Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells

Background

Mushrooms of the genus Agaricus are a common folk remedy against carcinoma. The active ingredients, polysaccharides and protein-polysaccharide complexes containing β-glucan, have been isolated and shown to have indirect tumor-suppressing activity via an immunological activation.

Methods

The diffusible fraction of a hot-water extract of Agaricus blazei Murrill (ABM) powder was fractionated by HPLC based on the anti-tumor activity against leukemic cells in vitro. The structure of the anti-tumor substance was determined by NMR and MS analyses.

Results

We purified a tumorcidal substance from the diffusible fraction of ABM and identified it as agaritine, β-N-(γ-l(+)-glutamyl)-4-(hydroxymethyl) phenylhydrazine, having a molecular mass of 267 Da. This compound inhibited the proliferation of leukemic cell lines such as U937, MOLT4, HL60 and K562 with IC₅₀ values of 2.7, 9.4, 13.0, and 16.0 μg/mL, respectively, but showed no significant effect on normal lymphatic cells at concentrations up to 40 μg/mL. Although agaritine has been suspected of having genotoxic or carcinogenic properties, agaritine did not activate the umu gene of Salmonella, which reacts to carcinogens.

General significance

The results indicate that agaritine from ABM has direct anti-tumor activity against leukemic tumor cells in vitro. This is in contrast to the carcinogenic activity previously ascribed to this compound. Our results also show that this activity is distinct from that of β-glucan, which indirectly suppresses proliferation of tumor cells.     

Liposomal temocene (m-THPPo) photodynamic treatment induces cell death by mitochondria-independent apoptosis

Background

The cell death pathway activated after photodynamic therapy (PDT) is controlled by a variety of parameters including the chemical structure of the photosensitizer, its subcellular localization, and the photodynamic damage induced. The present study aims to characterize a suitable m-THPPo liposomal formulation, to determine its subcellular localization in HeLa cells and to establish the cell death mechanisms that are activated after photodynamic treatments.

Methods

Liposomes containing m-THPPo were prepared from a mixture of DPPC and DMPG at a 9:1 molar ratio. In order to procure the best encapsulation efficiency, the m-THPPo/lipid molar ratio was considered. HeLa cells were incubated with liposomal m-THPPo and the subcellular localization of m-THPPo was studied. Several assays such as TUNEL, annexin V/propidium iodide and Hoechst-33258 staining were performed after photodynamic treatments. The apoptotic initiation was assessed by cytochrome c and caspase-2 immunofluorescence.

Results

m-THPPo encapsulated in liposomes showed a decrease of the fluorescence and singlet oxygen quantum yields, compared to those of m-THPPo dissolved in tetrahydrofuran. Liposomal m-THPPo showed colocalization with LysoTracker® and it induced photoinactivation of HeLa cells by an apoptotic mechanism. In apoptotic cells no relocalization of cytochrome c could be detected, but caspase-2 was positive immediately after photosensitizing treatments.

Conclusions

Photodynamic treatment with liposomal m-THPPo leads to a significant percentage of apoptotic morphology of HeLa cells. The activation of caspase-2, without the relocalization of cytochrome c, indicates a mitochondrial-independent apoptotic mechanism.

General significance

These results provide a better understanding of the cell death mechanism induced after liposomal m-THPPo photodynamic treatment.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.     

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells

Background

The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood.

Methods

In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)-induced L929 cell apoptosis.

Results

Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis.

General significance

These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.     

Ubiquitination and SUMOylation of annexin A1 and helicase activity

Background

While annexin A1 in nuclei is proposed to be involved in cell transformation, its functions remain poorly understood. Since annexin A1 has the consensus motif, ¹⁶⁰LKRD, for SUMOylation as well as Ks, acceptors for ubiquitination that regulates localization and functions of proteins, we investigated SUMOylation and ubiquitination of annexin A1.

Methods

SUMOylation and ubiquitination of bovine annexin A1 were biochemically tested in vitro by purified proteins, and were confirmed by cell experiments with L5178 lymphoma cells. Effects of the modifications on DNA helicase activity were measured by ssDNA binding activity and by dsDNA unwinding activity.

