C. Wahlestedt - Academia.edu (original) (raw)

Papers by C. Wahlestedt

BioTechniques, 2001

The deletion of specific genomic sequences is believed to influence the pathogenesis of certain d... more The deletion of specific genomic sequences is believed to influence the pathogenesis of certain diseases such as cancer. Identification of these sequences could provide novel therapeutic avenues for the treatment of disease. Here, we describe a simple and robust method called cloning of deleted sequences (CODE), which allows the selective cloning of deleted sequences from complex human genomes. Briefly, genomic DNA from two sources (human normal and tumor samples) was digested with restriction enzymes (e.g., BamHI, BglII, and BclI), then ligated to special linkers, and amplified by PCR. Tester (normal) DNA was amplified using a biotinylated primer and dNTPs. Driver (tumor) DNA was amplified using a non-biotinylated primer, but with dUTP instead of dTTP. After denaturation and hybridization, all the driver DNA was destroyed with uracil-DNA glycosylase (UDG), and all imperfect hybrids were digested with mung bean nuclease. Sequences deleted from the driver DNA but present in the teste...

The Journal of Physiology, 1988

1. Neuropeptide Y (NPY), peptide YY (PYY) and, to a lesser extent, human pancreatic polypeptide (... more 1. Neuropeptide Y (NPY), peptide YY (PYY) and, to a lesser extent, human pancreatic polypeptide (HPP) reduced short-circuit current (SCC) in a concentrationdependent manner in epithelial preparations of rat jejunum and descending colon. 2. From concentration-response curves in the jejunum EC50 values of 3 nm for PYY and 10 nM for NPY were obtained. HPP was much less potent, the threshold concentration being around 100 nm, and NPY 13-36 was inactive. 3. Repeated exposure of jejunal preparations to either NPY or PYY led to a rapid desensitization. Cross-desensitization to the actions of these two peptides was also observed. Neither tetrodotoxin (TTX) nor phentolamine affected responses to either NPY or PYY on the jejunum. 4. Responses to both peptides were inhibited by the presence of transport inhibitors, particularly diphenylamine-2-carboxylate (DPC, a chloride channel blocker) and piretanide (Na+-K+-2Clco-transport inhibitor). These results may indicate that the reduction in SCC caused by the neuropeptides is due to a net increase in chloride movement in the apical to basolateral direction. 5. 36C1-flux studies identified an inhibition of chloride secretion as the predominant mechanism of action of NPY and PYY, together with a smaller stimulation of chloride absorption. No significant changes in the movement of 22Na were seen in either direction. 6. The cyclo-oxygenase inhibitors piroxicam (5 gM) and indomethacin (5 /tM) significantly reduced the responses to both NPY and PYY in rat jejunum. From this and other evidence it was concluded that the peptides depended for their effect on the endogenous formation of eicosanoids, the prevention of which attenuated the SCC reduction due to the peptides.

Brain Research, 2016

Growing evidence indicates that targeting nociceptin receptor (NOP) signaling may have therapeuti... more Growing evidence indicates that targeting nociceptin receptor (NOP) signaling may have therapeutic efficacy in treating alcohol and opioid addiction. However, little is known about the therapeutic value of selective NOP agonists for the treatment of cocaine dependence. Recently, we identified a highly selective, brain-penetrant NOP small molecule agonist (SR-8993), and using this compound, we previously showed that nociceptin receptor activation attenuated consolidation of fear-related memories. Here, we sought to determine whether SR-8993 also affects the rewarding properties of cocaine. Using a conditioned place preference (CPP) procedure, we show that SR-8993 (3 or 10 mg/kg) failed to disrupt acquisition or expression of cocaine CPP (7.5 or 15 mg/kg) in C57BL/6 mice. Additionally, SR-8993 did not affect rate of extinction or reinstatement (yohimbine-and cocaine-induced) of cocaine CPP. These studies indicate that selective activation of NOP may not be sufficient in reducing behavioral responses to cocaine.

