Biomedical subjects

T Nordström

Publications and source records attributed to T Nordström.

At least 19 recordsLinked to original sources

PubMed2001

Development of a time-resolved fluorescence resonance energy transfer assay (cell TR-FRET) for protein detection on intact cells.

An assay named Cell TR-FRET based on time-resolved fluorescence resonance energy transfer, here utilized for detection of receptor proteins on intact cells, is described. In this assay, intact membrane-biotinylated Sf9 cells expressing human interleukin-2Ralpha due to infection with a recombinant baculovirus were prelabeled with a streptavidin-europium (Eu(3+)) chelate, the donor. These prelabeled cells were used in a homogeneous assay by addition of a fluorochrome-labeled anti-hIL-2Ralpha-specific antibody, 7G7B6-Cy5, the acceptor. Binding of 7G7B6-Cy5 to hIL-2Ralpha expressed on the cell surface and europium-labeled streptavidin to surface biotin esters brings the donor and the acceptor in close proximity, allowing transfer of energy from the excited state donor to the acceptor. This energy transfer was specifically inhibited by unlabeled antibody and by free biotin. The described assay constitutes a general method since no specific component of the cell membrane is labeled, thereby allowing a number of binding studies on the cell membrane, including receptor density determinations, to be performed. In addition, due to the rapid fashion in which the Cell TR-FRET assay is accomplished, it can be a valuable method not only for identifying novel membrane-associated proteins, but also for drug screening of large samples in high-throughput format.

Animals↗

PubMed2001

Method enabling fast partial sequencing of cDNA clones.

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.

Base Sequence↗

PubMed2001

Production and partial characterization of mouse monoclonal antibodies recognizing common cytokine receptor gamma chain (gammac) of human, mouse and primate origin.

Monoclonal antibodies specific for the common cytokine receptor gamma chain, gammac, were produced using traditional hybridoma technology. Fusion of P3X63-Ag8.653 myeloma cells with splenocytes from Balb/c mice immunized with Spodoptera frugiperda insect cells infected with the recombinant baculovirus VL1392-hIL-2Rgamma resulted in several hybridoma cell clones producing monoclonal gammac-specific antibodies. Four of these antibody-producing clones, IIIC3, IIIE8, IG3 and IF10C5, were further characterized by immunoblotting, flow cytometry and ELISA. Data are presented demonstrating that the generated monoclonal antibodies can identify the extracellular domain of the common cytokine receptor gamma chain of human and mouse origin, and two of the antibodies recognize gammac of primate origin as well.

Animals↗

PubMed2001

Generation of hydroxyl radicals by the antiviral compound phosphonoformic acid (foscarnet).

Phosphonoformic acid (foscarnet) is an antiviral agent that is used to treat severe cytomegalovirus infections in AIDS patients. We demonstrate by using the ferrous iron indicator Ferrozine and ascorbic acid (vitamin C) that foscarnet can chelate ferric iron and form a redox-active iron complex. By using the hydroxyl radical indicator coumarin-3-carboxylic acid we found that the foscarnet-Fe3 complex formed can readily catalyze hydroxyl radical (.OH) generation by the Fenton reaction: (Fe2+ + H2O2-4Fe3+ + .OH + -OH) if hydrogen peroxide and ascorbic acid are present. Hydroxylation of coumarin-3-carboxylic acid could be blocked by addition of known hydroxyl radical scavengers such as mannitol, sucrose, glucose and dimethyl sulfoxide. Moreover, by using a DNA nicking assay, we found that foscarnet catalyzed hydroxyl radicals can induce single strand brakes in DNA. The potency of the hydroxyl radicals formed to induce damage could also be demonstrated in a phosphate-free buffer where the hydroxyl radicals formed attacked and liberated phosphate from the foscarnet molecule. Our results indicate that foscarnet catalyzed hydroxyl radical formation might take place during conditions where a peroxide generating system(s), vitamin C and transitions metals are present.

Antiviral Agents↗

PubMed2000

Method enabling pyrosequencing on double-stranded DNA.

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.

Alkaline Phosphatase↗

PubMed2000

Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing.

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

Alleles↗

PubMed1999

Detection of baculovirus-infected insect cells by flow cytometric side-scatter analyses.