Results

SUMOylation of annexin A1 was catalyzed by Ubc9, while its ubiquitination was by Rad6-Rad 18. Ubiquitinated annexin A1 had higher affinity for damaged DNA, and promoted in vitro translesion DNA synthesis by Pol β. In mouse lymphoma L5178Y tk(+/−) cells, levels of SUMOylated annexin A1 decreased by DNA damaging agents, MMS or As³⁺, whereas those of ubiquitinated annexin A1 increased under the same conditions.

Conclusion

These observations suggest but do not necessarily prove that ubiquitinated annexin A1 in nuclei may be involved in DNA damage response, while SUMOylated annexin A1 functions in proliferation–differentiation.

Significance

Ubiquitination of annexin A1 may play an important role in mutagenesis, an initial step of cell transformation.     

5′-Nitro-indirubinoxime induces G1 cell cycle arrest and apoptosis in salivary gland adenocarcinoma cells through the inhibition of Notch-1 signaling

Background

5′-Nitro-indirubinoxime (5′-NIO) is a new derivative of indirubin that exhibits anti-cancer activity in a variety of human cancer cells. However, its mechanism has not been fully clarified.

Methods

Human salivary gland adenocarcinoma (SGT) cells were used in this study. Western blot and RT-PCR analyses were performed to determine cellular Notch levels. The cell cycle stage and level of apoptosis were analyzed using flow cytometry analysis.

Results

5′-NIO significantly inhibited the mRNA levels of Notch-1 and Notch-3 and their ligands (Delta1, 2, 3, and Jagged-2) in SGT cells. Immunocytochemistry analysis showed that 5′-NIO specifically decreased the level of Notch-1 in the nucleus. In addition, 5′-NIO induced G1 cell cycle arrest by reducing levels of CDK4 and CDK6 in SGT cells. Using flow cytometry and immunoblotting analysis, we found that 5′-NIO induces apoptosis following the secretion of cytochrome c and the activation of caspase-3 and caspase-7. Intracellular Notch-1 overexpression led to a decrease in G1 phase arrest and an inhibition of 5′-NIO-induced apoptosis.

Conclusion

These observations suggest that 5′-NIO induces cell cycle arrest and apoptosis by down-regulating Notch-1 signaling.

General significance

This study identifies a new mechanism of 5′-NIO-mediated anti-tumor properties. Thus, 5′-NIO could be used as a candidate for salivary gland adenocarcinoma therapeutics.     

Selective cytotoxicity of intense nanosecond-duration electric pulses in mammalian cells

Background

Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.

Methods

Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.

Results

10-ns EP caused apoptotic or necrotic death within 2–20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S = αD^(−K), where coefficients K and α determined the slope and the “shoulder” of the survival curve. K was similar in all groups, whereas α was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.

Conclusions

1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD₅₀ 1–3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD₅₀ 50–80 J/g for Jurkat and 400–500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores (”nanopores”), triggering different cell death mechanisms.

General significance

Nanosecond EP can selectively target certain cells in medical applications like tumor ablation.     

Glycated human serum albumin enhances macrophage inflammatory protein-1β mRNA expression through protein kinase C-δ and NADPH oxidase in macrophage-like differentiated U937 cells

Background:

In a previous report (Higai K et al., Biol Pharm Bull, 2007), glycated human serum albumin (Glc-HSA) was found to induce interleukin-8 (IL-8) mRNA expression in human monocyte-derived U937 cells through a reactive oxygen species (ROS)-dependent pathway; however, Glc-HSA signaling has not been elucidated in macrophages.

Methods:

U937 cells were differentiated by treatment with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 2 days and the macrophage-like differentiated U937 (differentiated U937) cells were stimulated with Glc-HSA and glycolaldehyde dimer-modified HSA (GA-HSA) in the presence of various signaling inhibitors. Macrophage inflammatory protein-1β (MIP-1β) mRNA expression was determined by real-time PCR. Intracellular ROS generation was estimated by confocal laser microscopy.