Molecular Psychiatry, 2016

Epigenetic processes have been implicated in the pathophysiology of alcohol dependence, but the s... more Epigenetic processes have been implicated in the pathophysiology of alcohol dependence, but the specific molecular mechanisms mediating dependence-induced neuroadaptations remain largely unknown. Here, we found that a history of alcohol dependence persistently decreased the expression of Prdm2, a histone methyltransferase that monomethylates histone 3 at the lysine 9 residue (H3K9me1), in the rat dorsomedial prefrontal cortex (dmPFC). Downregulation of Prdm2 was associated with decreased H3K9me1, supporting that changes in Prdm2 mRNA levels affected its activity. Chromatin immunoprecipitation followed by massively parallel DNA sequencing showed that genes involved in synaptic communication are epigenetically regulated by H3K9me1 in dependent rats. In non-dependent rats, viral-vector-mediated knockdown of Prdm2 in the dmPFC resulted in expression changes similar to those observed following a history of alcohol dependence. Prdm2 knockdown resulted in increased alcohol self-administration, increased aversion-resistant alcohol intake and enhanced stress-induced relapse to alcohol seeking, a phenocopy of postdependent rats. Collectively, these results identify a novel epigenetic mechanism that contributes to the development of alcohol-seeking behavior following a history of dependence.

Advanced Drug Delivery Reviews, 2015

Long non-coding RNAs (lncRNA), a class of non-coding RNA molecules recently identified largely du... more Long non-coding RNAs (lncRNA), a class of non-coding RNA molecules recently identified largely due to the efforts of FANTOM, and later GENCODE and ENCODE consortia, have been a subject of intense investigation in the past decade. Extensive efforts to get deeper understanding of lncRNA biology have yielded evidence of their diverse structural and regulatory roles in protecting chromosome integrity, maintaining genomic architecture, X chromosome inactivation, imprinting, transcription, translation and epigenetic regulation. Here we wil briefly review the recent studies in the field of lncRNA biology focusing mostly on mammalian species and discuss their therapeutic implications.

Regulatory Peptides, 1996

The mouse adrenocortical Y-1 cell line expresses a high level of neuropeptide Y1 receptor (NPY-Y1... more The mouse adrenocortical Y-1 cell line expresses a high level of neuropeptide Y1 receptor (NPY-Y1). Moreover the receptor density can be up-regulated by dexarnethasone or down-regulated by cAMP. To determine whether such regulation occurs at the level of gene expression, Y1 receptor mRNA was measured using a reverse transcriptase-competitive PCR method. Dexamethasone treatment increased Yl mRNA in Y-1 cells, whereas the cAMP and ACTH decreased it. We also observed that the amount of Y1 receptor RNA was unaffected by phorbol 12-mylistate 13-acetate, a protein kinase C stimulator, but was abolished in a cell line expressing apolipoprotein E (apoE). The results indicated that NPY-Y 1 receptor mRNA in Y-1 cells is highly regulated by several intracellular messengers. The role of apoE in such regulation is of particular interest in view of evidence that the isoform of the molecule is highly correlated to the age of onset of Alzheimer's disease. The effect observed in the Y-1 cell line which expresses apoE may implicate a possible role of this protein in the process of neuronal death that occurred in the Alzheimer's disease.

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The Journal of pharmacology and experimental therapeutics, 2000

The cloned delta-opioid receptor (DOR) is being investigated as a potential target for novel anal... more The cloned delta-opioid receptor (DOR) is being investigated as a potential target for novel analgesics with an improved safety profile over mu-opioid receptor agonists such as morphine. The current study used antisense techniques to evaluate the role of DOR in mediating supraspinal antinociception in rats. All of the opioid agonists tested (delta-selective: deltorphin II, DPDPE, pCl-DPDPE, SNC80; mu-selective: DAMGO; i.c.v.) provided significant, dose-dependent antinociception in the paw pressure assay. Administration of a phosphodiester antisense oligonucleotide (i.c.v. ) targeted against DOR inhibited antinociception in response to SNC80, deltorphin II, and pCl-DPDPE compared with mismatch and saline-treated controls. However, antisense treatment did not inhibit the response to DPDPE or DAMGO. In contrast, the highly selective mu-antagonist CTOP blocked antinociception in response to ED(80) concentrations of DAMGO and DPDPE, reduced the response to pCl-DPDPE, and did not alter th...

