Suppression of the Antioxidant System and PI3K/Akt/mTOR Signaling Pathway in Cisplatin-Resistant Cancer Cells by Quercetin.

We studied the effect of quercetin on ovarian adenocarcinoma SKOV-3 cell line and isogenic subline SKOV-3/CDDP resistant to the anticancer drug cisplatin. It was found that in resistant cells, quercetin in a concentration of 100 µM that causes a decrease in the cell viability suppressed the expression of genes encoding the key antioxidant enzymes (SOD2, CAT, GPX1, and HO-1), transcription factor Nrf2, and kinases of the PI3K/Akt/mTOR signaling pathway. In parental cells, quercetin, on the contrary, increased the expression of these genes. The results confirm the redox-dependent regulation induced by quercetin and its opposite nature in cisplatin-sensitive and cisplatin-resistant cancer cells.

Suppression of PI3K/Akt/mTOR Signaling Pathway and Antioxidant System and Reversal of Cancer Cells Resistance to Cisplatin under the Effect of Curcumin.

The effect of curcumin on the resistance of SKOV-3 human ovarian adenocarcinoma cells to cisplatin was studied. It was found that curcumin induced "reversal" of cancer cells resistance, which was associated with suppression of the expression of genes encoding the key antioxidant enzymes (SOD1, SOD2, CAT, GPX1, and HO-1) and transcription factor Nrf2 and a decrease in the expression of genes encoding kinases of the PI3K/Akt/mTOR signaling pathway. The obtained results confirm the role of redox-dependent regulation in the "reversal" of cancer cells resistance to cisplatin.

Being Born Large for Gestational Age is Associated with Increased Global Placental DNA Methylation.

Being born small (SGA) or large for gestational age (LGA) is associated with adverse birth outcomes and metabolic diseases in later life of the offspring. It is known that aberrations in growth during gestation are related to altered placental function. Placental function is regulated by epigenetic mechanisms such as DNA methylation. Several studies in recent years have demonstrated associations between altered patterns of DNA methylation and adverse birth outcomes. However, larger studies that reliably investigated global DNA methylation are lacking. The aim of this study was to characterize global placental DNA methylation in relationship to size for gestational age. Global DNA methylation was assessed in 1023 placental samples by LC-MS/MS. LGA offspring displayed significantly higher global placental DNA methylation compared to appropriate for gestational age (AGA; p < 0.001). ANCOVA analyses adjusted for known factors impacting on DNA methylation demonstrated an independent association between placental global DNA methylation and LGA births (p < 0.001). Tertile stratification according to global placental DNA methylation levels revealed a significantly higher frequency of LGA births in the third tertile. Furthermore, a multiple logistic regression analysis corrected for known factors influencing birth weight highlighted an independent positive association between global placental DNA methylation and the frequency of LGA births (p = 0.001).

Mineral trioxide aggregate pulpotomy for permanent molars with clinical signs indicative of irreversible pulpitis: a preliminary study.

AIM: To prospectively investigate the clinical and radiographic success rates of pulpotomy in permanent molars with clinical signs and symptoms suggestive of irreversible pulpitis using mineral trioxide aggregate (MTA) as a pulp dressing agent. METHODOLOGY: Sixteen patients with 23 restorable permanent molars exhibiting signs and symptoms indicative of irreversible pulpitis were enrolled. A standardized operative procedure was followed for all participants. All teeth were isolated with a dental dam and caries was removed, and then, pulpotomy performed with a sterile round and/or flame shape diamond burs. Haemostasis was achieved with 5% sodium hypochlorite (NaOCl). A mixture of MTA was placed against the wound, and a moistened cotton pellet was placed over the MTA. Teeth were temporized with a glass-ionomer restoration. Three to ten days later, the interim restoration was removed and setting of MTA was evaluated. Teeth were restored with stainless steel crowns. Follow-up evaluations were scheduled at 3, 6, 12 months and annually thereafter. Descriptive statistics were used to assess outcomes. RESULTS: The age of patients at time of pulpotomy ranged between 7.6 and 13.6 years (mean = 10.7± 1.7 yrs). The majority of teeth (91%) had clinical signs and symptoms consistent with a diagnosis of symptomatic irreversible pulpitis and symptomatic apical periodontitis (78%). The follow-up examination period ranged from 18.9 to 73.6 months. Clinically and radiographically, all pulpotomies were considered successful at the end of the follow-up period. Radiographically, a hard tissue barrier was noticed in 13 (57%) teeth. CONCLUSION: In children, MTA was associated with high clinical and radiographic success as a pulpotomy agent in permanent teeth with clinical signs and symptoms suggestive of irreversible pulpitis.

