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PubMed2003

Hypothalamic digoxin, hemispheric chemical dominance, and interstitial lung disease.

The isoprenoid pathway produces three key metabolites--endogenous digoxin, dolichol, and ubiquinone. This was assessed in patients with idiopathic pulmonary fibrosis and in individuals of differing hemispheric dominance to find out the role of hemispheric dominance in the pathogenesis of idiopathic pulmonary fibrosis. All 15 cases of interstitial lung disease were right-handed/left hemispheric dominant by the dichotic listening test. The isoprenoidal metabolites--digoxin, dolichol, and ubiquinone, RBC membrane Na(+)-K+ ATPase activity, serum magnesium, tyrosine/tryptophan catabolic patterns, free radical metabolism, glycoconjugate metabolism, and RBC membrane composition--were assessed in idiopathic pulmonary fibrosis as well as in individuals with differing hemispheric dominance. In patients with idiopathic pulmonary fibrosis there was elevated digoxin synthesis, increased dolichol and glycoconjugate levels, and low ubiquinone and elevated free radical levels. There was also an increase in tryptophan catabolites and a reduction in tyrosine catabolites. There was an increase in cholesterol phospholipid ratio and a reduction in glycoconjugate level of RBC membrane in patients with idiopathic pulmonary fibrosis. Isoprenoid pathway dysfunction con tributes to the pathogenesis of idiopathic pulmonary fibrosis. The biochemical patterns obtained in interstitial lung disease are similar to those obtained in left-handed/right hemispheric chemically dominant individuals by the dichotic listening test. However, all the patients with interstitial lung disease were right-handed/left hemispheric dominant by the dichotic listening test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. Interstitial lung disease occurs in right hemispheric chemically dominant individuals and is a reflection of altered brain function.

Adenosine Triphosphatases↗

PubMedDate not supplied

Synergistic growth inhibition and induction of apoptosis by a novel mixed backbone antisense oligonucleotide targeting CRIPTO in combination with C225 anti-EGFR monoclonal antibody and 8-Cl-cAMP in human GEO colon cancer cells.

We have evaluated the antiproliferative effect of a novel mixed backbone antisense oligonucleotide generated against the 5'-coding region of the human CRIPTO mRNA in GEO human colon cancer cells. We have also evaluated the effects of this anti-CRIPTO antisense oligonucleotide in combination with a chimeric anti-human epidermal growth factor receptor (EGFR) monoclonal antibody (MAb C225) and with 8-Cl-cAMP, a cAMP analog that specifically inhibits type I protein kinase A (PKAI), since a functional EGFR-driven autocrine pathway is operative and PKAI is overexpessed in GEO colon cancer cells. Treatment with a single agent at low doses determined a 15-35% growth inhibition. A synergistic antiproliferative effect was observed when combinations of two agents were used with a co-operativity quotient ranging between 1.5 and 2.2. Furthermore, the combined treatment with all three drugs caused an almost complete suppression of the ability of GEO cells to form colonies in soft agar. We next evaluated whether any combination of 8-Cl-cAMP, the anti-CRIPTO antisense oligonucleotide and MAb C225 could induce programmed cell death in GEO cells. Treatment with each agent alone at all doses tested did not cause DNA fragmentation. The treatment with any combination of two agents was not able to induce apoptosis. In contrast, treatment with all three compounds determined an approximately three-fold increase in DNA fragmentation. In conclusion, the combination of selective antineoplastic agents directed against different but related key signal tranduction pathways efficiently inhibits cell growth and causes apoptosis in human colorectal cancer cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2001

Insights into Wnt binding and signalling from the structures of two Frizzled cysteine-rich domains.

