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Biomedical subjects

E Persson

Publications and source records attributed to E Persson.

At least 37 records · Page 2Linked to original sources
PubMed2003

Influence of post-ovulatory insemination on sperm distribution, pregnancy and the infiltration by cells of the immune system, and the distribution of CD2, CD4, CD8 and MHC class II expressing cells in the sow endometrium.

This study investigates the distribution of leucocytes, CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following post-ovulatory insemination in relation to clinical findings and pregnancy outcome. Crossbred multiparous sows were inseminated once either at 15-20 h after ovulation [experiment 1, slaughtered at 20-25 h (5-6 h after artificial insemination (AI), group 1-A, n = 4), at 70 h after ovulation (group 1-B, n = 4), on day 11 (group 1-C, n = 4, first day of standing oestrus = day 1) or on day 19 (group 1-D, n = 4)] or 30 h after ovulation [experiment 2, slaughtered at 5-6 h after AI (group 2-A, n = 4) or on day 19 (group 2-D, n = 3)]. The uterine horns were flushed to control for the presence of spermatozoa and neutrophils and/or for recovery of oocytes and/or embryos. Mesometrial uterine samples were plastic embedded and stained. Cryofixed uterine samples were analysed by immunohistochemistry using mAbs to lymphocyte subpopulations and MHC class II molecules. Light microscopy was used to examine surface (SE) and glandular epithelia (GE), and connective tissue layers, both subepithelially (SL) and glandular (GL). In experiment 1, group 1-A, only one sow had spermatozoa in the utero-tubal junction (UTJ). Marked/moderated numbers of neutrophils and spermatozoa were observed in the flushings of two sows. In group 1-B, altogether 23 of 48 oocytes were cleaved. Day 11 (1-C), embryos with small diameter were observed. Day 19 (1-D), no embryos were found but small pieces of foetal membrane were observed in one of the sows. In group 1-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. For T lymphocyte subpopulations, in the SE, most CD2+ cells were found in group 1-A. For both SE and GE in all groups, the number of CD8+ cells was significantly larger than that of CD4+ cells. In experiment 2, group 2-A, no sow had spermatozoa in the UTJ or in the uterine flushings. At day 19, no sow was pregnant. In group 2-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. At day 19, high E2 levels showed a hormonal prooestrous stage but the endometrial neutrophil infiltration normally expected at pro-oestrus was absent. In conclusion, post-ovulatory insemination (about 18 h after ovulation) resulted in impaired spermatozoa transport within the uterus and embryonic degeneration. In sows post-ovulatory inseminated at a later stage (30 h after ovulation), no sow was pregnant. In both experiments, disturbed immune cell patterns were observed in some individuals.

Animals↗
PubMed2003

Immunohistochemical studies on oestrogen receptor alpha (ER alpha) and the proliferative marker Ki-67 in the sow uterus at different stages of the oestrous cycle.

In order to better understand physiological changes during the different stages of the oestrous cycle, immunohistochemistry was used in the present study to investigate the distribution of oestrogen receptor alpha (ER alpha) as well as the proliferative marker Ki-67, in the sow uterus during the oestrous cycle. Uterine samples were collected from multiparous sows with normal reproductive performance at selected stages of the oestrous cycle: at late dioestrus (d 17), prooestrus (d 19), oestrous (d 1), early dioestrus (d 4) and dioestrus (d 11-12), respectively. The tissue samples were fixed in 10% formaldehyde, embedded in paraffin and subjected to immunohistochemistry using monoclonal antibodies against ER alpha (C-311) and Ki-67 (MM-1). In general, the immunostaining of both ER alpha and Ki-67 was confined to nuclei of the target cells. Variations were seen, not only at the different stages of the oestrous cycle, but also in the different tissue compartments of the uterus. In the epithelia, the strongest ER alpha staining and highest amount of positive Ki-67 cells were found at early dioestrus. In the myometrium, the highest levels of staining of both ER alpha and Ki-67 positive cells were found at pro-oestrus and oestrus. For the proliferative marker, Ki-67, no positive cells were found at dioestrus and late dioestrus in the epithelium and myometrium. In the connective tissue stroma (subepithelial layer), the highest number of ER alpha positive cells were found at oestrus, which was significantly different compared with other stages (p< or = 0.05), whereas the levels of Ki-67 positive cells were relatively low and did not differ between the stages examined. Significant correlations between the number of ER alpha positive cells in the stroma and Ki-67 positive cells in the epithelia were observed. This suggests indirect regulatory mechanisms on epithelial proliferation via ER alpha in the stroma. In conclusion, these findings in the sow uterus show that the presence of ER alpha as well as Ki-67 protein varies not only between different stages of the oestrous cycle but also between different tissue compartments of the uterus. These findings indicate various regulatory mechanisms and stress the importance of localising ER alpha and proliferating cells in different uterine tissues.

