Biomedical subjects

K Lundstrom

Publications and source records attributed to K Lundstrom.

At least 19 recordsLinked to original sources

PubMed2006

Structural genomics for membrane proteins.

Structure-based drug discovery has proven useful in improving and shortening the drug development process. The approach of structural genomics to study a large number of targets in parallel has been commonly applied to protein families and even whole genomes. Paradoxically, although membrane proteins represent the largest type of drug targets, up to 70% today, determination of their structure has been modest compared to that of soluble proteins. Because membrane proteins are important for drug discovery an emphasis has been placed on developing technologies and methods to determine membrane protein structures. Several structural genomics initiatives have been established, focusing on the structural biology of membrane proteins.

Animals↗

PubMed2005

Biology and application of alphaviruses in gene therapy.

The broad host cell range and high expression levels of transgenes are features that have made alphaviruses attractive for gene expression studies and gene therapy applications. Particularly, Semliki Forest virus vectors have been applied for large-scale production of recombinant membrane proteins for drug screening purposes and structural biology studies. The high preference of expression in neuronal cells has led to many applications of alphavirus vectors in neuroscience. Studies on localization and transport of recombinant proteins as well as electrophysiological recording have become feasible in primary cultures of neurons and hippocampal slice cultures. Alphaviruses have frequently been used as vaccine vectors for expression of antigens against viruses and tumors. Administration of recombinant viral particles, DNA plasmids or in vitro transcribed RNA has resulted in protection against challenges against lethal viruses and tumors in rodent and primate models. Intratumoral injections of alphavirus vectors expressing reporter and immunostimulatory genes have led to significant tumor regression in mouse models. Modifications of the viral envelope structure have generated targeted Sindbis virus vectors. Astonishingly, conventional Sindbis vectors have demonstrated tumor-specific targeting in animal models due to the high density of laminin receptors on cancer cells. Moreover, encaspulation of Semliki Forest virus vectors in liposomes has provided a means of achieving tumor targeting and protection against the host immune response. Much attention has also been given to the engineering of novel mutant alphavirus vectors with properties such as reduced cytotoxicity, prolonged duration of transgene expression and improved survival of host cells.

Alphavirus↗

PubMed2004

Structural genomics on membrane proteins: mini review.

Structural genomics, structure-based analysis of gene products, has so far mainly concentrated on soluble proteins because of their less demanding requirements for overexpression, purification and crystallisation compared to membrane proteins. This so-called "low-hanging fruit" approach has generated more than 25,000 structures deposited in databases. In contrast, the substantially more complex membrane proteins, in relation to all steps from overexpression to high-resolution structure determination, represent less than 1% of available crystal structures. This is in sharp contrast to the importance of this type of proteins, particularly G protein-coupled receptors (GPCRs), as today 60-70% of the current drug targets are based on membrane proteins. The key to improved success with membrane protein structural elucidation is technology development. The most efficient approach constitutes parallel studies on a large number of targets and evaluation of various systems for expression. Next, high throughput format solubilisation and refolding screening methods for a wide range of detergents and additives in numerous concentrations should be established. Today, several networks dealing with structural genomics approaches of membrane proteins have been initiated, among them the Membrane Protein Network (MePNet) programme that deals with the pharmaceutically important mammalian GPCRs. In MePNet, three overexpression systems have been employed for the evaluation of 101 GPCRs, which has generated large quantities of numerous recombinant GPCRs, compatible for structural biology applications.

Animals↗

PubMed2002

Development of a scintiplate assay for recombinant human alpha(2B)-adrenergic receptor.

A high-throughput solid-phase platform for ligand-binding assays using microtiter plates (Scintiplates) has been developed using the scintillation proximity assay principle. The system has been developed using human alpha(2B)-adrenergic receptor (alpha(2B)-AR) expressed from Semliki Forest virus vectors in CHO cells. Alpha(2B)-AR bind natural (adrenaline and noradrenaline) and synthetic ligands with different affinities to mediate a variety of physiological and pharmacological responses. Antagonist radioligands were used for the binding experiments, and the values obtained for the binding constants with the Scintiplate system are in good agreement with those obtained by the traditional filter-binding assay system. The Scintiplate assay offers the advantages of a high-throughput format over the filter-binding assay and is amenable for screening many compounds rapidly for generation of leads.

Animals↗

PubMed2001

Recombinant Semliki Forest virus for over-expression and pharmacological characterisation of the histamine H(2) receptor in mammalian cells.

