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At least 667 records · Page 37Linked to original sources
PubMed2004

Long-term hypoxia alters calcium regulation in near-term ovine myometrium.

Previous studies showed that long-term hypoxia (LTH) during pregnancy alters myometrial contractility. The present study was designed to test the hypothesis that LTH during pregnancy suppresses myometrial contractility in sheep by affecting the calcium signaling cascade. Pregnant sheep were maintained at high altitude (3820 m) from Day 30 to Day 139 of gestation, when the animals were killed for collection of myometrial tissue. Tissue was also collected from age-matched, normoxic controls. Circular and longitudinal layers were separated, and strips from each layer were mounted in a muscle bath. After pretreatment with 10(-8) M oxytocin, the strips were exposed to increasing half- or quarter-log doses of nifedipine (L-type calcium-channel blocker), ruthenium red, ryanodine (blockers of inositol 1,4,5-trisphosphate-insensitive calcium stores), or 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; phospholipase C inhibitor). Area under the contraction curve was analyzed, and pD(2) (log of concentration yielding 50% of maximum response) values and maximum relaxation responses were calculated. The maximum relaxation response to nifedipine was increased in both longitudinal (P < 0.01) and circular (P < 0.05) myometrial layers from LTH compared to control tissue, whereas no difference was observed in response to ruthenium red or ryanodine. The maximum relaxation response to NCDC was lower in the LTH circular layer (P < 0.05). Together, these data are indicative of an increase in the dependence of ovine uterine smooth muscle on extracellular calcium influx through the L-type, voltage-gated calcium channels following LTH. This appears to occur not through an increase in L-type calcium channels but, rather, through a possible decline in importance of the oxytocin-induced, phospholipase C-mediated pathway, resulting in a greater proportion of extracellular calcium contributing to contraction. Layer-dependent differences also exist between the circular and longitudinal myometrium in response to phospholipase C inhibition.

Animals↗
PubMed2004

Multiple myeloma.

High-dose therapy with stem cell transplantation (SCT) and novel targeted therapies (thalidomide, its more potent analogues, and bortezomib) represent two approaches for overcoming resistance of multiple myeloma (MM) cells to conventional therapies. While it is now clear that dose-intensification improves the outcome in younger patients, long-term remissions are obtained in a minority of patients. Therefore, the impact of novel agents as part of front-line therapy is the objective of ongoing trials. Gene expression profiling (GEP) will help to improve the management of MM not only by identifying prognostic subgroups but also by defining molecular pathways that are associated with these subgroups and that are possible targets for future therapies. In Section I, Dr. John Shaughnessy describes recent data obtained with GEP of CD138-purified plasma cells from patients with MM. His group has already shown that overexpression of the Wnt signaling inhibitor DKK1 by MM plasma cells blocks osteoblast differentiation and contributes to the development of osteolytic bone lesions. Recent data allow identification of four subgroups of MM in which GEP is highly correlated not only with different clinical characteristics and outcome but also with different cytogenetic abnormalities. In addition, abnormal expression of only three genes (RAN, ZHX-2, CHC1L) is associated with rapid relapses. In the context of intensive therapy with tandem autotransplantations, this model appears to be more powerful than current prognostic models based on standard biologic variables and cytogenetics. Understanding why the dysregulation of these three genes is associated with a more aggressive behavior of the disease will help to define new therapeutic strategies. In Section II, Dr. Jean-Luc Harousseau presents recent results achieved with tandem autologous SCT (ASCT) and with reduced intensity conditioning (RIC) allogeneic SCT. ASCT is now considered as the standard of care in patients up to 65 years of age. The IFM (Intergroupe Francophone du Myelome) has recently shown that double ASCT is superior to single ASCT. Current results of three other randomized trials confirm that double ASCT is superior, at least in terms of event-free survival. However, patients with poor prognostic features do poorly even after tandem ASCT. Strategies to further improve the outcome of ASCT include more intensive therapies and the use of novel agents such as thalidomide and immunomodulatory analogs (IMiDs) or bortezomib. Results of allogeneic SCT remain disappointing in MM even with T cell-depleted grafts. Preliminary results of a strategy combining ASCT to reduce tumor burden and RIC allogeneic SCT are encouraging, although the follow-up is still short. However, again, patients with chromosome 13 deletions have poor results with RIC. Longer follow-up of ongoing multicentric studies will help to clarify the indications of RIC. In Section III, Dr. Paul Richardson summarizes current knowledge of novel targeted therapies in MM. A better understanding of interactions between MM cells and bone marrow stromal cells and of the signaling cascades whereby cytokines mediate proliferation, survival, drug resistance and migration of MM cells provide the rationale for testing novel agents in relapsed/refractory MM. Increased angiogenesis coupled with the known anti-angiogenesis activity of thalidomide justified its use in refractory MM. The remarkable responses initially achieved prompted a number of clinical studies in different indications and the development of more potent IMIDs. Among them CC-5013 (Revlimid) has been tested in Phase I/II studies and a randomized Phase III study has just been completed. Blockade of NF-kappa B using the proteasome inhibitor bortezomib (Velcade) may mediate anti-MM activity by inhibiting interleukin (IL)-6 production in stromal cells and other mechanisms of action have been shown in preclinical studies. Based on the promising results of the Phase II trial, a large randomized trial of bortezomib versus dexamethasone has been completed. Studies of bortezomib combined with other drugs are ongoing. Arsenic trioxide has a number of properties showing that it targets MM cells interacting with the microenvironment. Clinical studies are ongoing as well. Other agents in MM have already been or will probably be translated soon from the bench to the bedside.

