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Interfacial failure in carbon fiber-reinforced epoxy (CFRE) laminates is a prominent mode of failure, attracting significant research attention. The large surface-energy mismatch between carbon fiber (CF) and epoxy results in a weaker interface. This study presents a facile yet effective method for enhancing the interfacial adhesion between CF and epoxy with self-healable interfaces. Two variants of a designer sizing agent, poly(ether imide) (PEI), were synthesized, one without a self-healing property termed BO, and the second one by incorporating disulfide metathesis in one of its monomers that renders self-healing properties at the interface-mediated by network reconfiguration, termed BA. 0.25 wt % of CF was found to be the optimum amount of BO and BA sizing agents. The surface free energy of CF drastically increased and became quite close to the surface energy of epoxy after the deposition of both sizing agents and the higher surface roughness. The improved surface wettability, presence of functional groups, and mechanical interlocking worked in tandem to strengthen the interface. The interlaminar shear strength (ILSS) and flexural strength (FS) of CFRE laminate sized with BO consequently increased by 35% and 22% and of CFRE laminate sized with BA increased by 26% and 19%, respectively. Fractography analysis revealed outstanding bonding between epoxy and PEI-CF, indicating that matrix fracture is the predominant mode of failure. The self-healable interfaces due to the preinstalled disulfide metathesis in the sizing agent resulted in 51% self-healing efficiency in ILSS for BA-sized CFRE laminate. Interestingly, the functional properties, deicing, and EMI shielding effectiveness were not compromised by modification of the interface with this designer sizing agent. This study opens new avenues for interfacial modification to improve the mechanical properties while retaining the key functional properties of the laminates.

The peripheral adenomatoid odontogenic tumour is an uncommon subtype of adenomatoid odontogenic tumour (AOT).It has a female preponderance commonly in theage group of 3–25 years. Anterior maxilla is themost frequent site of involvement. Clinically, it is manifested as soft tissue mass on gingiva with infrabony pocket and minimum bone involvement. So this lesion is easily misdiagnosed by the clinician as simple gingival growth.Histopathological features are identical to that of their intra-osseous counterpart.We present a case of 27 years old female who had her lesion on the gingiva of right maxillary canine region which is notausual site of involvement. Few cases have been reported in the literature but all exclusively involved the gingiva of maxillary incisors. In this context extensive study is needed to figure out the exact site distribution and clinical presentation of the disease.

In this paper, we study some properties of the ring C(X)Fof all real valued functions which are continuous except on some finite subsets of a topological space X. We show that C(X)Fis closed under uniform limit if and only if the set of all non-isolated points of Xis finite. We also initiate and investigate the zero divisor graph of the ring C(X)F.

In this study, a magnetic nanocomposite of hematite Fe2O3and magnetite Fe3O4has been synthesized by the sol-gel method and then conjugated with the Gd2O3, activated charcoal and the drug metformin (5mol%)) by mechanical alloying the powders. The synthesized nanocomposite (without drug) and the drug-conjugated nanocomposite have been well characterized using several experimental procedures like the analysis of the XRD patterns, FTIR, and UV–Vis spectra. The average particle sizes of the samples have been obtained by analyzing FESEM and TEM images. The elemental compositions and their distribution over the localized surface have been revealed from EDS spectra and elemental mapping. The magnetic properties of the samples have also been characterized by analyzing the MH loops and the MT curves. The biocompatibility of the samples has been tested against the human normal lung fibroblast WI38 cells by MTT assay. The enhanced activity of the drug-conjugated nanocomposite to inhibit the growth of the carcinoma cells has been confirmed against the A549 cells and HeLa cells by calculating the IC50dose. It has been revealed that the drug-conjugated nanocomposite and the pure 100% metformin have almost similar efficacy against human carcinoma cells. In the case of the drug-conjugated sample, only 5 mol% drug was present. Thus, a significant enhancement of the drug efficacy has been confirmed after conjugating it with the nanocomposite sample. It is an entirely new method by which the effectiveness of metformin to inhibit the growth of human carcinoma cells has been enhanced significantly by conjugating it with a biocompatible nanocomposite. The drug-conjugated magnetic nanocomposite will reduce the side effects of the drug for its prolonged use.

We herein synthesize a bisindole enyne using the iron-catalyzed environmentally benign dimerization method. The enyne kills cancer cells by arresting the cell cycle at the G2/M phase via the necroptosis pathway, a non-apoptotic form of cell death that is also considered a promising and attractive pathway to induce cancer cell death. The inherent fluorescent property has been used to study its localization in cells and its binding interactions with genomic DNA. This work demonstrates bisindole enyne motifs could be a new pharmacophore for developing anticancer agents.

Self-assembled graphene oxide lyotropic liquid crystal (GO LLC) structures are mostly formed in aqueous medium; however, most GO derivatives are water insoluble, so processing GO LLCs in water poses a practical limitation. The use of polar aprotic solvent (like dimethyl sulfoxide) for the formation of GO LLC structures would be interesting, because it would allow incorporating additives, like photoinitiators or cross-linkers, or blending with polymers that are insoluble in water, which hence would expand its scope. The well-balanced electrostatic interaction between DMSO and GO can promote and stabilize the GO nanosheets’ alignment even at lower concentrations. With this in mind, herein we report mechanically robust, chlorine-tolerant, self-assembled nanostructured GO membranes for precise molecular sieving. Small-angle X-ray scattering and polarized optical microscopy confirmed the alignment of the modified GO nanosheets in polar aprotic solvent, and the LLC structure was effectively preserved even after cross-linking under UV light. We found that the modified GO membranes exhibited considerably improved salt rejection for monovalent ions (99%) and water flux (120 LMH) as compared to the shear-aligned GO membrane, which is well supported by forward osmosis simulation studies. Additionally, our simulation studies indicated that water molecules traveled a longer path while permeating through the GO membrane compared to the GO LLC membrane. Consequently, salt ions permeate slowly across the GO LLC membrane, yielding higher salt rejection than the GO membrane. This begins to suggest strong electrostatic repulsion with the salt ions, causing higher salt rejection in the GO LLC membrane. We foresee that the ordered cross-linked GO sheets contributed to excellent mechanical stability under a high-pressure, cross-flow, chlorine environment. Overall, these membranes are easily scalable, exhibit good mechanical stability, and represent a breakthrough for the potential use of polymerized GO LLC membranes in practical water remediation applications.