Members of the Frizzled family of seven-pass transmembrane proteins serve as receptors for Wnt signalling proteins. Wnt proteins have important roles in the differentiation and patterning of diverse tissues during animal development, and inappropriate activation of Wnt signalling pathways is a key feature of many cancers. An extracellular cysteine-rich domain (CRD) at the amino terminus of Frizzled proteins binds Wnt proteins, as do homologous domains in soluble proteins-termed secreted Frizzled-related proteins-that function as antagonists of Wnt signalling. Recently, an LDL-receptor-related protein has been shown to function as a co-receptor for Wnt proteins and to bind to a Frizzled CRD in a Wnt-dependent manner. To investigate the molecular nature of the Wnt signalling complex, we determined the crystal structures of the CRDs from mouse Frizzled 8 and secreted Frizzled-related protein 3. Here we show a previously unknown protein fold, and the design and interpretation of CRD mutations that identify a Wnt-binding site. CRDs exhibit a conserved dimer interface that may be a feature of Wnt signalling. This work provides a framework for studies of homologous CRDs in proteins including muscle-specific kinase and Smoothened, a component of the Hedgehog signalling pathway.

Alanine↗

PubMed1996

Cytokine regulation of experimental intestinal inflammation in genetically engineered and T-lymphocyte reconstituted rodents.

Phenotypically similar intestinal inflammation and systemic wasting develops following overexpression or targeted deletion of several key immunoregulatory molecules and transfer of lymphocyte subsets into immunodeficient recipients. Although many of these recently described models of intestinal inflammation have not been thoroughly investigated, several consistent features are present which provide important insights into the role of cytokines in the pathogenesis of intestinal inflammation: (i) entirely distinct genetic alterations of cytokine expression and T-lymphocyte activity can lead to phenotypically similar intestinal inflammation, suggesting that human inflammatory bowel disease could have marked genetic heterogeneity; (ii) dysregulation of any of a number to immunoregulatory molecules can result in intestinal inflammation, illustrating the complexity of the mucosal immune response; (iii) active immunosuppression is critical to maintaining mucosal homeostasis; (iv) interferon-gamma and CD4+ lymphocytes, probably of the TH1 phenotype, are required for progression of chronic intestinal inflammation; (v) monokines are consistently upregulated, but tumour necrosis factor blockade is only partially protective. These models represent powerful new tools to understand better the basic mechanisms of mucosal immunoregulation and to develop novel therapeutic approaches of enhancing endogenous immunosuppressive pathways and blocking key regulatory cytokines and cells.

Animals↗

PubMed2004

Distinct roles of Ca2+, calmodulin, and protein kinase C in H2O2-induced activation of ERK1/2, p38 MAPK, and protein kinase B signaling in vascular smooth muscle cells.

We have shown earlier that extracellular signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), two key mediators of growth-promoting and proliferative responses, are activated by hydrogen peroxide (H(2)O(2)) in A10 vascular smooth muscle cells (VSMC). In the present studies, using a series of pharmacological inhibitors, we explored the upstream mechanisms responsible for their activation in response to H(2)O(2). H(2)O(2) treatment of VSMC stimulated ERK1/2, p38 mitogen-activated protein kinase (MAPK), and PKB phosphorylation in a dose- and time-dependent fashion. BAPTA-AM and EGTA, chelators of intracellular and extracellular Ca(2+), respectively, inhibited H(2)O(2)-stimulated ERK1/2, p38 MAPK, and PKB phosphorylation. Fluphenazine, an antagonist of the Ca(2+)-binding protein calmodulin, also suppressed the enhanced phosphorylation of ERK1/2, p38 MAPK, and PKB. In contrast, the protein kinase C (PKC) inhibitors Gö 6983 and Rö 31-8220 attenuated H(2)O(2)-induced ERK1/2 phosphorylation, but had no effect on p38 MAPK and PKB phosphorylation. Taken together, these data demonstrate that the activation of Ca(2+)/calmodulin-dependent pathways represents a key component mediating the stimulatory action of H(2)O(2) on ERK1/2, p38 MAPK, and PKB phosphorylation. On the other hand, PKC appears to be an upstream modulator of the increased ERK1/2 phosphorylation, but not of p38 MAPK and PKB in response to H(2)O(2) in VSMC.

