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Stefan Nilsson

Publications and source records attributed to Stefan Nilsson.

At least 19 recordsLinked to original sources
PubMed2006

The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria.

In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins.

Antibodies↗
PubMed2006

Triazolo-tetrahydrofluorenones as selective estrogen receptor beta agonists.

Several tetrahydrofluorenones with a triazole fused across C7-C8 showed high levels of ERbeta-selectivity and were found to be potent ERbeta-agonists. As a class they demonstrate improved oral bioavailability in the rat over a parent class of 7-hydroxy-tetrahydrofluorenones. The most selective agonist displayed 5.7 nM affinity and 333-fold selectivity for ERbeta.

Animals↗
PubMed2005

Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different.

Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F(1)beta pre-sequence, peptides derived from the F(1)beta pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F(1)beta pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P(1) position and small, uncharged amino acids or serine residues in the P'(1) position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the alpha-helical elements of the F(1)beta pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic alpha-helix and positively charged amino acid residues and degraded the F(1)beta pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F(1)beta pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.

Amino Acid Sequence↗
PubMed2005

Catalysis, subcellular localization, expression and evolution of the targeting peptides degrading protease, AtPreP2.

We have previously identified a zinc metalloprotease involved in the degradation of mitochondrial and chloroplast targeting peptides, the presequence protease (PreP). In the Arabidopsis thaliana genomic database, there are two genes that correspond to the protease, the zinc metalloprotease (AAL90904) and the putative zinc metalloprotease (AAG13049). We have named the corresponding proteins AtPreP1 and AtPreP2, respectively. AtPreP1 and AtPreP2 show significant differences in their targeting peptides and the proteins are predicted to be localized in different compartments. AtPreP1 was shown to degrade both mitochondrial and chloroplast targeting peptides and to be dual targeted to both organelles using an ambiguous targeting peptide. Here, we have overexpressed, purified and characterized proteolytic and targeting properties of AtPreP2. AtPreP2 exhibits different proteolytic subsite specificity from AtPreP1 when used for degradation of organellar targeting peptides and their mutants. Interestingly, AtPreP2 precursor protein was also found to be dual targeted to both mitochondria and chloroplasts in a single and dual in vitro import system. Furthermore, targeting peptide of the AtPreP2 dually targeted green fluorescent protein (GFP) to both mitochondria and chloroplasts in tobacco protoplasts and leaves using an in vivo transient expression system. The targeting of both AtPreP1 and AtPreP2 proteases to chloroplasts in A. thaliana in vivo was confirmed via a shotgun mass spectrometric analysis of highly purified chloroplasts. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that AtPreP1 and AtPreP2 are differentially expressed in mature A. thaliana plants. Phylogenetic evidence indicated that AtPreP1 and AtPreP2 are recent gene duplicates that may have diverged through subfunctionalization.

Amino Acid Sequence↗
PubMed2005

Oestrogen receptors and selective oestrogen receptor modulators: molecular and cellular pharmacology.

The early termination of the two arms of the Women's Health Initiative Trials has led to an increased interest and demand for selective oestrogen receptor modulators because of their potential to retain the benefits of hormone replacement therapy (oestrogen plus a gestagen) and at the same time avoid most of its severe adverse events. Selective oestrogen receptor modulators are a class of oestrogen receptor binding, small organic molecules that take advantage of the plasticity of the oestrogen receptors (alpha and beta, respectively), modulating the surface conformation of the oestrogen receptors upon binding in the respective ligand binding cavity. By doing so they affect the binding of various co-factors to the surface of the oestrogen receptors that, at least in part, explains why selective oestrogen receptor modulators may mimic the activity of oestrogen in some tissues where so desired, while opposing its activity in tissues where oestrogen-like activity is undesirable. Although selective oestrogen receptor modulators have many properties in common they also display unique activities including oestrogen receptor surface modulation and regulation of target gene expression. Selective oestrogen receptor modulators therefore offer the opportunity to develop pharmaceuticals with very distinct pharmacology and mechanism of action. Furthermore, these modulators offer the advantage of decreased risk for the development of breast and endometrial cancer and circumvent the need for combination with a gestagen. Most selective oestrogen receptor modulators in development bind with roughly equal affinity to both oestrogen receptor alpha and beta (balanced) and our view is that it is unlikely that a balanced selective oestrogen receptor modulator will inherit all desired effects of oestrogen (e.g. 17beta-oestradiol) and at the same time be devoid of all undesired effects. We therefore propose that the development of oestrogen receptor-subtype (alpha and beta, respectively) selective pharmaceuticals for specific applications (designer drugs) would better provide the benefits of hormone replacement therapy without its associated risks.