Animals↗
PubMed2003

Enhanced in vitro procoagulant and antifibrinolytic potential of superactive variants of recombinant factor VIIa in severe hemophilia A.

BACKGROUND: Recombinant coagulation factor VIIa (rFVIIa) is generally accepted for treatment of patients with inhibitor-complicated hemophilia. Recently, rFVIIa variants with a specific enhancement of the tissue factor (TF)-independent proteolytic activity have been described. OBJECTIVES: The procoagulant and [thrombin-activatable fibrinolysis inhibitor (TAFI)-dependent] antifibrinolytic potentials of two superactive rFVIIa variants were compared with those of wild-type rFVIIa in a hemophilic setting. PATIENTS AND METHODS: Clot lysis assays were performed in plasma from six patients with inhibitor-complicated hemophilia A or in antibody-induced factor VIII-deficient platelet-rich plasma in the presence of different concentrations of the rFVIIa variants. RESULTS AND DISCUSSION: In the plasma model, M298Q-rFVIIa had a moderately increased procoagulant and antifibrinolytic potential, whereas V158D/E296V/M298Q/K337A-rFVIIa had a strongly increased procoagulant and antifibrinolytic activity compared with wild-type rFVIIa. The increased antifibrinolytic potential of the rFVIIa variants was completely dependent on enhancement of TAFI activation. In the platelet-rich plasma model similar results were obtained. The presence of TF was mandatory for clot formation in the absence of exogenous rFVIIa. At lower concentrations of rFVIIa (wild-type or variants), clot formation did occur but was significantly slower when TF activity was blocked. At increasing concentrations of rFVIIa, clotting times were no longer dependent on TF. In conclusion, should a TF-independent mechanism be involved in the efficacy of rFVIIa in patients with hemophilia, the superactive rFVIIa variants studied here might be clinically advantageous, as both procoagulant and antifibrinolytic potencies are significantly enhanced compared with those of wild-type rFVIIa. This ought to result in more efficient cessation of bleeding episodes and reduced risk of rebleeding.

Antifibrinolytic Agents↗
PubMed2002

Corrigendum to "The sow endometrium at different stages of the oestrus cycle: studies on morphological changes and infiltration by cells of the immune system." [Anim. Reprod. Sci. 65 (2001) 95-114].