The present study reveals a simple and cost-effective method to enhance the antibacterial activity of a model antibiotic drug azithromycin which can also be used as an inhibitor of protein-arginine deiminase type-4 (PADI4). At first, molecular docking has been used for obtaining binding modes and binding affinities of this drug to prove its role as an effective inhibitor for protein-arginine deiminase type-4 (PADI4). In the next step, a biocompatible nanocarrier has been synthesized for successful targeting of the drug azithromycin to enhance its efficiency to a greater extent. For the formation of the nanocarrier, Cu–Ag–Fe2O3nanocomposite has been synthesized by mechanical alloying the Cu, Ag and Fe2O3powder for 3 h. After the successful formation of the nanocomposite, 10 wt% of the drug has been conjugated with the nanocomposite by further milling for 1 h under the same experimental condition. The nanocomposite (NC) and drug conjugated nanocomposite (NC-DC) samples are well-characterized by the Rietveld refinement of the XRD pattern of the NC-DC sample, analyzing FTIR, EDS spectra, and FESEM images. It has been observed that both the samples are non-toxic to human normal lung fibroblast WI38 cells. The antibacterial activity of the drug conjugated nanocomposite sample has been enhanced significantly compared to the pure drug azithromycin. These investigations have been made by agar cup assay and minimum inhibitory concentrations (MIC) studies. As a minimal amount of drug is required for the conjugation, side effects and the cost of an expensive drug can be minimized to a great extent following the proposed synthesis route. The enhancement of the antibacterial activity of azithromycin conjugated nanocomposite gives a possibility that drug conjugated nanocomposite may have enhanced activity in the inhibition of PADI4. It may cure the harmful effects of COVID-19 or inhibit the growth of tumour cells more efficiently by improving the cell membrane penetration power of the drug due to its conjugation with the nanocomposite.

Arsenic contamination of ground water is emerging as a potential threat to human health. A report from our laboratory showed that aqueous garlic extract (AGE) had an extensive modulatory effect on arsenic toxicity. Moreover AGE formed a precipitate when incubated with NaAsO2overnight. Mass spectral analysis showed arginine to be one component of AGE responsible for forming the precipitate with NaAsO2. We further confirmed the role of arginine in mitigation of arsenic toxicity in A375 cells. Incubation of A375 cells with NaAsO2(10 μM) for 24 h caused a reduction in cell viability, enhanced reactive oxygen species (ROS) production, a reduction of the activities of the intracellular enzymatic as well as non-enzymatic antioxidants and also an enhancement of lipid peroxidation. Arginine (60 μM) along with NaAsO2almost normalized the altered cell viability, modulated ROS level and activities of antioxidant indices. Arginine also blunted arsenic induced genotoxicity and elevated the expression of poly-ADP ribose polymerase.

Epithelial myoepithelial carcinoma (EMC) is a rare low-grade biphasic tumour of salivary gland. Most common site of origin is the parotid gland with female predominance. We present a case of a 51-year-old male patient with symptomless slow-growing 2-year-old lump on the floor of the mouth. Histopathological diagnosis is corroborative with tubular variant of epithelial myoepithelial carcinoma. The floor of the mouth is the rare site for this tumour. In this literature, we intend to discuss the clinical and pathological aspect of EMC located on the floor of the mouth.

A majority of tissue factor (TF) on cell surfaces exists in a cryptic form (ie, coagulation function inactive) but retains its functionality in cell signaling. Recent studies have suggested that cryptic TF contains unpaired cysteine thiols and that activation involves the formation of the disulfide bond Cys186-Cys 209 and that protein disulfide isomerase (PDI) regulates TF coagulant and signaling activities by targeting this disulfide bond. This study was carried out to investigate the validity of this novel concept. Although treatment of MDA 231 tumor cells, fibroblasts, and stimulated endothelial cells with the oxidizing agent HgCl2 markedly increased the cell-surface TF coagulant activity, the increase is associated with increased anionic phospholipids at the cell surface. Annexin V, which binds to anionic phospholipids, attenuated the increased TF coagulant activity. It is noteworthy that treatment of cells with reducing agents also increased the cell surface TF activity. No evidence was found for either detectable expression of PDI at the cell surface or association of TF with PDI. Furthermore, reduction of PDI with the gene silencing had no effect on either TF coagulant or cell signaling functions. Overall, the present data undermine the recently proposed hypothesis that PDI-mediated disulfide exchange plays a role in regulating TF procoagulant and cell signaling functions.

