PubMed Health⌕ Search

Biomedical subjects

Claes Wahlestedt

Publications and source records attributed to Claes Wahlestedt.

52 records · Page 3Linked to original sources
PubMed2003

Small GTP-binding protein Rac is an essential mediator of vascular endothelial growth factor-induced endothelial fenestrations and vascular permeability.

BACKGROUND: Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces both angiogenesis and vascular permeability. Although its angiogenic activity has been well characterized, the signaling pathways of VEGF-induced permeability remain poorly understood. METHODS AND RESULTS: Using the mouse corneal micropocket assay, Miles assay, and a combination of cytochemical, electron microscopic, and biochemical assays, we demonstrate that VEGF-induced vascular leakage partly can be separated from its angiogenic activity. VEGF but not FGF-2 induced capillaries with a highly fenestrated endothelium, a feature linked with increased vascular permeability. A cell-permeable Rac antagonist (TAT-RacN17) converted VEGF-induced, leaky vascular plexuses into well-defined vascular networks. In addition, this Rac mutant blocked formation of VEGF-induced endothelial fenestrations and vascular permeability but only partially inhibited angiogenesis. Studies on endothelial cell cultures further revealed that VEGF stimulated phosphorylation of VEGF receptor-2 (VEGFR-2), leading to activation of Rac as well as increased phosphorylation of phospholipase Cgamma (PLCgamma), protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), and extracellular regulated kinase (Erk1/2). We further found that phosphatidylinositol-3-OH kinase (PI3K) acted upstream of Rac and Akt-eNOS in VEGF/VEGFR-2 signaling. CONCLUSIONS: Our findings indicate that the small GTP-binding protein Rac is a key component in mediation of VEGF-induced vascular permeability but less so in neovascularization. This may have conceptual implications for applying Rac antagonists in treatment and prevention of VEGF-induced vascular leakage and edema in connection with ischemic disorders.

Animals↗
PubMed2003

Activity-induced and developmental downregulation of the Nogo receptor.

The three axon growth inhibitory proteins, myelin associated glycoprotein, oligodendrocyte-myelin glycoprotein and Nogo-A, can all bind to the Nogo-66 receptor (NgR). This receptor is expressed by neurons with high amounts in regions of high plasticity where Nogo expression is also high. We hypothesized that simultaneous presence of high levels of Nogo and its receptor in neurons confers a locked state to hippocampal and cortical microcircuitry and that one or both of these proteins must be effectively and temporarily downregulated to permit plastic structural changes underlying formation of long-term memory. Hence, we subjected rats to kainic acid treatment and exposed rats to running wheels and measured NgR mRNA levels by quantitative in situ hybridization at different time points. We also studied spinal cord injuries and quantified NgR mRNA levels in spinal cord and ganglia during a critical postnatal period using real-time PCR. Strikingly, kainic acid led to a strong transient downregulation of NgR mRNA levels in gyrus dentatus, hippocampus, and neocortex during a time when BDNF mRNA was upregulated instead. Animals exposed to running wheels for 3 and 7, but not 1 or 21, days showed a significant downregulation of NgR mRNA in cortex, hippocampus and the dentate gyrus. NgR mRNA levels decreased from high to low expression in spinal cord and ganglia during the first week of life. No robust regulation of NgR was observed in the spinal cord following spinal cord injury. Together, our data show that NgR levels in developing and adult neurons are regulated in vivo under different conditions. Strong, rapid and transient downregulation of NgR mRNA in response to kainic acid and after wheel running in cortex and hippocampus suggests a role for NgR and Nogo-A in plasticity, learning and memory.

Animals↗
PubMed2003

Functional interactions between delta- and mu-opioid receptors in rat thermoregulation.

The selective delta-opioid receptor agonist deltorphin II (25.0-100.0 microg, i.c.v.) produced biphasic effects on core temperature in rats, in which hypothermia was followed by hyperthermia. Pretreatment with the selective delta-opioid receptor antagonist, naltrindole (25.0 microg, i.c.v.), blocked hypothermia produced by deltorphin II and had a tendency to potentiate the hyperthermic effect of deltorphin II. The non-selective opioid receptor antagonist naloxone (1.5 mg kg(-1), s.c.) potentiated hypothermia, and blocked hyperthermia, produced by deltorphin II (100.0 microg). Also, naloxone potentiated hypothermia produced by a lower dose of deltorphin II (25.0 microg), which did not produce hyperthermia. A similar pattern was found for the selective mu-opioid receptor antagonist, beta-funaltrexamine (5.0 microg, i.c.v.), which potentiated and blocked deltorphin II-induced hypo- and hyperthermia, respectively. The selective kappa-opioid receptor antagonist nor-binaltorphimine (20.0 microg, i.c.v.) had no effects on deltorphin II-induced temperature changes. The present results suggest that deltorphin II produces hypothermia through activation of delta-opioid receptors, whereas the hyperthermic effect of deltorphin II involves activation of mu-opioid receptors. This mu-opioid receptor stimulatory effect of deltorphin II is furthermore more pronounced than was anticipated based on the reported in vitro properties of this compound. The biphasic effect of deltorphin II implies a negative interaction between delta- and mu-opioid receptors in thermoregulation in rats.

