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Intracellular delivery of large molecule cargo via cell penetrating peptides (CPPs) is an inefficient process and despite intense efforts in past decades, improvements in efficiency have been marginal. Utilizing a standardized and comparative analysis of the delivery efficiency of previously described cationic, anionic, and amphiphilic CPPs, we demonstrate that the delivery ceiling is accompanied by irreparable plasma membrane damage that is part of the uptake mechanism. As a consequence, intracellular delivery correlates with cell toxicity and is more efficient for smaller peptides than for large molecule cargo. The delivery of pharmaceutically relevant cargo quantities with acceptable toxicity thus seems hard to achieve with the CPPs tested in our study. Our results suggest that any engineered intracellular delivery system based on conventional cationic or amphiphilic CPPs, or the design principles underlying them, needs to accept low delivery yields due to toxicity limiting efficient cytoplasmic uptake. Novel peptide designs based on detailed study of uptake mechanisms are required to overcome these limitations. [ABSTRACT FROM AUTHOR]

9-(2-Phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cpr-PMEDAP) is an acyclic nucleotide analog of the [9-(2-phosphonylmethoxyethyl)-] (PME) series containing a cyclopropyl substituent on the N6 position of the 2,6-diaminopurine (DAP) base. Growth inhibition assays in a broad range of tumor cell lines demonstrated that this analog had potent antiproliferative activity with IC50 values similar to those of the structurally related guanine analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG). A substantially lower growth inhibitory effect was observed for the 2,6-diaminopurine analog, PMEDAP. To dissect the basis for these varying potencies, the metabolism of the three analogs was examined in a human pancreatic carcinoma cell line, BxPC-3. HPLC analysis of the intracellular metabolites demonstrated that the cpr-PMEDAP was deaminated to PMEG and subsequently phosphorylated to PMEG mono- and diphosphates (PMEGp and PMEGpp). The level of PMEGpp generated from cpr-PMEDAP-treated cells was 50% greater than the level generated from cells incubated with PMEG. The presence of PMEG in the DNA of cells incubated with cpr-PMEDAP confirmed that the cpr-PMEDAP was converted to PMEG. In contrast, PMEDAP was not deaminated to PMEG, but directly phosphorylated to PMEDAPp and PMEDAPpp. The adenylate deaminase inhibitor 2'-deoxycoformycin (dCF) inhibited the conversion of cpr-PMEDAP in a rat liver cytosolic extract and increased the IC50 value for growth inhibition by 40-fold. The antiproliferative activities of PMEG and PMEDAP were unaffected by dCF. Thus, it appears that cpr-PMEDAP, but not PMEDAP, is converted by an adenylate deaminase-like enzyme and functions as a prodrug of PMEG.

PMEG (9-(2-phosphonylmethoxyethyl)guanine) is an acyclic nucleotide analog being evaluated for its anti-proliferative activity. We examined the inhibitory effects of PMEG diphosphate (PMEGpp) toward DNA polymerases (pol) delta and epsilon and found it to be a competitive inhibitor of both these enzymes. The apparent Ki values for PMEGpp were 3-4 times lower than the Km values for dGTP. The analog was shown to function as a substrate and to be incorporated into DNA by both enzymes. Examination of the ability of pol delta and pol epsilon to repair the incorporated PMEG revealed that pol epsilon could elongate PMEG-terminated primers in both matched and mismatched positions with an efficiency equal to 27 and 85% that observed for dGMP-terminated control template-primers. Because PMEG acts as an absolute DNA chain terminator, the elongation of PMEG-terminated primers is possible only by cooperation of the 3'-5'-exonuclease and DNA polymerase activities of the enzyme. In contrast to pol epsilon, pol delta exhibited negligible activity on these template-primers, indicating that pol epsilon, but not pol delta, can repair the incorporated analog.

Previous alanine scanning mutagenesis of thrombin revealed that substitution of residues W50, K52, E229, and R233 (W60d, K60f, E217, and R221 in chymotrypsinogen numbering) with alanine altered the substrate specificity of thrombin to favor the anticoagulant substrate protein C. Saturation mutagenesis, in which residues W50, K52, E229, and R233 were each substituted with all 19 naturally occurring amino acids, resulted in the identification of a single mutation, E229K, that shifted the substrate specificity of thrombin by 130-fold to favor the activation of the anticoagulant substrate protein C over the procoagulant substrate fibrinogen. E229K thrombin was also less effective in activating platelets (18-fold), was resistant to inhibition by antithrombin III (33-fold and 22-fold in the presence and absence of heparin), and displayed a prolonged half-life in plasma in vitro (26-fold). Thus E229K thrombin displayed an optimal phenotype to function as a potent and specific activator of endogenous protein C and as an anticoagulant in vivo. Upon infusion in Cynomolgus monkeys E229K thrombin caused an anticoagulant effect through the activation of endogenous protein C without coincidentally stimulating fibrinogen clotting and platelet activation as observed with wild-type thrombin. In addition, E229K thrombin displayed enhanced potency in vivo relative to the prototype protein C activator E229A thrombin. This enhanced potency may be attributable to decreased clearance by antithrombin III, the principal physiological inhibitor of thrombin.

Protein purification has been made significantly easier by the use of affinity tags that can be genetically engineered at either the amino- or carboxyl-terminus of recombinant proteins. One of the most widely used tags is six consecutive histidine residues or 6His tag. These residues bind with high affinity to metal ions immobilized on chelating resins even in the presence of denaturing agents and can be mildly eluted with imidazole. We report the methodology for the immobilization of six histidine-containing proteins onto microtiter plates. A derivative of nitrilotriacetic acid (NTA) was prepared. This derivative, N,N-bis[carboxymethyl]lysine (BCML), was easily coupled to a maleic anhydride-activated polystyrene microtiter plate. The plate was then charged with Ni2+ for the capture of the 6His-tagged proteins. Using two different recombinant proteins with the 6His tag at either the N- or C-terminus, we demonstrated that the binding to the Ni(2+)-NTA plate was specific for six histidine-containing proteins. Proteins lacking the 6His tags did not bind to the plate. The plate was used in a modified enzyme-linked immunoabsorbent assay format to quantitate protein concentrations and determine the affinity of protein-ligand interactions. The technology can also be extended to include high-throughput screening assays for antagonists of protein-protein interactions.

