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M Osterlund

Publications and source records attributed to M Osterlund.

15 recordsLinked to original sources
PubMed2001

Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa.

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF--FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.

Amino Acid Chloromethyl Ketones↗
PubMed2001

Spin and fluorescent probing of the binding interface between tissue factor and factor VIIa at multiple sites.

The specific complex between the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa) was chosen as a model for studies of the binding interface between two interacting proteins. Six surface-exposed positions in sTF, residues known to contribute to the sTF-FVIIa interaction, were selected for cysteine mutation and site-directed labeling with spin and fluorescent probes. The binding interface was characterized by spectral data from electron paramagnetic resonance (EPR) and steady-state and time-domain fluorescence spectroscopy. The labels reported on compact local environments at positions 158 and 207 in the interface region between sTF and the gamma-carboxyglutamic acid (Gla) domain of FVIIa, and at positions 22 and 140 in the interface region between sTF and the first epidermal growth factor-like (EGF1) domain of FVIIa. The tightness of the local interactions in these parts of the interface is similar to that seen in the interior of globular proteins. This was further emphasized by the reduced local polarity detected by the fluorescent label upon FVIIa binding, especially in the sTF-Gla region. There were indications of structural rigidity also at positions 45 and 94 in the interface region between sTF and the protease domain (PD) of FVIIa, despite the perturbed cofactor function of these sTF variants. The results of the present study indicate that the multi-probing approach enables comparison of the tightness and characteristics of interaction along the binding interface of a protein complex. This approach also increases the probability of acquiring reliable structural data that are descriptive of the wild-type proteins.

Amino Acid Substitution↗
PubMed2000

Spectroscopic probing of the influence of calcium and the gla domain on the interaction between the first EGF domain in factor VIIa and tissue factor.

The binding of factor VIIa (FVIIa) to tissue factor (TF) initiates blood coagulation. The binary complex is dependent on Ca2+ binding to several sites in FVIIa and is maintained by multiple contacts distributed throughout the various domains. Although the contributions from various residues and domains, including the Ca2+ coordination, to the global binding energy have been characterized, their importance for specific local interactions is virtually unknown. To address this aspect, we have attached four spectroscopic probes to an engineered Cys residue replacing Phe140 in soluble TF (sTF). This allows the monitoring of local changes in hydrophobicity and rigidity upon complex formation at the interface between the first epidermal growth factor-like (EGF1) domain of FVIIa and sTF. The fluorescent labels used sense a more hydrophobic environment and the spin labels are dramatically immobilized when FVIIa binds sTF. The results obtained with a 4-carboxyglutamic acid (Gla)-domainless derivative of FVIIa indicate that the Gla domain has no or minimal influence on the interaction between EGF1 and sTF. However, there is a difference in local Ca2+ dependence between Gla-domainless and full-length FVIIa.

Amino Acid Substitution↗
PubMed1999

Properties of spin and fluorescent labels at a receptor-ligand interface.

Site-directed labeling was used to obtain local information on the binding interface in a receptor-ligand complex. As a model we have chosen the specific association of the extracellular part of tissue factor (sTF) and factor VIIa (FVIIa), the primary initiator of the blood coagulation cascade. Different spectroscopic labels were covalently attached to an engineered cysteine in position 140 in sTF, a position normally occupied by a Phe residue previously characterized as an important contributor to the sTF:FVIIa interaction. Two spin labels, IPSL [N-(1-oxyl-2,2,5, 5-tetramethyl-3-pyrrolidinyl)iodoacetamide] and MTSSL [(1-oxyl-2,2,5, 5-tetramethylpyrroline-3-methyl)methanethiosulfonate], and two fluorescent labels, IAEDANS [5-((((2-iodoacetyl)amino) ethyl)amino)naphthalene-1-sulfonic acid] and BADAN [6-bromoacetyl-2-dimethylaminonaphthalene], were used. Spectral data from electron paramagnetic resonance (EPR) and fluorescence spectroscopy showed a substantial change in the local environment of all labels when the sTF:FVIIa complex was formed. However, the interaction was probed differently by each label and these differences in spectral appearance could be attributed to differences in label properties such as size, polarity, and/or flexibility. Accordingly, molecular modeling data suggest that the most favorable orientations are unique for each label. Furthermore, line-shape simulations of EPR spectra and calculations based on fluorescence depolarization measurements provided additional details of the local environment of the labels, thereby confirming a tight protein-protein interaction between FVIIa and sTF when the complex is formed. The tightness of this local interaction is similar to that seen in the interior of globular proteins.