Reinforcement learning for routing in cognitive radio ad hoc networks.

Cognitive radio (CR) enables unlicensed users (or secondary users, SUs) to sense for and exploit underutilized licensed spectrum owned by the licensed users (or primary users, PUs). Reinforcement learning (RL) is an artificial intelligence approach that enables a node to observe, learn, and make appropriate decisions on action selection in order to maximize network performance. Routing enables a source node to search for a least-cost route to its destination node. While there have been increasing efforts to enhance the traditional RL approach for routing in wireless networks, this research area remains largely unexplored in the domain of routing in CR networks. This paper applies RL in routing and investigates the effects of various features of RL (i.e., reward function, exploitation, and exploration, as well as learning rate) through simulation. New approaches and recommendations are proposed to enhance the features in order to improve the network performance brought about by RL to routing. Simulation results show that the RL parameters of the reward function, exploitation, and exploration, as well as learning rate, must be well regulated, and the new approaches proposed in this paper improves SUs' network performance without significantly jeopardizing PUs' network performance, specifically SUs' interference to PUs.

Application of reinforcement learning in cognitive radio networks: models and algorithms.

Cognitive radio (CR) enables unlicensed users to exploit the underutilized spectrum in licensed spectrum whilst minimizing interference to licensed users. Reinforcement learning (RL), which is an artificial intelligence approach, has been applied to enable each unlicensed user to observe and carry out optimal actions for performance enhancement in a wide range of schemes in CR, such as dynamic channel selection and channel sensing. This paper presents new discussions of RL in the context of CR networks. It provides an extensive review on how most schemes have been approached using the traditional and enhanced RL algorithms through state, action, and reward representations. Examples of the enhancements on RL, which do not appear in the traditional RL approach, are rules and cooperative learning. This paper also reviews performance enhancements brought about by the RL algorithms and open issues. This paper aims to establish a foundation in order to spark new research interests in this area. Our discussion has been presented in a tutorial manner so that it is comprehensive to readers outside the specialty of RL and CR.

Longevity of band and loop space maintainers using glass ionomer cement: a prospective study.

AIM: This was to prospectively investigate the success and median survival rate of band and loop space maintainers using glass ionomer luting cement for attachment. METHODS: A total of 40 children (22 females and 18 males) between the ages of 3.4 and 7.3 years participated in the study. Each patient received only one band and loop space maintainer. For each child, the same paediatric dentist carried out all diagnosis, band selection, and impression taking and appliance cementation. The same dental technician fabricated all appliances. The luting cement used was Ketac-Cem-Maxicap. Regular follow up appointments were scheduled at 4-6 months intervals. Variables, which might have affected the median survival time for the appliances were tested using Log-Rank and Chi-square tests. RESULTS: 40% of the band and loop space maintainers were successful and 57.5% failed during the study period (40 months). The most common cause of failure was decementation (82% of all failed cases). The overall median survival time was 19.9 months. Appliances fitted in the maxillary and mandibular left side of the mouth showed a statistically higher survival rate than those fitted in the right side (maxillary left quadrant = 35 months, mandibular left quadrant = 28 months, maxillary right quadrant = 14 months, mandibular right quadrant = 16 months) (p<0.008). CONCLUSIONS: Although the overall median survival time was clinically acceptable (19.9 months), the failure rate of the band and loop space maintainers in general was high (57.5%). The main reason for failure was decementation of the band. Further studies are required to compare glass ionomer cements with more recent resin modified luting cements.

