stable disease while receiving topotecan. Topotecan was well tolerated. The most commonly observed toxicities were myelosuppression (dose-limiting) and nausea and vomiting. Intrapatient dose escalations were performed in 68% of the patients who received more than one cycle of topotecan, and 1.3 mg/m2/day was tolerated by 79% of the patients who received the higher dose and were evaluable for hematological toxicity. In conclusion, topotecan administered as a 72-h continuous infusion every 21 days is inactive (objective response rate, < 20%) in children with refractory or recurrent neuroblastoma and sarcomas of soft tissue or bone.

Adolescent↗
PubMed1997

Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4.

Aquaporin-4 (AQP4) is a mercurial-insensitive, water-selective channel that is expressed in astroglia and basolateral plasma membranes of epithelia in the kidney collecting duct, airways, stomach, and colon. A targeting vector for homologous recombination was constructed using a 7-kb SacI AQP4 genomic fragment in which part of the exon 1 coding sequence was deleted. Analysis of 164 live births from AQP4[+/-] matings showed 41 [+/+], 83 [+/-], and 40 [-/-] genotypes. The [-/-] mice expressed small amounts of a truncated AQP4 transcript and lacked detectable AQP4 protein by immunoblot analysis and immunocytochemistry. Water permeability in an AQP4-enriched brain vesicle fraction in [+/+] mice was high and mercurial insensitive, and was decreased by 14-fold in [-/-] mice. AQP4 deletion did not affect growth or tissue morphology at the light microscopic level. Northern blot analysis showed that tissue-specific expression of AQPs 1, 2, 3, and 5 was not affected by AQP4 deletion. Maximum urine osmolality after a 36-h water deprivation was (in mosM, n = 15) [+/+] 3,342+/-209, [+/-] 3, 225+/-167, and [-/-] 2,616+/-229 (P < 0.025), whereas urine osmolalities before water deprivation did not differ among the genotypes. Rotorod analysis of 35- 38-d-old mice revealed no differences in neuromuscular function (performance time in s, n = 8): [+/+] 297+/-25, [+/-] 322+/-28, [-/-] 288+/-37. These results indicate that AQP4 deletion in CD1 mice has little or no effect on development, survival, growth, and neuromuscular function, but produces a small defect in urinary concentrating ability consistent with its expression in the medullary collecting duct.

Animals↗
PubMed1997

The expression of voltage-dependent calcium channel beta subunits in human cerebellum.

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.

Aged↗
PubMed1997

Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channel subunits.

Neuronal voltage-dependent calcium channels (VDCCs) each comprising of alpha 1, alpha 2 delta, and beta subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation Studies have shown the expression of several alpha 1 and beta subtypes within neuronal tissue. The comparative distribution of these in normal human brain is largely unknown. The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain. Previous studies have defined DNA sequences specific for each subunit Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E. coli. Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns. The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding alpha 1, alpha 2 delta and beta subunits.

Animals↗