Tissue factor (TF) is a transmembrane protein that functions in the initiation of blood coagulation in vivo. At sites of vascular injury, TF serves as a cell-surface receptor for the serine protease factor VIIa (FVIIa), forming an enzyme--cofactor complex and enhancing the catalytic activity of FVIIa. Tissue factor, along with the receptors for alpha- and gamma-interferons, is a member of the class 2 cytokine receptor superfamily. Crystallographic analysis demonstrated that the extracellular domain of TF consists of two immunoglobulin-like domains joined by a linker region. Each domain is comprised of two antiparallel beta-sheets containing seven conserved beta-strands separated by more variable loop regions. Extensive mutagenesis has been performed in order to map the FVIIa binding site on TF. Results indicated that the discontinuous binding site for FVIIa lies at the domain--domain interface and includes residues from extended loops and beta-strands within both the N- and C-terminal domains. Our previous study provided evidence that three consecutive residues (D44, W45, K46) within the TF loop region between beta-strands C and C' of the N-terminal domain were important for interactions with FVIIa. We have presently extended our alanine-scanning mutagenesis to include the residues within the flanking beta-strands. Thirteen sTF mutants were screened for their ability to enhance FVIIa activity. Three residues within strand C (Y34, Q37, I38) and two residues within C' (K48, Y51) were shown to be important for TF cofactor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue factor (TF) is a membrane-bound glycoprotein that functions as a cofactor for coagulation factor VIIa (VIIa) and initiates blood coagulation at sites of vascular injury. On the basis of sequence alignments, TF was predicted to be a member of the cytokine receptor superfamily. Utilizing the structural information available for the cytokine receptor superfamily, we have used site-directed mutagenesis to identify the binding site on TF for VIIa. The predicted loop regions in TF were systematically replaced with the homologous loops from the gamma-interferon receptor (gamma-IFN-R), the protein most related to TF in the superfamily of cytokine receptors. Six discontinuous regions (residues 16-20, 40-46, 60-69, 101-111, 129-151, 193-207) were identified that are required for interaction with VIIa and enhancement of activity. Individual substitution of 68 residues within these loops with alanine revealed eight residues (K20, D44, W45, K46, Q110, R135, F140, V207) that are required for cofactor activity. These residues fall into two groups, those that are required only for interactions with VIIa (K46, Q110, R135, F140, V207) and those that are also required to induce the conformational change in VIIa required for enhanced activity (K20, D44, W45). The discontinuous regions of TF required for interactions with VIIa form a single binding surface for VIIa that is analogous to the interface defined by the crystal structure of the complex between growth hormone and its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.

The biochemical behavior and peptide binding properties of a soluble form of the human class II DR4Dw4 molecule (PI-DR4Dw4) were compared to DR4Dw4 molecules containing the transmembrane and cytoplasmic domains that were purified both from B and transfected chinese hamster ovary cells. Recombinant and B cell-derived DR4Dw4 molecules bound monoclonal anti-DR4Dw4 antibodies with different affinities and varied in their stability in the presence of sodium dodecyl sulfate. The three forms of DR4Dw4 bound peptides with a similar apparent affinity constant, but soluble class II molecules bound up to ten times more peptide than DR4Dw4 containing a transmembrane region. Peptide binding kinetics for soluble DR4Dw4 molecules were 10-20 times faster than for the other two forms of DR4Dw4 molecules. Finally, soluble PI-DR4Dw4/peptide complexes were shown to stimulate T cell proliferation.

Full-length tissue factor (263 rTF) and three truncated forms have been expressed in human kidney 293 cells; 1) 243 rTF, which lacks the cytoplasmic tail, is fully functional in the chromogenic assay and has a specific activity comparable with that of the full-length molecule, 263 rTF; 2) 219 rTF, which lacks both the transmembrane and cytoplasmic domains, is not functional; 3) the third variant, referred to as TF-PI, is a fusion protein containing the extracellular domain of TF (amino acids 1-219) fused to the last 37 amino acids of decay-accelerating factor which contain a signal for attachment of a phosphatidylinositol membrane anchor (PI). TF-PI is a membrane-bound protein expressed on the cell surface. The PI anchor restores TF activity lost when the transmembrane domain is deleted from the 219 rTF variant. The ability of the PI anchor to restore activity to 219 rTF clearly demonstrates that while the transmembrane domain is not required for TF activity, lipid association is required.

The possible self-association of tissue factor molecules was investigated by treating cells expressing tissue factor with bifunctional cross-linking agents. The two reagents chosen were 3,3'-dithiobis(sulfosuccinimidylpropionate) and sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate, both of which are membrane-impermeable and thiol-cleavable. A human bladder carcinoma cell line, J82, and a transfected human kidney cell line expressing high amounts of recombinant tissue factor were used in these studies. Exposure of the intact cells to the crosslinking reagents was found to result in the formation of multimeric tissue factor-containing complexes, the extent of which appeared to be dependent upon the amount of tissue factor expressed by the cell. The self-association of tissue factor was prevented in a variant tissue factor molecule harboring a non-homologous transmembrane domain.

Recombinant human tissue factor (rTF) purified from transfected mammalian cells is a glycoprotein that contains N-linked, but not O-linked oligosaccharides. Two of the three potential N-linked sites in the extracellular portion are fully glycosylated, while one site is approximately 90% utilized. These sites have complex-type oligosaccharides attached. The potential N-linked site in the cytoplasmic domain near the C-terminus is not glycosylated. Characterization of the tryptic map of rTF confirmed most of the proposed amino acid sequence. In addition, the disulfide bonds (between Cys-49 and Cys-57 and between Cys-186 and Cys-209) were demonstrated by FAB-MS analysis of cysteine-containing fragments obtained from the tryptic map.