The aim of this study was to investigate the distribution of leukocytes and the morphological changes of the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire), with an average parity number of 3.4+/-0.7 (mean+/-S.D.) were used. Blood samples were collected from the jugular vein 1 h before slaughter for analyses of oestradiol-17beta and progesterone levels. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus, prooestrus, oestrus, early dioestrus and dioestrus, were fixed, embedded in plastic resin and stained with toluidine blue. The surface and glandular epithelium as well as subepithelial and glandular connective tissue layers were examined by light microscopy (LM). The significantly highest surface and the glandular epithelium were observed at oestrus and dioestrus, respectively. The largest number of capillaries (underneath the surface epithelium) was found at oestrus. In the surface epithelium, the largest number of intraepithelial lymphocytes (IELs, round nucleus) was found at early dioestrus. The largest number of lymphocytes and macrophages within the glandular epithelium were found at early dioestrus and oestrus, respectively. In the subepithelial connective tissue layer, the most common type of leukocytes during all stages was the lymphocyte. The largest numbers of lymphocytes and neutrophils were found at oestrus while the largest number of eosinophils was found at dioestrus. The dominating cells of the immune system in the connective tissue of the glandular layer were lymphocytes and macrophages. The significantly largest numbers of lymphocytes and plasma cells were found at early dioestrus and dioestrus, respectively. The number of lymphocytes in the connective tissue of the glandular layer and the number of plasma cells in the subepithelial layer were positively correlated with the plasma level of progesterone (P < or = 0.05). The numbers of capillaries and neutrophils in the subepithelial layer underneath the surface epithelium as well as the number of macrophages in both surface and glandular epithelium were positively correlated with the plasma level of oestradiol-17beta (P < or = 0.05). In conclusion, the present study showed a variation om the infiltration and distrobution of lymphocytes, neutrophils, eosinophils, macrophages, mast cells, and plasma cells in the sow endometrium during different stages of the oestrous cycle. Also morphological parameters (e.g. height of surface and glandular epithelium, capillaries density and degree of oedema) varied throughout the oestrous cycle.

Animals↗
PubMed2002

Corrigendum to "The sow endometrium at different stages of the oestrous cycle: studies on the distribution of CD2, CD4, CD8 and MHC class II expressing" cells. [Anim. Reprod. Sci. 68 (2001) 99-109].

The aim of this study was to investigate the distribution of CD2(+), CD4(+), CD8(+) lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire), with an average parity number of 3.4 +/- 0.7 (mean +/- S.D.) were used. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus (day 17, n = 3), prooestrus (day 19, n = 3), oestrus (day 1, n = 3), early dioestrus (day 4, n = 3) and dioestrus (days 11-12, n = 3), were stored in a freezer at -70 degrees C until analysed by immunohistochemistry with an avidin-biotin peroxidase method using monoclonal antibodies to lymphocyte subpopulations and MHC class II molecules. The surface and glandular epithelium as well as connective tissue layers in subepithelial and glandular areas were examined by light microscopy. For the T lymphocyte subpopulations, all oestrous cycle stages and different tissue layers taken together, the most commonly observed cell type was CD2(+) cells. The largest number of CD2(+) cells within the surface and glandular epithelium were observed at oestrus and early dioestrus. In the surface epithelium, a larger number of CD8(+) cells compared with CD4(+) cells were observed and no CD4(+) cells were found within the glandular epithelium at any stage of the oestrous cycle. In the subepithelial and glandular connective tissue layers, during the oestrus cycle stages, a larger number of CD4(+) cells compared with CD8(+) cells were found. Endothelial cells in the connective tissue generally expressed MHC class II. However, no obvious differences between oestrous cycle stages were observed. For other cells than endothelial cells, the result was as follows. In the surface epithelium, a large number of MHC class II expressing cells was observed at oestrus compared with the other stages. No MHC class II expressing cells were found at late dioestrus and dioestrus. MHC class II expressing cells were also found in the glandular epithelium, and in the subepithelial and glandular connective tissue layers during all oestrous cycle stages but with no significant differences between stages. In conclusion, the present study showed a variation in the distribution of T lymphocyte subpopulations (CD2(+), CD4(+) and CD8(+)) and MHC class II expressing cells in the sow endometrium during different stages of the oestrous cycle. Also a variation between different tissue layers was found. It is suggested that helper and cytotoxic function of the immune system have primary locations in different tissue layers of the endometrium.

Animals↗
PubMed2002

Effective Hamiltonian for a microwave billiard with attached waveguide.