Molecular pharmacology, 1995

The mouse adrenocortical Y-1 cell line has been found to express high affinity binding sites for ... more The mouse adrenocortical Y-1 cell line has been found to express high affinity binding sites for neuropeptide Y (NPY). Pharmacological studies have shown that these NPY binding sites are of the Y1 type. Reverse transcription-polymerase chain reaction using primers specific for the rat Y1 receptor revealed that the NPY Y1 receptor mRNA is present in Y-1 cells. The Kd of the receptor for NPY was found to be 1.75 +/- 0.20 nM and the Bmax was 265 +/- 18 fmol/mg. The NPY Y1 receptors in this adrenocortical cell line were shown to be coupled to pertussis toxin-sensitive G proteins. Stimulation of Y1 receptors resulted in the inhibition of forskolin- and adrenocorticotropic hormone (ACTH)-stimulated cAMP synthesis. NPY had no effect on basal steroid release from the Y-1 cells. At an ACTH concentration of 0.1 microM, NPY did not affect ACTH-stimulated steroid release, although NPY did inhibit cAMP production under the same hormonal conditions. cAMP profoundly affected the density of the NPY...

Medical biology, 1986

Neuropeptide Y (NPY) is widely distributed in central and peripheral neurons. In sympathetic post... more Neuropeptide Y (NPY) is widely distributed in central and peripheral neurons. In sympathetic postganglionic neurons, NPY coexists with noradrenaline. NPY and its structural relative peptide YY (PYY) appear to exert three principally different effects at the sympathetic neuroeffector junction. Firstly, NPY has a direct postjunctional effect; this effect is manifested as a vasoconstriction when studied on the guinea pig iliac vein. Secondly, NPY has an indirect postjunctional effect in that it potentiates the response to various vasoconstrictors; this was studied on the rabbit femoral artery and vein, using noradrenaline and histamine, respectively, as vasoconstrictors. Thirdly, NPY acts prejunctionally in that it suppresses the release of noradrenaline from sympathetic nerve terminals; this was studied in the rat vas deferens. The aim of the investigation was to examine whether the three effects of NPY were mediated by the same type of receptor. For this purpose, we examined the effe...

Primer on Cerebrovascular Diseases, 1997

This chapter aims to compare the antisense and knockout techniques for investigating the function... more This chapter aims to compare the antisense and knockout techniques for investigating the function of target genes in the central nervous system. Antisense technology is founded on the premise that any cloned gene can be specifically targeted as a consequence of Watson–Crick base pairing. Antisense “knockdown” techniques reduce gene expression by disrupting the flow of information from gene to protein. Antisense molecules prevent gene expression by hybridizing with a complementary target sequence to inhibit factors required for processes involved in the flow of information from gene to protein. Alternatively, homologous recombination “knockout” techniques eliminate gene expression entirely by inactivating the target gene. These methods share a common premise that gene function is best observed in vivo by preventing gene expression and observing the phenotypic changes in these animals in comparison to controls. Gene disruption knockout is performed in order to both identify the function of the target gene and monitor the ability of the animal to compensate for the loss of the gene. As the initial technical challenges with respect to the efficiency of recombination in embryonic stem cells have been largely overcome, the popularity of this technique has been rapidly growing.

International Journal of Alzheimer's Disease, 2011

Background. Alzheimer's disease (AD) is a devastating neurological disorder and the main caus... more Background. Alzheimer's disease (AD) is a devastating neurological disorder and the main cause of dementia in the elderly population worldwide. Adult neurogenesis appears to be upregulated very early in AD pathogenesis in response to some specific aggregates of beta-amyloid (Aβ) peptides, exhausting the neuronal stem cell pools in the brain. Previously, we characterized a conserved nonprotein-coding antisense transcript forβ-secretase-1 (BACE1), a critical enzyme in AD pathophysiology. We showed that the BACE1-antisense transcript (BACE1-AS) is markedly upregulated in brain samples from AD patients and promotes the stability of the (sense) BACE1 transcript. In the current paper, we examine the relationship between BACE1, BACE1-AS, adult neurogenesis markers, and amyloid plaque formation in amyloid precursor protein (APP) transgenic mice (Tg-19959) of various ages.Results. Consistent with previous publications in other APP overexpressing mouse models, we found adult neurogenesis ...

