Biomedical subjects

C Wahlestedt

Publications and source records attributed to C Wahlestedt.

At least 19 recordsLinked to original sources

PubMed2005

Antisense transcription in the mammalian transcriptome.

Antisense transcription (transcription from the opposite strand to a protein-coding or sense strand) has been ascribed roles in gene regulation involving degradation of the corresponding sense transcripts (RNA interference), as well as gene silencing at the chromatin level. Global transcriptome analysis provides evidence that a large proportion of the genome can produce transcripts from both strands, and that antisense transcripts commonly link neighboring "genes" in complex loci into chains of linked transcriptional units. Expression profiling reveals frequent concordant regulation of sense/antisense pairs. We present experimental evidence that perturbation of an antisense RNA can alter the expression of sense messenger RNAs, suggesting that antisense transcription contributes to control of transcriptional outputs in mammals.

Animals↗

PubMed2004

A 68930 and dihydrexidine inhibit locomotor activity and d-amphetamine-induced hyperactivity in rats: a role of inhibitory dopamine D(1/5) receptors in the prefrontal cortex?

The behavioral and biochemical effects of the full dopamine D(1/5) receptor agonists, dihydrexidine and (1R,3S)-1-aminomethyl-5,6-dihydroxy-3-phenylisochroman HCl (A 68930), were examined in rats. Both A 68930 (0-4.6 mg kg(-1), s.c.) and dihydrexidine (0-8.0 mg kg(-1), s.c.) caused a dose-dependent suppression of locomotor activity, as assessed in an open-field. This locomotor suppression was dose-dependently antagonized by the selective dopamine D(1/5) receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine HCl (SCH 23390; 0-5.0 microg kg(-1), s.c.), but not by the selective dopamine D(2/3) receptor antagonist raclopride (0-25.0 microg kg(-1), s.c.). Furthermore, A 68930 and dihydrexidine did not cause any locomotor activity in habituated rats that displayed a very low base-line activity. Neither did A 68930 nor dihydrexidine produce any excessive stereotypies that could possibly interfere with and mask ambulatory activity. In fact, both A 68930 and dihydrexidine potently blocked hyperactivity produced by d-amphetamine (0-4.0 mg kg(-1), s.c.). Such findings traditionally would be interpreted as a sign of potential antipsychotic properties of A 68930 and dihydrexidine. Examination of neuronal activation, as indexed by the immediate early gene c-fos, showed that A 68930 and dihydrexidine caused a highly significant expression of c-fos in the medial prefrontal cortex. This c-fos expression was sensitive to treatment with SCH 23390, but not with raclopride. The effects of A 68930 and dihydrexidine on c-fos expression in caudate putamen or nucleus accumbens were less marked, or undetectable. The results indicate that stimulation of dopamine D(1/5) receptors, possibly in the medial prefrontal cortex, is associated with inhibitory actions on locomotor activity and d-amphetamine-induced hyperactivity. Assuming an important role of prefrontal dopamine D(1/5) receptors in schizophrenia, such inhibitory actions of dopamine D(1/5) receptor stimulation on psychomotor activation may have interesting clinical implications in the treatment of schizophrenia.

Animals↗

PubMed2003

Lack of efficacy of 'naked' small interfering RNA applied directly to rat brain.

UNLABELLED: The intrastriatal infusions of 'naked' small interfering RNA (siRNA) targeted to dopamine D1 receptors (1.0-10.0 nmol over 3 days) did not reduce dopamine D1 receptor messenger RNA levels or receptor protein, assessed by [125I] SCH 23982 binding in intact rats. This was in contrast to results in vitro where a 76% reduction in dopamine D1 receptor ligand binding could be observed. CONCLUSION: The results suggest that synthetic siRNA, when applied directly to rat brain, is not capable of inducing RNA interference.

Animals↗

PubMed2003

Characterization of lacZ complementation deletions using membrane receptor dimerization.

We describe the screening of lacZ deletions in mammalian cells and the discovery of a novel pair of lacZ deletions that can undergo alpha-complementation only when they are fused to peptides that interact with each other. The two lacZ deletions, delta N 11-75 and delta C 82-1023, were first characterized by fusing to two small interacting peptides and were then further analyzed by fusing to three membrane receptors (G protein-coupled receptors alpha 2cAR and D2DRL and receptor tyrosine kinase insulin receptor) that were known to form homodimers in the membrane. Histochemical and quantitative FACS assays demonstrated that the novel deletions have much lower level of association with each other, thus offering a much lower background in monitoring membrane protein interactions compared to previously published lacZ deletions. Furthermore, our method has the exciting potential to monitor simultaneously membrane receptor dimerization and localization to the cell surface of living cells.