In a recent work the resonance widths in a microwave billiard with attached waveguide were studied in dependence on the coupling strength [E. Persson et al., Phys. Rev. Lett. 85, 2478 (2000)], and resonance trapping was experimentally found. In the present paper an effective Hamiltonian is derived that depends exclusively on billiard and waveguide geometry. Its eigenvalues give the poles of the scattering matrix provided that the system and environment are defined adequately. Further, we present the results of resonance trapping measurements where, in addition to our previous work, the position of the slit aperture within the waveguide was varied. Numerical simulations with the derived Hamiltonian qualitatively reproduce the experimental data.

Journal Article↗
PubMed2002

The influence of pre- and post-ovulatory insemination on sperm distribution in the oviduct, accessory sperm to the zona pellucida, fertilisation rate and embryo development in sows.

The aim of present study was to investigate the influence of pre-compared with post-ovulatory insemination, on the distribution of spermatozoa in the oviduct, the accessory sperm counts on the zona pellucida and early embryonic development. Thirty-six crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire) were artificially inseminated once either at 20-15 h before (group AIB) or at 15-20 h after (group AIA) ovulation by using a pooled semen of two boars. Thereafter, they were randomly allocated to one of five groups: slaughter at 5-6h after AI (group I-AIB), at 20-25 h after ovulation (groups II-AIB and II-AIA), at 70 h after ovulation (groups III-AIB and III-AIA), on day 11 (groups IV-AIB and IV-AIA, first day of standing oestrus=day 1) and on day 19 (groups V-AIB and V-AIA). The plasma levels of oestradiol-17beta and progesterone differed significantly (P<or=0.05 and P<or=0.001, respectively) between AI before (group AIB) and after (group AIA) ovulation. All sows inseminated before ovulation (group I-AIB) had spermatozoa in the UTJ but this was found only in one of the late inseminated sows (group II-AIA). The number of oocytes with spermatozoa in the ZP differed significantly (P<or=0.001) between 'group II-AIB' and 'group II-AIA'. Comparing fertilisation rate, the fertilisation rate in 'group III-AIB' was significantly (P<or=0.001) higher than in 'group III-AIA'. A larger number of recovered embryos (on days 11 and 19) per sow in groups IV-AIB and V-AIB compared with IV-AIA and V-AIA were found. The embryos in group IV-AIB were also larger than embryos in group IV-AIA. In group V-AIA, no embryos were found.In conclusion, the results of this study showed that if insemination was performed at 15-20 after ovulation in sows that still were in standing oestrus, the transport of spermatozoa to the UTJ and oviduct was impaired and a lower proportion of oocytes with accessory spermatozoa to the zona pellucida was found compared with insemination before ovulation. In the late inseminated sows, fertilised oocytes and developed embryos were observed up to day 11 but no embryos were found at day 19.

Animals↗
PubMed2002

Study of the distribution of inflammatory cells in the sow endometrium: effect of intravenous administration of adrenocorticotropin hormone.

Seventeen multiparous cross-bred sows (Swedish Land-race x Swedish Yorkshire) were inseminated in their second oestrus after weaning and divided into two groups. One group (ACTH, n = 9) was given an intravenous injection of adrenocorticotropin hormone (ACTH) every 6 h commencing 4-8 h after ovulation, whereas another group (control, n = 8) was given saline solution at the same times. The sows were slaughtered 35-53 h after ovulation. Uterine samples, taken from the mesometrial side of the uterine horns immediately after slaughter, were fixed, embedded in plastic resin and stained with toluidine blue. The endometrium was then examined by light microscopy. There was no significant effect of the ACTH treatment on the distribution of lymphocytes and macrophages, but there was a tendency of an effect on the distribution of neutrophils (P = 0.1) in the sow endometrium.

Adrenocorticotropic Hormone↗
PubMed2002

Septicaemia and meningitis in neonates and during early infancy in the Göteborg area of Sweden.

