Biomedical subjects

K Lundstrom

Publications and source records attributed to K Lundstrom.

At least 19 recordsLinked to original sources

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↗

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↗

PubMed1999

Recombinant Semliki Forest virus and Sindbis virus efficiently infect neurons in hippocampal slice cultures.

Gene transfer into nervous tissue is a powerful tool for the analysis of gene function. By using a rat hippocampal slice culture preparation, we show here that Semliki Forest virus (SFV) and Sindbis virus (SIN) vectors are useful for the effective infection of neurons. The stratum pyramidale and/or the granular cell layer were injected with recombinant virus encoding beta-galactosidase (LacZ) or green fluorescent protein (GFP). By using low concentrations of injected SFV-LacZ or SIN-LacZ, we detected LacZ staining of pyramidal cells, interneurons, and granule cells. About 60% of the infected cells showed clear neuronal morphology; thus, relatively few glial cells expressed the transgene. Expression of GFP from SFV and SIN vectors gave similar results, with an even higher percentage (>90%) of the GFP-positive cells identified as neurons. Infected pyramidal cells were readily recognized in living slices, displaying GFP fluorescence in dendrites of up to fourth order and in dendritic spines. They appeared morphologically normal and viable at 1-5 days postinfection. We conclude that both SFV and SIN vectors efficiently transfer genes into neurons in hippocampal slice cultures. In combination with the GFP reporter, SFV and SIN vectors will allow the physiological examination of identified neurons that have been modified by overexpression or suppression of a specific gene product.

Alphavirus Infections↗

PubMed1999

Cloning and functional expression of alternative spliced variants of the human metabotropic glutamate receptor 8.

Two new spliced variants of the human metabotropic glutamate receptor 8 (HmGluR8), designated HmGluR8b and HmGluR8c, were identified in a human fetal brain cDNA library. The HmGluR8b and c differ from previously reported HmGluR8a by the out-of-frame insertions of 55-bp and 74-bp, respectively. The 55-bp insertion which contains a stop codon resulted in substitution of the last 16 amino acids in the C-terminus of HmGluR8a with 16 different amino acids in HmGluR8b. The 74-bp insertion introduces a frame shift in the predicted translation resulting in termination of the polypeptide before the putative seven transmembrane domains. Thus, the predicted HmGluR8c protein is 501 amino acids long and could represent a secreted isoform of the receptor. The pattern of mRNA expression of mGluR8 variants in human brain were analyzed by RT-PCR, Northern blot and in situ hybridization. Both HmGluR8a and b are expressed with similar abundance in fetal and adult brains. The in situ hybridization results indicate a predominantly glial cell expression of HmGluR8c in human brain. The three isoforms were transiently expressed in CHO cells from Semliki Forest Virus vectors. [3H]l-AP4 binding was performed on the cell membranes and the saturation curves showed the presence of a binding site with KD values of 249 and 182 nM and Bmax values of 13.6 and 10.5 pmoles/mg protein for HmGluR8a and b, respectively. For the six mGluR ligands studied, a similar rank order of potency was observed on both HmGluRa and b: l-AP4>l-SOP=l-CCG I>l-glutamate>DCG IV>LY 354740.

Adult↗

PubMed1999

Effect of the umami peptides on the ligand binding and function of rat mGlu4a receptor might implicate this receptor in the monosodium glutamate taste transduction.

1. The effect of several metabotropic ligands and di- or tripeptides were tested on the binding of [3H]-L(+)-2-amino-4-phosphonobutyric acid ([3H]-L-AP4) on rat mGlu4 receptor. For selected compounds, the functional activity was determined on this receptor using the guanosine-5'[gamma-35S]-thiotriphosphate [gamma-35S]-GTP binding assay. 2. Using the scintillation proximity assay, [3H]-L-AP4 saturation analysis gave binding parameters K(D) and Bmax values of 150 nM and 9.3 pmoles mg-1 protein, respectively. The specific binding was inhibited concentration-dependently by several mGlu receptor ligands, and their rank order of affinity was established. 3. Several peptides inhibited the [3H]-L-AP4 binding with the following rank order of potency: glutamate-glutamate>glutamate-glutamate-leucine=aspartate - glutamate>>glutamate - glutamate-aspartate>lactoyl-glutamate>>aspartate-aspartate. Aspartate-phenylalanine-methyl ester (aspartame) was inactive up to 1 mM and guanosine-5'-monophosphate and inosine-5'-monophosphate were inactive up to 100 micronM. 4. The [gamma-35S]-GTP binding functional assay was used to determine the agonist activities of the different compounds. For the rat mGlu4 agonists, L-AP4 and L-glutamate, the correlation between their occupancy and activation of the receptor was close to one. The peptides, Glu-Glu, Asp-Glu and Glu-Glu-Asp failed to stimulate the [gamma-35S]-GTP binding at receptor occupancy greater than 80% and Glu-Glu-Leu appeared to be a weak partial agonist. These peptides did not elicit a clear dose-dependent umami perception. However, Glu-lac showed a good correlation between its potency to stimulate the [gamma-35S]-GTP binding and its affinity for displacement of [3H]-L-AP4 binding. These data are in agreement with the peptide taste assessment in human subjects, which showed that the acid derivatives of glutamate had characteristics similar to umami.

Adult↗

PubMed1998

Hypericum perforatum inhibits the binding of mu- and kappa-opioid receptor expressed with the Semliki Forest virus system.

The effects of Hypericum perforatum extracts on in vitro [3H]naloxone binding to the human mu- and rat kappa-opioid receptors were studied in chinese hamster ovary (CHO) cells using the Semliki Forest virus (SFV) expression system. Binding of [3H]naloxone to the mu- and kappa-opioid receptor was inhibited in the presence of Hypericum extracts showing IC50 values of approximately 25 and 90 micrograms/ml, respectively. In contrast, extracts of Valeriana officinalis did not inhibit binding to the mu-opioid receptor. Also, single constituents of H. perforatum like the flavonoids quercetin and kaempferol and the glycosilated flavonoid quercitrin did not inhibit [3H]naloxone binding to the mu-opioid receptor up to a concentration of 10 microM. The present in vitro data may suggest a new possible mechanism for the anti-depressant effect of H. perforatum.

Animals↗

PubMed1997

Effect of single point mutations of the human tachykinin NK1 receptor on antagonist affinity.

Molecular modelling and site-directed mutagenesis were used to identify eleven amino acid residues which may be involved in antagonist binding of the human tachykinin NK1 receptor. Recombinant receptors were expressed in mammalian cells using the Semliki Forest virus system. Wild type and mutant receptors showed similar expression levels in BHK and CHO cells, verified by metabolic labelling. Binding affinities were determined for a variety of tachykinin NK1 receptor antagonists in SFV-infected CHO cells. The binding affinity for GR203040, CP 99,994 and CP 96,345 was significantly reduced by mutant Q165A. The mutant F268A significantly reduced the affinity for GR203040 and CP 99,994 and the mutant H197A had reduced affinity for CP 96,345. All antagonists seemed to bind in a similar region of the receptor, but do not all rely on the same binding site interactions. Functional coupling to G-proteins was assayed by intracellular Ca2+ release in SFV-infected CHO cells. The wild type receptor and all mutants except A162L and F268A responded to substance P stimulation.

Animals↗