We describe a mammalian cell expression system used to rapidly produce microgram quantities of a membrane protein used as an immunogen. A fusion protein expression vector was constructed which contained the signal sequence and 27 amino acids of the Herpes simplex virus glycoprotein D (gD), followed by a factor VIII (fVIII) thrombin cleavage site and the mature tissue factor (TF) sequence. This fusion protein was transiently expressed and then purified using an antibody to gD. The purified fusion protein, gDTF, was incubated with thrombin to remove the gD-fVIII moiety and the resulting rTF served as antigen for the generation of TF-specific antibodies. The antibodies produced were then used for a comparison of the turnover rates of the constitutively and transiently produced fusion protein. In addition, sensitivity to glycosidases indicated that the transiently and constitutively produced recombinant proteins do not contain identical carbohydrate structures.

Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.

We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.

In this paper a novel model, based on the smoothed particle hydrodynamics (SPH) method, is proposed to simulate thrombus formation. This describes the main phases of the coagulative cascade through the balance of four biochemical species and three type of platelets. SPH particles can switch from fluid to solid phase when specific biochemical and physical conditions are satisfied. The interaction between blood and the forming blood clot is easily handled by an innovative monolithic FSI approach. Fluid-solid coupling is modelled by introducing elastic binds between solid particles, without requiring detention and management of the interface between the two media. The proposed model is able to realistically reproduce the thromboembolic process, as confirmed by the comparison of numerical results with experimental data available in the literature. [ABSTRACT FROM AUTHOR]

Background: The recognition that human tumors stimulate the production of autoantibodies has initiated the use of this immune response as serological markers for the early diagnosis and management of cancer. The enzyme-linked immunosorbent assay (ELISA) is the most common method used in detecting autoantibodies, which involves coating the microtiter plate with the tumor associated antigen (TAA) of interest and allowing serum antibodies to bind. The patient's sample is directly in contact with the coating antigen so the protein used for coating must be pure to avoid non-specific binding. In this study, a simplified method to selectively and specifically immobilize TAAs onto microtiter plates in order to detect circulating autoantibodies in cancer patients without prior purification process was described. Wild type full-length p53 protein was produced in fusion with biotin carboxyl carrier peptide (BCCP) or hexahistidine [(His)6] using pAK400 and pET15b(+) vectors, respectively. The recombinant p53 fusion protein produced was then subjected to react with either a commercial p53 monoclonal antibody (mAb) or sera from lung cancer patients and healthy volunteers in an enzyme-linked immunosorbent assay (ELISA) format. Results: Both of the immobilized p53 fusion proteins as well as the purified (His)6-p53 fusion protein had a similar dose response of detection to a commercial p53 mAb (DO7). When the biotinylated p53-BCCP fusion protein was used as an antigen to detect p53 autoantibodies in clinical samples, the result showed that human serum reacted strongly to avidin-coated microwell even in the absence of the biotinylated p53-BCCP fusion protein, thus compromised its ability to differentiate weakly positive sera from those that were negative. In contrast, the (His)6-p53 protein immobilized directly onto Ni+ coated microplate was able to identify the p53 autoantibody positive serum. In addition, its reactivity to clinical serum samples highly correlated with those obtained from using purified p53 as an antigen (R = 0.9803, p < 0.0001). Moreover, this directly immobilized p53 antigen can clearly differentiate p53 autoantibody positive sera in cancer patients from healthy volunteers' sera. Conclusion: A method of coating directly and specifically TAAs onto a microtiter plate without the purification processes was developed in this study. Although in this study only one tumor antigen was examined, the simplicity and the ability of coated antigens to identify p53 specific autoantibodies in serum accurately might enable a larger panel of TAAs specific autoantibodies to be explored as serological markers for cancer. [ABSTRACT FROM AUTHOR]

Since the invention of the first biosensors 70 years ago, they have turned into valuable and versatile tools for various applications, ranging from disease diagnosis to environmental monitoring. Traditionally, antibodies have been employed as the capture probes in most biosensors, owing to their innate ability to bind their target with high affinity and specificity, and are still considered as the gold standard. Yet, the resulting immunosensors often suffer from considerable limitations, which are mainly ascribed to the antibody size, conjugation chemistry, stability, and costs. Over the past decade, aptamers have emerged as promising alternative capture probes presenting some advantages over existing constraints of immunosensors, as well as new biosensing concepts. Herein, we review the employment of antibodies and aptamers as capture probes in biosensing platforms, addressing the main aspects of biosensor design and mechanism. We also aim to compare both capture probe classes from theoretical and experimental perspectives. Yet, we highlight that such comparisons are not straightforward, and these two families of capture probes should not be necessarily perceived as competing but rather as complementary. We, thus, elaborate on their combined use in hybrid biosensing schemes benefiting from the advantages of each biorecognition element. [ABSTRACT FROM AUTHOR]

Effective and safe hemodialysis is essential for patients with acute kidney injury and chronic renal failures. However, the development of effective anticoagulant agents with safe antidotes for use during hemodialysis has proven challenging. Here, we describe DNA origami-based assemblies that enable the inhibition of thrombin activity and thrombus formation. Two different thrombin-binding aptamers decorated DNA origami initiates protein recognition and inhibition, exhibiting enhanced anticoagulation in human plasma, fresh whole blood and a murine model. In a dialyzer-containing extracorporeal circuit that mimicked clinical hemodialysis, the origami-based aptamer nanoarray effectively prevented thrombosis formation. Oligonucleotides containing sequences complementary to the thrombin-binding aptamers can efficiently neutralize the anticoagulant effects. The nanoarray is safe and immunologically inert in healthy mice, eliciting no detectable changes in liver and kidney functions or serum cytokine concentration. This DNA origami-based nanoagent represents a promising anticoagulant platform for the hemodialysis treatment of renal diseases. Safe haemodialysis is essential for patients with acute kidney injury and renal failure. Here the authors present a DNA origami-based approach with high affinity and specificity to thrombin, inhibiting coagulation. [ABSTRACT FROM AUTHOR]