Adult↗
PubMed2005

High throughput screening in agrochemical research.

The demand for new herbicides, insecticides and fungicides led to a steady increase in the number of compounds being tested to find novel market products. To keep pace with the rising workload, high throughput screening (HTS) technologies have been introduced. In agrochemical research miniaturised in vivo tests on whole real target organisms are now possible and are an integral part of the screening cascade. A complementary target based in vitro HTS has also been established in agrochemical research. Target based HTS allows a directed approach towards untouched market shares by novel modes of action. Selection of the best suited targets is the most crucial issue in this approach. Genomic methods thereby deliver many essential genes as candidate targets. Consideration of further criteria such as druggability notably narrows down the number of promising targets. Though target to hit to lead progression still is as in pharmaceutical research a complex and therefore risky process, the implementation of novel bioscience technologies has entailed the transition to an integrated innovative agrochemical research perspective.

Agrochemicals↗
PubMed2000

High molecular weight kininogen adsorption on hemodialysis membranes: influence of pH and relationship with contact phase activation of blood plasma. influence of pre-treatment with poly(ethyleneimine).

Protein adsorption is an essential parameter for the evaluation of the hemocompatibility of biomedical materials. In effect, protein adsorption often generates unfavorable, complex, biochemical reactions and precedes cell adhesion on artificial interfaces. It is therefore necessary to modify the surface in contact with blood in order to minimize the onset of undesirable, biochemical, cascade reactions. Adsorption of high molecular weight kininogen (HK), which participates in the contact phase activation of the endogenous blood coagulation cascade, was evaluated at 37 degrees C. This paper also presents the results of contact phase activation tests carried out in vitro with 1:20 diluted human plasma flowing through minidialyzers containing hollow fibers of synthetic hemodialysis membranes. These tests showed that contact phase activation is strongly dependent on pH around the physiological value (ranging from 7.35 to 7.80) for negatively-charged membranes. The same pH effect was observed on the AN69 membrane as regards HK adsorption from binary labeled solutions of 125I-HK and 131I-Fibrinogen at concentrations corresponding to 1% diluted plasma. It is suggested that the influence of pH could be related to imidazole pKa values in histidine residues of kininogen D5H domain. The same study was also conducted with hemodialysis membranes pre-treated with poly(ethyleneimine). Both HK adsorption and contact phase activation proved to be greatly reduced, irrespective of the pH value (between 7.0 and 7.8). Hence, positively-charged poly(ethyleneimine) adsorbed on the membrane through strong ionic interactions with sulfonate groups of the surface probably constitutes a repelling, water-swollen layer for the kininogen molecule. In addition, the advantages of the high levels of adsorbance of small molecules on the AN69 membrane leading to blood epuration, due to its high porosity and for some of them to its charge density, should not be lost by such a surface treatment.

Acrylic Resins↗
PubMed2005

[Current and perspective treatment of chronic lymphocytic leukemia].

Chronic lymphocytic leukemia represents the most frequent hematological malignancy in the western population. Important role in its pathogenesis has the inhibition of apoptosis, disturbances in differentiation of B-lymphocytes, slightly increased proliferation and induction of angiogenesis. Current treatment options (chemotherapy and immunotherapy) can improve the quality of life and prolong disease-free survival, but the overall survival is not significantly influenced. The only curative treatment alternative is the allogeneic hematopoietic stem cell transplantation. However, this treatment is accompanied by the risk of peritransplantation mortality and its use is limited for younger patients. Number of new compounds is clinically tested. New drugs can influence intracellular events such as induction of apoptosis or inhibition of the transduction cascade. Review summarises treatment alternatives and presents an algorithm for their clinical application.