A majority of tissue factor (TF) on cell surfaces exists in a cryptic form (ie, coagulation function inactive) but retains its functionality in cell signaling. Recent studies have suggested that cryptic TF contains unpaired cysteine thiols and that activation involves the formation of the disulfide bond Cys186-Cys 209 and that protein disulfide isomerase (PDI) regulates TF coagulant and signaling activities by targeting this disulfide bond. This study was carried out to investigate the validity of this novel concept. Although treatment of MDA 231 tumor cells, fibroblasts, and stimulated endothelial cells with the oxidizing agent HgCl2markedly increased the cell-surface TF coagulant activity, the increase is associated with increased anionic phospholipids at the cell surface. Annexin V, which binds to anionic phospholipids, attenuated the increased TF coagulant activity. It is noteworthy that treatment of cells with reducing agents also increased the cell surface TF activity. No evidence was found for either detectable expression of PDI at the cell surface or association of TF with PDI. Furthermore, reduction of PDI with the gene silencing had no effect on either TF coagulant or cell signaling functions. Overall, the present data undermine the recently proposed hypothesis that PDI-mediated disulfide exchange plays a role in regulating TF procoagulant and cell signaling functions.

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa), and the formation of TF-FVIIa complexes on cell surfaces triggers the activation of the coagulation cascade and the cell signaling. Our recent studies have shown that a majority of TF resides in various intracellular compartments, predominantly in the Golgi, and that FVIIa binding to cell surface TF induces TF endocytosis and mobilizes the Golgi TF pool to translocate it to the cell surface. This present study is aimed to elucidate the mechanisms involved in TF endocytosis and its mobilization from the Golgi. Activation of protease-activated receptor 1 (PAR1) and PAR2 by specific peptide agonists and proteases, independent of FVIIa, mobilized TF from the Golgi store and increased the cell surface expression of TF. Blocking PAR2 activation, but not PAR1, with neutralizing antibodies fully attenuated the FVIIa-induced TF mobilization. Consistent with these data, silencing the PAR2 receptor, and not PAR1, abrogated the FVIIa-mediated TF mobilization. In contrast to their effect on TF mobilization, PAR1 and PAR2 activation, in the absence of FVIIa, had no effect on TF endocytosis. However, PAR2 activation is found to be critical for the FVIIa-induced TF endocytosis. Overall the data herein provide novel insights into the role of PARs in regulating cell surface TF expression.

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa), and the formation of TF-FVIIa complexes on cell surfaces triggers the activation of the coagulation cascade and the cell signaling. Our recent studies have shown that a majority of TF resides in various intracellular compartments, predominantly in the Golgi, and that FVIIa binding to cell surface TF induces TF endocytosis and mobilizes the Golgi TF pool to translocate it to the cell surface. This present study is aimed to elucidate the mechanisms involved in TF endocytosis and its mobilization from the Golgi. Activation of protease-activated receptor 1 (PAR1) and PAR2 by specific peptide agonists and proteases, independent of FVIIa, mobilized TF from the Golgi store and increased the cell surface expression of TF. Blocking PAR2 activation, but not PAR1, with neutralizing antibodies fully attenuated the FVIIa-induced TF mobilization. Consistent with these data, silencing the PAR2 receptor, and not PAR1, abrogated the FVIIa-mediated TF mobilization. In contrast to their effect on TF mobilization, PAR1 and PAR2 activation, in the absence of FVIIa, had no effect on TF endocytosis. However, PAR2 activation is found to be critical for the FVIIa-induced TF endocytosis. Overall the data herein provide novel insights into the role of PARs in regulating cell surface TF expression.

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa). The formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. In the present study, we characterized the subcellular distribution of TF and its transport in fibroblasts by dual immunofluorescence confocal microscopy and biochemical methods. Our data show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. Tissue factor at the cell surface is localized in cholesterol-rich lipid rafts and extensively colocalized with caveolin-1. FVIIa binding to TF induces the internalization of TF. Of interest, we found that TF-FVIIa complex formation at the cell surface leads to TF mobilization from the Golgi with a resultant increase in TF expression at the cell surface. This process is dependent on FVIIa protease activity. Overall, the present data suggest a novel mechanism for TF expression at the cell surface by FVIIa. This mechanism could play an important role in hemostasis in response to vascular injury by increasing TF activity where and when it is needed.

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa). The formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. In the present study, we characterized the subcellular distribution of TF and its transport in fibroblasts by dual immunofluorescence confocal microscopy and biochemical methods. Our data show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. Tissue factor at the cell surface is localized in cholesterol-rich lipid rafts and extensively colocalized with caveolin-1. FVIIa binding to TF induces the internalization of TF. Of interest, we found that TF-FVIIa complex formation at the cell surface leads to TF mobilization from the Golgi with a resultant increase in TF expression at the cell surface. This process is dependent on FVIIa protease activity. Overall, the present data suggest a novel mechanism for TF expression at the cell surface by FVIIa. This mechanism could play an important role in hemostasis in response to vascular injury by increasing TF activity where and when it is needed.

Abstract: Placental protein/peptides as biological response modifier are well documented, but not much known about melanogenesis. We possibly for the first time, demonstrated melanogenesis in B16F10 mouse melanoma by a placental protein/peptide fraction (PPPF) prepared from a hydroalcoholic extract of fresh term human placenta. This study described the effect of PPPF on the induction of tyrosinase; the key enzyme of melanogenesis to investigate the basis of PPPF induced pigmentation in primary melanocyte and B16F10 melanoma. Tyrosinase induction by PPPF in B16F10 cells was found dose- and time dependent at the level of activity. Tyrosinase, at the level of transcription and protein expression when assessed by RT-PCR and Western blot analyses found to have considerable induction over untreated control. PPPF led to enhanced activation of tyrosinase promoter resulting higher transcription thus substantiating the role of PPPF as a stimulator of melanogenesis. Actinomycin D, the transcriptional inhibitor of protein synthesis, blocked the stimulatory action of PPPF since the induction of tyrosinase and melanin was markedly reduced in presence of this inhibitor. Thus the results suggested that PPPF mediated increase in tyrosinase expression occurred through transcriptional upregulation to stimulate melanogenesis in B16F10 cells and in primary melanocyte also. (Mol Cell Biochem xxx: 1–10, 2004)

Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-β-cyclodextrin–reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding.

Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-β-cyclodextrin–reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding.

Tissue factor (TF), the cellular receptor for factor VIIa (FVIIa), besides initiating blood coagulation, is believed to play an important role in tissue repair, inflammation, angiogenesis, and tumor metastasis. Like TF, the chemokine interleukin-8 (IL-8) is shown to play a critical role in these processes. To elucidate the potential mechanisms by which TF contributes to tumor invasion and metastasis, we investigated the effect of FVIIa on IL-8 expression and cell migration in a breast carcinoma cell line, MDA-MB-231, a cell line that constitutively expresses abundant TF. Expression of IL-8 mRNA in MDA-MB-231 cells was markedly up-regulated by plasma concentrations of FVII or an equivalent concentration of FVIIa (10 nM). Neither thrombin nor other proteases involved in hemostasis were effective in stimulating IL-8 in these cells. Increased transcriptional activation of the IL-8 gene is responsible for increased expression of IL-8 in FVIIa-treated cells. PAR-2–specific antibodies fully attenuated TF-FVIIa–induced IL-8 expression. Additional in vitro experiments showed that TF-FVIIa promoted tumor cell migration and invasion, active site–inactivated FVIIa, and specific antibodies against TF, PAR-2, and IL-8 inhibited TF-FVIIa–induced cell migration. In summary, the studies described herein provide insight into how TF may contribute to tumor invasion. (Blood. 2004;103:3029-3037)

Tissue factor (TF), the cellular receptor for factor VIIa (FVIIa), besides initiating blood coagulation, is believed to play an important role in tissue repair, inflammation, angiogenesis, and tumor metastasis. Like TF, the chemokine interleukin-8 (IL-8) is shown to play a critical role in these processes. To elucidate the potential mechanisms by which TF contributes to tumor invasion and metastasis, we investigated the effect of FVIIa on IL-8 expression and cell migration in a breast carcinoma cell line, MDA-MB-231, a cell line that constitutively expresses abundant TF. Expression of IL-8 mRNA in MDA-MB-231 cells was markedly up-regulated by plasma concentrations of FVII or an equivalent concentration of FVIIa (10 nM). Neither thrombin nor other proteases involved in hemostasis were effective in stimulating IL-8 in these cells. Increased transcriptional activation of theIL-8gene is responsible for increased expression of IL-8 in FVIIa-treated cells. PAR-2–specific antibodies fully attenuated TF-FVIIa–induced IL-8 expression. Additional in vitro experiments showed that TF-FVIIa promoted tumor cell migration and invasion, active site–inactivated FVIIa, and specific antibodies against TF, PAR-2, and IL-8 inhibited TF-FVIIa–induced cell migration. In summary, the studies described herein provide insight into how TF may contribute to tumor invasion. (Blood. 2004;103:3029-3037)

Abstract: A hydroalcoholic extract of fresh term human placenta was found to be mitogenic as well as melanogenic on B16F10 mouse melanoma in anin vitro culture. The extract, a reservoir of a large number of bioactive molecules, was resolved to get the lipid fraction. Its activity was evaluated on B16F10 mouse melanoma by assessing the change in cellular morphology, growth and melanin induction. The lipid fraction, placental total lipid fraction (PTLF) tested in the study employed doses of 0.01 to 200 μg/ml; optimum growth and melanization accompanied by morphological changes were recorded at 10 and 100 μg/ml respectively. At intermediate doses growth and melanization were found to show a pattern of change over between growth and melanization and finally reached at an inverse relation at the respective optimal dose of response. Compared with defined sphingolipids, C2 ceramide and sphingosine-1-phosphate, the results were mostly corroborative. The duality of biological response of sphingolipids as reported in numerous studies was comparable for the PTLF suggesting that its active component is a sphingolipid and showing its use for pigment recovery in vitiligo.

This study reveals a new strategy to enhance the activity of an antibacterial drug by conjugating it with a Cu–Ag-based nanocarrier. The Cu–Ag–Co3O4–TiO2nanocomposite is successfully synthesized by mechanical alloying and applied for biocompatible targeting of an antibacterial drug by conjugating the drug (10 wt%) with the nanocarrier. The samples have been well-characterized by the Rietveld refinement of the XRD pattern and by analyzing TEM, FESEM images, and FTIR spectra. The successful formation of the drug conjugated nanocomposite sample has also been verified from the EDS and UV–Vis absorption spectra. Both the samples are non-toxic to the normal human cells which have been confirmed by cell viability assay on human normal lung fibroblast WI38 cells. Antibacterial activities of both the samples against the bacteria E. colihave been investigated by the agar cup diffusion method and minimum inhibitory concentration (MIC) study. The 10 wt% amoxicillin conjugated nanocomposite shows the same effect as the pure (100 wt %) drug. The enhanced activity of the drug conjugated nanocomposite is due to the more significant interaction of the drug conjugated nanocomposite with the cell wall and membrane of the bacteria as compared to the pure drug. It is confirmed by measuring the change of conductivity and total protein leakage in the culture filtrate of the nanocomposite, pure drug, and drug conjugated nanocomposite treated culture of Bacillus subtilis. This strategic protocol is found to have great importance for enhancing the efficacy of a standard antibiotic drug.