Tissue factor (TF) is the primary initiator of the coagulation cascade, though its effects extend well beyond hemostasis. When TF binds to Factor VII, the resulting TF:FVIIa complex can proteolytically cleave transmembrane G protein-coupled protease-activated receptors (PARs). In addition to activating PARs, TF:FVIIa complex can also activate receptor tyrosine kinases (RTKs) and integrins. These signaling pathways are utilized by tumors to increase cell proliferation, angiogenesis, metastasis, and cancer stem-like cell maintenance. Herein, we review in detail the regulation of TF expression, mechanisms of TF signaling, their pathological consequences, and how it is being targeted in experimental cancer therapeutics. [ABSTRACT FROM AUTHOR]

Significant progress has been made in understanding the complex interactions between the coagulation system and inflammation and autoimmunity. Increased blood-brain-barrier (BBB) permeability, a key event in the pathophysiology of multiple sclerosis (MS), leads to the irruption into the central nervous system of blood components that include virtually all coagulation/hemostasis factors. Besides their cytotoxic deposition and role as a possible trigger of the coagulation cascade, hemostasis components cause inflammatory response and immune activation, sustaining neurodegenerative events in MS. Early studies showing the contribution of altered hemostasis in the complex pathophysiology of MS have been strengthened by recent studies using methodologies that permitted deeper investigation. Fibrin(ogen), an abundant protein in plasma, has been identified as a key contributor to neuroinflammation. Perturbed fibrinolysis was found to be a hallmark of progressive MS with abundant cortical fibrin(ogen) deposition. The immune-modulatory function of the intrinsic coagulation pathway still remains to be elucidated in MS. New molecular details in key hemostasis components participating in MS pathophysiology, and particularly involved in inflammatory and immune responses, could favor the development of novel therapeutic targets to ameliorate the evolution of MS. This review article introduces essential information on coagulation factors, inhibitors, and the fibrinolytic pathway, and highlights key aspects of their involvement in the immune system and inflammatory response. It discusses how hemostasis components are (dys)regulated in MS, and summarizes histopathological post-mortem human brain evidence, as well as cerebrospinal fluid, plasma, and serum studies of hemostasis and fibrinolytic pathways in MS. Studies of disease-modifying treatments as potential modifiers of coagulation factor levels, and case reports of autoimmunity affecting hemostasis in MS are also discussed. [ABSTRACT FROM AUTHOR]

Cardiovascular implantable devices alter the biofluid dynamics and biochemistry of the blood in which they are placed. These perturbations can lead to thrombus formation which may or may not be desired, depending on the application. In this work, a computational model is developed that couples biofluid dynamics and biochemistry to predict the clotting response of blood to such devices. The model consists of 28 advection-diffusion-reaction partial differential equations to track proteins in the blood involved in clotting and utilizes boundary flux terms to model the initiation of the intrinsic clotting pathway at thrombogenic device surfaces. We use this model to simulate the transient clot growth within a 2D idealized bifurcation aneurysm filled with various distributions of bare metal coils with similar packing densities. The clot model predicts initial clot formation to occur in areas along coil surfaces where flow is minimal and where time-averaged shear rates are the smallest. Among the six coil-filled aneurysm cases simulated, maximum thrombus occlusion ranged between 80.8 and 92.2% of the post-treatment aneurysm volume and was achieved 325-450 s after treatment. With further refinement and validation, the computational clotting model will be a valuable engineering tool for evaluating and comparing the relative performance of cardiovascular implantable devices. [ABSTRACT FROM AUTHOR]

Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk. [ABSTRACT FROM AUTHOR]

Background Standard of care treatment for multicentric lymphoma in dogs remains doxorubicin (DOX)-based combination chemotherapy, but owners may hesitate to commit the time and financial resources to complete such a protocol, typically requiring 12-16 visits. Rabacfosadine (RAB), a double prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl) guanine, has substantial single-agent activity in dogs with lymphoma, and a different mechanism of action than DOX. Hypothesis/Objectives Our objective was to evaluate the efficacy and adverse effect (AE) profile of alternating doses of RAB and DOX in dogs with naïve multicentric lymphoma. Animals Fifty-four dogs with previously untreated lymphoma. Methods Open-label, multicenter prospective clinical trial. Dogs received alternating RAB (1.0 mg/kg IV weeks 0, 6, 12) and DOX (30 mg/m2 IV weeks 3, 9, 15). Dogs that achieved complete response (CR) were followed by monthly evaluations. Complete clinicopathological evaluation and assessment of remission and AEs were performed every 21 days. Results The overall response rate was 84% (68%; CR; 16%; partial response [PR)]. The overall median progression-free interval (PFI) was 194 days (216 for CR and 63 for PR). Most AEs were mild and self-limiting: gastrointestinal and hematologic AEs were most common. Thirteen dogs experienced dermatologic AEs, and 2 dogs developed grade 5 pulmonary fibrosis. Conclusions and Clinical Importance Alternating RAB/DOX generally was well tolerated and resulted in PFIs comparable to standard DOX-based multi-agent protocols, with fewer treatment visits. Most adverse events were mild or moderate and self-limiting. Further studies are warranted to explore long-term outcome and other RAB chemotherapy combinations. [ABSTRACT FROM AUTHOR]