Animals↗
PubMed2004

Identification of endogenous glucocorticoid repressed genes differentially regulated by a glucocorticoid receptor mutant able to separate between nuclear factor-kappaB and activator protein-1 repression.

Glucocorticoids are commonly used in the clinic, but long-term treatment is often associated with severe side effects. One way to reduce unwanted effects is to restrict glucocorticoid receptor (GR) signaling through defined pathways. In this study, we examine endogenous target genes regulated by a GR mutant that in contrast to the wild-type GR is unable to repress stimulated nuclear factor-kappaB (NF-kappaB) activity, whereas repression of activator protein-1 (AP-1) activity is maintained. This GR mutant (GRR488Q) harbors a point mutation in the second zinc finger of the DNA binding domain. Its ability to distinguish between NF-kappaB and AP-1 repression is defined using reporter genes regulated by both simple and natural promoters. The inability of GRR488Q to repress NF-kappaB was not related to its inability to activate target genes through a glucocorticoid response element. Furthermore, the discriminating property was observed in three different cell lines, suggesting that this is not a cell-specific effect. These results show that different receptor surfaces or mechanisms are involved in repression of NF-kappaB and AP-1, respectively. It is interesting that the GRR488Q still interacted physically with NF-kappaB. Gene expression profiling of human embryonic kidney 293 cells, which express the wild-type GR and the GRR488Q mutant allowed identification of endogenous genes preferentially repressed by GR interference with NF-kappaB activity. The genes differentially regulated by GRR488Q mutant versus the wild-type GR after 2 h of treatment seem mainly to be involved in control of transcription and cell growth. At 8 h, no such distinction could be seen.

Animals↗
PubMed2004

Identification of target genes involved in the antiproliferative effect of glucocorticoids reveals a role for nuclear factor-(kappa)B repression.

Glucocorticoid hormones (GCs) exert an antiproliferative effect on most cells. However, the molecular mechanism is still largely unclear. We investigated the antiproliferative mechanism by GCs in human embryonic kidney 293 cells with stably introduced glucocorticoid receptor (GR) mutants that discriminate between cross-talk with nuclear factor-(kappa)B (NF-(kappa)B) and activator protein-1 signaling, transactivation and transrepression, and antiproliferative vs. non-antiproliferative responses. Using the GR mutants, we here demonstrate a correlation between repression of NF-(kappa)B signaling and antiproliferative response. Gene expression profiling of endogenous genes in cells containing mutant GRs identified a limited number of genes that correlated with the antiproliferative response. This included a GC-mediated up-regulation of the NF-(kappa)B-inhibitory protein I(kappa)B(alpha), in line with repression of NF-(kappa)B signaling being important in the GC-mediated antiproliferative response. Interestingly, the GC-stimulated expression of I(kappa)B(alpha) was a direct effect despite the inability of the GR mutant to transactivate through a GC-responsive element. Selective expression of I(kappa)B(alpha) in human embryonic kidney 293 cells resulted in a decreased percentage of cells in the S/G2/M phase and impaired cell proliferation. These results demonstrate that GC-mediated inhibition of NF-(kappa)B is an important mechanism in the antiproliferative response to GCs.

Blotting, Northern↗
PubMed2004

Thyroxine-thyroid hormone receptor interactions.

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3), 3,5,3'-triiodo-l-thyronine) with high affinity, and its precursor thyroxine (T(4), 3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless, T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH, our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design.

Animals↗
PubMed2004

Identification of a functional variant of estrogen receptor beta in an African population.

In this study, we identified five novel polymorphisms in the estrogen receptor beta (ERbeta) gene in an African population. Interestingly, two of these variants are expected to change the amino acid sequence of the ERbeta protein. These changes correspond to an isoleucine to valine substitution at amino acid position 3 (I3V) and a valine to glycine substitution at position 320 (V320G), respectively. The functional consequences of these amino acid substitutions were determined in different in vitro assays. The I3V mutation displayed no differences with regard to transcriptional activity in a reporter assay, as compared with the wild-type receptor. The V320G mutation, however, showed significantly decreased maximal transcriptional activity in a reporter assay, although its binding affinity for 17beta-estradiol was not affected. A pull-down assay indicated that the interaction of full-length TIF2 with hERbetaV320G was weaker than with hERbetawt. Moreover, surface plasmon resonance analysis revealed reduced interaction of the V320G ERbeta variant with the NR box I and II modules of TIF2. To our knowledge, this represents the first identification of a functional polymorphism in the ERbeta gene. This novel polymorphism provides a tool for human genetic studies of diseases in the African population.