Anthropometric profiles of elite Asian female handball players.

AIM: Handball has increased in status as a sport since its introduction in 1972 into the Summer Olympic Games. Whereas anthropometric profiles of female athletes have been reported for certain sports, data for elite handball players are limited. The current study was based on anthropometric measurements of 60 female Asian handball players competing in the continental championship, the aim being to identify any differences between countries and between playing positions. METHODS: The setting was the 12(th) Asian Games in Hiroshima, Japan. Anthropometric data were obtained from 60 players including teams from China, Japan, Kazakhstan and South Korea. Measurements included height, mass, skinfold thicknesses: from these measures percent body fat and muscle mass were estimated. Profiles were compared between 4 nations and 4 positional roles by means of ANOVA. RESULTS: Overall, mean (SD) values were 1.708 (0.068) m, 64.6 (7.7) kg, 20.8% (4.4%), 39.6% (5.2%) for stature, mass, percent body fat and percent muscle mass, respectively. There were small differences between players from different countries but no significant (P>0.05) influence of playing position. Players from Japan were shortest, lightest and lowest in adiposity. The Chinese players were tallest and had the greatest muscle mass. CONCLUSION: These female international handball players differed in some respects in anthropometric characteristics according to their country of origin. The Asian players were found to be relatively homogeneous across the different positions.

Fluid loss and body composition of elite Kuwaiti soccer players during a soccer match.

AIM: The aim of this study was to assess the effect of the hot weather conditions of Kuwait on fluid loss (FL) and body composition of Kuwaiti soccer players. METHODS: During 5 preseason soccer games, 10 elite Kuwaiti soccer players participated in this study. The age and physical characteristics (mean+/-SD) of the subjects were: age 24+/-4.7 years; height 173.4+/-5.2 cm; mass 68.2+/-7 kg; and the percent body fat 12.3+/-3.7%, the mean temperature 45.4+/-2 degrees C, mean humidity was 23.6+/-4.2%. Measurements were taken at the beginning, half time, and the end of each game; FL was measured by body weight changes. Urine specific gravity was used to determine the state of hydration. Body composition was measured using Biodynamics Model 310e Body Composition Analyzer. RESULTS: There was a significant difference in body weight changes between 1st and 2nd half at P>0.05. The mean value of the FL at the end of the game was 3.1+/-1.4 L; indicating no significant difference in FL between 1st and 2nd half at P>0.05). The mean value urine specific gravity at the end game was 1.026+/-0.002; there was a significant difference between first, and second half for urine specific gravity (P>0.05). CONCLUSIONS: Results of this study indicated that the subjects did not consume enough fluid to offset FL. Players and coaches involved with activities in hot humid environments should pay closer attention to FL and body weight changes which occur during physical activity. Special care should always be taken to insure players consume extra water prior to each training session or game.

Developing peptide inhibitors to thrombin activation of platelets from bradykinin analogs.

Investigations identified peptide, platelet-selective thrombin inhibitors. Three peptides (MAP4-RPPGF, RGKWC and RGDWC) were relatively selective inhibitors of thrombin-induced platelet activation and calcium mobilization. MAP4-RPPGF at 35.5+/-0.03 microM inhibits gamma-thrombin-induced platelet aggregation 100% and alpha-thrombin-induced calcium mobilization in fibroblasts 84%. RGKWC at 800+/-400 microM inhibits gamma-thrombin-induced platelet aggregation 100% and calcium mobilization 63%. RGDWC at 140+/-100 microM inhibits gamma-thrombin-induced platelet aggregation 100% and calcium mobilization 32%. RGDWC also inhibits ADP-induced platelet aggregation, whereas MAP4-RPPGF and RGKWC do not. RGKWC prolongs the activated partial thromboplastin time (APTT) but not the prothrombin time (PT) or thrombin clotting time (TCT). RGKWC uniquely inhibits alpha-thrombin activation of human factor XI. Single amino acid substitutions in peptide pentamers result in differences in potency and mechanism(s) of inhibition of platelet and fibroblast activation by thrombin.