Proliferative vitreoretinopathy (PVR) is a blinding eye disease and there is no effective pharmacological measure to prevent PVR development. The difficulty comes from lack of potent antiproliferative agent and lack of sustained delivery to cover high-risk time window for PVR to develop. Lipid prodrug of PMEG, hexadecyloxypropyl 9-[(2-phosphonomethoxy)ethyl]guanine (HDP-PMEG), was prepared and was evaluated as a pharmacological adjuvant to surgical management of PVR. A dose-escalation study determined that the highest nontoxic dose for intravitreal use in pigmented rabbits was 3 µg per eye. The genotoxicity of HDP-PMEG was harnessed as a perioperative preventative measure against PVR in a rabbit eye model while the sustained intravitreal pharmacological effect was evaluated on a laser-induced fibrovascular model in rat eye. After intravitreal 3 µg, HDP-PMEG particles in the rabbit vitreous was visible for at least 6 weeks. A single 50-min intravitreal infusion of HDP-PMEG demonstrated significant inhibition of PVR formation when compared with the eyes infused with only BSS (BSS vs. HDP-PMEG: estimate = 1.14, OR = 3.1,p = .027). A single intravitreal 104 ng (equivalent to 3 µg for rabbit eye) of HDP-PMEG significantly inhibit laser-induced fibrovascular proliferation in rat eye by 55% (least square mean pixel, BSS = 4763569.5 vs. HDP-PMEG = 2148129.7,p < .0001, generalized estimating equation [GEE]). Retinal fluorescein angiography showed the odds for BSS intervened eyes to have higher-rated FA leaking grades were 38.5 times compared with HDP-PMEG treated eyes (p < .0001, GEE). Our study results indicate that single intravitreal HDP-PMEG may be a promising ocular drug delivery as a perioperative intervention to prevent PVR reoccurrence following primary surgical management. [ABSTRACT FROM AUTHOR]

ABSTRACT Proteases regulate many biological processes through their ability to activate or inactive their target substrates. Because proteases catalytically turnover proteins and peptides, they present unique opportunities for use in biotechnological and therapeutic applications. However, many proteases are capable of cleaving multiple physiological substrates. Therefore their activity, expression, and localization are tightly controlled to prevent unwanted proteolysis. Currently, the use of protease therapeutics has been limited to a handful of proteases with narrow substrate specificities, which naturally limits their toxicity. Wider application of proteases is contingent upon the development of methods for engineering protease selectivity, activity, and stability. Recent advances in the development of high-throughput, bacterial and yeast-based methods for protease redesign have yielded protease variants with novel specificities, reduced toxicity, and increased resistance to inhibitors. Here, we highlight new tools for protease engineering, including methods suitable for the redesign of human secreted proteases, and future opportunities to exploit the catalytic activity of proteases for therapeutic benefit. Biotechnol. Bioeng. 2017;114: 33-38. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In normal hemostasis, the blood clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to the integral membrane protein, human tissue factor (TF). The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the soluble extracellular domain of TF (sTF) exist; however, these structures are missing electron density in functionally relevant regions of the protein. In this context, NMR can provide complementary structural information as well as dynamic insights into enzyme activity. The resolution and sensitivity for NMR studies are greatly enhanced by the ability to prepare multiple milligrams of protein with various isotopic labeling patterns. Here, we demonstrate high-yield production of several isotopically labeled forms of recombinant sTF, allowing for high-resolution NMR studies both in the solid and solution state. We also report solution NMR spectra at sub-mM concentrations of sTF, ensuring the presence of dispersed monomer, as well as the first solid-state NMR spectra of sTF. Our improved sample preparation and precipitation conditions have enabled the acquisition of multidimensional NMR data sets for TF chemical shift assignment and provide a benchmark for TF structure elucidation. [ABSTRACT FROM AUTHOR]

Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aβ), the main pathological component of Alzheimer’s disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bβ-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions. [ABSTRACT FROM AUTHOR]

Aptamers are single strand DNA or RNA molecules, selected by an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Due to various advantages of aptamers such as high temperature stability, animal free, cost effective production and its high affinity and selectivity for its target make them attractive alternatives to monoclonal antibody for use in diagnostic and therapeutic purposes. Aptamer has been generated against vesicular endothelial growth factor 165 involved in age related macular degeneracy. Macugen was the first FDA approved aptamer based drug that was commercialized. Later other aptamers were also developed against blood clotting proteins, cancer proteins, antibody E, agents involved in diabetes nephropathy, autoantibodies involved in autoimmune disorders, etc. Aptamers have also been developed against viruses and could work with other antiviral agents in treating infections. [ABSTRACT FROM AUTHOR]

There are currently over 2.5 million breast cancer survivors in the United States and, according to the American Cancer Society, 10 to 20 percent of these women will develop recurrent breast cancer. Early detection of recurrence can avoid unnecessary radical treatment. However, self-examination or mammography screening may not discover a recurring cancer if the number of surviving cancer cells is small, while biopsy is too invasive and cannot be frequently repeated. It is therefore important to identify non-invasive biomarkers that can detect early recurrence. The present paper develops a mathematical model of cancer recurrence. The model, based on a system of partial differential equations, focuses on tissue biomarkers that include the plasminogen system. Among them, only uPAR is known to have significant correlation to its concentration in serum and could therefore be a good candidate for serum biomarker. The model includes uPAR and other associated cytokines and cells. It is assumed that the residual cancer cells that survived primary cancer therapy are concentrated in the same location within a region with a very small diameter. Model simulations establish a quantitative relation between the diameter of the growing cancer and the total uPAR mass in the cancer. This relation is used to identify uPAR as a potential serum biomarker for breast cancer recurrence. [ABSTRACT FROM AUTHOR]

It is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of the flowing blood to cell-surface protein, such as tissue factor (TF). Vascular injury and/or atherosclerotic plaque complication is responsible for this exposure, leading to clinical manifestations such as acute coronary syndromes. For many years, TF has been considered one of the major determinants of plaque-related thrombosis. However, the discoveries of different pools of TF that circulates in the blood as cell-associated TF, microparticles bound, and as soluble form have changed this dogma. Recent evidence suggests that an increased circulating TF activity may potentiate the initial thrombogenic stimulus related to vessel wall-associated TF, leading to the formation of larger and/or more stable thrombus and thus more severe clinical manifestations. Different pathological conditions, such as inflammatory status, enhance TF expression and activity. Conversely, TF upregulation may facilitate inflammation by formation of proinflammatory fibrin fragments and coagulation proteases generation, including FVIIa, FXa, and thrombin. Furthermore, the biology of TF has become more complex by the demonstration that, apart from its known effects on blood coagulation, it is involved in intracellular signaling, proliferation, angiogenesis, and tumor metastasis. [ABSTRACT FROM AUTHOR]