Animals↗

PubMed2002

Chaperoning signaling pathways: molecular chaperones as stress-sensing 'heat shock' proteins.

Heat shock proteins interact with multiple key components of signaling pathways that regulate growth and development. The molecular relationships between heat shock proteins, various signaling proteins and partner proteins appear to be critical for the normal function of signal transduction pathways. The relative levels of these proteins may be important, as too little or too much Hsp70 or Hsp90 can result in aberrant growth control, developmental malformations and cell death. Although the functions of heat shock proteins as molecular chaperones have been well characterized, their complementary role as a 'stress-induced' proteins to monitor changes and alter the biochemical environment of the cell remains elusive. Genetic and molecular interactions between heat shock proteins, their co-chaperones and components of signaling pathways suggest that crosstalk between these proteins can regulate proliferation and development by preventing or enhancing cell growth and cell death as the levels of heat shock proteins vary in response to environmental stress or disease.

Cell Differentiation↗

PubMed2004

A molecular beacon assay for measuring base excision repair activities.

The base excision repair (BER) pathway plays a key role in protecting the genome from endogenous DNA damage. Current methods to measure BER activities are indirect and cumbersome. Here, we introduce a direct method to assay DNA excision repair that is suitable for automation and industrial use, based on the fluorescence quenching mechanism of molecular beacons. We designed a single-stranded DNA oligonucleotide labelled with a 5'-fluorescein (F) and a 3'-Dabcyl (D) in which the fluorophore, F, is held in close proximity to the quencher, D, by the stem-loop structure design of the oligonucleotide. Following removal of the modified base or incision of the oligonucleotide, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Several modified beacons have been used to validate the assay on both cell-free extracts and purified proteins. We have further developed the method to analyze BER in cultured cells. As described, the molecular beacon-based assay can be applied to all DNA modifications processed by DNA excision/incision repair pathways. Possible applications of the assay are discussed, including high-throughput real-time DNA repair measurements both in vitro and in living cells.

Animals↗

PubMed2001

Molecular cloning and characterization of human WNT5B on chromosome 12p13.3 region.

WNT signaling pathway plays a key role in carcinogenesis and embryogenesis. We have cloned and characterized the human WNT5B. Overlapping WNT5B cDNAs, containing 1080-bp ORF, were isolated. WNT5B encoded a 359-amino-acid polypeptide with the N-terminal signal peptide, four N-linked glycosylation sites, and consensus amino-acid residues conserved among the WNT family. WNT5B showed 80.5% total-amino-acid identity with WNT5A. Comparison between nucleotide sequence of WNT5B cDNA and human genome draft sequences revealed that the WNT5B gene, consisting of 4 exons, was located on human chromosome 12p13.3 region. Northern blot analyses with W5B2 probe detected the 2.8- and 2.4-kb WNT5B mRNAs. WNT5B was moderately expressed in adult prostate and fetal brain, and weakly expressed in fetal lung, kidney, adult liver, ovary, and small intestine. Among human cancer cell lines, WNT5B was expressed in gastric cancer cell lines MKN7, MKN45, KATO-III, and a teratocarcinoma cell line NT2. WNT5B might be implicated in human carcinogenesis through activation of the WNT-beta-catenin-TCF signaling pathway, just like Wnt5a.

Amino Acid Sequence↗

PubMed2004

Persistent progenitors at the retinal margin of ptc+/- mice.