Animals↗
PubMed2003

Anti-cataleptic effects of clozapine, but not olanzapine and quetiapine, on SCH 23390- or raclopride-induced catalepsy in rats.

The present study investigated potential anti-cataleptic properties of the prototype atypical antipsychotic clozapine and two newly developed atypical antipsychotics, olanzapine and quetiapine, which are structurally related and display similar pharmacological profiles to clozapine. Clozapine (2.5 mg kg(-1), s.c.), but not olanzapine (2.0 mg kg(-1), s.c.) and quetiapine (20.0 mg kg(-1), s.c.), blocked catalepsy induced either by the dopamine D(1/5) receptor antagonist SCH 23390 (50.0 microg kg(-1), s.c) or the selective dopamine D(2/3) receptor antagonist raclopride (4.0 mg kg(-1), s.c.). Such findings are consistent with the beneficial effects of clozapine in the management of drug-induced psychosis in parkinsonian patients, and suggest that neither olanzapine nor quetiapine may be a safe alternative to clozapine in this field. Furthermore, the results indicate that clozapine has a unique pharmacological profile that distinguishes it from olanzapine and quetiapine. The mechanisms underlying anti-cataleptic or anti-parkinsonian properties of clozapine are unclear but may be related to dopamine D(1) receptor agonism of clozapine.

Animals↗
PubMed2003

NPY Leu7Pro and alcohol dependence in Finnish and Swedish populations.

BACKGROUND: Neuropeptide Y (NPY) is a modulator of alcohol intake in animal models of alcoholism, and is potentially involved in alcohol dependence. A coding Leu7Pro polymorphism in the signal peptide of preproNPY has been described, and the Pro7 allele has been reported to correlate with increased alcohol consumption in non-dependent Finnish males. Recently, this polymorphism was also reported to be associated with an actual diagnosis of alcohol dependence. METHODS: We compared Pro7 allele frequencies in one Finnish (n = 135) and one Swedish (n = 472) population of alcohol dependent individuals, and ethnically matched controls (Finns: n = 213; Swedes: n = 177) in whom alcohol dependence was established, or any diagnosis of substance disorder was excluded, respectively, through the use of structured face-to-face interviews. RESULTS: Pro7 frequencies in alcoholics were 5.2 and 4.1% in Finns and Swedes, respectively, similar to the 5.0-5.5% recently reported in European Americans in a Yale study. However, corresponding frequencies in the control populations were similar, at 6.1 and 5.9% in Finns and Swedes, respectively, yielding no association, in contrast with the Yale study, where an association was reported based on a 2.0% Pro7 frequency in European American controls. A meta-analysis of available data yields Pro7 frequencies of 4.7% both in Caucasian alcoholics and Caucasian controls. CONCLUSIONS: Pro7 does not seem to be associated with a diagnosis of alcoholism in Caucasian populations.

Alcoholism↗
PubMed2003

GeneLynx mouse: integrated portal to the mouse genome.

GeneLynx Mouse is a meta-database providing an extensive collection of hyperlinks to mouse gene-specific information in diverse databases available via the Internet. The GeneLynx project is based on the simple notion that given any gene-specific identifier (e.g., accession number, gene name, text, or sequence), scientists should be able to access a single location that provides a set of links to all the publicly available information pertinent to the specified gene. The recent climax in the mouse genome and RIKEN cDNA sequencing projects provided the data necessary for the development of a gene-centric mouse information portal based on the GeneLynx ideals. Clusters of RIKEN cDNA sequences were used to define the initial set of mouse genes. Like its human counterpart, GeneLynx Mouse is designed as an extensible relational database with an intuitive and user-friendly Web interface. Data is automatically extracted from diverse resources, using appropriate approaches to maximize the coverage. To promote cross-database interoperability, an indexing utility is provided to facilitate the establishment of hyperlinks in external databases. As a result of the integration of the human and mouse systems, GeneLynx now serves as a powerful comparative genomics data mining resource. GeneLynx Mouse can be freely accessed at http://mouse.genelynx.org.

