Propidium Iodide Binding Research Articles

Diminished amyloid-β uptake by mouse microglia upon treatment with quantum dots, silver or cerium oxide nanoparticles: Nanoparticles and amyloid-β uptake by microglia.

Alzheimer's disease (AD) is a chronic neurodegenerative disease leading to progressive dementia in elderly people. The disease is characterized, among others, by formation of amyloid-β (Aβ) polypeptide plaques in the brain. Although etiology of the disease is not fully understood, recent research suggest that nanomaterials may affect AD development. Here, we described the consequences of exposure of mouse BV-2 microglia to silver nanoparticles (AgNPs, 50 µg/mL), cerium oxide nanoparticles (CeO2NPs, 100 µg/mL), and cadmium telluride quantum dots (CdTeQDs, 3 or 10 µg/mL) in the context of its ability to clear Aβ plaques. The brain microglial cells play an important role in removing Aβ plaques from the brain. Cell viability and cycle progression were assessed by trypan blue test and propidium iodide binding, respectively. The uptake of Aβ and NPs was measured by flow cytometry. Secretion of proinflammatory cytokines was measured with the use of cytometric bead array. Aβ (0.1 μM) did not affect viability, whereas NPs decreased microglia growth by arresting the cells in G1 phase (CdTeQDs) or in S phase (AgNPs and CeO2NPs) of cell cycle. The uptake of Aβ was significantly reduced in the presence of AgNPs and CeO2NPs. In addition, the least toxic CeO2NPs induced the release of proinflammatory cytokine, tumor necrosis factor α. In summary, each of the NPs tested affected either the microglia phagocytic activity (AgNPs and CeO2NPs) and/or its viability (AgNPs and CdTeQDs) that may favor the occurrence of AD and accelerate its development.

Significance of apoptosis and autophagy of leukemic blasts for the outcomes of acute myeloid leukemia patients

Cytostatic treatment induces apoptosis or other types of cell death like autophagy, necrosis, mitotic catastrophe, etc. Autophagy can play a role in the drug resistance of neoplastic cells, allowing the survival of blast cells under stressful conditions, such as the use of cytostatics. Studies on apoptosis and autophagy 12-24 h after the start of treatment have not been conducted until now. The study aimed to investigate the predictive and prognostic significance of autophagy and apoptosis in patients with acute myeloid leukemia (AML). The study included 38 patients. Blood was collected before and 12-24 h after the start of treatment, since at that time point, the appropriate blast cell count was still available. Autophagy was measured with the expression of the ATG5, MAP1L3, LC3-I, and LC3-II proteins. The percentage of mononuclear cells in early and late apoptosis was evaluated with flow cytometry, using the annexin V and propidium iodide (PI) binding assay. The percentage of apoptotic blast cells before treatment was not associated with the response. However, in the remission group, the overall percentage of apoptotic cells measured 12-24 h after the start of treatment was higher than in non-remission patients, which was statistically significant. In neither group we found any difference in the level of autophagy before and 12-24 h after the start of treatment. Nevertheless, we observed an increasing tendency of the MAP1LC3 protein expression (not statistically significant) in the remission group 12-24 h after the start of treatment. Patients with a higher percentage of blast cells in apoptosis and with a higher expression of MAP1LC3 protein measured 12-24 h after the start of the therapy had longer overall survival (OS). A higher percentage of apoptotic as well as autophagic blast cells measured 12-24 h after the start of the chemotherapy is an independent factor associated with better outcomes.

Proteomic identification and purification of seed proteins from native Amazonian species displaying antifungal activity.