Results:

Glc-HSA and GA-HSA markedly enhanced MIP-1β mRNA expression in differentiated U937 cells. Enhanced MIP-1β mRNA expression was completely suppressed by the ROS scavenger N-acetyl-l-cysteine, the NADPH oxidase inhibitors diphenylene iodonium and apocynin, and the protein kinase C (PKC)-δ inhibitor rottlerin. Furthermore, ROS generation was suppressed completely by rottlerin but not by the PKC-γ inhibitor Ro318425 or the PKC-α, -β1 and -μ inhibitor Go6976.

Conclusion:

Glc-HSA and GA-HSA enhance MIP-1β mRNA expression in differentiated U937 cells through PKC-δ-dependent activation of NADPH oxidase.     

PKC activation contributes to caspase-mediated eIF2α phosphorylation and cell death

Back ground

Stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2α), involved in translation, promotes cell suicide or survival. Since multiple signaling pathways are implicated in cell death, the present study has analyzed the importance of PKC activation in the stress-induced eIF2α phosphorylation, caspase activation and cell death in the ovarian cells of Spodoptera frugiperda (Sf9) and in their extracts.

Methods

Cell death is analyzed by flow cytometry. Caspase activation is measured by Ac-DEVD-AFC hydrolysis and also by the cleavage of purified recombinant PERK, an endoplasmic reticulum-resident eIF2α kinase. Status of eIF2α phosphorylation and cytochrome c levels are analyzed by western blots.

Results

PMA, an activator of PKC, does not promote cell death or affect eIF2α phosphorylation. However, PMA enhances late stages of UV-irradiation or cycloheximide-induced caspase activation, eIF2α phosphorylation and apoptosis in Sf9 cells. PMA also enhances cytochrome c-induced caspase activation and eIF2α phosphorylation in cell extracts. These changes are mitigated more efficiently by caspase inhibitor, z-VAD-fmk, than by calphostin, an inhibitor of PKC. In contrast, tunicamycin-induced eIF2α phosphorylation that does not lead to caspase activation or cell death is unaffected by PMA, z-VAD-fmk or by calphostin.

Conclusions

While caspase activation is a cause and consequence of eIF2α phosphorylation, PKC activation that follows caspase activation further enhances caspase activation, eIF2α phosphorylation, and cell death in Sf9 cells.

General significance

Caspases can activate multiple signaling pathways to enhance cell death.     

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells

Background

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity.

Methods

MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting.

Results

BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α₁, α₆ and β₁ integrin subunit expression, and increases α₅ subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21.

Conclusion

BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells.

General significance

Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer.     

Regulatory role of nitric oxide in the reduced survival of erythrocytes in visceral leishmaniasis

Background

Nitric oxide (NO) plays a vital role in maintaining the survivability of circulating erythrocytes. Here we have investigated whether NO depletion associated with visceral leishmaniasis (VL) is responsible for the reduced survival of erythrocytes observed during the disease.

Methods

Infected hamsters were treated with standard anti-leishmanial sodium stibogluconate (SAG) and NO donor isosorbide dinitrate (ISD). Erythrophagocytosis by macrophages was determined by labelling the cells with FITC followed by flow cytometry. Aggregation of band3 was estimated from band3 associated EMA fluorescence. Caspase 3 activity was measured using immunosorbent assay kit. Phosphatidylserine (PS) externalization and cell shrinkage were determined using annexin V. Aminophspholipid translocase and scramblase activities were measured following NBD-PS and NBD-PC internalization, respectively.

Results

Impairment of both synthesis and uptake of NO resulted in decreased bioavailability of this signaling molecule in erythrocytes in VL. NO level was replenished after simultaneous treatment with ISD and SAG. Combination treatment decreased red cell apoptosis in infected animals by deactivating caspase 3 through s-nitrosylation. Drug treatment prevented infection-mediated ATP depletion and altered calcium homeostasis in erythrocytes. Improved metabolic environment effectively amended dysregulation of aminophospholipid translocase and scramblase, which in turn reduced cell shrinkage, and exposure of phosphatidylserine on the cell surface under the diseased condition.

Conclusion and general significance

In this study, we have identified NO depletion to be an important factor in promoting premature hemolysis with the progress of leishmanial infection. The study implicates NO to be a possible target for future drug development towards the promotion of erythrocyte survival in VL.     

