Autoantibodies that react with GRP78 expressed on the cell-surface of many tumor cell lines occur in the sera of patients with prostate cancer, melanoma, and ovarian cancer. These autoantibodies are a negative prognostic factor in prostate cancer, and when purified, stimulate tumor cell proliferation in vitro. It is unclear, however, whether these IgGs are merely a biomarker, or if they actually promote tumor growth in vivo. We immunized C57Bl/6 mice with recombinant GRP78 and then implanted the B16F1 murine melanoma cell line as flank tumors. We employed the antisera from these mice for in vitro cell signaling and proliferation assays. The immunodominant epitope in human cancer patients was well represented in the antibody repertoire of these immunized mice. We observed significantly accelerated tumor growth, as well as shortened survival in GRP78-immunized mice as compared to controls. Furthermore, antisera from these mice, as well as purified anti-GRP78 IgG from similarly immunized mice, stimulate Akt phosphorylation and proliferation in B16F1 and human DM6 melanoma cells in culture. These studies demonstrate a causal link between a humoral response to GRP78 and the progression of cancer in a murine melanoma model. They support the hypothesis that such autoantibodies are involved in the progression of human cancers and are not simply a biomarker. Because GRP78 is present on the surface of many types of cancer cells, this hypothesis has broad clinical and therapeutic implications.
Autoantibodies to GRP78; B16 Melanoma; Cell-surface GRP78; syngeneic tumor model
Kinetic analyses of GroE-assisted folding provide a dynamic sequence of molecular events that underlie chaperonin function. We used stopped-flow analysis of various fluorescent GroEL mutants to obtain details regarding the sequence of events that transpire immediately after ATP binding to GroEL and GroEL with prebound unfolded proteins. Characterization of GroEL CP86, a circularly permuted GroEL with the polypeptide ends relocated to the vicinity of the ATP binding site, showed that GroES binding and protection of unfolded protein from solution is achieved surprisingly early in the functional cycle, and in spite of greatly reduced apical domain movement. Analysis of fluorescent GroEL SR-1 and GroEL D398A variants suggested that among other factors, the presence of two GroEL rings and a specific conformational rearrangement of Helix M in GroEL contribute significantly to the rapid release of unfolded protein from the GroEL apical domain.
Elevation of serum homocysteine (Hcy) levels is a risk factor for cardiovascular diseases. Previous studies suggested that Hcy interferes with copper (Cu) metabolism in vascular endothelial cells. The present study was undertaken to test the hypothesis that Hcy-induced disturbance of Cu homeostasis leads to endothelial cell injury. Exposure of human umbilical vein endothelial cells (HUVECs) to concentrations of Hcy at 0.01, 0.1 or 1 mM resulted in a concentration-dependent decrease in cell viability and an increase in necrotic cell death. Pretreatment of the cells with a final concentration of 5 µM Cu in cultures prevented the effects of Hcy. Hcy decreased intracellular Cu concentrations. HPLC-ICP-MS analysis revealed that Hcy caused alterations in the distribution of intracellular Cu; more Cu was redistributed to low molecular weight fractions. ESI-Q-TOF detected the formation of Cu-Hcy complexes. Hcy also decreased the protein levels of Cu chaperone COX17, which was accompanied by a decrease in the activity of cytochrome c oxidase (CCO) and a collapse of mitochondrial membrane potential. These effects of Hcy were all preventable by Cu pretreatment. The study thus demonstrated that Hcy disturbs Cu homeostasis and limits the availability of Cu to critical molecules such as COX17 and CCO, leading to mitochondrial dysfunction and endothelial cell injury.
To determine if a no-carbohydrate ketogenic diet (NCKD) and lactate transporter inhibition can exert a synergistic effect on delaying prostate tumour growth in a xenograft mouse model of human prostate cancer.
MATERIALS AND METHODS
120 nude athymic male mice (aged 6–8 weeks) were injected s.c. in the flank with 1.0 x 105 LAPC-4 prostate cancer cells.