Animals↗

PubMed2002

Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

Alternative Splicing↗

PubMed2002

The single nucleotide polymorphism T1128C in the signal peptide of neuropeptide Y (NPY) was not identified in a Korean population.

OBJECTIVE: 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. METHODS: Genotyping was conducted by pyrosequencing, a newly developed real-time high-throughput SNP scoring technique. RESULTS: All 242 Korean subjects showed T1128/T1128 genotype. CONCLUSIONS: The T1128C SNP does not appear to exist in this Korean population.

Adult↗

PubMed2001

Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF.

Most endocrine hormones are produced in tissues and organs with permeable microvessels that may provide an excess of hormones to be transported by the blood circulation to the distal target organ. Here, we investigate whether leptin, an endocrine hormone, induces the formation of vascular fenestrations and permeability, and we characterize its angiogenic property in the presence of other angiogenic factors. We provide evidence that leptin-induced new blood vessels are fenestrated. Under physiological conditions, capillary fenestrations are found in the leptin-producing adipose tissue in lean mice. In contrast, no vascular fenestrations were detected in the adipose tissue of leptin-deficient ob/ob mice. Thus, leptin plays a critical role in the maintenance and regulation of vascular fenestrations in the adipose tissue. Leptin induces a rapid vascular permeability response when administrated intradermally. Further, leptin synergistically stimulates angiogenesis with fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF), the two most potent and commonly expressed angiogenic factors. These findings demonstrate that leptin has another new function-the increase of vascular permeability.

Adipose Tissue↗

PubMed2001

Initial isolation and analysis of the human Kv1.7 (KCNA7) gene, a member of the voltage-gated potassium channel gene family.

A novel human potassium channel gene was identified 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 classified as the ortholog of the mouse Kcna7 and given the name Kv1.7 (KCNA7). A structural prediction identified 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 fluorescence in situ hybridization, the gene was mapped to chromosomal band 19q13.4 (269.13 cR(3000)). A genomic sequence was identified 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-specific 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.

Amino Acid Sequence↗

PubMed2001

A two-dimensional protein map of Caenorhabditis elegans.

A protein map of Caenorhabditis elegans was constructed by using two-dimensional gel electrophoresis followed by peptide mass fingerprinting. A whole worm extract of a mixed population was separated on immobilized pH gradient strips 4-7 L, 3-10 NL, 6-11 L and 12% sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) gels. Gels were stained with colloidal Coomassie blue and 286 spots representing 152 proteins were subsequently identified by matrix-assisted laser desorption/ionization-mass spectrometry after in-gel digestion with trypsin. Most of the identified proteins with known cellular function were enzymes related to carbohydrate and lipid metabolism, or structural proteins with subcellular locations in the cytoplasm, mitochondria or cytoskeleton.

Animals↗

PubMed2001

Cloning of deleted sequences (CODE): A genomic subtraction method for enriching and cloning deleted sequences.

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 d7TP 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 tester DNA were purified with streptavidin magnetic beads, and the cycle was repeated three more times. This procedure resulted in the rapid isolation and efficient cloning of genomic sequences homozygously deleted from the driver DNA sample, but present in the tester DNA fraction.

Base Sequence↗

PubMed2000

A visual intracellular classification strategy for uncharacterized human proteins.

The human cDNA and genomic sequencing projects will result in the identification and isolation of some 140,000 genes, the majority of which lack predicted functions and for which the cellular localizations are not known. The identification and characterization of protein components of specific cell structures and machineries are essential steps not only toward defining functions of genes but also toward understanding cell function and regulation. We describe here a new approach, termed PROLOC, which uses full-length cDNAs for systematic classification of novel proteins as a functional pointer. We have PCR-amplified 25 uncharacterized human genes and expressed the encoded proteins as GFP fusions in a human cell line. This pilot project has identified novel proteins associated with the nucleolus, mitochondria, the ER, the ER-Golgi-intermediate compartment (ERGIC), the GC, the plasma membrane, and cytoplasmic foci. This visual classification approach may be scaled up to handle a large number of novel genes and permit the generation of a global cellular protein localization map. Such information should be valuable for many aspects of functional genomics and cell biology.

Carcinoma, Hepatocellular↗

PubMed2000

Altered behaviour following RNA interference knockdown of a C. elegans G-protein coupled receptor by ingested double stranded RNA.