Binding Sites↗
PubMed1998

Differential distribution and regulation of estrogen receptor-alpha and -beta mRNA within the female rat brain.

In the present study, estrogen receptor (ER)alpha and ER beta genes were found to be differentially expressed in discrete subregions of the rat amygdaloid complex. The amygdala nuclei showing predominant ER alpha mRNA expression included the posterolateral cortical nucleus, amygdala hippocampal area, and lateral dorsolateral nucleus, whereas the amygdala areas with predominant ER beta mRNA expression were the medial anterodorsal and central nuclei. Both ER alpha and ER beta mRNAs were highly expressed in the medial posterodorsal nucleus. In addition to the discrete anatomical expression patterns, there appeared to be a differential regulation by estradiol of the ER alpha and ER beta mRNAs. Two weeks of estradiol (170 microgram total) treatment decreased ER alpha mRNA expression levels in the arcuate, ventromedial hypothalamus, and posterolateral cortical amygdala nucleus, but increased ER beta mRNA in the arcuate. In the medial amygdala nuclei, only ER beta mRNA levels were altered (reduced) by estradiol treatment. These results suggest that estrogen can modulate behaviors and functions mediated by the amygdala and hypothalamus via differentially regulated ER subtypes.

Animals↗
PubMed1997

Autoradiographic comparison of [125I]epidepride and [125I]NCQ 298 binding to human brain extrastriated dopamine receptors.

Extrastriatal D2-dopamine receptors can be visualised in the monkey and human brain using the benzamides [11C]- and [76Br]FLB 457 in PET and [123I]epidepride in SPECT but not with the salicylamide analogues [76Br]FLB 463 and [123I]NCQ 298. To clarify the background for the differences in binding seen in vivo, we have compared the in vitro binding of [125I]epidepride and [123I]NCQ 298, using human whole hemisphere autoradiography. The images obtained with any radioligand showed detailed distribution with very dense binding in the putamen and the caudate nucleus and with the same detailed extrastriatal distribution. Thus, the divergent results obtained in vivo cannot be explained by different binding properties of the extrastriatal receptors.

Autoradiography↗
PubMed1997

Serum leptin concentrations in a normal population and in GH deficiency: negative correlation with testosterone in men and effects of GH treatment.

OBJECTIVE: To study relationships between leptin and factors regulating body composition as well as metabolic risk factors. Furthermore, to study the effects of GH on leptin. DESIGN: Cross-sectional and population-based. Regarding the effects of GH, prospective and interventional. PATIENTS: One hundred and eleven women and 107 men, 20-70 years old, randomly selected from the population registry in the community of Linköping, Sweden. Ten GH-deficient subjects were given GH until normalization of IGF-I levels. MEASUREMENTS: Venous blood was drawn in the fasting state. Serum leptin and hormones were analysed by immunoassay. RESULTS: In the population sample the natural logarithm of leptin (in(leptin)) correlated with body mass index (BMI) (men, r = 0.67), P < 0.0001; women, r = 0.71, P < 0.0001). The median value of leptin was 4.6 micrograms/l in men and 12.3 micrograms/l in women (P < 0.0001). Levels of in(leptin) did not correlate with plasma neuropeptide Y (men, P = 0.13; women, P = 0.35). In men only there was an inverse relationship between in(leptin) and testosterone (r = -0.46, P < 0.0001, after correction for BMI standardized r = -0.26, P = 0.03) as well as IGF-I (r = -0.20, P = 0.048). Although BMI was similar, smoking men had higher leptin levels than non-smoking men (median, 6.6 and 4.2 micrograms/l, respectively; Mann-Whitney; P = 0.006). In the GH-deficient subjects leptin levels were elevated and, although GH treatment did not change BMI, leptin levels decreased (median before GH, 21 micrograms/l and after 15 micrograms/l, respectively; P = 0.017). CONCLUSION: Serum leptin concentration is closely associated with BMI in the population with a gender difference in absolute levels and a strong negative correlation with testosterone in men. Serum leptin is elevated in GH deficiency and lowered by GH substitution.

Adult↗
PubMed1991

Analysis of polyomavirus enhancer-effect on DNA replication and early gene expression.