Mice were randomized to one of four treatment groups: Western diet (WD, 35% fat, 16% protein, 49% carbohydrate) and vehicle (Veh) treatment; WD and mono-carboxylate transporter-1 (MCT1) inhibition via α-cyano-4-hydroxycinnamate (CHC) delivered through a mini osmotic pump; NCKD (84% fat, 16% protein, 0% carbohydrate) plus Veh ; or NCKD and MCT1 inhibition.
Mice were fed and weighed three times per week and feed was adjusted to maintain similar body weights.
Tumour size was measured twice weekly and the combined effect of treatment was tested via Kruskal – Wallis analysis of all four groups. Independent effects of treatment (NCKD vs. WD and CHC vs. Veh) on tumour volume were tested using linear regression analysis.
All mice were killed on Day 53 (conclusion of pump ejection), and serum and tumour sections were analysed for various markers. Again, combined and independent effects of treatment were tested using Kruskal – Wallis and linear regression analysis, respectively.
There were no significant differences in tumour volumes among the four groups (P=0.09).
When testing the independent effects of treatment, NCKD was significantly associated with lower tumour volumes at the end of the experiment (P=0.026), while CHC administration was not (P=0.981). However, CHC was associated with increased necrotic fraction (P<0.001).
Differences in tumour volumes were observed only in comparisons between mice fed a NCKD and mice fed a WD.
MCT1 inhibition did not have a significant effect on tumour volume, although it was associated with increased necrotic fraction.
prostate cancer; carbohydrate; ketogenic; lactate
cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-AktThr-308, and p-AktSer-473 in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1•GTP, p-ERK1/2, p-B-Raf, p110α catalytic subunit of PI 3-kinase, p-PDK, and p-p70s6k. Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of AktThr-308 protein kinase activity and p-AktThr-308 levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced AktSer-473 kinase activity and p-AktSer-473 levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.
Cyclic AMP generation in macrophages; 8-CPT-2-O-Me-cAMP and cyclic AMP-dependent regulation in macrophages; Akt protein kinase activation; Epac1 and Akt protein kinase activation
Numerous dietary factors elevate serum levels of insulin and insulin-like growth factor I (IGF-I), both potent prostate cancer mitogens. We tested whether varying dietary carbohydrate and fat, without energy restriction relative to comparison diets, would slow tumor growth and reduce serum insulin, IGF-I, and other molecular mediators of prostate cancer in a xenograft model.
Individually caged male severe combined immunodeficient mice (n = 130) were randomly assigned to one of three diets (described as percent total calories): very high-fat/no-carbohydrate ketogenic diet (NCKD: 83% fat, 0% carbohydrate, 17% protein), low-fat/high-carbohydrate diet (LFD: 12% fat, 71% carbohydrate, 17% protein), or high-fat/moderate-carbohydrate diet (MCD: 40% fat, 43% carbohydrate, 17% protein). Mice were fed to maintain similar average body weights among groups. Following a preliminary feeding period, mice were injected with 1 × 106 LNCaP cells (day 0) and sacrificed when tumors were ≥1,000 mm3.
Two days before tumor injection, median NCKD body weight was 2.4 g (10%) and 2.1 g (8%) greater than the LFD and MCD groups, respectively (P < 0.0001). Diet was significantly associated with overall survival (log-rank P = 0.004). Relative to MCD, survival was significantly prolonged for the LFD (hazard ratio, 0.49; 95% confidence interval, 0.29–0.79; P = 0.004) and NCKD groups (hazard ratio, 0.59; 95% confidence interval, 0.37–0.93; P = 0.02). Median serum insulin, IGF-I, IGF-I/IGF binding protein-1 ratio, and IGF-I/IGF binding protein-3 ratio were significantly reduced in NCKD relative to MCD mice. Phospho-AKT/total AKT ratio and pathways associated with antiapoptosis, inflammation, insulin resistance, and obesity were also significantly reduced in NCKD relative to MCD tumors.