Humans↗
PubMed1988

Comparative activity of leukotriene D4, 5,6-dihydroxy-eicosatetraenoic acid and lipoxin A on guinea pig lung parenchyma and ileum smooth muscle.

Various compounds derived from the 5-lipoxygenase pathway of arachidonic acid (Leukotriene D4 [LTD4], 5S,6R-dihydroxy-7,9,11,14- eicosatetraenoic acid [5S,6R-DiHETE], 5S,6S-DiHETE, 5S,6R-Lipoxin A [5S,6R-Lx A], 5S,6S-Lx A) were tested on guinea-pig lung parenchyma strips (G.P.L.P.S.) and ileum smooth muscles (G.P.I.S.M.) mounted in a cascade superfusion system. These products induced contractile responses on G.P.L.P.S., whereas only the LTD4 was active on G.P.I.S.M. FPL-55712 inhibited almost totally their myotropic activity. The 5S,6R compounds were more active than the 5S,6S ones, with an order of potency of LTD4 greater than 5,6-DiHETE greater than Lx A. It is suggested that 5,6-DiHETE and Lx A isomers may act via the lung LTD4 receptors which appear less specific than ileum LTD4 receptors.

Animals↗
PubMed1982

[Mutagenic effect of polycyclic aromatic hydrocarbons (PAH) and polar organic compounds (POC) in urban aerosols].

The concentrations of the polycyclic aromatic hydrocarbons (PAH) and the polar organic fraction (polar organic compounds = POC) as part of the aerosol particles, collected on fiberglass filters with a six-stage high volume cascade impactor from 17.7. to 9.10. 1981 from Berlin (West) air, were determined.--The two fractions were tested for their mutagenicity by using the Salmonella typhimurium bioassay (Ames-test). According to their site of deposition in the human respiratory system the aerosol particles were fractioned into two classes: "A" (Dae = 1.3-10.2 microns) and "B" (Dae = 0.4-1.3 microns). The following data were obtained: 1. The ether extractable organic matter (EEOM) contains 8.9-12.0% PAH and 40.1-65.8% POC. 2. PAH and POC extracted from particles with a Dae:1.3-10.2 microns exhibit nearly the same mutagenic activity after metabolic activation whereas the POC derived from particles with a Dae:0.4-1.3 microns (lung penetrating fraction) result in a higher mutagenicity than the PAH. 3. The POC show a higher mutagenic activity without metabolic activation than the PAH. 4. Generally extracts derived from particles with a Dae:0.4-1.3 microns exhibit higher mutagenic effects than those derived from particles with a Dae:1.3-10.2 microns.

Aerosols↗
PubMed2006

Post-treatment with the Ca(2+)-Mg(2+)-endonuclease inhibitor aurintricarboxylic acid prevents peroxynitrite-induced DNA damage and death of murine astrocytes.

Oxidative stress plays critical roles in aging, cell death, and many diseases. Peroxynitrite is one of the major reactive oxygen species which mediates cell injury in a number of illnesses. It is of importance to identify the downstream events in peroxynitrite-initiated cell death cascade for preventing peroxynitrite toxicity. Ca(2+)-Mg(2+)-endonucleases have been suggested as the endonucleases that execute DNA fragmentation in several apoptotic cascades. In this study, we determined if astrocytes and neurons express the genes of Ca(2+)-Mg(2+)-endonucleases. We also tested our hypothesis that post-treatment with the Ca(2+)-Mg(2+)-endonuclease inhibitor aurintricarboxylic acid can decrease peroxynitrite-induced DNA damage and death of astrocytes. We found that both astrocytes and neurons express DNase I-like endonuclease-a major isoform of Ca(2+)-Mg(2+)-endonucleases. Treatment of astrocytes with aurintricarboxylic acid either before or after peroxynitrite exposures can profoundly decrease peroxynitrite-induced DNA damage and cell death. These results suggest that Ca(2+)-Mg(2+)-endonucleases may be a key downstream component in peroxynitrite-initiated cell death cascade in astrocytes and some other cell types, and aurintricarboxylic acid could be used to decrease peroxynitrite-induced DNA damage at delayed phases.

Animals↗
PubMed1988

[Chronic urticaria--still a vexing problem? Progress in diagnosis and therapy].