Decreased 13-S-hydroxyoctadecadienoic acid levels and 15-lipoxygenase-1 expression in human colon cancers.

13-S-Hydroxyoctadecadienoic acid (13-S-HODE), the product of 15-lipoxygenase (15-LOX) metabolism of linoleic acid, enhances cellular mitogenic responses to certain growth factors. Other observations have questioned whether 13-S-HODE has tumorigenic effects. Our study evaluated the hypothesis that 15-LOX-1 is overexpressed in colon cancers resulting in an increase in intracellular 13-S-HODE. 15-LOX-1 and 13-S-HODE were quantified using western blots, ELISA and immunohistochemistry in 18 human colon cancers with paired normal colonic mucosa. Additionally, 15-LOX-1 expression was measured by western blots in three transformed colonic cell lines and in a human umbilical vein endothelial cell line. Next, we evaluated 13-S-HODE effects on cellular proliferation, cell cycle distribution and apoptosis in a transformed colonic cell line (RKO). Cell cycle distributions were measured by flow cytometry and apoptosis was assessed by phase contrast microscopy, electron microscopy, flow cytometry and DNA fragmentation assay. 15-LOX-1 immunohistochemistry staining scores were reduced in tumor tissues (P

Thrombostatin inhibits induced canine coronary thrombosis.

Thrombostatin (RPPGF), an angiotensin converting enzyme metabolite of bradykinin, is an inhibitor of alpha-thrombin's ability to activate platelets. We examined the in vivo pharmacokinetics and pharmacodynamics of thrombostatin in rabbits and its ability to inhibit coronary thrombosis induced by electrolytic injury in dogs. Plasma half-life of thrombostatin had a t1/2alpha of 2.6 min and a t1/2beta of 24 min in rabbits. Ligating the renal arteries did not prolong clearance (t1/2alpha = 2.4 min; t1/2beta = 12 min). Thrombostatin produced a prolonged in vivo antiplatelet effect. At 30 min after a single intravenous administration in rabbits, thrombostatin's plasma concentration was <8.7 microM (5 microg/ml). However, ex vivo 20 and 40 nM gamma-thrombin-induced platelet aggregation of these rabbits' platelets was inhibited 40% for 2.75 and 1 h, respectively. In vitro, flow cytometry studies revealed that thrombostatin specifically bound to human platelets and washed human platelets treated with thrombostatin were less responsive to gamma-thrombin than control platelets. Using electrolytic injury to induce coronary artery thrombosis, dogs treated with thrombostatin, aspirin, or combined thrombostatin and aspirin occluded in 62+/-25 (mean +/- SD), 62+/-36, or 89+/-32 min versus untreated animals which occluded at 39+/-27 min, (p<0.01, p<0.01 and p<0.001, respectively). These studies show that thrombostatin binds to platelets and can delay coronary occlusion in vivo.

Factor XII does not initiate prekallikrein activation on endothelial cells.