These results support further preclinical exploration of carbohydrate restriction in prostate cancer and possibly warrant pilot or feasibility testing in humans.
Epac1 is a cAMP-stimulated guanine exchange factor that activates Rap1. The protein product of the T cell leukemia 1 (TCL1) proto-oncogene binds to Akt enhancing its kinase activity. TCL1 and Epac promote cellular proliferation because of their activating effects on Akt. Employing macrophages, we have studied the mechanisms whereby these proteins function in the regulation of Akt kinase activity. Cells were treated with 8-CPT-2-O-Me-cAMP, a cAMP analog which acts selectively and specifically via Epac1. Epac1 co-immunoprecipitated with TCL1 in plasma membrane and nuclear fractions of 8-CPT-2-O-Me-cAMP-stimulated macrophages. Interaction of TCL1 and Epac1 was also observed in a [125I]GST-Epac1 pulldown assay. A two-threefold increase in AktThr-308 and AktSer-473 protein kinase activities and their phosphoprotein levels was observed in TCL1 immunoprecipitates of plasma membranes and nuclei of the treated cells. Elevated AktThr-308 protein kinase activity and its phosphoprotein levels were significantly reduced in TCL1 immunoprecipitates of plasma membranes of 8-CPT-2-O-Me-cAMP treated cells where Epac1 gene expression was silenced. In contrast, AktSer-473 protein kinase activity and its phosphoprotein levels were reduced only in plasma membranes. Our studies suggest that a ternary complex of TCL1, Epac1, and Akt forms in activated macrophages both promoting Akt activation and regulating intracellular distribution of Akt.
Cyclic AMP generation in macrophages; 8-CPT-2-O-Me-cAMP and cyclic AMP-dependent regulation in macrophages; Akt protein kinase activation; Epac-1 and TCL1 interaction; protein kinases Akt in nuclei and plasma membranes
α2-macroglobulin (α2M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an ~40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in α2M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in α2M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of α2M complexes.
α2M; α2-macroglobulin; α2-macroglobulin and proteinase incorporation; α2-macroglobulin and non-proteolytic incorporation of lysozyme; α2-macroglobulin stabilization
Previous studies indicate that carbohydrate intake influences prostate cancer biology, as mice fed a no-carbohydrate ketogenic diet (NCKD) had significantly smaller xenograft tumors and longer survival than mice fed a Western diet. As it is nearly impossible for humans to consume and maintain NCKD, we determined whether diets containing 10% or 20% carbohydrate kcal showed similar tumor growth as NCKD. A total of 150 male severe combined immunodeficient mice were fed a Western diet ad libitum, injected with the human prostate cancer cell line LAPC-4, and then randomized 2 weeks later to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate diets. Ten mice not injected were fed an ad libitum low-fat diet (12% fat kcal) serving as the reference in a modified-paired feeding protocol. Mice were sacrificed when tumors reached 1,000 mm3. Despite consuming extra calories, all mice receiving low-carbohydrate diets were significantly lighter than those receiving a low-fat diet (P < 0.04). Among the low-carbohydrate arms, NCKD-fed mice were significantly lighter than the 10% or 20% carbohydrate groups (P < 0.05). Tumors were significantly larger in the 10% carbohydrate group on days 52 and 59 (P < 0.05), but at no other point during the study. Diet did not affect survival (P = 0.34). There were no differences in serum insulin-like growth factor-I or insulin-like growth factor binding protein-3 at sacrifice among the low-carbohydrate arms (P = 0.07 and P = 0.55, respectively). Insulin was significantly lower in the 20% carbohydrate arm (P = 0.03). LAPC-4 xenograft mice fed a low-carbohydrate diet (10–20% carbohydrate kcal) had similar survival as mice consuming NCKD (0% carbohydrate kcal).