This study reveals a cost-effective route (mechanical alloying) to synthesize an antibacterial drug conjugated copper-silver-titanium oxide nanocomposite (10 wt% cefixime conjugated Cu–Ag–TiO2) for successful targeting of the drug with enhanced antibacterial activity. For the nanocomposite (NC) and drug conjugated nanocomposite (NC-DC), detailed structural and microstructural characterizations have been carried out by analyzing the FESEM-EDS spectra, TEM images, and the Rietveld refinement of the XRD patterns. The NC-DC sample is completely biocompatible, as reflected by the MTT assay. The Antibacterial activity of the samples has been investigated by agar cup diffusion assay and minimum inhibitory concentration (MIC) studies. These studies indicate that the NC sample itself has significant antibacterial activity and NC-DCsample has four times greater antibacterial activity than the pure drug cefixime, as again supported by fluorescence microscopic studies. The mode of action of NC and NC-DC samples has been investigated in detail employing conductivity measurement and total protein leakage assay. The significant enhancement of the antibacterial activity of the drug seems to be very important for an antibacterial drug, as a low dose of the conjugated drug would result in better antibacterial activity with minimal side effects.

A mild and efficient method for etherification of cyclic ß-diketones with alcohols has been developed using a catalytic amount of cerium(IV) ammonium nitrate at room temperature to afford the corresponding ß-keto enol ethers in good to excellent yields. The deprotections of enol ethers in water–acetonitrile (1:1) using a catalytic amount (10 mol %) of cerium(IV) ammonium nitrate have also been achieved.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa) and the formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. TF is constitutively expressed in many extravascular cells, including fibroblasts and pericytes in and surrounding blood vessel walls, and lung epithelial cells. Our recent studies (Blood2006; 107:4746–4753) show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. FVIIa binding to cell surface TF induces the internalization of TF, and interestingly, mobilizes the Golgi TF pool and transports it to the outer cell surface. This process is dependent on FVIIa protease activity. This present study is aimed to elucidate potential mechanisms involved in TF internalization and the mobilization from the Golgi. Since studies from our laboratory and others showed that TF-FVIIa could activate protease-activated receptor (PAR)-mediated cell signaling and FVIIa protease activity is required for FVIIa-dependent internalization and trafficking of TF, we hypothesize that TF-VIIa activation of PAR1 or PAR2 plays a role in TF internalization and trafficking. To test this hypothesis, we first examined the role of PAR activation in TF-internalization and trafficking. Lung fibroblasts (WI-38 cells) were exposed to a variety of PAR activators, PAR activating peptide agonists (AP) and various proteases, and TF internalization and trafficking was evaluated by measuring the cell surface TF antigen and activity levels, internalization of cell surface TF (by using biotinylation of cell surface receptors and immunoprecipitation techniques) and mobilization of TF from the Golgi (by immunofluorescence confocal microscopy). PAR1 AP and PAR2 AP treatments increased the TF activity and antigen levels at the cell surface by 20 to 50% whereas PAR3 AP and PAR4 AP had no effect on cell surface TF activity and antigen levels. Cell surface TF activity and antigen levels were also increased slightly in fibroblasts exposed to thrombin and trypsin. Confocal microscopic image analysis of distribution of TF and the Golgi protein (golgin-97) revealed that about 85% of the untreated cells possess intact Golgi TF pool with high degree of colocalization with golgin-97 whereas as only 20–30% of FVIIa, thrombin, trypsin, PAR1 AP or PAR2 AP-treated cells had TF pool in the Golgi. Plasmin and FXa had moderate effect on TF mobilization from the Golgi. No detectable differences were found between control (untreated) cells and cells treated with either FFR-FVIIa, APC, PAR3 AP or PAR4 AP. Next, we investigated the role of PAR1 and PAR2 activation in FVIIa-mediated TF internalization and trafficking. Pretreatment of fibroblasts with PAR2 but not PAR1 activation blocking antibodies attenuated FVIIa-mediated Golgi TF mobilization. Consistent with these data, silencing PAR2 receptor by siRNA technique completely blocked FVIIa-mediated Golgi TF mobilization whereas PAR1 siRNA transfection had no effect (in control studies, we showed PAR1 antibodies or PAR1si RNA transfection blocked thrombin-mediated TF mobilization). Additional studies showed a significant internalization of TF in cells exposed to FVIIa which was completely blocked by silencing PAR2 but not PAR1. Overall the data provided herein suggest a novel mechanism by which tissue factor expression is regulated at the cell surface.