Seeds of native species from the rain forest (Amazon) are source of chitinases and their protein extracts exhibited strong and broad antifungal activity. Numerous plant species native to the Amazon have not yet been chemically studied. Studies of seeds are scarcer, since adversities in accessing study areas and seasonality pose constant hurdles to systematic research. In this study, proteins were extracted from seeds belonging to endemic Amazon species and were investigated for the first time. Proteolytic activity, peptidase inhibitors, and chitinases were identified, but chitinolytic activity predominated. Four proteins were purified through chromatography and identified as lectin and chitinases by MS/MS analyses. The proteins were examined for inhibition of a phytopathogen (Fusarium oxysporum). Analyses by fluorescence microscopy suggested binding of propidium iodide to DNA of fungal spores, revealing that spore integrity was lost when accessed by the proteins. Further structural and functional analyses of defensive proteins belonging to species facing highly complex ecosystems such as Amazonia should be conducted, since these could provide new insights into specificity and synergism involving defense proteins of plants submitted to a very complex ecosystem.

The release of microparticles and mitochondria from RAW 264.7 murine macrophage cells undergoing necroptotic cell death in vitro

Microparticles (MPs) are small membrane-bound vesicles released from activated or dying cells. As shown previously, LPS stimulation of the RAW 264.7 macrophage cell line can induce MP release, with the caspase inhibitor Z-VAD increasing the extent of this process. Since combined treatment of cells with LPS and Z-VAD can induce necroptosis, we explored particle release during this form of cell death using flow cytometry to assess particle size, binding of annexin V and staining for DNA with propidium iodide (PI) and SYTO 13. The role of necroptosis was assessed by determining the effects of necrostatin, an inhibitor of RIP1, a kinase regulating this form of cell death. These studies demonstrated that, during necroptosis, RAW 264.7 cells release MPs that resemble those released from cells treated with staurosporine to induce apoptosis. The particles contained DNA as determined by binding of PI and SYTO 13, with PCR analysis demonstrating both chromosomal and mitochondrial DNA. The presence of mitochondria in the MP preparations was demonstrated by staining with MitoTracker Green. Flow cytometry indicated that purified mitochondria have properties of MPs. Together, these studies indicate that cells undergoing necroptosis can release MPs and that mitochondria can be components of MP preparations.

Evaluation of synergistic anticandidal and apoptotic effects of ferulic acid and caspofungin against Candida albicans

This study aimed to investigate the synergy between anticandidal and apoptotic effects of ferulic acid and caspofungin against Candida albicans and Candida glabrata, with the help of a quantitative checkerboard microdilution assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as a viability dye. Apoptotic effects of caspofungin and ferulic acid concentrations (alone and combined) were analyzed for C. albicans and C. glabrata based on annexin V–propidium iodide binding capacities using flow cytometric analysis. C. albicans showed a synergistic effect, represented by a fractional inhibitory concentration index of < 0.5, but C. glabrata showed no synergistic effect (fractional inhibitory concentration index > 0.5). Early and late apoptotic effects of caspofungin and ferulic acid concentrations (1 μg/mL and 1000 μg/mL) were calculated as 55.7% and 18.3%, respectively, while their necrotic effects were determined as 5.8% and 51.6%, respectively, using flow cytometric analyses. The apoptotic effects of the combination of caspofungin and ferulic acid at concentrations of 1 μg/mL and 1000 μg/mL on C. albicans and C. glabrata were 73.0% and 48.7%, respectively. Ferulic acid also demonstrated a synergistic effect in combination with caspofungin against C. albicans. Another possibility is to combine the existing anticandidal drug with phytochemicals to enhance the efficacy of anticandidal drug.

The mechanism of γ-aminobutyric acid inhibitory on the growth of cholangiocarcinoma cell line