We describe the use of recombinant Semliki Forest virus (SFV) vectors for efficient expression of the rat histamine H(2) (rH(2)) receptor in COS-7 (African green monkey kidney cells) cells. Recombinant SFV-infected COS-7 cells express the histamine rH(2) receptor in a time-dependent fashion with a maximum expression level of 50 pmol mg(-1) after 40 h. SFV-mediated histamine rH(2) receptor expression shows similar pharmacological properties as the receptor expressed transiently or stably in mammalian cells. In addition, we demonstrate the pharmacological and functional characterisation of the D(115)N mutated histamine rH(2) receptor. It has been shown that the D(115)N mutation renders the receptor constitutively active and structurally unstable. The rapid onset of and high maximal expression levels obtained from SFV-infected COS-7 cells enabled us to characterise this mutant receptor. We prove that recombinant SFV vectors are powerful tools for heterologous expression of G-protein-coupled receptors and that one can achieve both the high-level gene expression described for baculovirus-infected insect cells and the use of mammalian cells as hosts.

Animals↗

PubMed2001

Semliki Forest virus vectors: efficient vehicles for in vitro and in vivo gene delivery.

Rapidly generated high-titer Semliki Forest virus (SFV) vectors can infect numerous mammalian cell lines and primary cell cultures, and result in high levels of transgene expression. SFV-based expression of transmembrane receptors has been characterized by specific ligand-binding activity and functional responses. Adaptation of the SFV technology for mammalian suspension cultures has allowed the production of hundreds of milligrams of recombinant receptor for purification and structural studies. The same SFV stock solutions used for the infection of mammalian cells in culture have also been successfully applied for efficient transgene expression in organotypic hippocampal slices, as well as in vivo in rodent brain.

Animals↗

PubMed2001

Gene transfer into neurons from hippocampal slices: comparison of recombinant Semliki Forest Virus, adenovirus, adeno-associated virus, lentivirus, and measles virus.

Viral vectors are useful for transferring genes into neurons. Here, we characterized recombinant Semliki Forest virus (SFV), adenovirus type 5 (Ad5), adeno-associated virus type 2 (AAV), lentivirus, and measles virus (MV) by their expression of green fluorescent protein (GFP) in rat hippocampal slice cultures. SFV infected more neurons (>90% of all GFP-positive cells) than AAV, lentivirus, and MV (71, 69, and 62%, respectively), whereas no infected neurons were identified with Ad5. AAV-mediated GFP expression was neuron-specific when the platelet-derived growth factor beta-chain promoter rather than cytomegalovirus promoter was used. Transgene expression occurred rapidly but transiently for SFV, increased slowly but remained stable with AAV and lentivirus, and was fast with MV. Resting membrane potential and conductance, action potentials, firing accommodation, and H-current appeared normal in infected CA1 pyramidal cells. Thus, SFV is useful for short-term and AAV and lentivirus for long-term transduction of hippocampal slices, while MV constitutes a novel vector.

Adenoviridae↗

PubMed2001

Novel mutant Semliki Forest virus vectors: gene expression and localization studies in neuronal cells.

Semliki Forest virus vectors (SFV) are suitable for high-level transgene expression in neuronal tissue, both in vitro and in vivo. Cortical and hippocampal primary neurons in culture are efficiently infected resulting in 75-95% of GFP-positive cells, and injection of SFV vectors into hippocampal slice cultures revealed a highly neuron-specific expression pattern with more than 90% of the infected cells being neurons. Here, we present novel SFV vector mutants and describe their infection patterns obtained in cultures of baby hamster kidney (BHK) cells, dissociated hippocampal neurons, and organotypic hippocampal slices. A less cytotoxic vector SFV(PD), carrying two point mutations in the nsP2 gene, showed much higher GFP expression levels in primary hippocampal neurons compared to the wild-type SFV vector. A triple mutant vector SFV(PDE153) demonstrated a temperature-sensitive phenotype in both BHK cells and primary neurons. In hippocampal slices cultured at 36 degrees C, SFV(PDE153) showed a remarkably higher (ca 250-fold) preference for expression in interneurons rather than in pyramidal cells as compared to wild-type SFV. The quadruple mutant SFV(PDTE) led to substantially increased and prolonged GFP expression in primary neurons. Relative to SFV(PDE153), a more pronounced temperature-sensitive phenotype was found resulting in no virus production and no GFP expression at the non-permissive temperature (36-37 degrees C) in BHK cells, in dissociated neurons, and in organotypic hippocampal slices. The described novel SFV vectors will be useful for several specific applications in neurobiology.

Animals↗

PubMed2001

Coupling of endothelin receptors to the ERK/MAP kinase pathway. Roles of palmitoylation and G(alpha)q.

Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ET(A) and ET(B). To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential G(alpha) protein binding capacity. Endothelin-1 stimulation of wild-type ET(A) or ET(B) induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas full-length nonpalmitoylated ET(A) and ET(B) mutants failed to stimulate ERK. A truncated ET(B) lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of G(alpha)q, but not of G(alpha)i or G(alpha)s, is essential for endothelin-mediated ERK activation. Inhibition of protein kinases A and C or epidermal growth factor receptor kinase failed to prevent ET(A)- and ET(B)-mediated ERK activation whereas blockage of phospholipase C-beta completely abrogated endothelin-promoted ERK activation through ET(A) and ET(B) in recombinant COS-7 and native C6 cells. Complex formation of Ca2+ or inhibition of Src family tyrosine kinases prevented ET-1-induced ERK-2 activation in C6-cells. Our results indicate that endothelin-promoted ERK/MAPK activation criticially depends on palmitoylation but not on phosphorylation of ET receptors, and that the G(alpha)q/phospholipase C-beta/Ca2+/Src signaling cascade is necessary for efficient coupling of ET receptors to the ERK/MAPK pathway.

Amino Acid Sequence↗

PubMed2001

Treatment of resistant mycoplasma infection in immunocompromised patients with a new pleuromutilin antibiotic.

Patients with primary antibody deficiency (PAD) are prone to mycoplasma infection with unusual strains which may be resistant to conventional antibiotics. Mycoplasmas were isolated from the joint fluid (Ureaplasma urealyticum) of two PAD patients with arthritis and from the cerebral spinal fluid (Mycoplasma maculosum) in one with meningitis, the latter probably originating from the patient's dog. Combinations of doxycycline and quinolones or macrolides failed to clear the infections, but after demonstrating in-vitro sensitivity to the pleuromutilin, Econor, for two of the isolates, all three patients responded to oral treatment with Econor. The infection was completely eradicated in two patients, with the emergence of a resistant strain in the third. Mycoplasma infection should be considered in PAD patients with unexplained sepsis. Pleuromutilins such as Econor are powerful new anti-mycoplasmal agents which provide an additional therapeutic option when patients fail to respond to conventional antibiotics.

Adolescent↗

PubMed2001

Interaction of various Piper methysticum cultivars with CNS receptors in vitro.

Methanolic leaf and root extracts of the Hawaiian kava (Piper methysticum Forst.) cultivars, Mahakea, Nene, Purple Moi and PNG, were tested on binding affinities to CNS receptors including GABAA (GABA and benzodiazepine binding site), dopamine D2, opioid (mu and delta), serotonin (5-HT6 and 5-HT7) and histamine (H1 and H2). HPLC analysis was carried out in order to determine the amount of the main kavalactones kavain, 7,8-dihydrokavain, methysticin, 7,8-dihydromethysticin, yangonin and 5,6-demethoxyyangonin. The most potent binding inhibition was observed for leaf extracts to GABAA receptors (GABA binding site) with IC50 values of approximately 3 micrograms/ml, whereas root extracts were less active with IC50 values ranging from 5 micrograms/ml (Nene) to 87 micrograms/ml (Mahakea). Since the leaf extracts generally contained lower amounts of the kavalactones than the root extracts, there might exist additional substances responsible for these activities. Leaf extracts also inhibited binding to dopamine D2, opioid (mu and delta) and histamine (H1 and H2) receptors more potently than the corresponding root extracts with IC50 values ranging from 1 to 100 micrograms/ml vs. > or = 100 micrograms/l, respectively. Significant differences in the potential of binding inhibition were also observed between cultivars. Binding to serotonin (5-HT6 and 5-HT7) and benzodiazepine receptors was only weakly inhibited by both root and leaf extracts of all four cultivars. In conclusion, our investigation indicates that the GABAA, dopamine D2, opioid (mu and delta) and histamine (H1 and H2) receptors might be involved in the pharmacological action of kava extracts. Since the cultivars contained similar amounts of kavalactones, while their pharmacological activities differed markedly, other constituents may play a role in the observed activities. Additionally, leaves generally exhibited more potent binding inhibition than roots, therefore leaf of P. methysticum might be an interesting subject for further pharmacological studies.

Animals↗

PubMed2001

Neurochemical studies with St. John's wort in vitro.