Animals↗
PubMed2002

Quantitative codon optimisation of DNA libraries encoding sub-random peptides: design and characterisation of a novel library encoding transmembrane domain peptides.

Codons for amino acids sharing similar chemical properties seem to cluster on the genetic codon table. Such a geographical distribution of the codons was exploited to create chemically synthesised DNA that encodes peptide libraries containing only a subset of the 20 natural amino acids. The frequency of each amino acid in the subset was further optimised by quantitatively manipulating the ratio of the four phosphoamidites during chemical synthesis of the libraries. Peptides encoded by such libraries show a reduced complexity and could be enriched in peptides of a desired property, which are thus more suitable when screening for functional peptides. Proof of concept for the codon-biased design of peptide libraries was shown by design, synthesis, and characterisation of a transmembrane peptide library that contains >80% transmembrane peptides, representing a 160-fold enrichment compared with a fully randomised library.

Amino Acid Sequence↗
PubMed2002

NotI subtraction and NotI-specific microarrays to detect copy number and methylation changes in whole genomes.

Methylation, deletions, and amplifications of cancer genes constitute important mechanisms in carcinogenesis. For genome-wide analysis of these changes, we propose the use of NotI clone microarrays and genomic subtraction, because NotI recognition sites are closely associated with CpG islands and genes. We show here that the CODE (Cloning Of DEleted sequences) genomic subtraction procedure can be adapted to NotI flanking sequences and to CpG islands. Because the sequence complexity of this procedure is greatly reduced, only two cycles of subtraction are required. A NotI-CODE procedure can be used to prepare NotI representations (NRs) containing 0.1-0.5% of the total DNA. The NRs contain, on average, 10-fold less repetitive sequences than the whole human genome and can be used as probes for hybridization to NotI microarrays. These microarrays, when probed with NRs, can simultaneously detect copy number changes and methylation. NotI microarrays offer a powerful tool with which to study carcinogenesis.

Animals↗
PubMed2002

NotI flanking sequences: a tool for gene discovery and verification of the human genome.

A set of 22 551 unique human NotI flanking sequences (16.2 Mb) was generated. More than 40% of the set had regions with significant similarity to known proteins and expressed sequences. The data demonstrate that regions flanking NotI sites are less likely to form nucleosomes efficiently and resemble promoter regions. The draft human genome sequence contained 55.7% of the NotI flanking sequences, Celera's database contained matches to 57.2% of the clones and all public databases (including non-human and previously sequenced NotI flanks) matched 89.2% of the NotI flanking sequences (identity > or =90% over at least 50 bp, data from December 2001). The data suggest that the shotgun sequencing approach used to generate the draft human genome sequence resulted in a bias against cloning and sequencing of NotI flanks. A rough estimation (based primarily on chromosomes 21 and 22) is that the human genome contains 15 000-20 000 NotI sites, of which 6000-9000 are unmethylated in any particular cell. The results of the study suggest that the existing tools for computational determination of CpG islands fail to identify a significant fraction of functional CpG islands, and unmethylated DNA stretches with a high frequency of CpG dinucleotides can be found even in regions with low CG content.

Cell Line, Transformed↗
PubMed2002

hUNC93B1: a novel human gene representing a new gene family and encoding an unc-93-like protein.

We have identified a novel human gene UNC93B1 encoding a protein related to unc-93 of Caenorhabditis elegans. The combined sequence derived from several cDNA clones is 2282 bp and comparison with genomic sequence shows that the gene contains 11 exons. The longest open reading frame encodes a deduced sequence of 597 amino acids. Homology analysis shows that the hUNC93B1 gene is highly conserved and related to sequences in Arabidopsis thaliana, C. elegans, Drosophila melanogaster, chicken and mouse. Structural analysis of the deduced amino acid sequence of hUNC93B1 points to possible existence of multiple membrane-spanning domains. hUNC93B1 protein also displays some similarities to the bacterial ABC-2 type transporter signature and to ion transporters of Deinococcus radiodurans and Helicobacter pylori. As revealed by Northern analysis, the level of expression varies significantly between tissues, with the highest level detected in the heart. The gene was mapped to chromosomal band 11q13 by fluorescence in situ hybridization. We suggest that this gene is a member of a novel hUNC93B-related gene family.