BACKGROUND: The baculovirus expression vector system (BEVS), utilizing the Autographa californica nuclear polyhedrosis virus (AcNPV), has turned out to be an attractive alternative for high-level expression (<600 mg/l) of recombinant proteins. However, there is a shortage of reliable methods for monitoring the infection process in situations where marker proteins cannot be used. METHODS: Three recombinant baculoviruses, FastBac1-wtGFP, VTBac-GFP, and VL1392-hIL-2Ralpha, all having the genes inserted under the transcriptional control of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV), were used to infect Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-MB-0503) insect cells. The infection process of the recombinant baculoviruses was monitored by flow cytometric side-scatter and fluorescence intensity analyses over a period of 6-96 h. RESULTS: A clear correlation between the side-scatter (SSC) signal and the relative fluorescence was observed for both of the infected cell lines, compared to noninfected cells. Comparison of SSC histograms from noninfected insect cells with cells infected with the nonfluorescent recombinant baculovirus VL1392-hIL-2Ralpha showed a clear increase of SSC for the infected cells. CONCLUSIONS: The SSC parameter can therefore be utilized for flow cytometric monitoring of a baculovirus infection process in situations where suitable markers are not available.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

A microplate-based fluorometric assay for monitoring human cancer cell attachment to cortical bone.

A microplate-based fluorometric assay was developed for quantitation of cancer cell attachment to bone matrix. To evaluate this model system we used a panel of human cancer cell lines, including the human breast cancer cell line MDA-MB-231. For the assay, bovine cortical bone from the shaft of the femur was cut, turned, and sliced to 6-mm-diameter round 200-microm-thin disks and placed into the wells of a 96-well microplate. A fluorescent dye 2', 7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein (BCECF-AM) and a microplate fluorometer equipped with a standard filter set for fluorescein isothiocyanate were used to detect the cells attached to the bone disks. The fluorescence was enhanced during the measurement step by using a K+ solution (pH 8.0) containing the H+/K+ ionophore nigericin, which equilibrates internal and external pH. By taking advantage of the pH sensitivity of BCECF, the fluorescence intensity in the assay was increased 2.5 times compared to cells loaded with calcein. The specificity of the assay was demonstrated with a specific immuneserum raised against the MDA-MB-231 cell line. The assay can be completed in 1 h and permits the use of a large number of samples and is therefore useful for screening potential drug candidates that could block cancer cell attachment to bone material. Moreover, the assay described can easily be used to characterize molecular structures involved in cell attachment to bone.

Animals↗

PubMed1999

Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.

A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.

Adenosine Triphosphate↗

PubMed1998

The role of alpha2 beta1 and alpha3 beta1 integrin receptors in the initial anchoring of MDA-MB-231 human breast cancer cells to cortical bone matrix.

Molecular mechanisms involved in the rapid attachment of the human breast cancer cell line MDA-MD-231 to cortical bone matrix were studied. The attachment of MDA-MD-231 cells to cortical bone disks could be blocked by 75% when cells were pretreated with a monoclonal antibody to the beta1-subunit of the integrin family. Monoclonal antibodies against the alpha2, alpha3, and alpha5 integrin subunits inhibited the attachment by 76, 26, and 8 % respectively. Collagenase type I and collagen type I antibody blocked the cell attachment by 45 and 50 % whereas pretreatment of the cells with soluble collagen type I blocked the attachment by 85 %. Our study with a panel of cancer cell lines further showed a close correlation between alpha2 beta1 and alpha3 beta1 integrin receptor expression and the capability to attach to cortical bone. These receptors appear to be the key receptors utilized by cancer cells for the initial attachment to cortical bone, and this could facilitate the localization of alpha2 beta1 and alpha3 beta1 expressing cancer cells to the skeleton.

Animals↗

PubMed1997

Chronic extracellular acidosis induces plasmalemmal vacuolar type H+ ATPase activity in osteoclasts.

Proton extrusion into an extracellular resorption compartment is an essential component of bone degradation by osteoclasts. Chronic metabolic acidosis is known to induce negative calcium balance and bone loss by stimulating osteoclastic bone resorption, but the underlying mechanism is not known. The present studies were undertaken to evaluate whether chronic acidosis affects proton extrusion mechanisms in osteoclasts cultured on glass coverslips. Acidosis, mimicked experimentally by maintaining the cells at extracellular pH 6.5, rapidly lowered intracellular pH to 6.8. However, after 2 hours, a proportion of cells demonstrated the capacity to restore intracellular pH to near normal levels. To define the mechanism responsible for this recovery, the activity of individual H+ transport pathways was analyzed. We found that chronic acid treatment for up to 6 h did not significantly affect the cellular buffering power or Na+/H+ antiport activity. In contrast, chronic acidosis activated vacuolar H+ pumps in the osteoclasts. Although only approximately 5% of the control cells displayed proton pump activity, about 40% of cells kept at extracellular pH 6. 5 for 4-6 h were able to recover from the acute acid load by means of bafilomycin A1-sensitive proton extrusion. Conversely, the H+-selective conductance recently described in the plasma membrane of osteoclasts was clearly inhibited in the cells exposed to chronic acidosis. Following acid treatment, the activation threshold of the H+ conductance was shifted to more positive potentials, and the current density was significantly reduced. Considered together, these results suggest that induction of plasmalemmal vacuolar type ATPase activity by chronic acidosis, generated either systemically due to metabolic disease or locally at sites of inflammation, is likely to stimulate osteoclastic bone resorption and thus to promote bone loss.