The effect of extracts and constituents of St. John's wort, Hypericum perforatum, at various CNS receptors were studied by radioligand binding techniques in order to determine a profile of pharmacological activity in vitro. Binding inhibition was examined for the G-protein coupled opioid, serotonin (5-HT), histamine, neurokinin and corticotropin releasing factor (CRF) receptors, for the steroid estrogen-alpha receptor and for the ligand-gated ionchannel GABA(A) receptor. Hypericin showed the most potent binding inhibiton of all tested constituents to human CRF1 receptor with an IC50 value of 300 nM. Preliminary GTPgamma35S binding studies to CRF1 coupled G-protein indicated an antagonistic action for hypericin. The acylphloroglucinole hyperforin failed to inhibit 125I-astressin binding to hCRF, receptor up to 10 microM. Hyperforin inhibited binding to opioid and serotonin (5-HT) receptors at IC50 values between 0.4 and 3 microM, while hypericin and pseudohypericin inhibited with weaker potency. The biflavonoid I3,II8-biapigenin inhibited 3H-estradiol binding to the estrogen-alpha receptor with an IC50 value of 1 microM. The inhibition of 3H-muscimol binding to the GABA(A) receptor is likely to be exclusively due to GABA present in the extract. We therefore hypothesize that additive or synergistic actions of several ditsinct compounds may be responsible for the beneficial antidepressant effect of St. John's wort.

Animals↗

PubMed2001

Improved Semliki Forest virus vectors for receptor research and gene therapy.

We have modified Semliki Forest virus (SFV) vectors to broaden their application range. Here we describe a series of site-directed mutagenesis experiments on the SFV subgenomic 26S promoter to down-regulate the heterologous gene expression. Several mutants showed a dramatic effect on transgene expression levels in BHK cells. The luciferase activity was reduced to approximately 30%, 3%, and 1% compared to the wild type promoter. Similarly, a decrease in beta-galactosidase activity was observed in BHK cells and after injection into the striatum of male Wistar rats. Novel non-cytopathogenic and temperature-sensitive SFV vectors have recently been developed by introduction of point mutations in the viral nonstructural genes nsP2 and nsP4. These vectors do not show the typical shut down of host cell protein synthesis after SFV infections and therefore allow for a substantially prolonged survival of host cells. Both the mutant vectors demonstrating lower and more physiological expression levels and the non-cytopathogenic vectors should be valuable tools for various applications within receptor research. Furthermore, recent studies suggest that SFV vectors can be efficient gene delivery vehicles for gene therapy applications.

Amino Acid Sequence↗

PubMed2001

Alphavirus vectors for gene therapy applications.

High-titer alphavirus vectors have been generated for efficient gene delivery both in vitro and in vivo. Studies on CNS infection via intranasal and peripheral injections with virulent and avirulent replication-competent Semliki Forest virus (SFV) strains has demonstrated the potential of gene delivery. Replication-deficient alphavirus particles have shown high local transgene expression of a transient nature in rodent brain. Alphavirus vectors have been demonstrated to induce apoptosis in infected human tumor cell lines and SFV vectors expressing interleukin-12 resulted in tumor regression in a B16 murine melanoma model. Repeated SFV injections led to stronger anti-tumor effects without immunogenic response detected against SFV. It has also been shown that intra-tumoral SFV-injections into nude mice with implanted human lung carcinomas led to tumor regression. Likewise, injection of replicative SFV-LacZ RNA resulted in tumor response as well as prophylactic protection against tumor formation. Alphaviruses have also showed potential in vaccine production. Additionally, modifications in the envelope structure of Sindbis virus resulted in substantial change in host range and demonstrated the feasibility of targeting alphavirus vectors. Moreover, SFV has been used as an expression vector for the generation of high-titer retrovirus-like particles. Recent alphavirus vector development has introduced novel non-cytopathogenic vectors, tightly temperature-regulated vectors as well as replication-persistent forms that should prolong the duration of expression. Alphavirus vectors can therefore be considered as highly potential gene delivery vehicles for future gene therapy applications, especially where only short-term expression is required, or even preferred.

Alphavirus↗

PubMed2000

Characterization of [(3)H]-LY354740 binding to rat mGlu2 and mGlu3 receptors expressed in CHO cells using semliki forest virus vectors.