Tissue factor (TF) is the cellular receptor for plasma clotting factor VIIa, and the formation of TF-VIIa complexes on cell surfaces trigger the coagulation cascade and cell signaling. It is a well-known fact that only a small fraction of TF at the cell surface is coagulantly active whereas a majority of TF on the cell surface is non-functional (cryptic). However, it is unclear, at present, how the coagulant active TF differs from the cryptic form, and mechanisms involved in TF activation. Recent studies show that a thiol oxidizing agent, HgCl2, increases TF coagulant activity on the surface of HL-60 cells by several fold (Chen et al., Blood vol 106, abstract #684, 2005). Further, TF is shown to associate with protein disulfide isomerase (PDI) in HaCaT cells (Ahamed et al., Blood vol 106, abstract #685, 2005). Based on these and other observations, it has been proposed that switching between cryptic and coagulant TF involves cleavage and formation of allosteric disulfide bond (Cys186-Cys209) and PDI has been implicated in controlling the conversion of cryptic TF to the coagulant form and to act as a switch between TF-mediated signaling and coagulation. Although these data are interesting and novel, there is no fail-proof evidence that disulfide switching alone and not other potential changes, such as exposure of anionic phospholipids, at the cell surface is responsible for the TF activation associated with various treatments. Therefore we have examined the effect of HgCl2 and other treatments on TF activation in MDA 231 cells in relation to anionic phospholipids and also characterized the cellular expression of PDI in this and other cell types. As reported earlier, the HgCl2 treatment increased the cell surface TF coagulant activity (5-fold or higher). However, the HgCl2 treatment also increased the prothombinase activity by 3-fold. More importantly, annexin V, which binds to anionic phospholipids, markedly reduced the increased TF coagulant activity associated with the HgCl2 treatment whereas it had only minimal and insignificant effect on TF activity of the control cells. Further, pretreatment of cells with 5,5′-dithio-bis(2-nitronezoic acid) (DTNB), a sulfhydryl reagent that reacts with thiol groups and thus can block disulfide switching, failed to prevent the increase in TF activity associated with the HgCl2 treatment. Interestingly, we found that treatment of MDA 231 cells with glutathione (5 to 100 mM), a cell impermeable reducing agent, also increased the surface TF activity by about 2- to 3-fold. In additional studies, we found that PDI antibodies had no effect on either the TF coagulant activity or TF-mediated cell signaling. Further, we found no evidence for the expression of PDI at the cell surface in immunofluorescence confocal microscopy as both monoclonal and polyclonal PDI antibodies failed to stain nonpermeabilized cells whereas they brightly stained intracellular PDI in permeabilized cells. In contrast, TF antibodies stained intensely the surface of both nonpermeabilized and permeabilized cells. Exposure of tumor cells to various proteases failed to transport the intracellular PDI to the cell surface. The present data raise a valid question whether disulfide switching by PDI plays the predominant and general regulatory role in controlling the TF coagulant activity and signaling functions. Our data also suggest that other cellular changes, including increase in anionic phospholipids, may be responsible for increased TF coagulant activity associated with the thiol oxidizers and other treatments.

Tissue factor (TF) is a cellular receptor for clotting factor VIIa (VIIa) and the formation of TF-VIIa complexes on cell surfaces not only triggers the coagulation cascade but also transduces cell signaling via activation of protease-activated receptors (PARs), particularly PAR2. Although a number of recent studies provide valuable information on intracellular signaling pathways that are activated by TF-VIIa, the role of various cell surface components in mediating the interaction of TF-VIIa with PARs, and the subsequent signal transmittance are unknown. Unlike thrombin and trypsin, VIIa has to bind to its cellular receptor (TF) to activate PARs. The inability of TF-VIIa to effectively activate Ca2+ signaling and failure to desensitize the signaling to subsequently added trypsin suggest that the TF-VIIa is a poor activator of PAR2. Despite this, a number of studies have shown that VIIa is as effective as trypsin or PAR2 agonist peptide in activating intracellular signaling pathways and gene expression in cells expressing TF. Although the potential mechanism for this phenomenon is unknown, compartmentalization of TF, PAR2, and G-proteins in plasma membrane microdomains could facilitate a robust TF-VIIa-induced PAR2-mediated cell signaling. Although certain G-protein coupled receptors and G-proteins are known to be segregated into specialized membrane microdomains, lipid rafts and caveolae, little is known whether PARs are segregated into lipid rafts and caveolae, and how such segregation might influence their activation by TF-VIIa and the subsequent coupling to G-proteins. To obtain answers to some of these questions, in the present study, we have characterized TF and PAR2 distribution on tumor cell surfaces and investigated the role of lipid raft/caveolae in modulating the TF-VIIa signaling in tumor cells. Detergent extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF and PAR2 were distributed both in lipid rafts (low-density) and soluble fractions. Immunofluorescence confocal microscopy revealed that TF at the cell surface is localized in discrete plasma membrane microdomains, and colocalized with caveolin-1, a structural integral protein of caveolae, indicating caveolar localization of TF. Similar to TF, PAR2 also displayed significant punctuate staining and colocalization with caveloin-1. Further, a substantial fraction of TF and PAR2 was colocalized in caveolae. Disruption of lipid rafts/caveolae by ß-methyl cyclodextrin or filipin treatments reduced TF association with PAR2 in lipid rafts and caveolar fractions and impaired the TF-VIIa-induced cell signaling (PI hydrolysis and IL-8 gene expression). Additional studies showed that both mßCD and filipin treatments specifically impaired TF-VIIa cleavage of PAR2 and but had no significant effect on trypsin cleavage of PAR2. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling. In summary, the data presented herein demonstrate that TF localization at the cell membrane could influence different functions of TF differently. While caveolar localization of TF had no influence in propagating the procoagulant activity of TF, it is essential in supporting the TF-VIIa-induced cell signaling.