Regulatory Peptides, 1985

Human omental arteries and veins are supplied with nerve fibers containing noradrenaline (NA) and... more Human omental arteries and veins are supplied with nerve fibers containing noradrenaline (NA) and neuropeptide Y (NPY); these two agents probably co-exist in perivascular sympathetic nerve fibers. Substance P (SP)- or vasoactive intestinal peptide (VIP)-containing fibers could not be detected. In studies on isolated omental vessels NA produced constriction. The results of blockade experiments suggest that human omental arteries are equipped predominantly with alpha 1-adrenoceptors and omental veins with a mixture of alpha 1- and alpha 2-adrenoceptors. NPY at a concentration of 10(-7) M or higher had a weak contractile effect on veins and virtually no effect on arteries. NPY at a concentration of 3 X 10(-8) M shifted the NA concentration response curve to the left in arteries (pD2 = 5.8 for NA versus 6.6. for NA in the presence of NPY; P less than 0.001) but not in veins. Both SP and VIP relaxed arteries precontracted with NA or prostaglandin F2 alpha (PGF2 alpha). The potency of SP as a relaxant agent was similar in arteries and veins; the effect of VIP was elicited at lower concentrations in veins than in arteries.

Regulatory Peptides, 1986

Calcitonin gene-related peptide (CGRP) in the anterior uvea coexists with tachykinins (substance ... more Calcitonin gene-related peptide (CGRP) in the anterior uvea coexists with tachykinins (substance P and neurokinin A) in sensory nerve fibers deriving from the trigeminal ganglion. Mechanical or electrical stimulation of the intracranial part of the trigeminal nerve/ganglion in rabbits produced a marked hyperemia in the anterior segment of the eye, increased intraocular pressure, breakdown of the blood-aqueous barrier and miosis. Simultaneously, CGRP-like immunoreactivity was released into the aqueous humor. This suggests that the highly vasoactive CGRP can be released from sensory nerve fibers to participate in vascular responses. Unlike the tachykinins, CGRP per se was without effect on the pupillary diameter while disrupting the bloodaqueous barrier (resulting in aqueous flare) upon intravitreal injection. In addition, CGRP enhanced the aqueous flare evoked by a minimal eye trauma (infrared irradiation of the iris). The miosis evoked by the intravitreal injection of substance P was more pronounced when CGRP was injected simultaneously, and finally, substance P induced aqueous flare much more effectively when given together with a threshold dose of CGRP.

Regulatory Peptides, 1984

Neuropeptide Y (NPY)-immunoreactive nerve fibers were numerous around arteries and few around vei... more Neuropeptide Y (NPY)-immunoreactive nerve fibers were numerous around arteries and few around veins. NPY probably co-exists with noradrenaline in such fibers since chemical or surgical sympathectomy eliminated both NPY and noradrenaline from perivascular nerve fibers and since double staining demonstrated dopamine-beta-hydroxylase, the enzyme that catalyzes the conversion of dopamine to noradrenaline, and NPY in the same perivascular nerve fibers. Studies on isolated blood vessels indicated that NPY is not a particularly potent contractile agent in vitro. NPY greatly enhanced the adrenergically mediate contractile response to electrical stimulation and to application of adrenaline, noradrenaline or histamine, as studied in the isolated rabbit gastro-epiploic and femoral arteries. The potentiating effect of NPY on the response to electrical stimulation is probably not presynaptic since NPY affected neither the spontaneous nor the electrically evoked release of [3H]noradrenaline from perivascular sympathetic nerve fibers.

Journal of Pediatric Surgery, 1987

Specimens from aganglionic (constricted) and ganglionic (dilated) gut were obtained from nine pat... more Specimens from aganglionic (constricted) and ganglionic (dilated) gut were obtained from nine patients with Hirschsprung's disease. Transmural nerve stimulation of ganglionic smooth muscle in vitro evoked an initial relaxation followed by a contraction. This contraction was reduced but not abolished by atropine and it was further reduced by substance P antagonists. Guanethidine did not affect the electrically evoked responses. In aganglionic smooth muscle, an atropine-sensitive contraction but no initial relaxation was registered. Tetrodotoxin abolished all responses to electrical stimulation in both ganglionic and aganglionic specimens. Application of carbachol or substance P produced contraction and the adrenergic agonist isoprenaline or vasoactive intestinal peptide produced relaxation in ganglionic as well as aganglionic specimens. Two other gut neuropeptides, neuropeptide Y and galanin, were without effect. The results do not indicate a different receptor set up in ganglionic v aganglionic gut. The results are compatible with a lack of noncholinergic nonadrenergic inhibitory neurons in the aganglionic gut.