It is well known that on artificial surfaces, binding and autoactivation of factor XII (FXII) is the initiating event of plasma prekallikrein (PK) activation. We performed investigations to examine whether this mechanism was true for FXII activation on endothelial cells (HUVEC). Activation of PK on HUVEC required an optimal substrate and Zn2+ concentration, the latter of which varied with the buffer's carrier protein. Maximal PK activation required the addition of 250 microM or 10 microM Zn2+ to buffers containing bovine serum albumin (BSA) or gelatin, respectively. However, the actual free Zn2+ concentration in these buffers was the same at 8 microM. In both BSA- and gelatin-containing buffers and using two different chromogenic substrates for FXII, no autoactivation of FXII on HUVEC was seen when incubated for up to 60 min. Rather, initiation of FXII enzymatic activity required the presence of PK. FXII activation after PK activation contributed to the extent of measured enzymatic activity, but its role was secondary because treatment with corn trypsin inhibitor or a neutralizing antibody to FXIIa did not abolish the measured enzymatic activity. They also reduced the activity to the level seen with PK activation alone. Alternatively, soybean trypsin inhibitor abolished the proteolytic activity associated with PK and FXII activation on HUVEC. Further, only normal human and FXII-deficient plasmas, not PK-deficient plasma, had the ability to generate proteolytic activity when incubated over endothelial cells. In a purified system, maximal PK activation was measured after a 10-15 min incubation depending upon the concentration of reactants. When FXII was added with the PK, maximal activation occurred within 7.5-10 min. In normal human or FXII-deficient plasmas, but not in PK-deficient plasma, maximal activation was seen in 4 min. These data indicate that on HUVEC, unlike artificial surfaces, PK activation when bound to HK is the initiating activation event in this system. FXII activation is secondary to PK activation and contributes to the extent of measured enzymatic activity. These data challenge the accepted dogmas of "contact activation" and suggest that on biologic membranes a new notion as to how this system is activated needs to be considered.

Identification of cytokeratin 1 as a binding protein and presentation receptor for kininogens on endothelial cells.

A kininogen binding protein(s), a putative receptor, was identified on endothelial cells. A 54-kDa protein was isolated by a biotin-high molecular mass kininogen (HK) affinity column that, on aminoterminal sequencing of tryptic digests, was identified as cytokeratin 1. Multiple antibodies directed to cytokeratin 1 reacted with a 54-kDa band on immunoblot of lysates of endothelial cells. On laser scanning confocal microscopy, cytokeratin 1 antigen was found on the surface of endothelial cells. Cytokeratin 1 antigen also was detected on endothelial cell membranes by flow cytometry. Moreover, an antipeptide antibody to a sequence unique to cytokeratin 1 also specifically bound to nonpermeabilized endothelial cells. Biotin-HK specifically bound to cytokeratin only in the presence of Zn2+, and cytokeratin blocked biotin-HK binding to endothelial cells. Further, HK and low molecular mass kininogen, but not factor XII, blocked biotin-HK binding to cytokeratin, and peptides of each cell binding region of HK on domains 3,4, and 5 blocked biotin-HK binding to cytokeratin. gC1qR and soluble urokinase-like plasminogen activator receptor also inhibited biotin-HK binding to cytokeratin. These investigations identify a new function for cytokeratin 1 as a kininogen binding protein. Cytokeratins, members of the family of intermediate filament proteins, may represent a new class of receptors.

High molecular weight kininogen regulates prekallikrein assembly and activation on endothelial cells: a novel mechanism for contact activation.

The consequences of assembling the contact system of proteins on the surface of vascular cells has received little study. We asked whether assembly of these proteins on the surface of cultured human endothelial cells (HUVECs) results in the activation of prekallikrein (PK) and its dependent pathways. Biotinylated PK binds specifically and reversibly to HUVECs in the presence of high molecular weight kininogen (HK) (apparent Kd of 23 +/- 11 nmol/L, Bmax of 1.7 +/- 0.5 x 10(7) sites per cell [mean +/- SD, n = 5 experiments]). Cell-associated PK is rapidly converted to kallikrein. Surprisingly, the activation of cell-associated HK.PK complexes is entirely independent of exogenous factor XII (Km = 30 nmol/L, Vmax = 12 +/- 3 pmol/L/min in the absence v Km = 20 nmol/L, Vmax = 9.2 +/- 2.1 pmol/L/min in the presence of factor XII). Rather, kallikrein formation is mediated by an endothelial cell-associated, thiol protease. Cell-associated HK is proteolyzed during the course of prekallikrein activation, releasing kallikrein from the surface. Furthermore, activation of PK bound to HK on HUVECs promotes kallikrein-dependent activation of pro-urokinase, resulting in the formation of plasmin. These results indicate the existence of a previously undescribed, factor XII-independent pathway for contact factor activation on HUVECs that regulates the production of bradykinin and may contribute to cell-associated plasminogen activation in vivo.