Tissue factor (TF) is the cellular receptor for plasma clotting factor VIIa, and the formation of TF-VIIa complexes on cell surfaces trigger the coagulation cascade and cell signaling. It is a well-known fact that only a small fraction of TF at the cell surface is coagulantly active whereas a majority of TF on the cell surface is non-functional (cryptic). However, it is unclear, at present, how the coagulant active TF differs from the cryptic form, and mechanisms involved in TF activation. Recent studies show that a thiol oxidizing agent, HgCl2, increases TF coagulant activity on the surface of HL-60 cells by several fold (Chen et al., Blood vol 106, abstract #684, 2005). Further, TF is shown to associate with protein disulfide isomerase (PDI) in HaCaT cells (Ahamed et al., Blood vol 106, abstract #685, 2005). Based on these and other observations, it has been proposed that switching between cryptic and coagulant TF involves cleavage and formation of allosteric disulfide bond (Cys186-Cys209) and PDI has been implicated in controlling the conversion of cryptic TF to the coagulant form and to act as a switch between TF-mediated signaling and coagulation. Although these data are interesting and novel, there is no fail-proof evidence that disulfide switching alone and not other potential changes, such as exposure of anionic phospholipids, at the cell surface is responsible for the TF activation associated with various treatments. Therefore we have examined the effect of HgCl2and other treatments on TF activation in MDA 231 cells in relation to anionic phospholipids and also characterized the cellular expression of PDI in this and other cell types. As reported earlier, the HgCl2treatment increased the cell surface TF coagulant activity (5-fold or higher). However, the HgCl2treatment also increased the prothombinase activity by 3-fold. More importantly, annexin V, which binds to anionic phospholipids, markedly reduced the increased TF coagulant activity associated with the HgCl2treatment whereas it had only minimal and insignificant effect on TF activity of the control cells. Further, pretreatment of cells with 5,5′-dithio-bis(2-nitronezoic acid) (DTNB), a sulfhydryl reagent that reacts with thiol groups and thus can block disulfide switching, failed to prevent the increase in TF activity associated with the HgCl2treatment. Interestingly, we found that treatment of MDA 231 cells with glutathione (5 to 100 mM), a cell impermeable reducing agent, also increased the surface TF activity by about 2- to 3-fold. In additional studies, we found that PDI antibodies had no effect on either the TF coagulant activity or TF-mediated cell signaling. Further, we found no evidence for the expression of PDI at the cell surface in immunofluorescence confocal microscopy as both monoclonal and polyclonal PDI antibodies failed to stain nonpermeabilized cells whereas they brightly stained intracellular PDI in permeabilized cells. In contrast, TF antibodies stained intensely the surface of both nonpermeabilized and permeabilized cells. Exposure of tumor cells to various proteases failed to transport the intracellular PDI to the cell surface. The present data raise a valid question whether disulfide switching by PDI plays the predominant and general regulatory role in controlling the TF coagulant activity and signaling functions. Our data also suggest that other cellular changes, including increase in anionic phospholipids, may be responsible for increased TF coagulant activity associated with the thiol oxidizers and other treatments.

Tissue factor (TF) is a cellular receptor for clotting factor VIIa (VIIa) and the formation of TF-VIIa complexes on cell surfaces not only triggers the coagulation cascade but also transduces cell signaling via activation of protease-activated receptors (PARs), particularly PAR2. Although a number of recent studies provide valuable information on intracellular signaling pathways that are activated by TF-VIIa, the role of various cell surface components in mediating the interaction of TF-VIIa with PARs, and the subsequent signal transmittance are unknown. Unlike thrombin and trypsin, VIIa has to bind to its cellular receptor (TF) to activate PARs. The inability of TF-VIIa to effectively activate Ca2+signaling and failure to desensitize the signaling to subsequently added trypsin suggest that the TF-VIIa is a poor activator of PAR2. Despite this, a number of studies have shown that VIIa is as effective as trypsin or PAR2 agonist peptide in activating intracellular signaling pathways and gene expression in cells expressing TF. Although the potential mechanism for this phenomenon is unknown, compartmentalization of TF, PAR2, and G-proteins in plasma membrane microdomains could facilitate a robust TF-VIIa-induced PAR2-mediated cell signaling. Although certain G-protein coupled receptors and G-proteins are known to be segregated into specialized membrane microdomains, lipid rafts and caveolae, little is known whether PARs are segregated into lipid rafts and caveolae, and how such segregation might influence their activation by TF-VIIa and the subsequent coupling to G-proteins. To obtain answers to some of these questions, in the present study, we have characterized TF and PAR2 distribution on tumor cell surfaces and investigated the role of lipid raft/caveolae in modulating the TF-VIIa signaling in tumor cells. Detergent extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF and PAR2 were distributed both in lipid rafts (low-density) and soluble fractions. Immunofluorescence confocal microscopy revealed that TF at the cell surface is localized in discrete plasma membrane microdomains, and colocalized with caveolin-1, a structural integral protein of caveolae, indicating caveolar localization of TF. Similar to TF, PAR2 also displayed significant punctuate staining and colocalization with caveloin-1. Further, a substantial fraction of TF and PAR2 was colocalized in caveolae. Disruption of lipid rafts/caveolae by ß-methyl cyclodextrin or filipin treatments reduced TF association with PAR2 in lipid rafts and caveolar fractions and impaired the TF-VIIa-induced cell signaling (PI hydrolysis and IL-8 gene expression). Additional studies showed that both mßCD and filipin treatments specifically impaired TF-VIIa cleavage of PAR2 and but had no significant effect on trypsin cleavage of PAR2. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling. In summary, the data presented herein demonstrate that TF localization at the cell membrane could influence different functions of TF differently. While caveolar localization of TF had no influence in propagating the procoagulant activity of TF, it is essential in supporting the TF-VIIa-induced cell signaling.