Cordycepin induces cell cycle arrest and apoptosis by inducing DNA damage and up-regulation of p53 in Leukemia cells

Cordycepin, an adenosine analog derived from Cordyceps militaris has been shown to exert anti-tumor activity in many ways. However, the mechanisms by which cordycepin contributes to the anti-tumor still obscure. Here our present work showed that cordycepin inhibits cell growth in NB-4 and U937 cells by inducing apoptosis. Further study showed that cordycepin increases the expression of p53 which promotes the release of cytochrome c from mitochondria to the cytosol. The released cytochrome c can then activate caspase-9 and trigger intrinsic apoptosis. Cordycepin also blocks MAPK pathway by inhibiting the phosphorylation of ERK1/2, and thus sensitizes the apoptosis. In addition, our results showed that cordycepin inhibits the expression of cyclin A2, cyclin E, and CDK2, which leads to the accumulation of cells in S-phase. Moreover, our study showed that cordycepin induces DNA damage and causes degradation of Cdc25A, suggesting that cordycepin-induced S-phase arrest involves activation of Chk2-Cdc25A pathway. In conclusion, cordycepin-induced DNA damage initiates cell cycle arrest and apoptosis which leads to the growth inhibition of NB-4 and U937 cells.     

Pyruvate therapy for Leigh syndrome due to cytochrome c oxidase deficiency

Background

Recently we proposed the therapeutic potential of pyruvate therapy for mitochondrial diseases. Leigh syndrome is a progressive neurodegenerative disorder ascribed to either mitochondrial or nuclear DNA mutations.

Methods

In an attempt to circumvent the mitochondrial dysfunction, we orally applied sodium pyruvate and analyzed its effect on an 11-year-old female with Leigh syndrome due to cytochrome c oxidase deficiency accompanied by cardiomyopathy. The patient was administered sodium pyruvate at a maintenance dose of 0.5 g/kg/day and followed up for 1 year.

Results

The exercise intolerance was remarkably improved so that she became capable of running. Echocardiography indicated improvements both in the left ventricle ejection fraction and in the fractional shortening. Electrocardiography demonstrated amelioration of the inverted T waves. When the pyruvate administration was interrupted because of a gastrointestinal infection, the serum lactate level became elevated and the serum pyruvate level, decreased, suggesting that the pyruvate administration was effective in decreasing the lactate-to-pyruvate ratio.

Conclusions

These data indicate that pyruvate therapy was effective in improving exercise intolerance at least in a patient with cytochrome c oxidase deficiency.

General significance

Administration of sodium pyruvate may prove effective for other patients with cytochrome c oxidase deficiency due to mitochondrial or nuclear DNA mutations.     

Stimulation of autophagic activity in human glioma cells by anti-proliferative ardipusilloside I isolated from Ardisia pusilla

Aims

Ardipusilloside I (ADS-I), a triterpenoid saponin isolated from Ardisia pusilla A.DC (Myrsinaceae), has been recently tested for cancer treatment including brain cancer. However, the mechanism of its action remains elusive. The present study was to investigate the role of autophagy activation in the anti-tumor activities of ADS-I in human glioma cells.

Main methods

The tetrazolium dye (MTT) colorimetric assay was used for the measurement of cell proliferation in cultured glioma cells, transmission electron microscopy (TEM) for the examination of autophagic activity, flow cytometric analysis for the determination of cell cycle and apoptotic cells, and immunocytochemistry and Western blot for protein expression of microtubule-associated protein light-chain 3 (LC3) and Beclin 1.

Key findings

ADS-I significantly inhibited the proliferation of both U373 and T98G glioma cells in cultures in a dose-dependent manner. The cytotoxic activity of ADS-I against glioma cell growth was associated not only with the induction of cell cycle arrest at G₂/M phase and cell apoptosis in flow cytometric analysis, but also with the activation of autophagy, indicated by the formation of autophagosomes and up-regulated expression of both autophagic protein Beclin 1 and LC3 in glioma cells. Additionally, the treatment with chloroquine, an autophagy inhibitor, reduced ADS-1-mediated cell death.

Significance

These data suggest that the anti-proliferative activity of ADS-I in human glioma cells is associated with the activation of autophagy in addition to cell cycle arrest and apoptosis, and the antagonistic effect of chloroquine suggests an important role of autophagy in ADS-I-mediated cell death against tumor growth.     