The hedgehog signaling pathway is a key regulator of neural development, affecting both proliferation and differentiation of neural progenitors. Sonic hedgehog (Shh) is a mitogenic factor for retinal progenitors in vitro. To determine whether this signaling system is important in vivo for regulating retinal progenitor proliferation, we analyzed mice with a single functional allele of the Shh receptor patched (ptc). We found that ptc+/- mice had increased numbers of neural progenitors at every stage of retinal development that we examined. In addition, these mice had persistent progenitors at the retinal margin for up to 3 months of age, reminiscent of the ciliary marginal zone of lower vertebrates. To test whether the progenitors at the retinal margin of ptc+/- mice could be induced to regenerate retinal neurons in response to damage, we bred ptc+/- mice onto a retinal degeneration background (pro23his rhodopsin transgenic) and labeled newly generated cells with combined immunohistochemistry for bromodeoxyuridine and retinal neuron and photoreceptor-specific markers. We found newly generated neurons and photoreceptors at the retinal margin in ptc+/-;pro23his mice. We propose that the Shh pathway may act as a regulator of both prenatal and postnatal retinal growth.

Animals↗

PubMed1991

Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance.

Based on our previous finding that desensitization of the insulin-responsive glucose transport system (GTS) requires three components, glucose, insulin, and glutamine, we postulated that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance in primary cultured adipocytes. Two approaches were used to test this hypothesis. First, we assessed whether glucose-induced desensitization of the GTS could be prevented by glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-phosphate amidotransferase (GFAT) the first and the rate-limiting enzyme in hexosamine biosynthesis. Both O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxonorleucine inhibited desensitization in 18-h treated cells without affecting maximal insulin responsiveness in control cells. Moreover, close agreement was seen between the ability of azaserine to prevent desensitization of the GTS in intact adipocytes (70% inhibition, ED50 = 1.1 microM), its ability to inactivate GFAT in intact adipocytes (64% inhibition, ED50 = 1.0 microM) and its ability to inactivate GFAT activity in a cytosolic adipocyte preparation (ED50 = 1.3 microM). From these results we concluded that a glutamine amidotransferase is involved in the induction of insulin resistance. As a second approach, we determined whether glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, could induce cellular insulin resistance. When adipocytes were exposed to various concentrations of glucosamine for 5 h, progressive desensitization of the GTS was observed (ED50 = 0.36 mM) that culminated in a 40-50% loss of insulin responsiveness. Moreover, we estimated that glucosamine is at least 40 times more potent than glucose in mediating desensitization, since glucosamine entered adipocytes at only one-quarter of the glucose uptake rate, yet induced desensitization at an extra-cellular dose 10 times lower than glucose. In addition, we found that glucosamine-induced desensitization did not require glutamine and was unaffected by azaserine treatment. Thus, we conclude that glucosamine enters the hexosamine-desensitization pathway at a point distal to GFAT amidation. Overall, these studies indicate that a unique metabolic pathway exists in adipocytes that mediates desensitization of the insulin-responsive GTS, and reveal that an early step in this pathway involves the conversion of fructose 6-phosphate to glucosamine 6-phosphate by the first and rate-limiting enzyme of the hexosamine pathway, glutamine:fructose-6-phosphate amidotransferase.

Animals↗

PubMed1999

A novel Ggamma isolated from Drosophila constitutes a visual G protein gamma subunit of the fly compound eye.

Visual transduction in the compound eye of flies is a well established model system for the study of G protein-coupled transduction pathways. To characterize key components of the phototransduction cascade we performed substractive hybridization screening. We cloned the cDNA coding for the visual Ggamma (Ggamma(e)) subunit from Drosophila which had so far eluded identification at the molecular level. Northern blot analysis revealed the presence of a major, 1.4-kilobase(kb) Ggamma(e) transcript and two minor transcripts of 1.8 and 6 kb in size. The major 1.4-kb mRNA is expressed preferentially in the eye. The spatial expression pattern determined for Ggamma(e) as well as co-immunoprecipitation experiments demonstrated that Ggamma(e) dimerizes with Gbeta(e) to form the heterodimeric Gbetagamma subunit which functions in visual transduction in the Drosophila compound eye. Ggamma(e) shares common characteristics with the visual Ggamma subunits of human rod and cone photoreceptors although different classes of Galpha subunits are employed in vertebrate and invertebrate phototransduction. By the molecular cloning and characterization of the visual gamma subunit of Drosophila one of the few missing links in the well studied Drosophila phototransduction cascade has been characterized to complete our knowledge about the Drosophila visual transduction pathway.