Objective To explore the molecular mechanism of cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway in γ-aminobutyric acid inhibitory on the growth of cholangiocarcinoma QBC939 cell line. Methods QBC939 cells were cultured in different groups and treated withγ-aminobutyric acid (GABA), GABA+ 8 Br (cAMP agonists), GABA+ H89 (PKA antagonist) for 48 hours. (4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to determine the proliferation of QBC939 cells. Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) binding assay was used to detect apoptosis in the QBC939 cells. Enzymelinkedimmunosorbentassay (ELISA) assay was detected to the expression of cAMP and PKA. Western blotting was applied to check the expression of PKAI and PKAII and extracellular regulated protein kinases (ERK) proteins in different groups of QBC939 cells. Animal models of cholangiocarcinoma bearing nude mice were established by subcutaneous injection of QBC939 cells and randomized into 2 groups: control and GABA-treated groups. The effect of GABA was evaluated after 5 weeks, including tumor volume. The expression of PKA Ⅰ and PKA Ⅱ and ERK was detected by Western blotting in xenograft tumors. Results MTT and flow cytometry (FCM) assays all showed that the effect of GABA inhibitory on the proliferation and induced apoptosis of QBC939 cells could be antagonized by H89, but not 8 Br. GABA significantly increased the content of cAMP and PKA, the content of cAMP was enhanced by 8 Br, the content of PKA was antagonized by H89. Western blotting analysis showed that GABA significantly down-regulated the expression of PAK I protein (0.087 8±0.003 0 vs. 0.152 1±0.003 0, t=29.687, P<0.05), up-regulated the expression of PKAⅡ, and also decreased the expression of ERK protein protein (0.368 3±0.007 0 vs. 0.468 7±0.010 0, t=13.647, P<0.05). Xenograft tumor volume [ (0.500±0.020 vs. 0.320±0.030) cm3,t=15.354, P<0.05]. The expression of PKAI and ERK were significantly decreased, and PKA II was increased in GABA-treated group as compared with control group. Conclusion GABA may inhibit the growth of cholangiocarcinoma cells QBC939 through cAMP/PKA, down-regulated PAK I, up-regulated PKA II, and decreased the expression of the ERK perhaps is one of its anti-tumor mechanisms. Key words: Bile duct neoplasms; Gamma-aminobutyric acid; Cyclic adenosine monophosphate; Protein kinase A; Extracellular regulated protein kinases

RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines.

BackgroundCellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced “RNA disruption” is, the extent to which it is associated with drug response or what the underlying mechanisms are.MethodsOvarian (A2780, CaOV3) and breast (MDA-MB-231, MCF-7, BT474, SKBR3) cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor.ResultsAll chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3’-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption.ConclusionsSupporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2197-1) contains supplementary material, which is available to authorized users.

A new dammarane-type saponin from Gynostemma pentaphyllum induces apoptosis in A549 human lung carcinoma cells

Gynostemma pentaphyllum has been widely used as a traditional herb for its antioxidant and immunostimulatory activities. We have previously reported several useful dammarane-type saponins with cytotoxicity against A549 human lung cancer cells from heat-processed G. pentaphyllum. In this study, a new dammarane-type saponin, 20(S)-2α,3β,12β-tetrahydroxydammar-3-O-β-d-glucopyranoside (namely gypenoside Jh1), was isolated from the ethanol extract of heat-processed G. pentaphyllum using column chromatography and semi-preparative HPLC. Gypenoside Jh1 exhibited strong cytotoxicity against A549 cells in a concentration-dependent manner, which was associated with apoptotic cell death characterized by morphological changes, Hoechst 33258 nuclear staining, Annexin V and propidium iodide binding and mitochondrial potentials assay. Quantitative analysis using flow cytometry also showed that the proportion of apoptotic cells was increased after gypenoside Jh1 treatment. These findings indicated that gypenoside Jh1 showed antiproliferative effects on A549 cells and mitochondrial-dependent pathway is involved in gypenoside Jh1-induced apoptosis.

In situ studies of microbial inactivation during high pressure processing

ABSTRACTHigh pressure processing (HPP) has been shown to reduce microbial concentration in foods. The mechanisms of microbial inactivation by HPP have been associated with damage to cell membranes. The real-time response of bacteria to HPP was measured to elucidate the mechanisms of inactivation, which can aid in designing more effective processes. Different pressure cycling conditions were used to expose Enterobacter aerogenes cells to HPP. Propidium iodide (PI) was used as a probe, which fluoresces after penetrating cells with damaged membranes and binding with nucleic acids. A HPP vessel with sapphire windows was used for measuring fluorescence in situ. Membrane damage was detected during pressurization and hold time, but not during depressurization. The drop in fluorescence was larger than expected after pressure cycles at higher pressure and longer times. This indicated possible reversible disassociation of ribosomes resulting in additional binding of PI to exposed RNA under pressure and its release after depressurization.