Using a systemic and continuous delivery method based on feeding on a particular strain of transformed Escherichia coli to induce double stranded RNA-mediated interference, we targeted the product of the npr-1 gene, a putative Caenorhabditis elegans homologue of a neuropeptide Y receptor, a G-protein coupled receptor. We were able to reproduce the social behaviour observed for the naturally occurring npr-1 mutant when wild type N2 Bristol eggs developed in a lawn of bacteria producing double stranded RNA for npr-1. This facile approach may also be useful when studying the function of other worm G-protein coupled receptors.

Animals↗

PubMed2000

Potent and nontoxic antisense oligonucleotides containing locked nucleic acids.

Insufficient efficacy and/or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a high-affinity DNA analog, locked nucleic acid (LNA), confers several desired properties to antisense agents. Unlike DNA, LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. However, like DNA, the LNA/DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain. LNA/DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.

Animals↗

PubMed2000

NotI clones in the analysis of the human genome.

Not I linking clones contain sequences flanking Not I recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of Not I clones in genome research, high density grids with 50 000 Not I linking clones originating from six representative Not I linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of Not I sites in the human genome. A total of 3437 sequences flanking Not I sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity < or =90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of Not I sites with the first exon and suggest that the so-called 3' ESTs can actually be generated from 5'-ends of genes that contain Not I sites in their first exon.

Base Sequence↗

PubMed2000

Dopamine D(2) receptor ribozyme inhibits quinpirole-induced stereotypy in rats.

The injection of a dopamine D(2) receptor hammerhead ribozyme (20 microg) once daily for 5 days into the nucleus accumbens of rats resulted in an inhibition of stereotyped sniffing and locomotor activation produced by the selective dopamine D(2) receptor agonist, quinpirole (0.4 mg kg(-1) s.c.). The results suggest that ribozymes may be useful in the study of in vivo gene function in the brain.

Animals↗

PubMed2000

Cloning of polymorphisms (COP): enrichment of polymorphic sequences from complex genomes.

Here we describe a new procedure (cloning of polymorphisms, COP) for enrichment of single nucleotide polymorphisms (SNPs) that represent restriction fragment length polymorphisms (RFLPs). COP would be applicable to the isolation of SNPs from particular regions of the genome, e.g. CpG islands, chromosomal bands, YACs or PAC contigs. A combination of digestion with restriction enzymes, treatment with uracil-DNA glycosylase and mung bean nuclease, PCR amplification and purification with streptavidin magnetic beads was used to isolate polymorphic sequences from the genomes of two human samples. After only two cycles of enrichment, 80% of the isolated clones were found to contain RFLPs. A simple method for the PCR detection of these polymorphisms was also developed.

Cloning, Molecular↗

PubMed2000

CIS--cloning of identical sequences between two complex genomes.

Development of the methods permitting cloning of identical sequences between two sources of DNA can be very useful for many purposes, including isolation of disease genes. Here we describe a new method called CIS (cloning of identical sequences). A combination of digestion with MvnI, treatment with mung bean nuclease, UDG (uracil-DNA glycosylase) and PCR with 5'-methyl-dCTP and dUTP was used to isolate identical sequences between two micro-cell hybrid lines (MCH). In a control experiment, mouse MCH903.1 and MCH939.2 containing human chromosome 3 from different individuals, were compared using the CIS procedure. Only background fluorescence in-situ hybridization (FISH) was achieved. In another experiment, mouse MCH903.1, containing complete human chromosome 3, and rat MCH429.11, containing a part of human 3q from the same chromosome were compared. The experiment showed that the original MCH429.11 and the DNA purified using the CIS procedure had identical FISH patterns to human metaphase chromosomes, thus demonstrating the efficiency of CIS.

Animals↗

PubMed2000

Isolation and chromosomal localization of a new human retinoblastoma binding protein 2 homologue 1a (RBBP2H1A).

Using a NotI linking clone NR-025 as a probe, we isolated a novel putative member of the RB binding protein family, namely a human retinoblastoma binding protein 2 homologue (RBBP2H1A). The maximal open reading frame encodes a protein of 1681 amino acids. Homology analysis indicated that the predicted product has an overall 56% amino acid identity to RBBP2, which plays an important role in RB tumor suppressor regulation. Many extended regions are 100% identical in amino acids sequences. The degree of nucleotide identity is lower. The structure prediction analysis identified three DNA-binding zinc finger domains and two bipartite nuclear localization signals. Northern expression analysis revealed expression in all tissues; however, the level of expression significantly varied between tissues. The highest level of expression was detected in testis and the lowest in skeletal muscle. The mRNA sizes corresponding to two major products are around 6kb and 7kb. Using fluorescence in situ hybridization, we mapped the gene to chromosomal band 1q32.1.

Amino Acid Sequence↗