Amino Acid Sequence↗

PubMed2026

Stage-specific remodeling of wingless-related integration sites (WNT) signaling during oocyte-to-embryo transition in pigs.

The WNT signaling pathway is a central regulator of cell polarity, adhesion, cytoskeletal dynamics, and lineage specification during early embryonic development. Although its roles have been extensively studied in murine and human models, the temporal regulation and pathway architecture of WNT signaling during early porcine development remain poorly defined. Here, we performed a comprehensive transcriptomic analysis to characterize WNT pathway dynamics across key stages of pig in vitro development, including immature oocytes (IMO), mature oocytes (MO), zygotes (ZY), cleaved embryos (2-4 cells; CL), and blastocysts (BL). Global analyses revealed major transcriptomic transitions (FDR <0.05; |Fold Change| ≥2) during oocyte maturation and blastocyst formation, whereas zygotes and cleaved embryos exhibited highly similar expression profiles. Module-based and gene-level analyses showed that oocyte maturation is associated with increased expression of extracellular WNT antagonists and components of the β-catenin destruction complex, together with selective regulation of Frizzled receptors, consistent with tight control of canonical WNT signaling at the MII stage. Following fertilization, this inhibitory configuration was partially relieved, alongside transient upregulation of specific WNT ligands, transcriptional mediators, and adhesion-related components during zygotic genome activation and early cleavage. At the blastocyst stage, WNT signaling became increasingly associated with planar cell polarity and epithelial organization modules. Together, the data reveal a highly dynamic and stage-specific restructuring of WNT signaling during early porcine development. Our findings indicate that precise temporal modulation-rather than uniform activation-of WNT pathway components accompanies the porcine oocyte-to-embryo transition, providing a molecular framework to better understand early developmental regulation and offering insights relevant to reproductive biotechnology and developmental biology.

Wnt Signaling Pathway↗

PubMed2003

Mechanisms of pyruvate inhibition of oxidant-induced apoptosis in human endothelial cells.

We have recently demonstrated that the redox reactant pyruvate prevents hydrogen peroxide (H2O2)-induced endothelial apoptosis and that its anti-apoptotic feature is mediated partially through the mitochondrial compartment. However, little is known about molecular signal pathways that mediate the anti-apoptotic feature of pyruvate. A biochemical approach to elucidate such signal pathways was attempted in human umbilical vein endothelial cells (HUVECs). Effects of antioxidant pyruvate were compared with those of cytosolic reductant L-lactate, redox-neutral acetate, and malate-aspartate shuttle blocker aminooxyacetate. Various indices of endothelial apoptosis were correlated with cell viability. Submillimolar H2O2 caused >50% cell killing, as manifested by its oxidant insult. The massive cell death induced by H2O2 was inhibited by pyruvate but not by L-lactate or aminooxyacetate, suggesting a role of cytosolic NADH reducing equivalents, possibly via stimulated oxidant generation. The induction and nuclear translocation of p53 by H2O2 was blocked by pyruvate and appeared to be somewhat enhanced by L-lactate or aminooxyacetate in association with oxidant generation. Nuclear translocation of p53 accompanied the transactivation of bax and downregulation of bcl-2. The pyruvate-related redox manipulation inhibited the H2O2-induced p53 activation, restored the downregulated bcl-2 and the upregulated bax, and hence enhanced the bcl-2/bax expression ratio. In contrast, L-lactate, acetate, or aminooxyacetate had no such effect. These results indicate that pyruvate could modulate key regulatory signal pathways in cytosol and mitochondrial matrix, thereby inactivating endothelial death pathways. Furthermore, it is suggested that stabilizing the expression of bcl-2 and bax genes by metabolic antioxidants may be an effective strategy for endothelial protection against oxidative stress.

