525 resultados para BIOCHEMISTRY


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The lack of fundamental knowledge on the biological processes associated with wound healing represents a significant challenge. Understanding the biochemical changes that occur within a chronic wound could provide insights into the wound environment and enable more effective wound management. We report on the stability of wound fluid samples under various conditions and describe a high-throughput approach to investigate the altered biochemical state within wound samples collected from various types of chronic, ulcerated wounds. Furthermore, we discuss the viability of this approach in the early stages of wound sample protein and metabolite profiling and subsequent biomarker discovery. This approach will facilitate the detection of factors that may correlate with wound severity and/or could be used to monitor the response to a particular treatment.

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BACKGROUND: Melanoma is the most lethal form of skin cancer, but recent advances in molecularly targeted agents against the Ras/Raf/MAPK pathway demonstrate promise as effective therapies. Despite these advances, resistance remains an issue, as illustrated recently by the clinical experience with vemurafenib. Such acquired resistance appears to be the result of parallel pathway activation, such as PI3K, to overcome single-agent inhibition. In this report, we describe the cytotoxicity and anti-tumour activity of the novel MEK inhibitor, E6201, in a broad panel of melanoma cell lines (n = 31) of known mutational profile in vitro and in vivo. We further test the effectiveness of combining E6201 with an inhibitor of PI3K (LY294002) in overcoming resistance in these cell lines. RESULTS: The majority of melanoma cell lines were either sensitive (IC50 < 500 nM, 24/31) or hypersensitive (IC50 < 100 nM, 18/31) to E6201. This sensitivity correlated with wildtype PTEN and mutant BRAF status, whereas mutant RAS and PI3K pathway activation were associated with resistance. Although MEK inhibitors predominantly exert a cytostatic effect, E6201 elicited a potent cytocidal effect on most of the sensitive lines studied, as evidenced by Annexin positivity and cell death ELISA. Conversely, E6201 did not induce cell death in the two resistant melanoma cell lines tested. E6201 inhibited xenograft tumour growth in all four melanoma cell lines studied to varying degrees, but a more pronounced anti-tumour effect was observed for cell lines that previously demonstrated a cytocidal response in vitro. In vitro combination studies of E6201 and LY294002 showed synergism in all six melanoma cell lines tested, as defined by a mean combination index < 1. CONCLUSIONS: Our data demonstrate that E6201 elicits a predominantly cytocidal effect in vitro and in vivo in melanoma cells of diverse mutational background. Resistance to E6201 was associated with disruption of PTEN and activation of downstream PI3K signalling. In keeping with these data we demonstrate that co-inhibition of MAPK and PI3K is effective in overcoming resistance inherent in melanoma.

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The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 800 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 619 is Kallikrein-related Peptidase 15 (Prostinogen).

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We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of single-stranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely affected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold differently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic effect, which might have broad implications in the evolution of plants and their viruses.

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Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

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Background Post traumatic stress disorder (PTSD) and depressive disorder are over represented in combat veterans. Veterans with both disorders have an increased risk of suicide. The nitric oxide synthase 1 adaptor protein (NOS1AP) gene, which modulates stress-evoked N-methyl-D-aspartate (NMDA) activity, was investigated in combat veterans. Methods A comprehensive genetic analysis of NOS1AP and its association with PTSD was investigated in Vietnam combat veterans with PTSD (n=121) and a group of healthy control individuals (n=237). PTSD patients were assessed for symptom severity and level of depression using the Mississippi Scale for Combat-Related PTSD and the Beck Depression Inventory-II (BDI). Results The G allele of NOS1AP SNP rs386231 was significantly associated with PTSD (p = 0.002). Analysis of variance revealed significant differences in BDI-II and Mississippi scores between genotypes for rs386231 with the GG genotype associated with increased severity of depression (p = 0.002 F = 6.839) and higher Mississippi Scale for Combat-Related PTSD scores (p = 0.033). Haplotype analysis revealed that the C/G haplotype (rs451275/rs386231) was significantly associated with PTSD (p = 0.001). Limitations The sample sizes in our study were not sufficient to detect SNP associations with very small effects. In addition the study was limited by its cross sectional design. Conclusions This is the first study reporting that a variant of the NOS1AP gene is associated with PTSD. Our data also suggest that a genetic variant in NOS1AP may increase the susceptibility to severe depression in patients with PTSD and increased risk for suicide.