Journal of Clinical Pharmacy and Therapeutics, 2002

A single nucleotide polymorphism (SNP) T1128C, which causes an amino acid change from leucine(7) ... more A single nucleotide polymorphism (SNP) T1128C, which causes an amino acid change from leucine(7) to proline(7) in the signal peptide of neuropeptide Y (NPY), has been found to be associated with multiple clinical parameters or disease. However, this SNP was not identified in the Japanese population. We asked if this is also true in the Korean population. Genotyping was conducted by pyrosequencing, a newly developed real-time high-throughput SNP scoring technique. All 242 Korean subjects showed T1128/T1128 genotype. The T1128C SNP does not appear to exist in this Korean population.

Gene, 2001

A novel human potassium channel gene was identi®ed and isolated. The maximal open reading frame e... more A novel human potassium channel gene was identi®ed and isolated. The maximal open reading frame encodes a protein of 456 amino acids. The predicted product exhibits 91% amino acid identity to the murine voltage-gated potassium channel protein Kv1.7 (Kcna7), which plays an important role in the repolarization of cell membranes. Based on the high similarity, the human gene has been classi®ed as the ortholog of the mouse Kcna7 and given the name Kv1.7 (KCNA7). A structural prediction identi®ed a pore region characteristic of potassium channels and six membrane-spanning domains. Northern expression analysis revealed the gene is expressed preferentially in skeletal muscle, heart and kidney. However, it is expressed at lower level in other tissues, including liver. A single mRNA isoform was observed, with a size of approximately 4.5 kb. Using¯uorescence in situ hybridization, the gene was mapped to chromosomal band 19q13.4 (269.13 cR 3000). A genomic sequence was identi®ed in the database from this region, and the KCNA7 gene structure determined. Computational analysis of the genomic sequence reveals the location of a putative promoter and a likely muscle-speci®c regulatory region. Initial comparison to the published murine Kcna7 cDNA suggested a different N-terminal sequence for the human protein, however, further analysis suggests that the original mouse sequence contained an error or an unusual polymorphism.

European Journal of Pharmacology, 1985

Pilocarpine contracts the sphincter pupillae muscle via an effect on muscarinic receptors and phe... more Pilocarpine contracts the sphincter pupillae muscle via an effect on muscarinic receptors and phenylephrine contracts the dilator pupillae muscle via an effect on alpha-adrenergic receptors. These effects are thought to mimic the action of the parasympathetic and sympathetic nervous systems, respectively. Intracellular injection of substance P (SP) produces an atropine-resistant constriction of the pupil. This response is thought to mimic the effect of local sensory reflexes on the sphincter pupillae muscle, involving SP-containing trigeminal nerve endings. Repeated intraocular injections of tetrodotoxin, a general blocker of nervous conduction, over a period of 3 weeks produced supersensitivity to pilocarpine, phenylephrine and SP in the rabbit iris. These findings support the view that, like acetylcholine and noradrenaline, SP or an SP-like compound acts as a neurotransmitter in the iris. Also, long-term topical application of an SP antagonist, (D-Pro2,D-Trp7,9)-SP or (Arg5,D-Trp7,9)-SP5-11, to the rabbit eye produced supersensitivity to SP but not to pilocarpine, thus supporting the view that the SP antagonists interact specifically with the SP receptors. The isolated rabbit iris sphincter muscle responds to electrical stimulation with a cholinergic twitch followed by a slow non-cholinergic contraction that can be blocked by antagonists to SP. Analysis of the motor activity of the iris sphincter muscle after long-term topical treatment of the eye with an SP antagonist followed by an interval of 2 days after termination of treatment revealed a greatly enhanced non-cholinergic contraction compared with the cholinergic twitch, a finding that seems to be consistent with the idea that supersensitivity to SP had developed.