Interactions between tissue factor and factor VIIa are the primary initiators of coagulation in hemostasis and certain thrombotic diseases. Tissue factor, an integral membrane protein expressed extensively outside of the vasculature, is the regulatory protein cofactor for coagulation factor VIIa. Factor VIIa, a trypsin-like serine protease homologous with other blood coagulation proteases, is weakly active when free in solution and must bind its membrane-bound cofactor for physiologically relevant activity. Tissue factor allosterically activates factor VIIa by several mechanisms such as active site positioning, spatial stabilization, and direct interactions with the substrate. Protein-membrane interactions between tissue factor, factor VIIa, and substrates all play critical roles in modulating the activity of this enzyme complex. Additionally, divalent cations such as Ca2+ and Mg2+ are critical for correct protein folding, as well as protein-membrane and protein-protein interactions. The contributions of these factors toward tissue factor-factor VIIa activity are discussed in this review. [ABSTRACT FROM AUTHOR]

The plasma coagulation system in mammalian blood consists of a cascade of enzyme activation events in which serine proteases activate the proteins (proenzymes and procofactors) in the next step of the cascade via limited proteolysis. The ultimate outcome is the polymerization of fibrin and the activation of platelets, leading to a blood clot. This process is protective, as it prevents excessive blood loss following injury (normal hemostasis). Unfortunately, the blood clotting system can also lead to unwanted blood clots inside blood vessels (pathologic thrombosis), which is a leading cause of disability and death in the developed world. There are two main mechanisms for triggering the blood clotting, termed the tissue factor pathway and the contact pathway. Only one of these pathways (the tissue factor pathway) functions in normal hemostasis. Both pathways, however, are thought to contribute to thrombosis. An emerging concept is that the contact pathway functions in host pathogen defenses. This review focuses on how the initiation phase of the blood clotting cascade is regulated in both pathways, with a discussion of the contributions of these pathways to hemostasis versus thrombosis. [ABSTRACT FROM AUTHOR]

Background Canine cutaneous T-cell lymphoma ( CTCL) is an uncommon disease for which efficacious therapies are lacking. The novel anticancer nucleotide prodrug VDC-1101 (formerly known as GS-9219) has shown efficacy in dogs with multicentric lymphoma. One of the observed adverse effects with this drug was a skin change characterized by hair loss, erythema, and pruritus, implying delivery of VDC-1101 to the skin. Hypothesis/Objectives The primary study objective was to identify the objective response rate ( ORR) to VDC-1101 in canine CTCL; secondary objectives included characterization of progression-free survival ( PFS) and adverse events ( AEs). Animals Twelve dogs with chemotherapy-naïve or relapsed, histologically and immunohistochemically confirmed CTCL. Methods Dogs received VDC-1101 as a 30-minute IV infusion once every 21 days. Prednisone (1 mg/kg PO q48h) was administered concurrently. Results In 11 evaluable patients, responses included 1 complete response ( CR), 4 partial responses ( PR), 2 stable disease ( SD), and 4 progressive disease for an ORR of 45% and biologic response rate ( CR/ PR/ SD) of 64%. The median PFS was 37.5 days (26 to >399 days), which includes 1 durable and ongoing CR (>1 year). Gastrointestinal and hematologic AEs were mild; no dogs developed grade 3 or 4 AEs. Three dogs developed dermatopathies and 1 of these dogs was removed from the study as a result of this AE. Conclusions and Clinical Importance VDC-1101 has activity against canine CTCL and could provide another treatment option in a disease process with a poor prognosis. [ABSTRACT FROM AUTHOR]

Recombinant DNA methods are being widely used to express proteins in both prokaryotic and eukaryotic cells for both fundamental and applied research purposes. Expressed protein must be well characterized to be sure that it retains the same properties as the native one, especially when expressed protein will be used in the pharmaceutical field. In this aim, interfacial and kinetic properties of native, untagged recombinant and tagged recombinant forms of a pancreatic lipase were compared using the monomolecular film technique. Turkey pancreatic lipase (TPL) was chosen as model. A kinetic study on the dependence of the stereoselectivity of these three forms on the surface pressure was performed using three dicaprin isomers spread in the form of monomolecular films at the air-water interface. The heterologous expression and the N-His-tag extension were found to modify the pressure preference and decrease the catalytic hydrolysis rate of three dicaprin isomers. Besides, the heterologous expression was found to change the TPL regioselectivity without affecting its stereospecificity contrary to the N-tag extension which retained that regioselectivity and changed the stereospecificity at high surface pressures. The study of parameters, termed Recombinant expression Effects on Catalysis (REC), N-Tag Effects on Catalysis (TEC), and N-Tag and Recombinant expression Effects on Catalysis (TREC) showed that the heterologous expression effects on the catalytic properties of the TPL were more deleterious than the presence of an N-terminal tag extension. [ABSTRACT FROM AUTHOR]