Acidosis↗

PubMed1996

Activation of proton pumping in human neutrophils occurs by exocytosis of vesicles bearing vacuolar-type H+-ATPases.

Proton pump activity is not measurable in the plasma membrane of unstimulated neutrophils but becomes readily detectable upon activation by soluble agonists. The mechanism of pump activation was investigated in this report. V-type H+ pump activity, estimated as a bafilomycin A1-sensitive elevation of the cytosolic pH, was stimulated in suspended neutrophils by chemotactic peptides and by phorbol esters. Stimulation of pump activity induced by the agonists was greatly enhanced by cytochalasin B, an agent known to potentiate granular secretion in neutrophils. We therefore compared the rate and extent of pump activation with the pattern of exocytosis of the four types of secretory organelles present in neutrophils, using flow cytometry and enzyme-linked immunosorbent assay. The kinetics of exocytosis of secretory vesicles and secondary and tertiary granules but not primary granules paralleled the appearance of pump activity. The subcellular localization of the pump was defined by cellular fractionation and immunoblotting using an antibody to the C subunit of the V-type ATPase. The pump was abundant in tertiary granules, with significant amounts present also in primary granules and secretory vesicles. The pump was scarce in secondary granules and not detectable in the cytosol. Finally, the agonists failed to stimulate pump activity in neutrophil cytoplasts, which are intact cell fragments devoid of acidic granules. Together, our results suggest that the V-type H+-ATPase is not constitutively present in the plasma membrane of neutrophils but is delivered to the surface membrane by exocytosis during cellular activation. Tertiary granules and secretory vesicles are the most likely source of V-ATPases. Following insertion in the plasma membrane, the pump is poised to effectively extrude the excess metabolic acid that is generated during chemotaxis and bacterial killing.

Enzyme Activation↗

PubMed1996

Interleukin-1 increases vacuolar-type H+-ATPase activity in murine peritoneal macrophages.

Maintenance of cytoplasmic pH (pHi) within a narrow physiological range is crucial to normal cellular function. This is of particular relevance to phagocytic cells within the acidic inflammatory microenvironment where the pHi tends to be acid loaded. We have previously reported that a vacuolar-type H(+)-ATPase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pHi regulatory mechanism within the inflammatory milieu. Since this microenvironment is frequently characterized by the influx of cells known to release inflammatory cytokines, we performed studies to examine the effect of one such mediator molecule, interleukin-1 (IL-1), on pHi regulation in peritoneal macrophages. IL-1 caused a time- and dose-dependent increase in macrophage pHi recovery from an acute acid load. This effect was specific to IL-1 and was due to enhanced plasmalemmal V-ATPase activity. The increased V-ATPase activity by IL-1 occurred following a lag period of several hours and required de novo protein and mRNA synthesis. However, Northern blot analysis revealed that IL-1 did not exert its effect via alterations in the levels of mRNA transcripts for the A or B subunits of the V-ATPase complex. Finally, stimulation of both cAMP-dependent protein kinase and protein kinase C was required for the stimulatory effect of IL-1 on V-ATPase activity. Thus, cytokines present within the inflammatory milieu are able to modulate pHi regulatory mechanisms. These data may represent a novel mechanism whereby cytokines may improve cellular function at inflammatory sites.

Animals↗

PubMed1995

Regulation of cytoplasmic pH in osteoclasts. Contribution of proton pumps and a proton-selective conductance.

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pHi) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO(3-)-free solutions. When suspended in Na(+)-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na+/H+ antiporter. However, rapid recovery was also observed in Na(+)-free medium when K+ was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K+/H+ exchange. In contrast, pHi recovery in high K+ medium was largely attributed to a Zn(2+)-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H+ (equivalent) translocation across the membrane. The H+ current was greatly stimulated by reducing pHi, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H+ conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.

Animals↗

PubMed1994

Protein kinase C activation accelerates proton extrusion by vacuolar-type H(+)-ATPases in murine peritoneal macrophages.