The polyomavirus enhancer is located adjacent to the origin of DNA replication and the transcriptional promoters. It has a cis-acting essential function in the initiation of both viral DNA synthesis and early transcription. The enhancer is activated by the binding of protein factors to specific sites in DNA. Mutants with deletions of the A- or the B-segment of the enhancer were constructed. In mouse 3T6 cells, the transcription of the viral early region was significantly decreased by deletion of the A-segment, but not by deletion of the B-segment. In contrast, the two deletions had a similar, moderately negative effect on viral DNA synthesis. However, the presence of DNA with a wild-type enhancer in doubly transfected cells resulted in very strong interference with the replication, but not with the transcription, of deletion mutant DNA. DNA of the deletion mutants were subjected to site-directed mutagenesis of the remaining enhancer segment. Three non-viable mutants were isolated. All three had base-pair changes in the A-segment affecting immediately adjacent binding sites of cellular protein factors. The mutants had lost the enhancer activity on the early promoter, but only one of them with multiple base substitutions had lost the capacity of DNA replication. Together, the results suggest that different aspects of enhancer function determine the activity in initiation of transcription and replication.

Amino Acid Sequence↗
PubMed1989

Acetylcholine receptor expression in primary cultures of embryonic chick myotubes--I. Discoordinate regulation of alpha-, gamma- and delta-subunit gene expression by calcitonin gene-related peptide and by muscle electrical activity.

The neuropeptide calcitonin gene-related peptide calcitonin gene-related peptide, and muscle electrical activity regulate in opposite directions the content of nicotinic acetylcholine receptor alpha-subunit mRNA in primary cultures of chick embryonic myotubes. Indeed, treating the cells with calcitonin gene-related peptide or blocking the spontaneous activity of muscle cells by tetrodotoxin (an inhibitor of sodium channels) increases, although to different levels, the content of acetylcholine receptor alpha-subunit mRNA [Fontaine B., Klarsfeld A. and Changeux J. P. (1987) J. Cell Biol. 105, 1337-1342; Klarsfeld A. and Changeux J. P. (1985) Proc. natn. Acad. Sci. U.S.A. 82, 4558-4562]. In this paper, we demonstrate that, under these in vitro culture conditions, calcitonin gene-related peptide (0.1 microM) and tetrodotoxin (0.5 microM) regulate to a smaller extent (no more than 2.5-fold above control) the levels of acetylcholine receptor gamma- and delta-subunit mRNAs. No effect of either compound on acetylcholine receptor biosynthesis was observed during the initial three days of culture. The response to calcitonin gene-related peptide was already maximal when the cells were treated between days three and four after plating (about 3-fold increase of the alpha-subunit mRNA level). The effect of tetrodotoxin resulted in a six-fold increase of the acetylcholine receptor alpha-subunit mRNA level in cells treated between days three and four, and still increased when the cells were exposed to tetrodotoxin through days six and eight (up to a maximum of 20-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Acetylcholine receptor expression in primary cultures of embryonic chick myotubes--II. Comparison between the effects of spinal cord cells and calcitonin gene-related peptide.

Spinal cord cells co-cultured with primary chick myotubes caused a 1.5-3-fold increase in the number of muscle surface acetylcholine receptors assayed with [125I]alpha-bungarotoxin. This increase did not result from the metabolic stabilization of the acetylcholine receptor protein and was at least partially due to a stimulation of acetylcholine receptor biosynthesis up to the level of the accumulation of alpha-subunit mature and partially spliced precursor mRNAs. A medium conditioned by spinal cord cells also caused a rise in acetylcholine receptor number. This increase did not coincide with an augmentation of the intracellular cyclic AMP level as reported for the neuropeptide calcitonin gene-related peptide. In contrast, spinal cord cells and the medium conditioned by them potentiated the effect of calcitonin gene-related peptide on acetylcholine receptor number. Stimulation of acetylcholine receptor synthesis by the conditioned medium was blocked by the protein kinase C activator 12-O-tetradecanoyl phorbol-13-acetate and by the calcium ionophore A23187. These two compounds have already been reported to block the increase of acetylcholine receptor number produced by the voltage sensitive sodium channel antagonist tetrodotoxin which stimulates acetylcholine receptor biosynthesis by blocking spontaneous electrical activity of the cultured muscle cells. The possibility that different neural factors and second messenger systems are involved in the regulation of acetylcholine receptor biosynthesis during the development of the neuromuscular junction is discussed.

Animals↗
PubMed1988

Complex alternative splicing of acetylcholinesterase transcripts in Torpedo electric organ; primary structure of the precursor of the glycolipid-anchored dimeric form.