Background Multiple myeloma (MM) is an important human and canine cancer for which novel therapies remain necessary. VDC-1101 (formerly GS-9219), a novel double prodrug of the antiproliferative nucleotide analog 9-(2-phosphonylmethoxyethyl) guanine (PMEG), possesses potent cytotoxic activity in vitro in human lymphoblasts and leukemia cell lines and in vivo in spontaneous canine lymphoma. Given the similarity in lineage between lymphoma and MM, we hypothesized that VDC-1101 would be active against MM. Results We evaluated the in vitro antiproliferative effects of VDC-1101 against 3 human MM cell lines, and we performed a phase-II clinical trial in 14 dogs with spontaneous MM. Each dog was treated with a maximum of 6 doses of VDC-1101 monotherapy over 10-15 weeks. Dosedependent antiproliferative activity was observed in all evaluated cell lines. Major antitumor responses (reduction of serum paraprotein and resolution of hypercalcemia, peripheral cytopenias and bone marrow plasmacytosis) were observed in 9 of 11 evaluable dogs for a median of 172 days, including a durable stringent complete response (>1047 days) in a dog with melphalan-refractory disease. 2 dogs were euthanized due to presumed pulmonary fibrosis; there were no other dose-limiting toxicities encountered. Conclusions In conclusion, VDC-1101 has significant anti-tumor activity at well-tolerated doses in spontaneous canine MM. [ABSTRACT FROM AUTHOR]

The production of proteins in appropriate quantity and quality is an essential requirement of the present time. There appears to be a progressive increase in the application of mammalian cells for proteins production. Expression systems utilizing mammalian cells for recombinant proteins are able to introduce proper protein folding, post-translational modifications, and product assembly, which are important for complete biological activity. This review article is totally based on literature survey. In this article much emphasis has been done on the mammalian expression system. The author focused on different mammalian cell lines that express the gene. The different vector systems that transfer the gene into mammalian cells like plasmid based expression vectors, adenovirus vectors, vaccinia vectors, retroviral vector and baculovirus as vectors were explored. The processes for the transfer of gene into mammalian cells were also reviewed. Application and limitations of mammalian expression system were also focused. The purpose of research in writing this article is to create awareness in researchers, starting their career in gene expression related to mammalian cells. The principal result and major conclusion of this article is to make available the molecular technologies, expression system and applications of gene expression in mammalian cell lines. [ABSTRACT FROM AUTHOR]

Tissue factor (TF), a 47-kDa transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa (FVIIa), is expressed in several tumor types.TF has been shown to play a role in cell signaling, inflammation, angiogenesis, as well as tumor growth and metastasis. Activation of the TF signaling pathway has been implicated in mediating the function of many tumor cell types and has led to TF as a potential target in the treatment of several malignancies. Formation of the TFFVIIa complex in breast cancer cells has been shown to exert an antiapoptotic effect and play a key role in tumor growth and metastasis. Breast cancer growth is suppressed by inhibition of TF-mediated PAR2 signaling,and deficiency in PAR2 delays spontaneous breast cancer development in mice. TF is expressed in triple-negative breast cancer (TNBC), an aggressive type of breast cancer in which there is currently a paucity of available targets. Various methods of targeting TF have been investigated and include immunoconjugates or icons, anti-TF antibodies, TF pathway inhibitors, targeted photodynamic therapy, and microRNAs. These investigations may give way to promising clinical therapies for breast cancer, especially in TNBC, for which there are relatively few effective treatment options. [ABSTRACT FROM AUTHOR]

Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair. After wounding, the hemostatic system engages a plethora of vascular and extravascular receptors that act in concert with blood components to seal off the damage inflicted to the vasculature and the surrounding tissue. The first important component that contributes to hemostasis is the coagulation system, while the second important component starts with platelet activation, which not only contributes to the hemostatic plug, but also accelerates the coagulation system. Eventually, coagulation and platelet activation are switched off by blood-borne inhibitors and proteolytic feedback loops. This review summarizes new concepts of activation of proteases that regulate coagulation and anticoagulation, to give rise to transient thrombin generation and fibrin clot formation. It further speculates on the (patho)physiological roles of intra- and extravascular receptors that operate in response to these proteases. Furthermore, this review provides a new framework for understanding how signaling and adhesive interactions between endothelial cells, leukocytes, and platelets can regulate thrombus formation and modulate the coagulation process. Now that the key molecular players of coagulation and platelet activation have become clear, and their complex interactions with the vessel wall have been mapped out, we can also better speculate on the causes of thrombosis-related angiopathies. [ABSTRACT FROM AUTHOR]

Protease-activated receptors (PARs) are G protein-coupled receptors that are activated by proteolytical cleavage of the amino-terminus and thereby act as sensors for extracellular proteases. While coagulation proteases activate PARs to regulate hemostasis, thrombosis, and cardiovascular function, PAR2 is also activated in extravascular locations by a broad array of serine proteases, including trypsin, tissue kallikreins, coagulation factors VIIa and Xa, mast cell tryptase, and transmembrane serine proteases. Administration of PAR2-specific agonistic and antagonistic peptides, as well as studies in PAR2 knockout mice, identified critical functions of PAR2 in development, inflammation, immunity, and angiogenesis. Here, we review these roles of PAR2 with an emphasis on the role of coagulation and other extracellular protease pathways that cleave PAR2 in epithelial, immune, and neuronal cells to regulate physiological and pathophysiological processes. [ABSTRACT FROM AUTHOR]

Purpose: Acyclic nucleoside phosphonates possess unique antiviral and antineoplastic activities; however, their polar phosphonate moiety is associated with low ability to cross biological membranes. We explored the potential of transdermal and topical delivery of 2,6-diaminopurine derivative cPr-PMEDAP. Methods: In vitro diffusion of cPr-PMEDAP was investigated using formulations at different pH and concentration and with permeation enhancer through porcine and human skin. Results: Ability of 0.1-5% cPr-PMEDAP to cross human skin barrier was very low with flux values ~40 ng/cm/h, the majority of compound found in the stratum corneum. The highest permeation rates were found at pH 6; increased donor concentration had no influence. The permeation enhancer dodecyl 6-dimethylaminohexanoate (DDAK, 1%) increased flux of cPr-PMEDAP (up to 61 times) and its concentration in nucleated epidermis (up to ~0.5 mg of cPr-PMEDAP/g of the tissue). No deamination of cPr-PMEDAP into PMEG occurred during permeation studies, but N-dealkylation into PMEDAP mediated by skin microflora was observed. Conclusions: Transdermal or topical application of cPr-PMEDAP enabled by the permeation enhancer DDAK may provide an attractive alternative route of administration of this potent antitumor and antiviral compound. [ABSTRACT FROM AUTHOR]