Decline in mitochondrial bioenergetics and shift to ketogenic profile in brain during reproductive senescence

Background

We have previously demonstrated that mitochondrial bioenergetic deficits precede Alzheimer's pathology in the female triple transgenic Alzheimer's (3xTgAD) mouse model. Herein, we sought to determine the impact of reproductive senescence on mitochondrial function in the normal non-transgenic (nonTg) and 3xTgAD female mouse model of AD.

Methods

Both nonTg and 3xTgAD female mice at 3, 6, 9, and 12 months of age were sacrificed and mitochondrial bioenergetic profile as well as oxidative stress markers were analyzed.

Results

In both nonTg and 3xTgAD mice, reproductive senescence paralleled a significant decline in PDH, and Complex IV cytochrome c oxidase activity and mitochondrial respiration. During the reproductive senescence transition, both nonTg and 3xTgAD mice exhibited greater individual variability in bioenergetic parameters suggestive of divergent bioenergetic phenotypes. Following transition through reproductive senescence, enzymes required for long-chain fatty acid (HADHA) and ketone body (SCOT) metabolism were significantly increased and variability in cytochrome c oxidase (Complex IV) collapsed to cluster at a ∼ 40% decline in both the nonTg and 3xTgAD brain which was indicative of alternative fuel generation with concomitant decline in ATP generation.

Conclusions

These data indicate that reproductive senescence in the normal nonTg female brain parallels the shift to ketogenic/fatty acid substrate phenotype with concomitant decline in mitochondrial function and exacerbation of bioenergetic deficits in the 3xTgAD brain.

General significance

These findings provide a plausible mechanism for increased life-time risk of AD in postmenopausal women and suggest an optimal window of opportunity to prevent or delay decline in bioenergetics during reproductive senescence.     

Antitumor activity of methyl gallate by inhibition of focal adhesion formation and Akt phosphorylation in glioma cells

Background

Methyl gallate (MG) possesses a wide range of biological properties that include anti-oxidant, anti-inflammatory, and anti-microbial activities. However, its anti-tumor activity has not been extensively examined in cancer cells. Thus, we examined the effect of MG in both glutamate-induced rat C6 and human U373 glioma cell proliferation and migration.

Methods

MG was isolated from the stem bark of Acer barbinerve. Cell viability and migration were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch wound-healing assay, respectively. Focal adhesion formation was detected with immunofluorescence.

Results

Treatment of C6 and U373 glioma cells with MG significantly reduced cell viability, migration, and Akt phosphorylation level. Glutamate stimulation markedly increased the level of ERK1/2 phosphorylation. However, cells treated with MG displayed decreased ERK1/2 phosphorylation. Inhibition of ERK1/2 by MG or MEK1/2 inhibitor significantly inhibited paxillin phosphorylation at Ser⁸³ and focal adhesion turn-over produced inefficient glioma cell migration. In addition, activation of Akt and ERK1/2 upon glutamate stimulation was independently regulated by Ca^2 + and protein kinase C activity, respectively, via the α-amino-3-hydroxy-5-methy-4-isoxazolepropionate acid glutamate receptor and metabotropic glutamate receptor.

General significance

Our results clearly indicate that MG has a strong anti-tumor effect through the down-regulation of the Akt and ERK1/2 signaling pathways. Thus, methyl gallate is a potent anti-tumor and novel therapeutic agent for glioma.     

Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH

Background

In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O₂ is linked to proton uptake from solution to form H₂O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.

Methods

In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O₂. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.

Results

The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O₂ reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.

Conclusions

The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.

General significance

These results provide new insights into the function of cytochrome c oxidase.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

Background

Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells.

Results

In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G₀/G₁ and Sub-G₁ cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G₀/G₁ phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment.

Conclusions

The results demonstrated that HCT-induced G₀/G₁ phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells     

Purification,characterization and cloning of a ricin B-like lectin from mushroom Clitocybe nebularis with antiproliferative activity against human leukemic T cells

Background

Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.

Methods

Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.

Results

A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1–3(Fucα1–2)Galβ-containing carbohydrates, and GalNAcβ1–4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells.

Conclusions

The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.

General significance

CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.