In this paper, we show the existence of alternative splicing in the 3' region of the coding sequence of Torpedo acetylcholinesterase (AChE). We describe two cDNA structures which both diverge from the previously described coding sequence of the catalytic subunit of asymmetric (A) forms (Schumacher et al., 1986; Sikorav et al., 1987). They both contain a coding sequence followed by a non-coding sequence and a poly(A) stretch. Both of these structures were shown to exist in poly(A)+ RNAs, by S1 mapping experiments. The divergent region encoded by the first sequence corresponds to the precursor of the globular dimeric form (G2a), since it contains the expected C-terminal amino acids, Ala-Cys. These amino acids are followed by a 29 amino acid extension which contains a hydrophobic segment and must be replaced by a glycolipid in the mature protein. Analyses of intact G2a AChE showed that the common domain of the protein contains intersubunit disulphide bonds. The divergent region of the second type of cDNA consists of an adjacent genomic sequence, which is removed as an intron in A and Ga mRNAs, but may encode a distinct, less abundant catalytic subunit. The structures of the cDNA clones indicate that they are derived from minor mRNAs, shorter than the three major transcripts which have been described previously (14.5, 10.5 and 5.5 kb). Oligonucleotide probes specific for the asymmetric and globular terminal regions hybridize with the three major transcripts, indicating that their size is determined by 3'-untranslated regions which are not related to the differential splicing leading to A and Ga forms.

Amino Acid Sequence↗
PubMed1986

Mutational analysis of DNA sequences affecting the replication of defective polyomavirus variant D-50.

The genome of the defective polyomavirus variant D-50 consists of tandemly repeated DNA segments. The repeat unit corresponds to a 17% fragment of polyomavirus DNA (Griffin and Fried (1975) Nature 256, pp. 175-179). To allow mutational analysis, a monomer unit of D-50 DNA was cloned. After excision from the plasmid and ligation to form a random mixture of products, circular head to tail oligomers of the cloned segment were replicated in transfected cells. Those molecules had the same replication properties as original D-50 DNA. Deletion of base sequences assumed to be at the origin of DNA synthesis inhibited the replication completely, whereas a deletion of a segment at the junction of the tandem repeats had only a slight inhibitory effect. Mutation of potential coding sequences of the variant genome had a slight stimulatory effect on DNA synthesis, ruling out that D-50 expresses any protein that stimulates replication. In an attempt to construct variants similar to D-50, cells were transfected with polyomavirus DNA fragments including the sequences of the D-50 monomer unit. However, all these molecules replicated very slowly, suggesting the presence of an element that inhibited DNA synthesis. The combined data show that D-50 genomes can be reconstituted by ligation of monomer units and that the origin of DNA replication was the only essential element of the variant genome, whereas other elements had cis-acting auxiliary functions.

Base Sequence↗
PubMed1984

Inhibition of polyoma DNA synthesis by base pair substitutions at the replication origin.

The effect of base pair substitutions on the function of the polyoma virus origin of DNA replication was studied. The mutations were all C-G to T-A transitions, induced by bisulfite treatment of recombinant DNA molecules. The mutagenesis was directed to short single-stranded gaps in duplex DNA, or to loops in heteroduplex molecules. Modification of a 34 base pair sequence of dyad symmetry led to cis-acting inhibition of viral DNA synthesis, ranging from slight defects to total inactivation. One of the mutants was temperature sensitive. Mutants with base changes in an adjacent DNA segment, including an 18 base pair long purine-pyrimidine tract, had similar, but less severe, deficiences. In contrast to the effect of mutations in the homologous region of the simian virus 40 genome, there was no strict relationship between mutation of the putative large T-antigen-binding base sequence GPuGGC and defective viral DNA synthesis.

DNA Replication↗
PubMed1982

Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA.

We analyzed the effect of ethidium bromide (EtBr) on the cleavage of closed circular pBR322 DNA molecules by six restriction enzymes which make staggered or flush cuts (EcoRI, HindIII, BglI, PstI, HincII, PvuII). EtBr concentrations and reaction temperatures were determined at which DNA molecules with single-strand breaks were the major reaction product of digestion by all the enzymes. However, the amounts of intermediates which could be isolated differed for various enzymes. The results extend previous studies, showing that sequential cleavage of the DNA strands probably is a general property of restriction endonucleases.

DNA Restriction Enzymes↗