Despite the suggestion than an intolerance reaction could be a possible cause of chronic urticaria in some cases, the pathogenesis of this disease remains enigmatic. It is unlikely that an intolerance reaction to substances used in provocation testing is the cause of the daily rashes. It rather seems that the intolerance reaction is an indication of a certain instability of the mast cell membrane of other factors of the reaction cascade leading to the wheals. Fortunately, an effective symptomatic therapy is available in such cases where the cause cannot be detected and the trigger cannot be avoided. The modern, non-sedating H1-antihistamines have been tested in clinical-experimental studies as well as in a multicentric study in patients with chronic urticaria. In histamine-induced wheals, terfenadine was slightly superior to other non-sedating H1-blockers, when the grade of reduction of wheals and flare areas was taken as criterion. In clinical studies, another antihistamine, loratadine, was slightly, but statistically not significantly superior to terfenadine. In cases that cannot be effectively treated with H1-blockers alone, the additional administration of non-steroidal antiphlogistics (acetylsalicylic acid, indomethacin) is recommended. To avoid aggravation of the symptoms caused by the antiphlogistics, the drugs must be given in increasing dosages and after concurrent treatment with H1-blockers. With this combined therapy, about 50% of the patients present amelioration.

Chronic Disease↗
PubMed2005

Integrin signaling cascades are operational in adult hippocampal synapses and modulate NMDA receptor physiology.

Integrin class adhesion proteins are concentrated at adult brain synapses. Whether synaptic integrins engage kinase signaling cascades has not been determined, but is a question of importance to ideas about integrin involvement in functional synaptic plasticity. Accordingly, synaptoneurosomes from adult rat brain were used to test if matrix ligands activate integrin-associated tyrosine kinases, and if integrin signaling targets include NMDA-class glutamate neurotransmitter receptors. The integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) induced rapid (within 5 min) and robust increases in tyrosine phosphorylation of focal adhesion kinase, proline-rich tyrosine kinase 2 and Src family kinases. Increases were similarly induced by the native ligand fibronectin, blocked with neutralizing antibodies to beta1 integrin, and not obtained with control peptides, indicating that kinase activation was integrin-mediated. Both GRGDSP and fibronectin caused rapid Src kinase-dependent increases in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Tests of the physiological significance of the latter result showed that ligand treatment caused a rapid and beta1 integrin-dependent increase in NMDA receptor-mediated synaptic responses. These results provide the first evidence that, in adult brain, synaptic integrins activate local kinase cascades with potent effects on the operation of nearby neurotransmitter receptors implicated in synaptic plasticity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMedDate not supplied

The effects of drugs on the arachidonate cascade in experimentally burned rabbits.

Platelet reduction and renal dysfunction may occur after major thermal injury and be causally related to changes in the arachidonate cascade. In this article two new drugs, prostaglandin I2 analog OP-41483 (Venopirin) and thromboxane synthetase inhibitor OKY-046 (Cataclot), were tested in addition to aspirin in animal experiments with rabbits. The rabbits were divided into five groups and measurements made before and 8 and 24 hours after burn injury. The results indicate that platelet reduction and renal dysfunction occur within 8 hours after thermal injury in the no-therapy group and renal dysfunction could not be prevented completely even by adequate infusion therapy, but treatment with OP-41483 proved to suppress platelet reduction and improve renal function. Improvement in renal function was also noted in animals treated with aspirin or OKY-046, but platelet reduction could not be prevented by these drugs. Analysis of the time course of changes in thromboxane B2 level and 6-keto prostaglandin F1 alpha level disclosed that animals treated by infusion showed an elevation in thromboxane B2 and a significant elevation in the thromboxane B2/6-keto prostaglandin F1 alpha ratio, whereas animals treated with these three drugs showed a reduction in the thromboxane B2/6-keto prostglandin F1 alpha ratio. These results indicate that an effective measure for prevention of dysfunction of the organs after thermal injury is treatment with drugs that can modify the arachidonate cascade and cause an absolute or relative reduction in TXA2.

6-Ketoprostaglandin F1 alpha↗
PubMed1996

Extracellular matrix proteins: bystanders or active participants in the allograft rejection cascade?