α2-Macroglobulin (α2M) is a proteinase inhibitor that functions by a trapping mechanism which has been exploited such that the receptor-recognized, activated form (α2M*) can be employed to target antigens to antigen-presenting cells. Another potential use of α2M* is as a drug delivery system. In this study we demonstrate that guanosine triphosphate, labeled with Texas red (GTP-TR) formed complexes with α2M* following activation by proteolytic or non-proteolytic reactions. Optimal incorporation occurred with 20μM GTP-TR, pH 8.0 for 5h at 50°C. NaCl concentration (100mM or 200mM) had little effect on incorporation at this pH or temperature, but was significant at sub-optimum temperature and pH values. Maximum incorporation was 1.2 mol GTP-TR/mol α2M*. PAGE analysis showed that 70-90% of the GTP-TR is bound in a SDS/2-mercaptoethanol resistant manner. Guanosine, adenosine, and imidazole competed with GTP-TR to form complexes with α2M*.
α2-macroglobulin and drug delivery; α2-macroglobulin and non-proteolytic incorporation of nucleosides; α2-macroglobulin and incorporation of GTP-Texas red
Extracellular GRP94 (gp96) can initiate both innate and adaptive immune responses through interactions with antigen presenting cell surface receptors. Following the identification of CD91 as a receptor functioning in the cross-presentation of GRP94-associated peptides, scavenger receptors SR-A and SREC-I were demonstrated to function in GRP94 cell surface binding and endocytosis, lending controversy to the assignment of CD91 as the unique GRP94 endocytic receptor. To assess CD91 function in GRP94 surface binding and endocytosis, these parameters were examined in murine embyronic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via RNAi or eliminated by genetic disruption of the CD91 locus. Reduction or loss of CD91 expression abrogated the binding and uptake of the CD91 ligand receptor-associated protein (RAP); surface binding and uptake of an N-terminal domain of GRP94 (GRP94.NTD) was unaffected. GRP94.NTD surface binding was markedly suppressed following treatment of MEF cell lines with heparin, the sulfation inhibitor sodium chlorate, or heparinase II, demonstrating that heparin sulfate proteoglycans can function in GRP94.NTD surface binding. The role of CD91 in the cross-presentaton of GRP94-associated peptides was examined in the DC2.4 dendritic cell line. In DC2.4 cells, which express CD91, GRP94.NTD-peptide cross-presentation was insensitive to RAP or activated α2-macroglobulin and occurred primarily via a fluid phase uptake pathway. In summary, these data clarify conflicting data on CD91 function in GRP94 surface binding, endocytosis and peptide cross-presentation and identify HSPGs as novel GRP94 cell surface binding sites.
Scarring is a highly prevalent and multifactorial process, yet no studies to date have attempted to distinguish pathologic from non-pathologic scarring.
This article defines and proposes methods of classifying pathologic scarring as it pertains to clinical presentation.
We propose a new scar scale that incorporates pain and functional impairment.
The modified POSAS scar assessment scale is the first of its kind to factor in the functional deficits, pain and pruritus of scarring into measurements of associated morbidity. This has great potential in evaluating patient response to treatment and analyzing clinical outcomes.
Autistic children show elevated serum levels of autoantibodies to several proteins essential for the function of normal brains. The voltage-dependent anion channel, VDAC, and hexokinase-I, a VDAC protective ligand, were identified as targets of this autoimmunity in autistic children. These autoantibodies were purified using immunoaffinity chromatographic techniques. Both antibodies induce apoptosis of cultured human neuroblastoma cells. Because VDAC and hexokinase-I are essential for brain protection from ischemic damage, the presence of these autoantibodies suggests a possible causal role in the neurologic pathogenesis of autism.