The role of protein kinase C in the regulation of vacuolar-type H(+)-ATPase (V-ATPase) activity was studied in thioglycolate-elicited mouse peritoneal macrophages. Acid-loaded macrophages suspended in a Na(+)- and HCO(3-)-free K(+)-medium containing Zn2+, a H(+)-conductance blocker, exhibited an initial intracellular pH recovery rate of 0.33 +/- 0.04 pH/min (n = 9). Pretreatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) or mezerein for as little as 3 min induced a marked (82%) increase in the initial pH recovery rate. Stimulation was prevented by the V-ATPase inhibitor, bafilomycin A1 (200 nM) indicating that the effect of the protein kinase C agonist was via augmentation of proton pump activity. The protein kinase C inhibitor, staurosporine (100 nM) completely blocked the stimulatory effects of TPA and mezerein, suggesting involvement of protein kinase C. In keeping with this notion, the inactive analogue of TPA, 4-phorbol didecanoate did not stimulate recovery from an acid load. Extracellular pH determinations revealed that the observed increase in cytosolic pH recovery rate by the protein kinase C agonists was due to increased extrusion of protons from the cells, likely through V-ATPases located in the plasma membrane. Considered together, these data demonstrate regulation of plasmalemmal V-ATPase-mediated proton extrusion by protein kinase C.

Animals↗

PubMed1994

Cross-linking of surface IgG induces apoptosis in a bcl-2 expressing human follicular lymphoma line of mature B cell phenotype.

We have established three lymphoma cell lines, HF-1 from one follicular lymphoma (FL) patient, and HF-4 and HF-9 from another. All cell lines carry the characteristic t(14;18) chromosomal translocation and express constitutively the bcl-2 gene product (Bcl-2 protein). Cross-linking of their surface membrane Igs (sIgs) with relevant antibodies triggers a vigorous calcium signal in all three lines but only HF-1 is induced to apoptosis. Treatment with anti-Ig arrests the proliferation of HF-1 within 6-12 h, nucleosomal DNA fragmentation is evident in 18 h and a morphologically complete apoptosis is seen in 24-48 h. While bcl-2 was expressed at equal levels in all lines, the apoptosis-sensitive HF-1 line displayed a much lower expression of c-myc than seen in the apoptosis-resistant line. This finding challenges the concept that expression of bcl-2 per se renders resistance to apoptosis but that the balance between the expression of bcl-2 and c-myc may dictate the outcome of sIg cross-linking. HF-1 is a unique, phenotypically mature human B cell line expressing surface IgG. This cell line offers a new tool for investigations on apoptosis and induction of tolerance in mature B lymphocytes. Our results suggest that some FLs may be amenable to anti-cancer treatment based on anti-sIg antibody induced apoptosis.

Apoptosis↗

PubMed1994

Oxidant stress inhibits pH regulatory mechanisms in murine peritoneal macrophages.

BACKGROUND: Maintenance of cytoplasmic pH (pHi) close to the physiologic range is vital to normal cellular homeostasis. We have previously reported that a vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) situated in the plasma membrane of macrophages and poised to extrude protons from the cytoplasmic to the extracellular space is an important pHi regulatory mechanism. Since the inflammatory microenvironment is frequently characterized by the influx of cells known to release reactive oxygen metabolites, we performed studies to examine the effect of oxidant stress on pHi regulation in peritoneal macrophages. Specifically, the effect of hydrogen peroxide on V-ATPase-mediated proton extrusion from acid-loaded macrophages was investigated. METHODS: Thioglycollate-elicited murine peritoneal macrophages were exposed to varying concentrations of hydrogen peroxide and examined for their ability to recover from an acid-load. pHi was studied by preloading cells with the pH-sensitive fluorescent dye, bis-carboxyethyl-carboxyfluorescein, and monitoring changes in fluorescence under various conditions using a fluorescence spectrometer. RESULTS: Hydrogen peroxide caused a time- and dose-dependent decrease in proton pump-mediated pHi recovery in peritoneal macrophages. This effect occurred without cytotoxicity and was a specific effect as evidenced by the ability of catalase to reverse the inhibition. Since hydrogen peroxide is known to deplete intracellular ATP, a substrate for V-ATPase activity, we hypothesized that ATP depletion may underlie the effect. These studies showed that hydrogen peroxide-mediated ATP depletion was both necessary and sufficient for the effect. Finally, depletion of intracellular glutathione in vivo by using diethyl maleate increased the sensitivity of V-ATPase activity to oxidant stress. CONCLUSIONS: Oxidant stress within the inflammatory milieu impairs macrophage pHi regulation. This effect is magnified by depletion of intracellular antioxidants, as occurs during sepsis. This represents another mechanism whereby oxidants may contribute to cellular dysfunction associated with inflammatory states.

Adenosine Triphosphate↗