Amino Acid Sequence↗
PubMed2004

Comparison between different sheathless electrospray emitter configurations regarding the performance of nanoscale liquid chromatography-time-of-flight mass spectrometry analysis.

Four different sheathless electrospray ionization (ESI) configurations were investigated for a nano liquid chromatography (LC) system. The studied configurations were: a column with an integrated emitter, with the ESI potential applied before or after the column, and a column with separate emitter, with the ESI voltage applied at a union before the emitter or at the emitter tip. The results indicates that the efficiency of the LC system is rather independent of the configuration when using 95 microm i.d. columns, acetic mobile phase and standard peptides as a sample. Introduction of post column dead volume seems not to be a critical issue at least with flow rates down to 600 nl/min.

Nanotechnology↗
PubMed2004

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia.

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.

Enzyme Precursors↗
PubMed2004

Interference of the electrospray voltage on chromatographic separations using porous graphitic carbon columns.

The electrospray ionization (ESI) voltage is shown to interfere with liquid chromatographic separations performed with packed porous graphitic carbon (PGC) capillary columns. This interference is ascribed to the presence of an electric field over the conductive column in the absence of an earth point between the column and the ESI emitter. The current evolved alters the chromatographic behavior of the catecholamine metabolite 3-O-methyl-DOPA significantly, as both peak splitting and a dramatic decrease in the retention time were observed. Furthermore, the response from the mass spectrometer was decreased by 33% at the same time. A related compound, tyrosine, exhibited decreased retention times but no peak splitting, whereas no shifts in the retention times (or peak splitting) were seen for the less retained dopamine and noradrenaline. When the current through the PGC column was eliminated by the use of an earth point between the column and the ESI emitter, the chromatographic behavior of the column was found to return slowly to normal after hours of equilibration with 60 : 40 (v/v) methanol-ammonium formate buffer of pH 2.9. The behavior of the PGC column with and without the earth point was found to be highly reproducible during a period of 1 month. We propose that the effect of the ESI voltage on the chromatographic behavior of the PGC column is due to associated redox reactions affecting both the PGC particles and the analytes. It is concluded that (for analytical reasons), care should be taken to ensure that no current is flowing through the chromatographic system when interfacing PGC columns, and conducting parts in general, to ESI mass spectrometry.

Chromatography, Liquid↗
PubMed2004

Molecular mechanisms, physiological consequences and pharmacological implications of estrogen receptor action.

The estrogen receptors (ERs), ERalpha and ERbeta, play a central role in mediating the biological effects of estrogen. The transcription rate of estrogen target genes is determined by several parameters including the type of ligand, estrogen receptor subtype and isoform, as well as interactions with receptor-binding cofactor proteins. The ERs regulate gene expression by binding to specific response element sequences in the promoters of estrogen target genes. Alternative pathways have also been described in which the ERs modulate transcription indirectly, via protein : protein interactions. In this regulatory mode, which has been traced to activator protein (AP)-1-, cyclic adenosine monophosphate (cAMP)-, and Sp1-response elements, the ERs appear to be tethered to target gene promoters via heterologous transcription factors. It has been found that ERalpha and ERbeta have opposite effects on transcription mediated via the indirect mode of action. Moreover, recent studies suggest that ERbeta may inhibit the stimulatory effects of ERalpha on cellular proliferation. Estrogen is a key regulatory hormone that affects numerous physiological processes. Estrogen is required for female pubertal development and affects growth, differentiation and function of the female reproductive system. It has recently been suggested that estrogen also has an important role in the male urogenital tract. In addition, estrogens have profound effects in other tissues. For instance, in the skeleton estrogen prevents bone-resorption by inhibition of osteoclast function. Numerous reports have suggested that estrogen has a beneficial effect in the cardiovascular system and in the CNS; however, this has not been confirmed in randomized clinical trials. In fact, a large randomized trial on healthy postmenopausal women receiving oral estrogen plus progestin showed an increased incidence of cardiovascular disease. In addition, this study revealed an increased risk for dementia and impaired cognitive function in the group receiving oral estrogen/progestin. Additional clinical trials are required to determine which hormonal component causes these health risks or whether the effects were due to the combination of estrogen and progestin.

Female↗
PubMed2003

Sheathless electrospray from polymer microchips.