Elemental iron does repress transferrin, haemopexin and haemoglobin receptor expression in Haemophilus influenzae.

The iron repressible nature of Haemophilus influenzae transferrin binding proteins suggests a regulatory role for elemental iron in their expression. The existence of a Haemophilus ferric uptake repressor (Fur) binding motif identified in the promoter region of both tbpA and tbpB further supports this hypothesis. However, a recent study using brain heart infusion growth medium suggested that transferrin binding protein synthesis in H. influenzae was haem-rather than iron-regulated. The present study re-investigates this observation and using a chemically defined medium, we demonstrate that elemental iron haem or protoporphyrin IX can each regulate Haemophilus influenzae transferrin, haemopexin and haemoglobin receptor expression.

Conservation and antigenic cross-reactivity of the transferrin-binding proteins of Haemophilus influenzae, Actinobacillus pleuropneumoniae and Neisseria meningitidis.

Haemophilus influenzae acquires iron from the iron-transporting glycoprotein transferrin via a receptor-mediated process. This involves two outer-membrane transferrin-binding proteins (Tbps) termed Tbp1 and Tbp2 which show considerable preference for the human form of transferrin. Since the Tbps are attracting considerable attention as potential vaccine components, we used transferrin affinity chromatography to examine their conservation amongst 28 H. influenzae type b strains belonging to different outer-membrane-protein subtypes as well as six non-typable strains. Whole cells of all type b and non-typable strains examined bound human transferrin; whilst most strains possessed a Tbp1 of approximately 105 kDa, the molecular mass of Tbp2 varied from 79 to 94 kDa. Antisera raised against affinity-purified native H. influenzae Tbp1/Tbp2 receptor complex cross-reacted on Western blots with the respective Tbps of all the Haemophilus strains examined. When used to probe Neisseria meningitidis Tbps, sera from each of four mice immunized with the Haemophilus Tbp1/2 complex recognized the 68 kDa Tbp2 of N. meningitidis strain B16B6 but not the 78 kDa Tbp2 of N. meningitidis strain 70942. Serum from one mouse also reacted weakly with Tbp1 of strain B16B6. Apart from a weak reaction with the Tbp2 of a serotype 5 strain, this mouse antiserum failed to recognize the Tbps of the porcine pathogen A. pleuropneumoniae. However, a monospecific polyclonal antiserum raised against the denatured Tbp2 of Neisseria meningitidis B16B6 recognized the Tbps of all Haemophilus and Actinobacillus strains examined. Since H. influenzae forms part of the natural flora of the upper respiratory tract, human sera were screened for the presence of antibodies to the Tbps. Sera from healthy adults contained antibodies which recognized both Tbp1 and Tbp2 from H. influenzae but not N. meningitidis. Convalescent sera from meningococcal meningitis patients contained antibodies which, on Western blots, recognized the Tbps2s of both pathogens. These data demonstrate the existence of shared epitopes on the Tbps of H. influenzae, N. meningitidis and A. pleuropneumoniae despite their transferrin species specificity.

Bradykinin and its metabolite, Arg-Pro-Pro-Gly-Phe, are selective inhibitors of alpha-thrombin-induced platelet activation.

BACKGROUND: Plasma kininogens are selective inhibitors of alpha-thrombin activation of platelets and endothelial cells. In the present study, we localized the alpha-thrombin inhibitory sequence of kininogens and describe its mechanism of action. METHODS AND RESULTS: Bradykinin and an analogue, MKRPPGFSPFRSSRIG, inhibited alpha-thrombin-induced platelet aggregation and secretion with an IC50 of 0.25 and 1 mmol/L and of 0.23 and 0.5 mmol/L, respectively. The minimal inhibitory peptide was RPPGF. Bradykinin and its analogues did not inhibit ADP-, collagen-, U46619-, or SFLLRN-induced platelet activation or the ability of alpha-thrombin to cleave chromogenic substrates, clot fibrinogen, or block alpha-thrombin binding to platelets. Bradykinin, MKRPPGFSPFRSSRIG, and RPPGF abolished alpha-thrombin-induced (1 nmol/L) calcium mobilization. On flow cytometry, bradykinin and MKRPPGFSPFRSSRIG blocked alpha-thrombin from removing the epitope of its cleavage site on the cloned thrombin receptor. Furthermore, peptide RPPGF or high-molecular-weight kininogen prevented alpha-thrombin from cleaving the thrombin receptor peptide, NATLDPRSFLLR, between arginine and serine. CONCLUSIONS: These results indicate that bradykinin and its metabolites are selective antithrombins by preventing alpha-thrombin cleavage of the cloned thrombin receptor between arginine-41 and serine-42. These newly recognized antithrombin peptides, which are termed thrombostatins, contribute to the cardioprotective nature of kinins.