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria.

Effects of cyclic tension stress on the apoptosis of osteoclasts in vitro.

The aim of the present study was to investigate the effect of cyclic tension stress on osteoclast apoptosis in vitro using murine RAW264.7 cells treated with receptor activator of nuclear factor-κB. Using the EF3200 mechanical testing instrument with BioDynamic bioreactor system, cultured osteoclasts which were seeded in a silicone rubber membrane load carrier, were loaded with periodic cyclic stretch microstrain. The induced osteoclasts were subjected to 0, 5, 10 and 15% stretch microstrain for 1 h daily for three days. The number of tartrate-resistant acid phosphatase-positive osteoclasts and the resorption area were assessed. Osteoclast apoptosis was detected by the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide binding assay. The mRNA expression of Bcl-2, Bax, caspase-3 and cytochrome c was detected following force loading using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Compared with the cells under no cyclic tension stress, the number of osteoclasts and the resorption area were increased in the cells under 10 and 15% stretch microstrain. The Annexin V binding assay showed that the early apoptosis rate of the 5, 10 and 15% stretch microstrain groups was decreased compared with that of the control group. RT-qPCR results showed that the Bcl-2/Bax ratio was significantly increased in the cells subjected to 5, 10 and 15% stretch microstrain compared with that in the control cells (P<0.05), while the expression of cytochrome c in the 10 and 15% stretch microstrain groups was decreased significantly (P<0.05). No significant difference was observed between the cytochrome c expression of the 5% stretch microstrain group and that of the control group (P>0.05). The expression of caspase-3 in the 5, 10 and 15% stretch microstrain groups was decreased significantly compared with that in the control group (P<0.05). These data suggest that cyclic tension stress can inhibit apoptosis in osteoclasts, possibly by increasing the Bcl-2/Bax ratio, inhibiting the activity of caspase-3 and downregulating the expression of cytochrome c.

Impact of anemia treatment with methoxy polyethylene glycol-epoetin beta on polymorphonuclear cells apoptosis in predialysis patients with chronic kidney disease.

Some data in literature indicate increased apoptosis of polymorphonuclear cells (PMNs) in chronic kidney disease (CKD), what seems to be connected with anemia. Erythropoiesis-stimulating agents, used in anemia treatment in CKD may affect cells apoptosis. Aim of this study was to investigate impact of anemia treatment with methoxy polyethylene glycol-epoetin beta (CERA) on PMNs apoptosis in predialysis patients with CKD. Percentage of early and late apoptotic PMNs was measured by flow cytometry based on annexin V and propidium iodide binding. CD90 (Fas), CD95L (FasL), CD16 and CD11b expression on PMNs were evaluated by flow cytometry after incubation with respective monoclonal antibody. Percentage of PMNs in early and late apoptosis in CKD patients before CERA treatment was significantly higher to control group, which was accompanied by significantly higher Fas and Fas-L expression and significantly lower expression of CD16. CERA treatment downregulated significantly percentage of early, apoptotic PMNs but percentage of late apoptotic cells did not change and was still significantly higher to control group. In all investigated groups we observed a significant negative correlation between hemoglobin concentration and percentage of apoptotic PMNs, as well as Fas and FasL expression and significant positive correlation between Hb and CD16 expression. Our results indicate that PMNs apoptosis is increased in predialysis patients with CKD and anemia treatment with CERA may diminish readiness of PMNs to undergo apoptosis. This antiapoptotic impact of anemia treatment with CERA seems to concern early apoptotic PMNs before they undergo to late, irreversible stage of apoptosis.