autism; autoimmunity; voltage-dependent anion channel; hexokinase-I; apoptosis
C1q and members of the defense collagen family are pattern recognition molecules that bind to pathogens and apoptotic cells and trigger a rapid enhancement of phagocytic activity. Candidate phagocytic cell receptors responsible for the enhancement of phagocytosis by defense collagens have been proposed but not yet discerned. Engagement of phagocyte surface-associated calreticulin in complex with the large endocytic receptor, low-density lipoprotein receptor-related protein (LRP/CD91), by defense collagens has been suggested as one mechanism governing enhanced ingestion of C1q-coated apoptotic cells. To investigate this possibility, macrophages were derived from transgenic mice genetically-deficient in LRP resulting from tissue-specific loxP/Cre recombination. LRP-deficient macrophages were impaired in their ability to ingest beads coated with an LRP ligand when compared to LRP-expressing macrophages, confirming for the first time that LRP participates in phagocytosis. When LRP-deficient and -expressing macrophages were plated on C1q-coated slides, they demonstrated equivalently enhanced phagocytosis of sheep red blood cells suboptimally-opsonized with IgG or complement, compared to cells plated on control protein. In addition, LRP-deficient and -expressing macrophages ingested equivalent numbers of apoptotic Jurkat cells in the presence and absence of serum. Both LRP-deficient and -expressing macrophages ingested fewer apoptotic cells when incubated in the presence of C1q-deficient serum compared to normal mouse serum, and addition of purified C1q reconstituted uptake to control serum levels. These studies demonstrate a direct contribution of LRP to phagocytosis and indicate that LRP is not required for the C1q-triggered enhancement of phagocytosis, suggesting that other, still undefined, receptor(s) exist to mediate this important innate immune function.
Macrophage; phagocytosis; apoptosis; cell surface molecules; complement
Angiostatin binds to endothelial cell (EC)-surface F1-F0 ATP synthase, leading to inhibition of EC3 migration and proliferation during tumor angiogenesis. This has led to a search for angiostatin-mimetics specific for this enzyme. A naturally occurring protein that binds to the F1 subunit of ATP synthase and blocks ATP hydrolysis in mitochondria is Inhibitor of F1 (IF1). The present study explores the effect of IF1 on cell surface ATP synthase. IF1 protein bound to purified F1 ATP synthase and inhibited F1-dependent ATP hydrolysis consistent with its reported activity in studies of mitochondria. While exogenous IF1 did not inhibit ATP production on the surface of EC, it did conserve ATP on the cell surface, particularly at low extracellular pH. IF1 inhibited ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. In cell-based assays used to model angiogenesis in vitro, IF1 did not inhibit EC differentiation to form tubes and only slightly inhibited cell proliferation compared to angiostatin. From these data, we conclude that inhibition of ATP synthesis is necessary for an anti-angiogenic outcome in cell-based assays. We propose that IF1 is not an angiostatin-mimetic, but it can serve a protective role for EC in the tumor microenvironment. This protection may be overridden in a concentration-dependent manner by angiostatin. In support of this hypothesis, we demonstrate that angiostatin blocks IF1 binding to ATP synthase, and abolishes its ability to conserve ATP. These data suggest that there is a relationship between the binding sites of IF1 and angiostatin on ATP synthase and that IF1 could be employed to modulate angiogenesis.
angiostatin; angiogenesis; ATP synthesis; cell surface-associated ATP synthase; endothelial cells; Inhibitory Factor 1; F1-F0 ATP synthase
Dipeptidyl peptidase IV (DPP IV) (CD26) plays a critical role in the modulation and expression of autoimmune and inflammatory diseases. We recently reported that sera from patients with rheumatoid arthritis and systemic lupus erythematosus contained low levels of DPP IV and high titers of anti-DPP IV autoantibodies of the immunoglobulin A (IgA) and IgG classes and found a correlation between the low circulating levels of DPP IV and the high titers of anti-DPP IV autoantibodies of the IgA class. Since streptokinase (SK) is a potent immunogen and binds to DPP IV, we speculated that patients with autoimmune diseases showed higher DPP IV autoantibody levels than healthy controls as a consequence of an abnormal immune stimulation triggered by SK released during streptococcal infections. We assessed this hypothesis in a group of patients suffering from acute myocardial infarction, without a chronic autoimmune disease, who received SK as part of therapeutic thrombolysis. Concomitant with the appearance of anti-SK antibodies, these patients developed anti-DPP IV autoantibodies. These autoantibodies bind to DPP IV in the region which is also recognized by SK, suggesting that an SK-induced immune response is responsible for the appearance of DPP IV autoantibodies. Furthermore, we determined a correlation between high titers of DPP IV autoantibodies and an augmented clearance of the enzyme from the circulation. Serum levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) increased significantly after 30 days of SK administration, while the levels of soluble IL-2 receptor remained unchanged during the same period, suggesting a correlation between the lower levels of circulating DPP IV and higher levels of TNF-α and IL-6 in serum in these patients.