The role extracellular matrix (ECM) proteins may play in the host immune cascade triggered by organ transplantation is largely unknown. We have employed well-defined rat cardiac allograft model to test the hypothesis that the expression of ECM components, such as fibronectin (FN) or laminin (LN), represents an integral part of the host rejection response in transplant recipients. Indeed, treatment of rats with anti-LN Ab profoundly affected lymphocyte recirculation pathways, and selectively decreased deposition of alloreactive lymphocytes at the graft site and in host peripheral lymph nodes. Moreover, cardiac rejection was accompanied by markedly increased intragraft expression of both LN and FN. The infiltrating mononuclear cells, however, accumulated selectively in FN-rather than LN-rich cardiac areas. We also observed distinct temporal expression and spatial distribution patterns of FN variants in rat cardiac allografts and isografts. The local synthesis of FN, in both allografts and isografts was increased as early as at 3 h post-transplant. The expression of FN in cardiac allografts but not in isografts, remained high during later intervals to the point of ultimate rejection. The newly synthesized FNs derived from graft infiltrating macrophages and arterioles, and included EIIIA +, EIIIB +, and CS-I + splicing variants. The EIIIA was preferentially enriched in the earlier, whereas EIIIB and CS-I were selectively enhanced in the later, rejection phases. Finally, treatment of rat recipients with synthetic FN-CS-I peptides prevented the incidence of early (acute) and late (chronic) rejection, supporting the notion that distinct FNs may play a fundamental role in the host immune cascade triggered by organ transplantation. These data offer potential novel sites for intervention in the control of transplant rejection, and contribute to the development of refined therapeutic strategies based upon new concepts of host immunosuppression.

Animals↗
PubMed2004

Association testing in 9,000 people fails to confirm the association of the insulin receptor substrate-1 G972R polymorphism with type 2 diabetes.

The insulin receptor substrate (IRS)-1 is an important component of the insulin signal transduction cascade. Several reports suggest that a Gly-->Arg change in codon 972 is associated with type 2 diabetes and related traits, and a recent meta-analysis reported a modest but nominally significant association with type 2 diabetes (odds ratio [OR] 1.25 in favor of carriers of the Arg allele [95% CI 1.05-1.48). To test the reproducibility of the model in a recent meta-analysis, we examined genotype-phenotype correlation in three large Caucasian samples (not previously reported for this variant) totaling 9,000 individuals (estimated to have >95% power to obtain a P < 0.05 for the OR of 1.25 estimated in the meta-analysis). In our combined sample, comprising 4,279 case and 3,532 control subjects, as well as 1,189 siblings discordant for type 2 diabetes, G972R was not associated with type 2 diabetes (OR 0.96 [0.84-1.10], P = 0.60). Genotype at G972R had no significant effect on various measures of insulin secretion or insulin resistance in a set of Scandinavian samples in whom we had detailed phenotypic data. In contrast, the well-documented associations of peroxisome proliferator-activated receptor gamma P12A and Kir6.2 E23K with type 2 diabetes are both robustly observed in these 9,000 subjects, including an additional (previously unpublished) confirmation of Kir6.2 E23K and type 2 diabetes in the Polish and North American samples (combined OR 1.15 [1.05-1.26], P = 0.001). Despite genotyping 9,000 people and >95% power to reproduce the estimated OR from the recent meta-analysis, we were unable to replicate the association of the IRS-1 G972R polymorphism with type 2 diabetes.

Amino Acid Substitution↗
PubMed2006

Sudden cardiac death in the young: a strategy for prevention by targeted evaluation.

The annual incidence of sudden cardiac death (SCD) in the general population is estimated as 1 in a 1,000. Since survival rates from out-of-hospital cardiac arrests are poor, primary prevention is key to reducing the burden of SCD in the community. Prominent causes of SCD include ischaemic heart disease, anomalous coronary arteries, and the primary myocardial diseases: hypertrophic cardiomyopathy, dilated cardiomyopathy, and ar rhythmogenic right ventricular cardiomyopathy (ARVC). In 4% of sudden deaths in the 16-64 age group, post-mortem examination fails to identify a cause, yielding a default diagnosis of sudden arrhythmic death syndrome (SADS). The inherited arrhythmia syndromes (long QT, short QT, and Brugada syndromes, and familial catecholaminergic polymorphic ventricular tachycardia) may be implicated in SADS, owing to their propensity for producing ventricular tachyarrhythmia in the structurally normal heart. Monogenic disorders therefore predominate as causes of SCD in the young. The advent of effective therapies for these diseases, particularly implantable cardioverter defibrillators, has prompted calls for universal screening to enable timely diagnosis of occult cardiac disease. Since prospective cardiac assessment of the general population is not feasible, the solution may be to target high-risk subgroups, namely, patients with cardiac symptoms, relatives of SCD victims, and competitive athletes. The recommended preliminary work-up includes a 12-lead ECG, signal-averaged ECG, transthoracic echocardiogram, exercise test, and ambulatory ECG monitoring. Cardiovascular magnetic resonance is a useful adjunct in patients with suspected ARVC or anomalous coronary arteries. Provocative challenge with a sodium challenge blocker may be of value in unmasking the Brugada syndrome. Identification of disease-causing mutations in affected individuals facilitates cascade screening of families.