Sirtuin 6 protects the heart from hypoxic damage

Sirtuin 6 (SIRT6) is a protein associated with prolonged life expectancy. We investigated whether life extension is associated with cardioprotection against hypoxia. The proposed study is to develop approaches to reduce hypoxic damage through the use of the sirtuin pathway and to elucidate the mechanism involved. For that purpose we subjected cardiomyocytes from transgenic mice (TG) with over-expression of SIRT6, to hypoxic stress in cell cultures. We hypothesized that cardiomyocytes from transgenic mice subjected to prolonged hypoxia may release survival factors or fewer damage markers to protect them from hypoxic stress compared with wild type (WT) mice. Lactate dehydrogenase (LDH) and creatine kinase (CK) released to the medium and propidium iodide (PI) binding, were markedly decreased following hypoxia in TG cardiomyocytes. The protective mechanism of SIRT6 over-expression includes the activation of pAMPKα pathway, the increased protein level of B-cell lymphoma 2 (Bcl2), the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), the decrease of reactive oxygen species (ROS) and the reduction in the protein level of phospho-protein kinase B (pAkt) during hypoxia. Together, all these processes impede the necrosis/apoptosis pathways leading to the improved survival of cardiomyocytes following hypoxia, which might explain life extension.

Glutamate and GABA imbalance promotes neuronal apoptosis in hippocampus after stress

BackgroundPeople who experience traumatic events have an increased risk of post-traumatic stress disorder (PTSD). However, PTSD-related pathological changes in the hippocampus and prefrontal cortex remain poorly understood.Material/MethodsWe investigated the effect of a PTSD-like animal model induced by severe stress. The experimental rats received 20 inescapable electric foot shocks in an enclosed box for a total of 6 times in 3 days. The physiological state (body weight and plasma corticosterone concentrations), emotion, cognitive behavior, brain morphology, apoptosis, and balance of gamma-aminobutyric acid (GABA) and glutamate in the hippocampus and prefrontal cortex were observed. Cell damages were examined with histological staining (HE, Nissl, and silver impregnation), while apoptosis was analyzed with flow cytometry using an Annexin V and propidium iodide (PI) binding and terminal deoxynucleotidyl transferase mediated-dUTP nick end labeling (TUNEL) method.ResultsIn comparison with the sham litter-mates, the stressed rats showed decreased body weight, inhibition of hypothalamic-pituitary-adrenal (HPA) axis activation, increase in freezing response to trauma reminder, hypoactivity and anxiety-like behaviors in elevated plus maze and open field test, poor learning in Morris water maze, and shortened latency in hot-plate test. There were significant damages in the hippocampus but not in the prefrontal cortex. Imbalance between glutamate and GABA was more evident in the hippocampus than in the prefrontal cortex.ConclusionsThese results suggest that neuronal apoptosis in the hippocampus after severe traumatic stress is related to the imbalance between glutamate and GABA. Such modifications may resemble the profound changes observed in PTSD patients.

P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo

Extracellular nucleotides acting via P2 receptors play important roles in cardiovascular physiology/pathophysiology. Pyrimidine nucleotides activate four G protein-coupled P2Y receptors (P2YRs): P2Y2 and P2Y4 (UTP-activated), P2Y6, and P2Y14. Previously, we showed that uridine 5'-triphosphate (UTP) activating P2Y2R reduced infarct size and improved mouse heart function after myocardial infarct (MI). Here, we examined the cardioprotective role of P2Y2R in vitro and in vivo following MI using uridine-5'-tetraphosphate δ-phenyl ester tetrasodium salt (MRS2768), a selective and more stable P2Y2R agonist. Cultured rat cardiomyocytes pretreated with MRS2768 displayed protection from hypoxia [as revealed by lactate dehydrogenase (LDH) release and propidium iodide (PI) binding], which was reduced by P2Y2R antagonist, AR-C118925 (5-((5-(2,8-dimethyl-5H-dibenzo[a,d][7]annulen-5-yl)-2-oxo-4-thioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl)-N-(1H-tetrazol-5-yl)furan-2-carboxamide). In vivo, echocardiography and infarct size staining of triphenyltetrazolium chloride (TTC) in 3 groups of mice 24h post-MI: sham, MI, and MI+MRS2768 indicated protection. Fractional shortening (FS) was higher in MRS2768-treated mice than in MI alone (40.0 ± 3.1% vs. 33.4 ± 2.7%, p < 0.001). Troponin T and tumor necrosis factor-α (TNF-α) measurements demonstrated that MRS2768 pretreatment reduced myocardial damage (p < 0.05) and c-Jun phosphorylation increased. Thus, P2Y2R activation protects cardiomyocytes from hypoxia in vitro and reduces post-ischemic myocardial damage in vivo.