Acetates↗

PubMed2004

Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-kappaB transcription factor pathway.

Epstein-Barr virus (EBV)-associated malignancies display distinct patterns of virus latent gene expression that reflect the complex interplay between the virus and its host cell. In the EBV-associated epithelial tumor nasopharyngeal carcinoma (NPC), the virus-encoded latent membrane protein LMP2A is consistently expressed whereas the oncogenic LMP1 protein appears to be restricted to only a proportion of tumors. In an attempt to understand the contribution of LMP2A to the pathogenesis of NPC, we established carcinoma cell lines stably infected in vitro with either a wild-type recombinant EBV (rEBV) or a mutant rEBV in which LMP2A is deleted (rEBV-2A). An NPC-like pattern of EBV gene expression including LMP2A but not LMP1 was consistently observed in carcinoma cells infected with rEBV. However, carcinoma cells infected with rEBV-2A expressed high levels of LMP1 from the signal transducer and activator of transcription (STAT)-regulated L1-TR promoter. Consistent with this effect, basal STAT activity was reduced in rEBV-infected carcinoma cells, and this repression was relieved in the absence of LMP2A. This modulation of STAT activity correlated with the ability of LMP2A to inhibit the autocrine secretion of IL-6 from carcinoma cell lines. Exogenous IL-6 was able to induce expression of LMP1 by means of STAT3 activation both in rEBV-infected carcinoma cell lines and in the EBV-positive C666-1 NPC cell line. The LMP2A-mediated suppression of IL-6 was a consequence of NF-kappaB inhibition. These data reveal that LMP2A modulates two key transcription factor pathways in carcinoma cells and suggest that this finding may be important in the pathogenesis of EBV-associated tumors.

Base Sequence↗

PubMed2006

Acetyl-CoA carboxylase alpha is essential to breast cancer cell survival.

Activation of de novo fatty acid synthesis is a characteristic feature of cancer cells. We have recently described an interaction between acetyl-CoA carboxylase alpha (ACCalpha), a key enzyme in fatty acid synthesis, and BRCA1, which indicates a possible connection between lipid synthesis and genetic factors involved in susceptibility to breast and ovarian cancers. For this reason, we explored the role of ACCalpha in breast cancer cell survival using an RNA interference (RNAi) approach. We show that specific silencing of either the ACCalpha or the fatty acid synthase (FAS) genes in cancer cells results in a major decrease in palmitic acid synthesis. Depletion of the cellular pool of palmitic acid is associated with induction of apoptosis concomitant with the formation of reactive oxygen species (ROS) and mitochondrial impairment. Expression of a small interfering RNA (siRNA)-resistant form of ACCalpha mRNA prevented the effect of ACCalpha-RNAi but failed to prevent the effect of FAS gene silencing. Furthermore, supplementation of the culture medium with palmitate or with the antioxidant vitamin E resulted in the complete rescue of cells from both ACCalpha and FAS siRNA-induced apoptosis. Finally, human mammary epithelial cells are resistant to RNAi against either ACCalpha or FAS. These data confirm the importance of lipogenesis in cancer cell survival and indicate that this pathway represents a key target for antineoplastic therapy that, however, might require specific dietary recommendation for full efficacy.

Acetyl-CoA Carboxylase↗

PubMed2006

The mouse Ovol2 gene is required for cranial neural tube development.