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Rationale Although the advent of atypical, second-generation antipsychotics (SGAs) has resulted in reduced likelihood of akathisia, this adverse effect remains a problem. Extrapyramidal adverse effects are associated with increased drug occupancy of dopamine 2 receptors (DRD2). The A1 allele of the DRD2/ANKK1,rs1800497, is associated with decreased striatal DRD2 density. Objectives The aim of this study was to identify whether the A1(T) allele of the DRD2/ANKK1 was associated with akathisia (measured with the Barnes Akathisia Rating Scale) in a clinical sample of 234 patients treated with antipsychotics. Results Definite akathisia (a score≥ 2 for the global clinical assessment of akathisia) was significantly less common in subjects prescribed SGAs (16.8 %) than those prescribed FGAs (47.6%), p<0.0001. Overall, 24.1% of A1+ (A1A2/A1A1) patients treated with SGAs had akathisia compared to 10.8% of A1- (A2A2) patients. A1+ (A1A2/A1A1) patients administered SGAs also had higher global clinical assessment of akathisia scores than A1- subjects (p=0.01). SGAs maintained their advantage over FGAs regarding akathisia even in A1+ patients treated with SGAs. Conclusions These results strongly suggest that A1+ variants of the DRD2/ANKK1 Taq1A allele confer risk for akathisia in patients treated with SGAs and may explain inconsistencies across prior studies comparing FGAs and SGAs.

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The concept of the cellular glycoprotein vitronectin acts as a biological ‘glue’ and key controller of mammalian tissue repair and remodelling activity is emerging from nearly 50 years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin-knock-out mouse was found to be viable and to have largely normal phenotype. However, diligent observation revealed that the VN-KO animal exhibits delayed coagulation and poor wound healing. This is interpreted to indicate that vitronectin occupies a role in the earliest events of thrombogenesis and tissue repair. That role is as a foundation upon which the thrombus grows in an organised structure. In addition to closing the wound, the thrombus also serves to protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair mediators and provides a provisional scaffold for the repairing tissue. In the absence of vitronectin (e.g. VN-KO animal) this cascade is disrupted before it begins. Our data demonstrates that a wide variety of biologically active species associate with VN. While initial studies were focused on mitogens, other classes of bioactives (e.g. glycosaminoglycans, metalloproteinases) are now also known to specifically interact with VN. Many of these interactions are long-lived, often resulting in multi-protein complexes, while others are stable for prolonged periods. Multiprotein complexes provide several advantages: prolonging molecular interaction; sustaining local concentrations, facilitating co-stimulation of cell surface receptors and thereby enhancing cellular / biological responses. We contend that these, or equivalent, multi-protein complexes mediate vitronectin functionality in vivo. It is also likely that many of the species demonstrated to associate with vitronectin in vitro, also associate with vitronectin in vivo in similar multi-protein complexes. Thus the predominant biological function of vitronectin is that of a master controller of the extracellular environment; informing, and possibly instructing cells ‘where’ to behave, ‘when’ to behave, and ‘how’ to behave (i.e. appropriately for the current circumstance).

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Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

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We investigated the potential of an extract of Lycopodium obscurum L.; stigmastane-3-oxo-21-oic acid (SA), to enhance osteogensis of mouse osteoblastic MC3T3-E1 cells. SA at a concentration of 16 µM was found to have no significant effect upon the viability of the cells, thus concentrations of 8 µM and 16 µM of SA were used in all further experiments. Both concentrations of SA had an inhibitory affect upon alkaline phosphatase activity (ALP) after 8 days incubation, however, after 16 days activity was restored to control levels. However Alizarin red S staining showed increased levels of mineralization for both concentrations after 16 days culture. Real time PCR showed inhibition of genes Runx2 and Osterix genes responsible for the up-regulation of ALP. However early time point (8 days) up-regulation of bone matrix mineralization genes OPN and OCN, and late time point (16 days) up-regulation of both Jun-D and Fra-2 mRNA expression was significantly enhanced. These results suggest a potential me-chanism of SA in enhancing bone fracture healing is through the up-regulating bone matrix minera-lization.