Amino Acid Sequence↗
PubMed2002

A new approach to genome mapping and sequencing: slalom libraries.

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with approximately 46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of approximately 100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5-10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.

Chromosomes, Artificial, Human↗
PubMed2002

Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model.

Although implantation of fetal dopamine (DA) neurons can reduce parkinsonism in patients, current methods are rudimentary, and a reliable donor cell source is lacking. We show that transplanting low doses of undifferentiated mouse embryonic stem (ES) cells into the rat striatum results in a proliferation of ES cells into fully differentiated DA neurons. ES cell-derived DA neurons caused gradual and sustained behavioral restoration of DA-mediated motor asymmetry. Behavioral recovery paralleled in vivo positron emission tomography and functional magnetic resonance imaging data demonstrating DA-mediated hemodynamic changes in the striatum and associated brain circuitry. These results demonstrate that transplanted ES cells can develop spontaneously into DA neurons. Such DA neurons can restore cerebral function and behavior in an animal model of Parkinson's disease.

Adrenergic Agents↗
PubMed2001

A novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors.

The baculovirus Autographa californica multiple nucleopolyhedrosis virus causes non-productive infection in mammalian cells. Recombinant baculovirus therefore has the capability to transfer and express heterologous genes in these cells if a mammalian promoter governs the gene of interest. We have investigated the possibility of using baculovirus as a tool to produce recombinant adeno-associated virus (rAAV). AAV has become increasingly popular as a vector for gene therapy and functional genomics efforts, although its use is hampered by the lack of a simple and efficient vector production method. We show here that co-infection of mammalian producer cells with three viruses - a baculovirus containing the reporter gene flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a helper adenovirus expressing the AAV cap gene - produces infectious rAAV particles. This baculovirus-based chimeric vector method may in future improve large-scale rAAV vector preparations and circumvent present-day problems associated with rAAV production.

Baculoviridae↗
PubMed2000

Antisense PNA effects in Escherichia coli are limited by the outer-membrane LPS layer.

Antisense peptide nucleic acids (PNAs) can inhibit Escherichia coli gene expression and cell growth through sequence-specific RNA binding, and this opens possibilities for novel anti-infective agents and tools for microbial functional genomics. However, the cellular effects of PNAs are limited relative to effects in cell extracts, presumably because of cell barrier components such as the outer-membrane lipopolysaccharide (LPS) layer or drug efflux pumps, both of which function to exclude antibiotics and other foreign molecules. To evaluate the importance of such cellular factors on PNA effects, the authors developed a positive assay for antisense inhibition by targeting the lac operon repressor and compared PNA susceptibilities in mutant and wild-type E. coli by assessing lacZ induction. Strains with defective LPS (AS19 and D22) were more permeable to the antibiotic nitrocefin and more susceptible to PNA than the wild-type. Also, PNA potency was improved in wild-type cells grown in the presence of certain cell-wall-permeabilizing agents. In contrast, the activities of the Acr and Emr drug efflux pumps were not found to affect PNA susceptibility. The results show that the LPS layer is a major barrier against cell entry, but PNAs that can enter E. coli are likely to remain active inside cells.

Bacterial Proteins↗
PubMedDate not supplied

Decreased cerebrospinal fluid neuropeptide Y (NPY) in patients with treatment refractory unipolar major depression: preliminary evidence for association with preproNPY gene polymorphism.

Extensive animal studies suggest neuropeptide Y (NPY) to be involved in coping with a wide range of stressors, and that impaired central NPY signalling could be involved in the pathophysiology of anxiety and depression. Human studies of central NPY levels in depression have, however, been inconclusive. Here, we examined levels of NPY-like immunoreactivity (NPY-LI) in the cerebrospinal fluid (CSF) of medication-free subjects with treatment refractory unipolar depression. Patients were admitted to a research inpatient unit, examined under standardized conditions, and compared with a sample of volunteers in whom psychiatric morbidity was excluded. A robust suppression of NPY levels in patient CSF was found, while other putative CSF markers (monoamine metabolites, somatostatin) did not differ between the groups. We then explored whether this finding might be related to a recently described T1128C coding polymorphism which results in a Leu7-> Pro7 substitution of the signal peptide, and a previously not described T -399C polymorphism in the promoter region of the preproNPY gene. Preliminary evidence was found for an association of both markers with a diagnosis of depression, indicating the possibility of an underlying haplotype influencing the vulnerability for developing depressive illness. Our present findings are in line with an extensive animal literature, and further support the notion that impaired NPY function could contribute to depressive illness.

Adult↗