Conformational changes in low molecular weight kininogen alters its ability to bind to endothelial cells.

The plasma kininogens, high (HK) and low (LK) molecular weight kininogens, are the parent proteins for bradykinin, a potent vasoactive peptide that locally influences vascular biology. Binding of both HK and LK to the endovascular wall contributes to bradykinin delivery. Recently, we found one preparation of LK (LKd) which had reduced inhibition of biotin-HK binding to endothelium. The functional defect in LKd was not merely due to bradykinin loss because two preparations of bradykinin-free LK blocked biotin-HK binding. However, using two different particular monoclonal antibodies to bradykinin, LKd, but no other preparation of LK, had its epitope to bradykinin exposed on non-reduced samples on immunoblot. These data suggested that LKd had an altered conformation which exposed the amino terminal arginine of bradykinin to antigenic detection. The altered conformation of LKd allowed it to be more susceptible to trypsin proteolysis. On circular dichroism, the percentage of alpha-helix was significantly increased, indicating an alteration in the protein. This alteration in LKd was not due to a loss of molecular mass of the protein. On laser desorption mass spectroscopy, the molecular mass of LKd was similar to the other preparations of LK. Investigations were performed to ascertain the mechanism by which LKd had altered ability to bind to cells. LKd was found to be proteolyzed by an unknown protease at the beginning of domain 2 between threonine119 and alanine120. Reduction of functional LK with dithiothreitol to expose its bradykinin epitope did not produce the LKd defect. Proteolysis of functional LK with plasma kallikrein, elastase followed by plasma kallikrein, chymotrypsin, or bromelain also did not produce the defect seen in LKd. These combined data indicated that LK maintains a particular conformation that allows the protein to orient itself such that it can bind to endothelial cells. Proteolysis in the surface exposed region between domains 1 and 2 probably allows for the protein to unfold and contributes to its lost ability to bind to endothelial cells.

Mapping the cell binding site on high molecular weight kininogen domain 5.

Investigations mapped the region(s) on the light chain of high molecular weight kininogen (HK) that participates in cell binding. Sequential and overlapping peptides of domain 5 (D5H) were synthesized to determine its cell binding site(s). Three peptides from non-overlapping regions on D5H were found to inhibit biotin-HK binding to endothelial cells. Peptides GKE19 and HNL 21 weakly inhibited biotin-HK binding with IC50 of 792 and 215 microM, respectively. Peptide HKH20 inhibited biotin-HK binding with an IC50 of 0.2 microM. Two peptides, GGH18 and HVL24, which overlapped HKH20, also inhibited biotin-HK binding to endothelial cells with IC50 values of 108 and 0.8 microM, respectively. Biotinylated HKH20 directly bound to endothelial cells. HK and HKH20 bound at or near the same site on endothelial cells because HK inhibited biotin-HKH20 binding (IC50 = 0.2 microM). A polyclonal anti-HKH20 antibody also blocked biotin-HK binding. Peptides HKH20 and HVL24 and anti-HKH20 antibody also inhibited the procoagulant activity of plasma HK. These data indicated that the cell and artificial surface binding sites on D5H overlap. The orientation of HK on endothelial cells may be critical for the assembly and activation of contact system enzymes and the expression of kininogen's anti-thrombin activity.