The catabolic pathways of streptokinase, plasmin, and activator complex prepared with human plasminogen were studied in mice. 125I-streptokinase clearance occurred in the liver and was 50% complete in 15 min. Incubation with mouse plasma had no effect on the streptokinase clearance rate. Complexes of plasmin and α2-plasmin inhibitor were eliminated from the plasma by a specific and saturable pathway. Competition experiments demonstrated that this pathway is responsible for the clearance of injected plasmin. Streptokinase-plasminogen activator complex formed with either 125I-plasminogen or 125I-streptokinase cleared in the liver at a significantly faster rate than either of the uncomplexed proteins (50% clearance in <3 min). Streptokinase incubated with human plasma also demonstrated this accelerated clearance. p-Nitrophenyl-p′-guanidinobenzoate-HCl or pancreatic trypsin inhibitor-treated complex cleared slowly compared with untreated complex independent of which protein was radiolabeled. Significant competition for clearance was demonstrated between α2-macroglobulin-trypsin and activator complex only when the plasmin(ogen) was the radiolabeled moiety. Large molar excesses of α2-plasmin inhibitor-plasmin failed to retard the clearance of activator complex. Hepatic binding of streptokinase-plasmin, in liver perfusion experiments, was dependent upon prior incubation with plasma (8-10% uptake compared to a background of ∼ 2.5%). Substitution of human α2-macroglobulin for plasma also resulted in binding when the incubation was performed for 10 min at 37°C (7.5%). Electrophoresis experiments confirmed the transfer of 0.8 mol plasmin/mol α2-macroglobulin when activator complex was incubated at 37°C with α2-macroglobulin for 40 min. Streptokinase transfer from activator complex to α2-macroglobulin was negligible. The in vivo clearance of activator complex is proposed to involve active attack of the complex on the α2-macroglobulin “bait region,” resulting in facilitated plasmin transfer. Dissociated streptokinase is rapidly bound and cleared by sites in the liver.
Human factor VIII from normals and hemophiliacs was partially purified by ethanol and polyethylene glycol precipitations. Final purification was achieved by gel filtration on 2 or 4% agarose or ion exchange chromatography on diethylaminoethyl cellulose. Comparable amounts of highly purified protein were obtained from normal and hemophilic plasma following the agarose chromatography step. Highly purified factor VIII was not dissociated by 6 M guanidine hydrochloride or 1% sodium dodecyl sulfate. However, when reduced by β-mercaptoethanol and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, a single subunit species with an estimated 195,000 molecular weight was found for both normal and hemophilic factor VIII. By sedimentation equilibrium analysis, the normal factor VIII subunit was homogeneous and had an estimated molecular weight of 202,000. The subunit polypeptides from normal or hemophilic factor VIII contained carbohydrate. Each was homogeneous by isoelectric focusing. Immunodiffusion of purified normal and hemophilic factor VIII against rabbit antiserum to purified normal human factor VIII showed a single line of precipitation. Very low concentrations of purified human thrombin initially increased the activity of normal factor VIII about threefold and then progressively destroyed activity by 3 h. Only minimal activation occurred with hemophilic factor VIII. Both the activation and inactivation of normal and hemophilic factor VIII were unaccompanied by detectable changes in subunit molecular weight. These findings may have implications for the definition of the molecular defect in hemophilic factor VIII.