The binding properties of [(3)H]-LY354740 were characterized on rat metabotropic glutamate receptors mGlu2 and mGlu3 expressed in Chinese hamster ovary (CHO) cells using Semliki Forest virus vectors. The saturation isotherm gave K(D) values of 20+/-5 and 53+/-8 nM and B(max) values of 474+/-161 and 667+/-89 fmol/mg protein for mGlu2 and mGlu3 receptors, respectively. NMDA, CaCl(2), DHPG and kainate were inactive up to 1 mM, whereas LY341495, DCG IV and ibotenate inhibited [(3)H]-LY354740 binding with similar potencies on both receptors. L-CCG I, L-AP4, L-AP5, LY354740 and 1S,3R-ACPD were 2- to 4-fold more potent inhibitors of [(3)H]-LY354740 binding to mGlu2 than mGlu3 receptors. However, MPPG and L-AP3 had a 6-fold and DTT a 28-fold preference for mGlu2 over mGlu3. ZnCl(2), at 10 mM, inhibited more than 70% of [(3)H]-LY354740 binding to mGlu2 receptors. At the same concentration it did not affect significantly [(3)H]-LY354740 binding to mGlu3 receptors. On the contrary, glutamate, quisqualate, EGLU and NAAG showed a 3-, 5-, 7- and 12-fold preference for mGlu3 over mGlu2. Finally, GTPgammaS, which partially inhibited the binding on mGlu2 receptors, was inactive to inhibit [(3)H]-LY354740 binding on mGlu3 receptors.

Amino Acids↗

PubMed2000

A synthetic agonist at the orphanin FQ/nociceptin receptor ORL1: anxiolytic profile in the rat.

The biochemical and behavioral effects of a nonpeptidic, selective, and brain-penetrant agonist at the ORL1 receptor are reported herein. This low molecular weight compound [(1S,3aS)-8- (2,3,3a,4,5, 6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza- spiro[4. 5]decan-4-one] has high affinity for recombinant human ORL1 receptors and has 100-fold selectivity for ORL1 over other members of the opioid receptor family. It is a full agonist at these receptors and elicits dose-dependent anxiolytic-like effects in a set of validated models of distinct types of anxiety states in the rat (i.e., elevated plus-maze, fear-potentiated startle, and operant conflict). When given systemically, the compound has an efficacy and potency comparable to those of a benzodiazepine anxiolytic such as alprazolam or diazepam. However, this compound is differentiated from a classical benzodiazepine anxiolytic by a lack of efficient anti-panic-like activity, absence of anticonvulsant properties, and lack of effects on motor performance and cognitive function at anxiolytic doses (0.3 to 3 mg/kg i.p.). No significant change in intracranial self-stimulation performance and pain reactivity was observed in this dose range. Higher doses of this compound (>/=10 mg/kg) induced disruption in rat behavior. These data confirm the notable anxiolytic-like effects observed at low doses with the orphanin FQ/nociceptin neuropeptide given locally into the brain and support a role for orphanin FQ/nociceptin in adaptive behavioral fear responses to stress.

Acoustic Stimulation↗

PubMed2000

Alphavirus vectors: applications for DNA vaccine production and gene expression.

Replication-deficient alphavirus vectors have been developed for efficient high-level transgene expression. The broad host range of alphaviruses has allowed infection of a wide variety of mammalian cell lines and primary cultures. Particularly, G protein-coupled receptors have been expressed at high levels and subjected to binding and functional studies. Expression in suspension cultures has greatly facilitated production of large quantities of recombinant proteins for structural studies. Injection of recombinant alphavirus vectors into rodent brain resulted in local reporter gene expression. Highly neuron-specific expression was obtained in hippocampal slice cultures in vivo. Additionally, preliminary studies in animal models suggest that alphavirus vectors can be attractive candidates for gene therapy applications. Traditionally alphavirus vectors, either attenuated strains or replication-deficient particles, have been used to elicit efficient immune responses in animals. Recently, the application of alphaviruses has been extended to naked nucleic acids. Injection of DNA as well as RNA vectors has demonstrated efficient antigen production. In many cases, protection against lethal challenges has been obtained after immunization with alphavirus particles or nucleic acid vectors. Alphavirus vectors can therefore be considered as potentially promising vectors for vaccine production.

Alphavirus↗

PubMed2000

Recombinant Semliki forest virus infects and kills human prostate cancer cell lines and prostatic duct epithelial cells ex vivo.

Semliki Forest Virus (SFV) is a broad host range RNA virus capable of high-level recombinant protein expression and apoptosis induction in many cell types. We have successfully used a recombinant, replication deficient SFV vector to express the LacZ marker gene product in seven human prostate cell lines, as well as in human prostate tissue explants. Flow cytometry revealed that 40-60% of PPC-1 prostate cancer cells died 24-72 h after infection with SFV-LacZ virus. Most human prostate cancer cell lines expressed high levels of recombinant protein. Infection of human prostate tissue ex vivo led to similarly high expression levels but the recombinant beta-galactosidase was confined to duct epithelial cells. Infection of cell and tissue cultures resulted in detachment of adherent cells from the culture surface and detachment of epithelial cells from the basement membrane of tissue. Our results indicate that SFV may be useful in targeting recombinant protein expression and apoptosis to prostatic duct epithelial cells.

Apoptosis↗