Adolescent↗
PubMed2006

Na+/K+/Cl- cotransporter activates MAP-kinase cascade downstream to protein kinase C, and upstream to MEK.

In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl- cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl- cotransporter, is downstream of tyrosine kinase receptors, as well as of the G-protein-coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl- cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.

Animals↗
PubMedDate not supplied

[Determination of proteinase activity of complement system by immunoenzyme methods].

Modern ELISA for determination of functional activity of component C2 and factors B and D, proteinases of a complement system, and component C3, substrate C3-convertases, key complex enzymes of the complement, have been developed. Essential feature of C3-convertases classical (C4bC2a) and alternative (C3bBb) pathways of the complement activation is that their substrate C3 after proteolytic cleavage is converted into C3b, carrying on the surface thioester covalent bond linking C3b with nucleophilic acceptors that results in immobilization of this proteolytic product near the activating enzyme. Cascade character of an activation of complement system allows to create artificial deficit of separate components in the experimental system and to determine (by ELISA) covalently immobilized component C3 during activation, and also to determine functional activity of any of pre-exhausted components. Use of such approach resulted in the development of the ELISA systems suitable for determination of functional activity of component C2 of classical pathway and factors B and D of the alternative pathway by testing quantity of the immobilized C3b at excess C3. The developed methods allow to investigate mechanisms of functioning of complement, inhibition of the cascade activation by endogenic and exogenous inhibitors, and also to find functional deficiency of components in serum and other biological fluids.

Animals↗
PubMed1990

The first EGF-like domain from human factor IX contains a high-affinity calcium binding site.

It has been suggested that epidermal growth factor-like (EGF-like) domains, containing conserved carboxylate residues, are responsible for the high-affinity calcium binding exhibited by a number of vitamin K-dependent plasma proteins involved in the control of the blood coagulation cascade. These include the procoagulant factors IX and X, and the anticoagulants protein C and protein S. To test this hypothesis we have expressed the first EGF-like domain from human factor IX (residues 46-84) using a yeast secretion system, and examined calcium binding to the domain. Using 1H-NMR to measure a calcium-dependent shift assigned to Tyr69 we have detected a high-affinity calcium binding site (Kd = 200-300 microM). We suggest that other EGF-like domains of this type may have similar calcium binding properties. In addition, we have completely assigned the aromatic region of the NMR spectrum by NOESY and COSY analysis, and have used these data to discuss the effect of calcium and pH on the conformation of the domain with reference to a model based on the structure of human EGF.

Amino Acid Sequence↗
PubMed2000

Cytokines associated with the pathophysiology of aggressive fibromatosis.

The rare benign extra-abdominal desmoid tumor is characterized by aggressive invasion of normal tissue. Treatment is complicated by its recurrence, invasiveness, and persistence. The etiology is unknown and the pathophysiology is obscure. Because of exuberant fibroblastic proliferation with collagenous tissue being the primary tissue component, this desmoid tumor has been compared with keloids arising from excessive scar formation in healing wounds. Numerous cytokines are associated with signaling for growth and maintenance of mesenchymal cells. Altered expression of these proteins is associated with many pathologic conditions. It has been proposed that the enhanced expression of platelet-derived growth factor and its receptor characterize desmoid tumors. We tested the hypothesis that the exuberant fibrosis of desmoid tumors may have resulted from the initiation of the cascade of molecular events producing increased expression of cytokines. We used immunohistochemical analysis of cytokines in desmoid tumors compared with keloids and skin to localize the expression of cytokines. The results showed localized increased expression of the cytokines epidermal growth factor, transforming growth factor-beta, tumor necrosis factor-alpha, vascular endothelial growth factor, interleukin-1beta, and interleukin-6 in the endothelial cells of blood vessels in the tumors. Production of tumor necrosis factor-alpha and interleukin-1beta in tumor tissue was increased, but we did not find increased expression of platelet-derived growth factor. We concluded that the increased expression of cytokines associated with angiogenesis usually found in wound healing and invasive tumors may contribute to the pathophysiology of the desmoid tumor.

Adolescent↗