Fibrin formed in response to ancrod, reptilase, or thrombin was reduced by β-mercaptoethanol and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that ancrod progressively and totally digested the α-chains of fibrin monomers at sites different than plasmin; however, further digestion of fibrin monomers by either reptilase or thrombin was not observed. Highly purified ancrod did not activate fibrin-stabilizing factor (FSF); however, the reptilase preparation used in these experiments, like thrombin, activated FSF and thereby promoted cross-link formation. Fibrin, formed by clotting purified human fibrinogen with ancrod, reptilase, or thrombin for increasing periods of time in the presence of plasminogen, was incubated with urokinase and observed for complete lysis. Fibrin formed by ancrod was strikingly more vulnerable to plasmin digestion than was fibrin formed by reptilase or thrombin. The lysis times for fibrin formed for 2 hr by ancrod, reptilase, or thrombin were 18, 89, and 120 min, respectively. Evidence was also obtained that neither ancrod nor reptilase activated human plasminogen. These results indicate that fibrin formed by ancrod is not cross-linked and has significantly degraded α-chains: as expected, ancrod-formed fibrin is markedly susceptible to digestion by plasmin.
The formation of human fibrin from fibrinogen has been examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, a method which separates a mixture of proteins on the basis of differences in molecular weight. It has been found that the plasma from a patient with a congenital deficiency of fibrin-stabilizing factor forms clots lacking the cross links among the α- and γchains found in normal, cross-linked human fibrin. The addition of purified fibrin-stabilizing factor or normal plasma to the deficient plasma results in extensive cross-linking of the chains. Thus, the fibrinogen in the fibrin-stabilizing factor deficient plasma appears to be normal and forms fibrin which contains dimeric, cross-linked γ-chains and polymeric, high molecular weight forms of α-chains. By the use of these electrophoretic methods, it has also been possible to develop a highly sensitive method for measuring the content of fibrin-stabilizing factor in plasma. This method depends upon the use of urea-treated fibrinogen, which is completely devoid of fibrin-stabilizing factor, but which forms the usual cross-linked subunits after conversion to fibrin by thrombin in the presence of fibrin-stabilizing factor.
ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca2+ retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 μmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 μmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F0F1ATPase, significantly increased the effect of 50 μmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 μmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca2+-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.
Lotus nelumbo (LN) (Nelumbo nucifera Gaertn.) is an aquatic crop that is widely distributed throughout Asia and India, and various parts of this plant are edible and medicinal. It is noteworthy that different organs of this plant are used in traditional herbal medicine or folk recipes to cure different diseases and to relieve their corresponding symptoms. The compounds that are contained in each organ, which are named based on their chemical compositions, have led to their respective usages. In this work, a strategy was used to identify the difference ingredients and screen for Nuclear-factor-kappaB (NF-κB) inhibitors with anti-inflammatory ability in LN. Seventeen main difference ingredients were compared and identified from 64 samples of 4 different organs by ultra-performance liquid chromatography that was coupled with quadrupole/time of flight mass spectrometry (UPLC/Q-TOF-MS) with principal component analysis (PCA). A luciferase reporter assay system combined with the UPLC/Q-TOF-MS information was applied to screen biologically active substances. Ten NF-κB inhibitors from Lotus plumule (LP) extracts, most of which were isoquinoline alkaloids or flavone C-glycosides, were screened. Heat map results showed that eight of these compounds were abundant in the LP. In conclusion, the LP extracts were considered to have the best anti-inflammatory ability of the four LN organs, and the chemical material basis (CMB) of this biological activity was successfully validated by multivariate statistical analysis and biological research methods.