UNLABELLED: In a retrospective study of neonatal septicaemia and meningitis carried out in 1987-1996 in the Göteborg area of Sweden, 305 cases during the first 28 d of life were found. The incidence was 3.7/1000 live births, which was higher than that found in a study from 1975 to 1986 in the same area, 2.8/1000 live births. The most common pathogens were group B streptococci, aerobic gram-negative rods, Staphylococcus aureus and enterococci. The cases were approximately equally divided between very early, early and late onset infections. The case-fatality rate was lower in the present study (9%) than that in the previous study (15%). The case-fatality rate varied between 23% in neonates with a gestational age of < or = 29 wk and 3% in full-term neonates. Eighty-three very late onset infections occurred between days 28 and 120 after birth, corresponding to an incidence of 1.0/1000 live births. Of those, 47 occurred in preterm neonates. The most common organisms were aerobic gram-negative rods. Coagulase-negative staphylococci (CoNS) were studied separately. This organism was isolated in 60 and 32 cases during the first 28 d of life and between days 28 and 120, respectively, in neonates fulfilling the criteria of the Yale-New Haven Hospital for infections caused by commensal species. The incidence rates of CoNS infections were 0.7 and 0.4/1000 live births, respectively. CONCLUSION: The incidence of neonatal infections increased and the case-fatality rate decreased in the Göteborg area compared to the findings of a previous study. Very late onset infections should be added to the total burden of neonatal infections. CoNS are important pathogens but their role is difficult to determine since they are both pathogens and contaminants of cultures from blood and cerebrospinal fluid.

Age of Onset↗
PubMed2002

Quantification of myocardial hypoperfusion with 99m Tc-sestamibi in patients undergoing prolonged coronary artery balloon occlusion.

Percutaneous transluminal coronary angioplasty provides an excellent opportunity to investigate the location and quantity of hypoperfusion during sudden complete occlusion of one of the major coronary arteries. Thirty-five patients referred for elective percutaneous transluminal coronary angioplasty were injected intravenously with 99mTc-sestamibi during balloon inflation. To visualize and quantify the hypoperfused region, a map of perfusion was constructed from that occlusion study and from the control study performed on the following day. Patients were divided into groups according to proximal or distal occlusion within each of the three coronary arteries. The region of myocardium supplied by each coronary artery varied in location and extended outside the typical borders for all arteries, but most prominently for the left circumflex coronary artery. The quantities of hypoperfusion varied within each artery group, but the average hypoperfusion was greater for the left anterior descending coronary artery than for either the right coronary artery or the left circumflex coronary artery. It is concluded that the quantities of hypoperfusion were highly variable within each artery group. Occlusion of the left anterior descending coronary artery was associated with the largest ischaemic region. The area of hypoperfusion extended outside the typical borders, most prominently for the left circumflex coronary artery.

Adult↗
PubMed2001

Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity.

A trace amount of coagulation factor VII (FVII) circulates in the blood in the activated form, FVIIa (EC 3.4.21.21), formed by internal proteolysis. To avoid disseminated thrombus formation, FVIIa remains in a conformation with zymogen-like properties. Association with tissue factor (TF), locally exposed upon vascular injury, is necessary to render FVIIa biologically active and initiate blood clotting. We have designed potent mutants of FVIIa by replacing residues believed to function as determinants for the inherent zymogenicity. The TF-independent rate of factor X activation was dramatically improved, up to about 100-fold faster than that obtained with the wild-type enzyme and close to that of the FVIIa-soluble TF complex. The mutants appear to retain the substrate specificity of the parent enzyme and can be further stimulated by TF. Insights into the mechanism behind the increased activity of the mutants, presumably also pertinent to the TF-induced, allosteric stimulation of FVIIa activity, were obtained by studying their calcium dependence and the accessibility of the N terminus of the protease domain to chemical modification. The FVIIa analogues promise to offer a more efficacious treatment of bleeding episodes especially in hemophiliacs with inhibitory antibodies precluding conventional replacement therapy.

