Christer Lindqvist

Generation of a new protein purification matrix by loading ceramic hydroxyapatite with metal ions—demonstration with poly-histidine tagged green fluorescent protein

The gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, was inserted under transcriptional control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The baculovirus transfervector pBlueBacHisB was used for constructing the recombinant baculovirus, so that the green fluorescent protein could be tagged with a poly-histidine tail. This fusion protein was utilized as a marker for evaluating the properties of metal ion loaded ceramic hydroxyapatite as a matrix in protein purification. Ceramic hydroxyapatite loaded with Zn(II) was the best choice for purifying this poly-histidine tagged GFP, followed by Fe(III) of the metal ions tested. Ni(II) that is superior especially in many poly-histidine purification systems did not, when loaded to hydroxyapatite, have binding properties comparable to Zn(II) or Fe(III). Elution of poly-histidine tagged GFP was best performed with phosphate buffers or EDTA that could compete with the phosphate molecules in hydroxyapatite or complexly bind the metal ions, respectively.

Functional analysis of the human α2C-C4 adrenergic receptor in insect cells expressed by a luciferase-based baculovirus vector

A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human alpha 2-adrenergic receptor (alpha 2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-alpha 2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the alpha 2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of alpha 2-receptors at a level of 1.370 pmol/mg protein was obtained at 48 h after infection as determined by ligand-binding experiments using the alpha 2-receptor antagonist, [3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [3H]rauwolscine binding with Ki values 12.3 +/- 1.54 microM and 1.23 +/- 0.11 microM, respectively. The recombinant receptors were functionally intact since the agonists inhibited forskolin-stimulated cAMP production. Here, however, the maximal inhibition was obtained at 36 h after the infection. The results presented here, suggest that the baculovirus expression vector system (BEVS) provides a simple method for abundant expression of functional alpha 2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.

Autocrine Endocannabinoid Signaling through CB1 Receptors Potentiates OX1 Orexin Receptor Signaling

It has been proposed that OX1 orexin receptors and CB1 cannabinoid receptors can form heteromeric complexes, which affect the trafficking of OX1 receptors and potentiate OX1 receptor signaling to extracellular signal–regulated kinase (ERK). We have recently shown that OX1 receptor activity releases high levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), suggesting an alternative route for OX1-CB1 receptor interaction in signaling, for instance, in retrograde synaptic transmission. In the current study, we set out to investigate this possibility utilizing recombinant Chinese hamster ovary K1 cells. 2-AG released from OX1 receptor–expressing cells acted as a potent paracrine messenger stimulating ERK activity in neighboring CB1 receptor–expressing cells. When OX1 and CB1 receptors were expressed in the same cells, OX1 stimulation–induced ERK phosphorylation and activity were strongly potentiated. The potentiation but not the OX1 response as such was fully abolished by specific inhibition of CB1 receptors or the enzyme responsible for 2-AG generation, diacylglycerol lipase (DAGL). Although the results do not exclude the previously proposed OX1-CB1 heteromerization, they nevertheless unequivocally identify DAGL-dependent 2-AG generation as the pivotal determinant of the OX1-CB1 synergism and thus suggest a functional rather than a molecular interaction of OX1 and CB1 receptors.

The lamprey (Lampetra fluviatilis) erythrocyte; morphology, ultrastructure, major plasma membrane proteins and phospholipids, and cytoskeletal organization.

The aim of this study was to characterize the erythrocyte of the lamprey (Lampetra fluviatilis), a primitive vertebrate. The lamprey erythrocyte predominantly has a non-axisymmetric stomatocytelike shape. It has a nucleus and a haemoglobin-filled cytosol with a few organelles and vesicular structures. Surprisingly, there is no marginal band of microtubules. Sodium dodecylsulphate polyacrylamide gel electrophoresis followed by Coomassie blue staining of isolated plasma membranes revealed a single band at the level of the human spectrin doublet. Major bands also occurred at approximately 175 kDa and comigrating with human erythrocyte actin (approximately 45 kDa). The presence of spectrin, actin and vimentin was shown by immunoblotting. Band 3 protein, the anion exchanger in higher vertebrates, seemed to be highly deficient or lacking, as was also the case with ankyrin. Confocal laser scanning microscopy combined with immunocytochemical methods showed spectrin, actin and vimentin mainly to be localized around the nucleus, from where actin- and vimentin-strands extended out into the cytoplasm. Actin also seemed to be present at the plasma membrane. Phospholipid analyses of plasma membrane preparations showed the presence of the same four major phospholipid groups as in the human erythrocyte, although with higher and lower amounts of phosphatidylcholine and sphingomyelin, respectively. The low fluorescein isothiocyanate conjugated annexin V binding, as monitored by flow cytometry, indicated that phosphatidylserine is mainly confined to the inner membrane leaflet in the lamprey erythrocyte plasma membrane.

Green fluorescent protein as a tool for screening recombinant baculoviruses

The gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, ligated to the honeybee melittin signal peptide-encoding sequence, was inserted under transcriptional control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The recombinant green fluorescent protein was identified by SDS-PAGE gel electrophoresis followed by Coomassie blue staining of lysates from the recombinant baculovirus infected insect cells. Emission and excitation scanning of the recombinant baculovirus infected insect cells gave an emission maximum of 509 nm and excitation maximum of 398 nm. The GFP protein expressed was also detected in infected insect cells by a flow cytometer analysis.