In this study, sheathless electrospray from polymer microchips with conducting layer on the emitter tip is described for the first time. The electrospray emitter tips were fabricated directly from the end of the microchips that were made of polycarbonate or poly(methyl methacrylate) with injection molding. A variety of tip shapes and conducting coatings were evaluated using an electrospray time-of-flight mass spectrometer run in the sheathless mode. Stable electrospray was obtained both from hand-polished and machine-milled three-dimensional tips coated with either polymer-embedded gold particles or graphite particles as the conducting layer. Sputtered gold, on the other hand, suffered from poor stability mainly due to bad adhesion to the polymer tip. The durability of the different coatings was confirmed with electrochemical experiments under simulated electrospray conditions. The relative standard deviations of the response received from the ion current of the MS analysis were in the range of 3.5-12%. The detection limit for a standard mixture containing five neuropeptides was lower than 0.5 fmol. The low detection limit makes the emitter tips highly attractive for the analysis of low-abundance biological species.

Electrochemistry↗
PubMed2003

Gold-coated fused-silica sheathless electrospray emitters based on vapor-deposited titanium adhesion layers.

Gold-coated fused-silica electrospray (ES) emitters based on vapor-deposited adhesion layers of titanium have been manufactured to investigate the possibilities of producing durable ES emitters applicable in chip-based analytical devices. The stabilities of the emitters were studied by both electrospray and electrochemical experiments and a marked increase in the emitter lifetime, compared to that for Cr/Au coated emitters, was found for the Ti/Au emitters in the ES durability tests. This indicates that Ti (rather than Cr) adhesion layers should be used in association with large-scale fabrication of ES emitters by vapor-deposition techniques. The lifetime of about 500-700 hours also allowed the Ti/Au-coated emitter to be used as an integrated part of a capillary liquid chromatography column coupled to a mass spectrometer in a series of LC/MS experiments. The Ti/Au coating was further studied by electrochemical techniques and scanning electron microscopy in conjunction with X-ray spectroscopy. It is shown that the eventual failure of the Ti/Au emitters in ES experiments was due to an almost complete detachment of the gold layer. Experimental evidence suggests that the detachment of the gold coating was due to a reduced adhesion to the titanium layer during oxidation in positive electrospray. Most likely, this was caused by the formation of an oxide layer on the titanium film. It is thus shown that unlimited emitter stabilities are not automatically obtained even if the metallic adhesion layer is stabilized by an oxide formation under positive electrospray conditions.

Journal Article↗
PubMed2003

Radiological outcome after external fixation of 97 femoral shaft fractures in children.

The aim of this prospective, consecutive study was to assess overgrowth and malunion after anatomical reduction and external fixation of displaced femoral shaft fractures in children. Ninety-seven closed femoral fractures in 95 children aged 3-15 were fixed with a unilateral external fixator and followed with conventional radiographs and standardized orthograms for 1 year. To evaluate further overgrowth and remodelling a subgroup of 45 patients was also seen at 2 years. Parameters known to influence overgrowth and remodeling were recorded.Eighty-six percent of the femora had an angulation less than 10 degrees in varus/valgus or recurvatum at the time of healing. The remaining fractures remodeled nearly completely. Leg length discrepancy was on average 0.1cm at 1 year. The mean overgrowth was 0.3 cm at 1 year and 0.5 cm at 2 years. Overgrowth and remodeling was not correlated to whether the leg healed in a shortened or lengthened position, nor was it correlated to pin site infection or re-reduction.The overgrowth was far less than expected when compared with previous studies using traction. When external fixation is used in femoral fractures in children, we therefore, recommend fixing the fracture without shortening regardless of the age of the child, type of fracture, fracture level or injury.

Adolescent↗
PubMed2003

Estren is a selective estrogen receptor modulator with transcriptional activity.

It was recently reported that the synthetic compound estren increases bone mass without affecting reproductive organs or classic transcription. The aim of the present study was to further characterize the in vivo and in vitro effects of estren. We demonstrate that estren is a selective estrogen receptor modulator (SERM) with a strong effect on thymus, a moderate effect on uterus and trabecular bone, but no major effect on fat or cortical bone in 11-month-old ovariectomized mice. The effect of estren on trabecular bone and uterus is mediated via estrogen receptors (ERs) because no effect is seen in ER double-inactivated mice. Furthermore, with the use of ERalpha- and ERbeta-expressing reporter cell lines, we demonstrate that estren displays an agonistic effect on transcriptional activity of an estrogen-responsive element-driven reporter gene with a degree of agonism similar to that of 17beta-estradiol for both ERalpha and ERbeta. Thus, estren has the capacity to exert genomic effects via both ERalpha and ERbeta. We conclude, in contrast to what was previously reported by others, that estren is a SERM with transcriptional activity.

Animals↗