Antithrombins↗
PubMed2001

The sow endometrium at different stages of the oestrous cycle: studies on the distribution of CD2, CD4, CD8 and MHC class II expressing cells.

The aim of this study was to investigate the distribution of CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire), with an average parity number of 3.4+/-0.7 (mean+/-S.D.) were used. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus (day 17, n=3), prooestrus (day 19, n=3), oestrus (day 1, n=3), early dioestrus (day 4, n=3) and dioestrus (days 11-12, n=3), were stored in a freezer at -70 degrees C until analysed by immunohistochemistry with an avidin-biotin peroxidase method using monoclonal antibodies to lymphocyte subpopulations and MHC class II molecules. The surface and glandular epithelium as well as connective tissue layers in subepithelial and glandular areas were examined by light microscopy. For the T lymphocyte subpopulations, all oestrous cycle stages and different tissue layers taken together, the most commonly observed cell type was CD2+ cells. The largest number of CD2+ cells within the surface and glandular epithelium were observed at oestrus and early dioestrus. In the surface epithelium, a larger number of CD8+ cells compared with CD4+ cells were observed and no CD4+ cells were found within the glandular epithelium at any stage of the oestrous cycle. In the subepithelial and glandular connective tissue layers, during the oestrus cycle stages, a larger number of CD4+ cells compared with CD8+ cells were found. Endothelial cells in the connective tissue generally expressed MHC class II. However, no obvious differences between oestrous cycle stages were observed. For other cells than endothelial cells, the result was as follows. In the surface epithelium, a large number of MHC class II expressing cells was observed at oestrus compared with the other stages. No MHC class II expressing cells were found at late dioestrus and dioestrus. MHC class II expressing cells were also found in the glandular epithelium, and in the subepithelial and glandular connective tissue layers during all oestrous cycle stages but with no significant differences between stages. In conclusion, the present study showed a variation in the distribution of T lymphocyte subpopulations (CD2+, CD4+ and CD8+) and MHC class II expressing cells in the sow endometrium during different stages of the oestrous cycle. Also a variation between different tissue layers was found. It is suggested that helper and cytotoxic function of the immune system have primary locations in different tissue layers of the endometrium.

Animals↗
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

Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity.

Factor VII requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active factor VIIa (FVIIa) conformation. The former event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. We have designed a number of FVIIa mutants with the aim of mimicking the effect of TF, that is, creating molecules with increased intrinsic (TF-independent) enzymatic activity. Based on a possible structural difference between free and TF-bound FVIIa (Pike, A. C. W., Brzozowski, A. M., Roberts, S. M., Olsen, O. H., and Persson, E. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8925--8930), we focused on the helical region encompassing residues 307-312 and residues in its spatial vicinity. For instance, FVIIa contains Phe-374 and Leu-305, whereas a Phe/Tyr residue in the position corresponding to 374 in homologous coagulation serine proteases is accompanied by Val in the position corresponding to 305. This conceivably results in a unique orientation of this helix in FVIIa. Substitution of Val for Leu-305 in FVIIa resulted in a 3--4-fold increase in the intrinsic amidolytic and proteolytic activity as compared with wild-type FVIIa, whereas the activity in complex with soluble TF remained the same. In accordance with this, L305V-FVIIa exhibited an increased rate of inhibition as compared with wild-type FVIIa, both by d-Phe-Phe-Arg-chloromethyl ketone and antithrombin III in the presence of heparin. The increased FVIIa activity upon replacement of Leu-305 by Val may be mediated by a movement of the 307--312 helix into an orientation resembling that found in factors IXa and Xa and thrombin. The corresponding shortening of the side chain of residue 374 (Phe --> Pro) had a smaller effect (about 1.5-fold increase) on the intrinsic activity of FVIIa. Attempts to increase FVIIa activity by introducing single or multiple mutations at positions 306, 309, and 312 to stabilize the 307-312 helix failed.