Oxidative decomposition of vitamin C in drinking water

We have previously shown that vitamin C (ascorbic acid) can initiate hydroxyl radical formation in copper contaminated household drinking water. In the present study, we have examined the stability of vitamin C in copper and bicarbonate containing household drinking water. In drinking water samples, contaminated with copper from the pipes and buffered with bicarbonate, 35% of the added vitamin C was oxidized to dehydroascorbic acid within 15 min. After 3 h incubation at room temperature, 93% of the added (2 mM) ascorbic acid had been oxidized. The dehydroascorbic acid formed was further decomposed to oxalic acid and threonic acid by the hydrogen peroxide generated from the copper (I) autooxidation in the presence of oxygen. A very modest oxidation of vitamin C occurred in Milli-Q water and in household water samples not contaminated by copper ions. Moreover, addition of vitamin C to commercially sold domestic bottled water samples did not result in vitamin C oxidation. Our results demonstrate that ascorbic acid is rapidly oxidized to dehydroascorbic acid and further decomposed to oxalic- and threonic acid in copper contaminated household tap water that is buffered with bicarbonate. The impact of consuming ascorbic acid together with copper and bicarbonate containing drinking water on human health is discussed.

Measurement of ascorbic acid (vitamin C) induced hydroxyl radical generation in household drinking water.

Ascorbic acid (vitamin C) induced hydroxyl radical formation was measured in household drinking water samples using the hydroxyl radical sensitive probe coumarin-3-carboxylic acid. Vitamin C, a reducing agent that is commonly used as a food additive, triggered a significant hydroxyl radical generating reaction when added to the tap-water samples tested. The capacity of ascorbic acid to trigger hydroxyl radical formation in the tap-water samples was dependent on the flushing time before the samples were taken indicating that the water in the copper piping had been contaminated by copper ions. In line with this, high concentrations of copper were measured in the hydroxyl radical generating first-draw samples. Moreover, a strong correlation was found between the hydroxyl radical generation capacity seen in the coumarin-3-carboxylic acid based microplate assay and the DNA damage seen in an agarose gel assay using the pBluescript plasmid. In the water samples showing high capacity to hydroxylate coumarin-3-carboxylic acid, a rapid formation of the open circular form of the plasmid could also be seen indicating a copper assisted hydroxyl radical attack on the DNA. In conclusion, our results show that addition of vitamin C to household tap water that is contaminated with copper ions, results in Fenton type reactions that continuously generate harmful and reactive hydroxyl radicals.

Synthesis and processing of the rubella virus p110 polyprotein precursor in baculovirus-infected Spodoptera frugiperda cells

In order to study the processing of rubella virus (RV) structural proteins (capsid protein, of 33 kDa; E2 of 42-47 kDa; and E1 of 58 kDa) in Spodoptera frugiperda (fall armyworm) cells, a 24S cDNA encoding the polyprotein precursor, p110, was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during viral infection. By immunoblot analysis using antibodies directed against whole RV and the individual structural proteins, evidence is presented that polypeptides similar to those synthesized in RV-infected B-Vero cells are expressed in this lepidopteran insect cell line infected with the recombinant baculovirus, VL1392-RV24S. The identity of the recombinant proteins was further confirmed using human convalescent sera. By expressing the recombinant proteins in the presence and absence of tunicamycin, we have further demonstrated that the 24S transcription-translation unit of RV, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared to B-Vero cells.

Expression of Human IL‐2 Receptor α‐ and β‐Chains using the Baculovirus Expression System

The genes encoding the α‐ and β‐chains of the human interleukin‐2 receptor were expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding genes were inserted under the polyhedrin promoter of the Autographa california nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line during viral infection. The recombinant receptor proteins were identified in the insect cell lysates by using protein dot blot and ELISA techniques. At 36 h post infection the corresponding proteins were clearly detected using anti‐IL‐2 α‐ and β‐receptor‐specific antibodies. A large amount of the α‐chain was also found in the supernatant culture media at 72 h post infection and metabolic labelling with [35S]‐methionine indicated that it was proteolytieally cleaved into a 32 kDa soluble form. A similar soluble or secreted form of the β‐chain was. however, not observed. Both receptor proteins were expressed on the surface of the insect cells as determined by flow cytometry analysis. Studies performed with the different IL‐2 receptor forms (α‐ and β‐chains alone or in combination) in the presence or absence of rIL‐2 suggest that the receptor proteins when expressed in infected insect cells are non‐functional with respect to tyrosine phosphorylation.

Iron prevents ascorbic acid (vitamin C) induced hydrogen peroxide accumulation in copper contaminated drinking water

Ascorbic acid (vitamin C) induced hydrogen peroxide (H2O2) formation was measured in household drinking water and metal supplemented Milli-Q water by using the FOX assay. Here we show that ascorbic acid readily induces H2O2 formation in Cu(II) supplemented Milli-Q water and poorly buffered household drinking water. In contrast to Cu(II), iron was not capable to support ascorbic acid induced H2O2 formation during acidic conditions (pH: 3.5–5). In 12 out of the 48 drinking water samples incubated with 2 mM ascorbic acid, the H2O2 concentration exceeded 400 μM. However, when trace amounts of Fe(III) (0.2 mg/l) was present during incubation, the ascorbic acid/Cu(II)-induced H2O2 accumulation was totally blocked. Of the other common divalent or trivalent metal ions tested, that are normally present in drinking water (calcium, magnesium, zinc, cobalt, manganese or aluminum), only calcium and magnesium displayed a modest inhibitory activity on the ascorbic acid/Cu(II)-induced H2O2 formation. Oxalic acid, one of the degradation products from ascorbic acid, was confirmed to actively participate in the iron induced degradation of H2O2. Ascorbic acid/Cu(II)-induced H2O2 formation during acidic conditions, as demonstrated here in poorly buffered drinking water, could be of importance in host defense against bacterial infections. In addition, our findings might explain the mechanism for the protective effect of iron against vitamin C induced cell toxicity.