A sensitive label-free electrochemical aptasensor was successfully fabricated for thrombin detection with nafion@graphene as platform. With electrostatic interaction between nafion and methylene blue (MB), positive charged MB was successfully assembled on nafion@graphene modified electrode surface, which provided amounts of redox probes for electrochemical aptasensor. In the presence of thrombin, the thrombin aptamer (TBA) on the electrode surface would catch the target on the electrode interface, which made a barrier for electrons and inhibits the electro-transfer, resulting in the decreased differential pulse voltammetry signals of MB. As a result, the proposed approach showed a high sensitivity and a wider linearity to thrombin in the range 0.01-50 nM with a detection limit of 6 pM. [ABSTRACT FROM AUTHOR]

Conventional anticoagulants have proven efficacy in the management of thromboembolism. Their adverse effects and a narrow therapeutic window, necessitating regular need for monitoring, however, have long been an incentive for the development of safer anticoagulants without compromising efficacy. Over the last decade or so several new parenteral and oral anticoagulants have been launched with efficacy comparable with conventional agents. From fondaparinux to its long acting derivative idraparinux, and the factor Xa inhibitor rivaroxaban to the direct thrombin inhibitor dabigatran, the advent of new anticoagulants is radically changing anticoagulation. For conventional anticoagulants, despite their shortcomings, effective methods of reversing their anticoagulant effects exist. Moreover, strategies to deal with the occurrence of fresh thrombotic events in the face of therapeutic anticoagulation with the conventional agents have also been addressed. Nevertheless, for the new anticoagulants, the optimal management of these complications remains unknown. This review explores these issues in the light of current evidence. [ABSTRACT FROM AUTHOR]

Acute thrombus formation on disrupted atherosclerotic plaques plays a key role during the onset of acute coronary syndromes. Lesion disruption facilitates the interaction between circulating blood and prothrombotic substances, such as tissue factor (TF) present within the atherosclerotic lesion. For a long period of time, vessel-wall TF has been considered the major determinant of thrombosis. However, this old dogma has been recently changed owing to the discovery of a different pool of TF that circulates in flowing blood (blood-borne TF). Several studies have shown that blood-borne TF circulates in different pools that are associated with selected blood cells, such as monocytes, granulocytes and platelets in cell-derived microparticles, and as a soluble protein generated by alternative splicing of its full-length mRNA. Recent studies have identified a hypercoagulable state associated with an increased circulating TF activity, leading to the concept of 'vulnerable blood'. Part of the blood-borne TF circulates in an 'inactive' form and it is required to be 'activated' to exert its thrombogenic potential. Certain pathological conditions, such as smoking, hyperlipidemia and diabetes, show a higher incidence of thrombotic complications. These conditions are also characterized by the presence of high levels of circulating TF activity. Recent evidence may also suggest that an increased circulating TF activity may potentiate the initial thrombogenic stimulus represented by vessel wall-associated TF, leading to the formation of larger and/or more stable thrombus, and thus more severe acute coronary syndromes. It has been reported that inflammation increases TF expression and activity by different cell types. On the other hand, TF upregulation may facilitate inflammation by enhancing intravascular fibrin deposition, formation of proinflammatory fragments of fibrin, and by generating coagulation proteases, including FVIIa, FXa and thrombin, that activate protease-activated receptors. Furthermore, the biology of TF is know known to be more complex than previously thought by the demonstration that this protein, apart from its known effects on blood coagulation, can also function as a signaling receptor. INSET: Key issues. [ABSTRACT FROM AUTHOR]

Background/Aims: Tissue factor (TF) is a transmembrane protein that is essential for coagulation. TF is expressed on podocytes and its cytoplasmic domain has cell signalling functions in epithelial cells. Methods: Mice lacking the cytoplasmic domain of TF (TFCT-/- mice) were used to study its role in physiological albuminuria and pathological proteinuria following induction of glomerulonephritis (GN). Results: Absence of the cytoplasmic domain of TF was associated with increased albuminuria, podocyte effacement, reduced podocyte numbers and increased spontaneous glomerular tumour necrosis factor α(TNFα) production under physiological conditions. In mice developing GN, absence of the cytoplasmic domain of TF resulted in increased proteinuria and enhanced renal TNFα production without altering other parameters of renal inflammation and injury. Studies in TFCT-/- chimeric mice (created by bone marrow transplantation) showed increased proteinuria and renal TNFα mRNA in GN was associated with absence of the cytoplasmic domain of TF in the kidney and was independent of the leucocyte phenotype. Conclusion: These studies demonstrate that the cytoplasmic domain of TF contributes to renal albumin retention and its renal expression protects against proteinuria in leucocyte-mediated renal inflammation. Increased glomerular production of TNFα in the absence of cytoplasmic domain of TF may contribute to podocyte injury resulting in albuminuria and proteinuria. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) is one of the most active antiproliferative compounds in a series of acyclic nucleoside phosphonates and is active in intraperitoneal P388 tumors in mice. Methods: We synthesized octadecyloxyethyl (ODE) and hexadecyloxypropyl esters of PMEG and compared their antiproliferative activity with unmodified PMEG in primary human fibroblasts and CaSki, Me-180 and HeLa human cervical cancer cell lines in vitro. Results: ODE-PMEG had excellent antiproliferative activity in vitro in this panel of human cervical cancers. We compared the effects of ODE-PMEG and ODE-cidofovir (ODE-CDV) in a solid tumor model using Me-180 human cervical cancer cell lines in athymic nude mice. Intratumoral injection of 25 μg of ODE-PMEG or 100 μg of ODE-CDV daily for 21 days followed by observation for 20–35 days resulted in near-complete disappearance of measurable cervical cancers. Conclusion: ODE-PMEG may be suitable for local or topical treatment of cervical dysplasia. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]