Amino Acid Substitution↗
PubMed2001

Substitution of aspartic acid for methionine-306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor.

The enzyme factor VIIa (FVIIa) triggers the blood coagulation cascade upon association with tissue factor (TF). The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex, and Met-306 in the serine protease domain of FVIIa participates in this event. We have characterized FVIIa variants mutated in position 306 with respect to their ability to be stimulated by TF. The amidolytic activity of FVIIa mutants with Ser, Thr, and Asn in position 306 was stimulated 9-, 12-, and 7-fold, respectively, by soluble TF as compared to 22-fold for wild-type FVIIa. In contrast, the activity of Met306Asp-FVIIa only increased about 2-fold and that of Met306Asp/Asp309Ser-FVIIa increased about 1.5-fold. Modeling suggests that Asp in position 306 prevents the TF-induced stimulation of FVIIa by disrupting essential intermolecular hydrogen bonds. The ability of the FVIIa variants to catalyze factor X activation and the amidolytic activity were enhanced to a similar extent by soluble TF. This indicates that factor X does not promote its own activation through interactions with exosites on FVIIa made accessible upon FVIIa-TF assembly. Met306Asp-FVIIa binds soluble TF with a dissociation constant of 13 nM (about 3-fold higher than that of FVIIa), and, in sharp contrast to FVIIa, its binding kinetics are unaltered after inactivation with D-Phe-Phe-Arg chloromethyl ketone. We conclude that a single specific amino acid replacement, substitution of Asp for Met-306, virtually prevents the TF-induced allosteric changes which normally result in dramatically increased FVIIa activity and eliminates the effect of the active site inhibitor on TF affinity.

Allosteric Regulation↗
PubMed2001

The sow endometrium at different stages of the oestrous cycle: studies on morphological changes and infiltration by cells of the immune system.

The aim of this study was to investigate the distribution of leukocytes and the morphological changes of the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire), with an average parity number of 3.4 +/- 0.7 (mean +/- S.D.) were used. Blood samples were collected from the jugular vein 1h before slaughter for analyses of oestradiol-17beta and progesterone levels. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus, prooestrus, oestrus, early dioestrus and dioestrus, were fixed, embedded in plastic resin and stained with toluidine blue. The surface and glandular epithelium as well as subepithelial and glandular connective tissue layers were examined by light microscopy. The significantly highest surface and the glandular epithelium were observed at oestrus and dioestrus, respectively. The largest number of capillaries (underneath the surface epithelium) was found at oestrus. In the surface epithelium, the largest number of intraepithelial lymphocytes (IELs, round nucleus) was found at early dioestrus. The largest number of lymphocytes and macrophages within the glandular epithelium were found at early dioestrus and oestrus, respectively. In the subepithelial connective tissue layer, the most common type of leukocytes during all stages was the lymphocyte. The largest numbers of lymphocytes and neutrophils were found at oestrus while the largest number of eosinophils was found at dioestrus. The dominating cells of the immune system in the connective tissue of the glandular layer were lymphocytes and macrophages. The significantly largest numbers of lymphocytes and plasma cells were found at early dioestrus and dioestrus, respectively. The number of lymphocytes in the connective tissue of the glandular layer and the number of plasma cells in the subepithelial layer were positively correlated with the plasma level of progesterone (P < or = 0.05). The numbers of capillaries and neutrophils in the subepithelial layer underneath the surface epithelium as well as the number of macrophages in both surface and glandular epithelium were positively correlated with the plasma level of oestradiol-17beta (P < or = 0.05). In conclusion, the present study showed a variation in the infiltration and distribution of lymphocytes, neutrophils, eosinophils, macrophages, mast cells and plasma cells in the sow endometrium during different stages of the oestrous cycle. Also morphological parameters (e.g. height of surface and glandular epithelium, capillaries density and degree of oedema) varied throughout the oestrous cycle.

Animals↗
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↗