High occurrence ofin vitroapoptosis of lymphocytes induced by serum from systemic lupus erythematosus patients is associated with increased serum levels of anti-C1q autoantibodies

The ethiopathogenesis of increased apoptosis of lymphocytes in systemic lupus erythematosus (SLE) is still incompletely understood but anti-C1q autoantibodies have been shown to induce apoptosis in lymphocytes from healthy donors and certain cell lines. This study was undertaken to investigate the relationship between peripheral lymphocyte apoptosis and serum levels of anti-C1q autoantibodies in SLE patients. The sera of 124 patients with SLE involving 62 active SLE and 62 inactive SLE, fulfilling America College of Rheumatology (ACR) classification criteria for SLE (1997) were incubated with peripheral blood lymphocytes of healthy donors. The results were compared with 124 sex- and age-matched normal controls. Apoptotic lymphocytes (AL) were detected by flow cytometry using annexin V and propidium iodide binding. Anti-C1q autoantibodies were detected by an enzyme-linked immunoassay kit for all SLE patients. Results demonstrated that the percentage of AL in the peripheral blood of active SLE patients was significantly higher (n=62, 34.95±12.78%) than that of the inactive SLE patients (n=62, 30.69±10.13%, P=0.042, 95%CI=0.16-8.36) and normal controls (n=124, 27.92±10.22%, P=0.001, 95%CI=3.33-10.73). The percentage of AL significantly correlated with serum levels of anti-C1q autoantibodies in the active SLE patients (r=0.263, P=0.039) but not in the inactive SLE patients (r=0.170, P=0.185). The results of this study suggest that increased serum levels of anti-C1q autoantibodies are responsible for apoptosis and may play a pathogenic role in SLE patients, especially in active disease.

Antibody‐mediated glycophorin C coligation on K562 cells induces phosphatidylserine exposure and cell death in an atypical apoptotic process

Glycophorin C (GPC) is necessary in the maintenance of red blood cell structure. Severe autoimmune hemolytic anemia and hemolytic disease of the fetus and newborn (HDFN) have been associated with Gerbich (Ge) blood group system antigens expressed on GPC. Previous in vitro studies with cord blood progenitor cells have shown that anti-Ge suppresses erythropoiesis. Here, we evaluated the K562 erythroleukemic cell line to study the cellular effects of a murine anti-GPC. Cell proliferation was evaluated after treatment with anti-GPC. Flow cytometry was used to evaluate exofacial phosphatidylserine (PS) expression and cell viability (propidium iodide binding). Cell morphology was evaluated under light microscopy with cytospin preparations stained with May-Grünwald Giemsa. Anti-GPC dramatically inhibited K562 proliferation and increased PS expression, consistent with cytoplasmic blebbing, suggesting evidence of apoptosis. Z-VAD-FMK, an inhibitor of classical apoptosis, was unable to reverse the suppressive effect of anti-GPC. However, hemin was able to attenuate growth suppression. Together, the data suggest that anti-GPC suppresses erythroid proliferation through the induction of nonclassical apoptosis.