This study describes the investigation of the efficiency of conjugated linoleic acid (CLA) isomers in reducing cancer cells viability exploring the role of the oxidative stress and acylpeptide hydrolase (APEH)/proteasome mediated pathways on pro-apoptotic activity of the isomer trans10,cis12 (t10,c12)-CLA. The basal activity/expression levels of APEH and proteasome (β-5 subunit) were preliminarily measured in eight cancer cell lines and the functional relationship between these enzymes was clearly demonstrated through their strong positive correlation. t10,c12-CLA efficiently inhibited the activity of APEH and proteasome isoforms in cell-free assays and the negative correlation between cell viability and caspase 3 activity confirmed the pro-apoptotic role of this isomer. Finally, modulatory effects of t10,c12-CLA on cellular redox status (intracellular glutathione, mRNA levels of antioxidant/detoxifying enzymes activated through NF-E2-related factor 2, Nrf2, pathway) and on APEH/β-5 activity/expression levels, were investigated in A375 melanoma cells. Dose- and time-dependent variations of the considered parameters were established and the resulting pro-apoptotic effects were shown to be associated with an alteration of the redox status and a down-regulation of APEH/proteasome pathway. Therefore, our results support the idea that these events are involved in ROS-dependent apoptosis of t10,c12-CLA-treated A375 cells. The combined inhibition, triggered by t10,c12-CLA, via the modulation of APEH/proteasome and Nrf2 pathway for treating melanoma, is suggested as a subject for further in vivo studies.
Traditionally, GRP78 has been regarded as an endoplasmic reticulum (ER) lumenal protein due to its carboxyl KDEL retention motif. Recently, a subfraction of GRP78 is found to localize to the surface of specific cell types, serving as co-receptors and regulating signaling. However, the physiological relevance of cell surface GRP78 (sGRP78) expression in cancer and its functional interactions at the cell surface are just emerging. In this report, we combined biochemical, imaging and mutational approaches to address these issues. For detection of sGRP78, we utilized a mouse monoclonal antibody highly potent and specific for GRP78 or epitope-tagged GRP78, coupled with imaging and biochemical techniques that allowed detection of sGRP78 but not intracellular GRP78. Our studies revealed that breast and prostate cancer cells resistant to hormonal therapy actively promote GRP78 to the cell surface, which can be further elevated by a variety of ER stress-inducing conditions. We showed that sGRP78 forms complex with PI3K, and overexpression of sGRP78 promotes PIP3 formation, indicative of PI3K activation. We further discovered that an insertion mutant of GRP78 at its N-terminus domain, while retaining stable expression and the ability to translocate to the cell surface as the wild-type protein, exhibited reduced complex formation with p85 and production of PIP3. Thus, our studies provide a mechanistic explanation for the regulation of the PI3K/AKT signaling by sGRP78. Our findings suggest that targeting sGRP78 may suppress therapeutic resistance in cancer cells and offer a novel strategy to suppress PI3K activity.
Though plant metabolic changes are known to occur during interactions with bacteria, these were rarely challenged for pharmacologically active compounds suitable for further drug development. Here, the occurrence of specific chemicals with antiproliferative activity against human cancer cell lines was evidenced in hyperplasia (leafy galls) induced when plants interact with particular phytopathogens, such as the Actinomycete Rhodococcus fascians.
We examined leafy galls fraction F3.1.1 on cell proliferation, cell division and cytoskeletal disorganization of human cancer cell lines using time-lapse videomicroscopy imaging, combined with flow cytometry and immunofluorescence analysis. We determined the F3.1.1-fraction composition by gas chromatography coupled to mass spectrometry.
The leafy galls induced on tobacco by R. fascians yielded fraction F3.1.1 which inhibited proliferation of glioblastoma U373 cells with an IC50 of 4.5 µg/mL, F.3.1.1 was shown to increase cell division duration, cause nuclear morphological deformations and cell enlargement, and, at higher concentrations, karyokinesis defects leading to polyploidization and apoptosis. F3.1.1 consisted of a mixture of isomers belonging to the cembrenoids. The cellular defects induced by F3.1.1 were caused by a peculiar cytoskeletal disorganization, with the occurrence of fragmented tubulin and strongly organized microtubule aggregates within the same cell. Colchicine, paclitaxel, and cembrene also affected U373 cell proliferation and karyokinesis, but the induced microtubule rearrangement was very different from that provoked by F3.1.1. Altogether our data indicate that the cembrenoid isomers in F3.1.1 have a unique mode of action and are able to simultaneously modulate microtubule polymerization and stability.