Tissue factor (TF) is the cellular receptor for clotting factor VIIa (FVIIa) and the formation of TF-FVIIa complexes on cell surfaces triggers the activation of coagulation cascade and cell signaling. TF is constitutively expressed in many extravascular cells, including fibroblasts and pericytes in and surrounding blood vessel walls, and lung epithelial cells. Our recent studies (Blood 2006; 107:4746–4753) show that a majority of TF resides in various intracellular compartments, predominantly in the Golgi. FVIIa binding to cell surface TF induces the internalization of TF, and interestingly, mobilizes the Golgi TF pool and transports it to the outer cell surface. This process is dependent on FVIIa protease activity. This present study is aimed to elucidate potential mechanisms involved in TF internalization and the mobilization from the Golgi. Since studies from our laboratory and others showed that TF-FVIIa could activate protease-activated receptor (PAR)-mediated cell signaling and FVIIa protease activity is required for FVIIa-dependent internalization and trafficking of TF, we hypothesize that TF-VIIa activation of PAR1 or PAR2 plays a role in TF internalization and trafficking. To test this hypothesis, we first examined the role of PAR activation in TF-internalization and trafficking. Lung fibroblasts (WI-38 cells) were exposed to a variety of PAR activators, PAR activating peptide agonists (AP) and various proteases, and TF internalization and trafficking was evaluated by measuring the cell surface TF antigen and activity levels, internalization of cell surface TF (by using biotinylation of cell surface receptors and immunoprecipitation techniques) and mobilization of TF from the Golgi (by immunofluorescence confocal microscopy). PAR1 AP and PAR2 AP treatments increased the TF activity and antigen levels at the cell surface by 20 to 50% whereas PAR3 AP and PAR4 AP had no effect on cell surface TF activity and antigen levels. Cell surface TF activity and antigen levels were also increased slightly in fibroblasts exposed to thrombin and trypsin. Confocal microscopic image analysis of distribution of TF and the Golgi protein (golgin-97) revealed that about 85% of the untreated cells possess intact Golgi TF pool with high degree of colocalization with golgin-97 whereas as only 20–30% of FVIIa, thrombin, trypsin, PAR1 AP or PAR2 AP-treated cells had TF pool in the Golgi. Plasmin and FXa had moderate effect on TF mobilization from the Golgi. No detectable differences were found between control (untreated) cells and cells treated with either FFR-FVIIa, APC, PAR3 AP or PAR4 AP. Next, we investigated the role of PAR1 and PAR2 activation in FVIIa-mediated TF internalization and trafficking. Pretreatment of fibroblasts with PAR2 but not PAR1 activation blocking antibodies attenuated FVIIa-mediated Golgi TF mobilization. Consistent with these data, silencing PAR2 receptor by siRNA technique completely blocked FVIIa-mediated Golgi TF mobilization whereas PAR1 siRNA transfection had no effect (in control studies, we showed PAR1 antibodies or PAR1si RNA transfection blocked thrombin-mediated TF mobilization). Additional studies showed a significant internalization of TF in cells exposed to FVIIa which was completely blocked by silencing PAR2 but not PAR1. Overall the data provided herein suggest a novel mechanism by which tissue factor expression is regulated at the cell surface.

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The plasminogen activator/plasmin system plays an important role, in addition to fibrinolysis, in many pathophysiological processes. Our recent studies showed that plasmin, similar to factor VIIa/tissue factor and thrombin, upregulates Cyr61, a growth factor like immediate early response gene, in fibroblasts via protease-activated receptor-mediated signaling. In preliminary studies we found that when fibroblasts were treated with VIIa (100 nM), thrombin (10 nM) or plasmin (50 nM), in concentrations that were shown to induce Cyr61 expression maximally, VIIa and thrombin increased the DNA synthesis by only about 20–30% whereas plasmin increased the DNA synthesis by 200 to 500%. The present study was carried out to investigate potential mechanism(s) by which plasmin promotes the DNA synthesis in fibroblasts and the role of protease-activated receptors and Cyr61 in plasmin-induced DNA synthesis. The plasmin-induced DNA synthesis in fibroblasts was dependent on its protease activity as the addition of active site-inhibited plasmin had no effect; and tissue factor pathway inhibitor-2 (TFPI-2) completely abrogated the plasmin-induced response. Neutralizing antibodies against PAR-1 and not PAR-2 attenuated the plasmin-induced DNA synthesis. Cyr61 antibodies completely blocked the plasmin-induced DNA synthesis suggesting that Cyr61 acts as a down-stream mediator. Surprisingly, we found no detectable increase in Cyr61 protein in plasmin-treated cell lysates whereas Cyr61 protein levels were clearly increased in thrombin-treated cells. These data suggest that plasmin might be cleaving newly synthesized Cyr61, which accumulates in ECM. Studies performed with purified recombinant Cyr61 and tumor cells that constitutively express abundant Cyr61 provided support for this notion. To test the hypothesis that plasmin release of Cyr61 from ECM plays a role in promoting DNA synthesis, we investigated the effect of conditioned media from control, plasmin-, and thrombin-treated fibroblasts in promoting DNA synthesis. The data revealed that the conditioned media of plasmin-treated cells (the protease activity in the media was inactivated) and not others significantly increased 3H-thymidine incorporation in fibroblasts. Addition of Cyr61 antibodies to the conditioned media of plasmin-treated cells attenuated the enhancing effect of the conditioned media. In summary, the data presented herein provide a novel mechanism by which plasmin promotes cell proliferation. In this mechanism, plasmin first induces the expression of Cyr61 via activation of PAR-1 mediated signaling. Next, plasmin releases/cleaves newly synthesized Cyr61 from the ECM, making it accessible to cells. The ability of plasmin to induce mitogenesis, in addition to its pericellular proteolysis, may provide a coordinated spatiotemporal effect that is required for would healing and tissue remodeling.