A non-cyclic baboon θ-defensin derivative exhibiting antimicrobial activity against the phytopathogen Verticillium dahliae

θ-Defensins are the only natural cyclic proteins found in primates. They have strong antimicrobial activity related to their trisulfide ladders and macrocyclic conformation. A non-cyclic baboon θ-defensin (BTD) was synthesized by substituting valine with phenylalanine at position 17, at the C-terminal end of the BTD; this was termed "BTD-S." The antimicrobial activities of this synthetic peptide were investigated against Escherichia coli and two cotton phytopathogens: Verticillium dahliae and Fusarium oxysporum. The minimum inhibitory concentration (MIC) of BTD-S for E. coli was 10 μg/mL and for V. dahliae was 5 μg/mL, significantly lower than that for F. oxysporum (40.0 μg/mL). A time course analysis of fungal cultures indicated that the growth of V. dahliae was completely inhibited after 96 h of BTD-S treatment. Furthermore, hemolysis assays revealed that BTD-S was not toxic to mammalian cells as it could not induce lysis of sheep red blood cells even at ten times the MIC (50 μg/mL). Scanning electron microscopy and double-stained (calcofluor white and propidium iodide binding) fluorescence microscopy showed that exposure of spores of V. dahliae to BTD-S either disabled normal germination or disintegrated the spores. The size of cells exposed to BTD-S was significantly reduced compared with controls, and their number increased in a dose-dependent curve when measured by flow cytometry. These findings suggest that BTD-S has great potential to inhibit the growth of V. dahliae and can be utilized as an effective remedy to control economic losses caused by Verticillium wilt in the development of wilt-resistant cotton.

Photophysics of DNA staining dye Propidium Iodide encapsulated in bio-mimetic micelle and genomic fish sperm DNA

Photophysical processes in Propidium Iodide (PI), the well known DNA staining dye, have been exploited in homogeneous as well as heterogeneous medium by steady state and time resolved spectroscopy. The DNA staining dye PI exhibits intermolecular proton transfer reaction in aqueous and hydrogen-bonding acceptor solvents due to the formation of quinonoid structure which is acidic in nature. Time resolved emission spectroscopy also predicts the hydrogen bond donor ability of PI. The target dye interacts only with anionic surfactant sodium dodecylsulphate but not with cationic surfactant cetyltrimethylammonium bromide and neutral surfactant Triton X-100 and this interaction is found to be electrostatic in nature. We have further scrutinized the mode of binding of PI with fish sperm DNA. The effect of addition of urea, fluorescence quenching phenomenon, CD measurements reveal that the probe binds to DNA through intercalative style.

Changes in cyst’s nuclear chromatin resulting after experimental manipulation of Taenia crassiceps mice infections: Biological implications

During some estimations of the nuclear DNA content, based on determinations propidium iodide (PI) binding through fluorocytometry for Taenia crassiceps cysticerci, significant variation in the results were found. This initial observation led to a series of experiments designed to explain the variation. These changes could be induced by the diameter of the needles in the syringes used for the mouse to mouse transfer of the cysts. Nuclei from cysts transferred through 27-gauge needles showed 30% less PI staining than those transferred through 21 gauge needles, after 2months infections. Reduction in PI capture induced by 27-gauge needle was reversible when the cysts were maintained in their mice hosts during 5months. Moreover, variation in PI binding to cysticercal DNA was also found when comparing parasites grown in male versus female mice. The use of agents that homogenize the chromatin structure during PI staining, allowed demonstrating that variation were entirely due to differences in the chromatin relaxation/compaction. Additional experiments demonstrated that the higher compaction is accompanied by a reduced ability of cysts to grow in the peritoneal cavity of BALB/cAnN mice. Furthermore, proteomic analysis also showed that these changes in chromatin relaxation/compaction resulted in different levels and patterns of protein expression. Our results strongly suggest that chromatin is involved in several well characterized phenomena of the T. crassiceps murine model, and open new avenues for a detailed approach to understand such a complex host-parasite relationship.