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Ubiquitination involves the attachment of ubiquitin (Ub) to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Polyubiquitination through different lysines (seven) or the N-terminus of Ub can generate different protein-Ub structures. These include monoubiquitinated proteins, polyubiqutinated proteins with homotypic chains through a particular lysine on Ub or mixed polyubiquitin chains generated by polymerization through different Ub lysines. The ability of the ubiquitination pathway to generate different protein-Ub structures provides versatility of this pathway to target proteins to different fates. Protein ubiquitination is catalyzed by Ub-conjugating and Ub-ligase enzymes, with different combinations of these enzymes specifying the type of Ub modification on protein substrates. How Ub-conjugating and Ub-ligase enzymes generate this structural diversity is not clearly understood. In the current review, we discuss mechanisms utilized by the Ub-conjugating and Ub-ligase enzymes to generate structural diversity during protein ubiquitination, with a focus on recent mechanistic insights into protein monoubiquitination and polyubiquitination.

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Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

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The DNA damage response encompasses a complex series of signaling pathways that function to regulate and facilitate the repair of damaged DNA. Recent studies have shown that the repair of transcriptionally inactive chromatin, named heterochromatin, is dependent upon the phosphorylation of the co-repressor, Krüppel-associated box (KRAB) domain-associated protein (KAP-1), by the ataxia telangiectasia-mutated (ATM) kinase. Co-repressors, such as KAP-1, function to regulate the rigid structure of heterochromatin by recruiting histone-modifying enzymes, such HDAC1/2, SETDB1, and nucleosome-remodeling complexes such as CHD3. Here, we have characterized a phosphorylation site in the HP1-binding domain of KAP-1, Ser-473, which is phosphorylated by the cell cycle checkpoint kinase Chk2. Expression of a nonphosphorylatable S473A mutant conferred cellular sensitivity to DNA-damaging agents and led to defective repair of DNA double-strand breaks in heterochromatin. In addition, cells expressing S473A also displayed defective mobilization of the HP1-β chromodomain protein. The DNA repair defect observed in cells expressing S473A was alleviated by depletion of HP1-β, suggesting that phosphorylation of KAP-1 on Ser-473 promotes the mobilization of HP1-β from heterochromatin and subsequent DNA repair. These results suggest a novel mechanism of KAP-1-mediated chromatin restructuring via Chk2-regulated HP1-β exchange from heterochromatin, promoting DNA repair.

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Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis.

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The resection of DNA double-strand breaks (DSBs) to generate ssDNA tails is a pivotal event in the cellular response to these breaks. In the two-step model of resection, primarily elucidated in yeast, initial resection by Mre11-CtIP is followed by extensive resection by two distinct pathways involving Exo1 or BLM/WRN-Dna2. However, resection pathways and their exact contributions in humans in vivo are not as clearly worked out as in yeast. Here, we examined the contribution of Exo1 to DNA end resection in humans in vivo in response to ionizing radiation (IR) and its relationship with other resection pathways (Mre11-CtIP or BLM/WRN). We find that Exo1 plays a predominant role in resection in human cells along with an alternate pathway dependent on WRN. While Mre11 and CtIP stimulate resection in human cells, they are not absolutely required for this process and Exo1 can function in resection even in the absence of Mre11-CtIP. Interestingly, the recruitment of Exo1 to DNA breaks appears to be inhibited by the NHEJ protein Ku80, and the higher level of resection that occurs upon siRNA-mediated depletion of Ku80 is dependent on Exo1. In addition, Exo1 may be regulated by 53BP1 and Brca1, and the restoration of resection in BRCA1-deficient cells upon depletion of 53BP1 is dependent on Exo1. Finally, we find that Exo1-mediated resection facilitates a transition from ATM- to ATR-mediated cell cycle checkpoint signaling. Our results identify Exo1 as a key mediator of DNA end resection and DSB repair and damage signaling decisions in human cells.