The Ovo gene family encodes a group of evolutionarily conserved transcription factors and includes members that reside downstream of key developmental signaling pathways such as Wg/Wnt and BMP/TGF-beta. In the current study, we explore the function of Ovol2, one of three Ovo paralogues in mice. We report that Ovol2 is expressed during early-mid embryogenesis, particularly in the inner cell mass at E3.5, in epiblast at E6.5, and at later stages in ectodermally derived tissues such as the rostral surface (epidermal) ectoderm. Embryos in which Ovol2 is ablated exhibit lethality by E10.5, prior to which they display severe defects including an open cranial neural tube. The neural defects are associated with improper Shh expression in the underlying rostral axial mesoderm and localized changes of neural marker expression along the dorsoventral axis, as well as with expanded cranial neural tissue and reduced cranial surface ectoderm culminating in a lateral shift of the neuroectoderm/surface ectoderm border. We propose that these defects reflect the involvement of Ovol2 in independent processes such as regionalized gene expression and neural/non-neural ectodermal patterning. Additionally, we present evidence that Ovol2 is required for efficient migration and survival of neural crest cells that arise at the neuroectoderm/surface ectoderm border, but not for their initial formation. Collectively, our studies indicate that Ovol2 is a key regulator of neural development and reveal a previously unexplored role for Ovo genes in mammalian embryogenesis.

Animals↗

PubMed2003

Inhibition of JNK signaling diminishes early but not late cellular stress-induced apoptosis.

The human leukemic T-cell line Jurkat was used to define the role of the cellular stress pathway with its key player kinase JNK in cancer therapy-induced apoptosis. JNK activity was inhibited by stable transfection with a dominant negative mutant of the upstream kinase JNKK/MKK4 or with the novel, potent and selective JNK1, -2 and -3 inhibitor SP600125. Inhibition of JNK activity delayed the onset of apoptosis induced by cisplatin, doxorubicin, gamma-irradiation and CD95-L but did not prevent apoptosis per se. Early events during apoptosis such as induction of CD95-L, activation of caspase-8 and exposure of phosphatidylserine on the cell surface were strongly inhibited. Also, at early time points of apoptosis, loss of the mitochondrial membrane potential and release of cytochrome c were markedly impaired. However, late signaling events during apoptosis such as cleavage of PARP and DNA fragmentation apoptosis were only marginally affected. These findings are in accordance with the activity of initiator and effector caspases. Whereas activity of the initiator caspase-8 was strongly inhibited early and late after induction, an inhibition of caspase-3 activity was only observed early after induction of apoptosis. We therefore suggest that cellular stress signaling contributes to the initiation of apoptosis, whereas it might be dispensable for the progression of apoptosis. Dysfunction of this pathway under pathological conditions might contribute to therapy resistance of cancer cells.

Anthracenes↗

PubMed2005

Phospholipase A2 pathway association with macrophage-mediated polycarbonate-urethane biodegradation.

Activation of the phospholipase A2 (PLA2) pathway is a key cell signaling event in the inflammatory response. The PLA2 family consists of a group of enzymes that hydrolyze membrane phospholipids, resulting in the liberation of arachidonic acid (AA), a precursor to pro-inflammatory molecules. Given the well-documented activating role of biomaterials in the inflammatory response to medical implants, the present study investigated the link between PLA2 and polycarbonate-based polyurethane (PCNU) biodegradation, and the effect that material surface had on PLA2 activation in the U937 cell line. PCNUs were synthesized with poly(1,6-hexyl 1,2-ethyl carbonate)diol, 1,4-butanediol and one of two diisocyanates (hexane 1,6-diisocyanate or 4,4'-methylene bisphenyl diisocyanate) in varying stoichiometries and incubated with adherent U937 cells. PLA2 inhibiting agents resulted in significantly decreased PCNU biodegradation (p < 0.05). Moreover, when activation of PLA2 was assessed (3H-AA release), significantly more 3H-AA was released from PCNU-adherent U937 cells than polystyrene-adherent U937 cells (p < 0.05) which was significantly decreased in the presence of PLA2 inhibitors. The pattern of inhibition of U937 cell-mediated biodegradation and 3H-AA release that was modulated by PCNU surface differences, suggests a role for secretory PLA2 along with cytosolic PLA2. Understanding PCNU activation of intracellular pathways, such as PLA2, will allow the design of materials optimized for their intended use.

Absorbable Implants↗