Molecular mechanisms of <Emphasis Type="Italic">Bombyx batryticatus</Emphasis> ethanol extract inducing gastric cancer SGC-7901 cells apoptosis

Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.     

Pre-apoptotic activity of aqueous extracts of <Emphasis Type="Italic">Cynanchum sarcomedium</Emphasis> Meve &amp; Liede on cells of <Emphasis Type="Italic">Allium cepa</Emphasis> and human erythrocytes

Cynanchum sarcomedium Meve & Liede is a member of Apocynaceae, seen in dry and rocky areas. The present study highlights the cytotoxic potential of C. sarcomedium mediated by apoptosis on cells of Allium cepa and human red blood cells (RBCs). Cytogenetic changes in A. cepa and in situ visualization of cell death were revealed through acetocarmine and Evans blue staining techniques. Quantitative estimation of cell death was carried out at 600 nm in a spectrophotometer. Membrane characteristics of RBC in response to the treatment were evaluated by May-Grünwald-Giemsa staining and scanning electron microscopy (SEM). Cell membrane damage is a major factor for assessing apoptosis which is observed in the present study (90.91 %). Cell shrinkage, cytoplasmic fragmentation, condensed chromatin and presence of apoptotic bodies were the common cytological changes in A. cepa associated with apoptosis. Blebs in RBC evidenced by SEM revealed the membrane damage potential of the plant. Results obtained hereby suggest that the plant is an effective source to be used in toxicological studies and anti-cancer therapy.     

Assessment of the in vitro cytotoxicity and in vivo anti-tumor activity of the alcoholic stem bark extract/fractions of Mimusops elengi Linn.

Various parts of Mimusops elengi Linn. (Sapotaceae) have been used widely in traditional Indian medicine for the treatment of pain, inflammation and wounds. The study was conducted to explore the use of stem bark of M. elengi on pharmacological grounds and to evaluate the scientific basis of cytotoxic and anti-tumor activity. Extract/fractions were prepared and in vitro cytotoxicity was assessed using SRB assay. Most effective fractions were subjected to fluorescence microscopy based acridine orange/ethidium bromide (AO/EB) and Hoechst 33342 staining to determine apoptosis induction and DNA fragmentation assay. Comet and micronuclei assay were performed to assess genotoxicity. Cell cycle analysis was also performed. In vivo anti-tumor potential was evaluated by Ehrlich ascites carcinoma (EAC) model in mice. The alcoholic stem bark extract of M. elengi along with four fractions showed potential in vitro cytotoxicity in SRB assay. Of these, dichloromethane and ethyl acetate fractions were selected for further studies. The fractions revealed apoptosis inducing potential in AO/EB and Hoechst 33342 staining, which was further confirmed by DNA fragmentation assay. Genotoxic potential was revealed by comet and micronuclei assay. Fractions also exhibited specific cell cycle inhibition in G₀/G₁ phase. In EAC model, ethyl acetate fraction along with the standard (cisplatin) effectively reduced the increase in body weight compared to control and improved mean survival time. Both fractions were able to restore the altered hematological and biochemical parameters. Hence, M. elengi stem bark may be a possible therapeutic candidate having cytotoxic and anti-tumor potential.     

Fermentative metabolism impedes p53-dependent apoptosis in a Crabtree-positive but not in Crabtree-negative yeast

Tumour cells distinguish from normal cells by fermenting glucose to lactate in presence of sufficient oxygen and functional mitochondria (Warburg effect). Crabtree effect was invoked to explain the biochemical basis of Warburg effect by suggesting that excess glucose suppresses mitochondrial respiration. It is known that the Warburg effect and Crabtree effect are displayed by Saccharomyces cerevisiae, during growth on abundant glucose. Beyond this similarity, it was also demonstrated that expression of human pro-apoptotic proteins in S. cerevisiae such as Bax and p53 caused apoptosis. Here, we demonstrate that p53 expression in S. cerevisiae (Crabtree-positive yeast) causes increase in ROS levels and apoptosis when cells are growing on non-fermentable carbon sources but not on fermentable carbon sources, a feature similar to tumour cells. In contrast, in Kluyveromyces lactis (Crabtree-negative yeast) p53 causes increase in ROS levels and apoptosis regardless of the carbon source. Interestingly, the increased ROS levels and apoptosis are correlated to increased oxygen uptake in both S. cerevisiae and K. lactis. Based on these results, we suggest that at least in yeast, fermentation per se does not prevent the escape from apoptosis. Rather, the Crabtree effect plays a crucial role in determining whether the cells should undergo apoptosis or not.     

Necrotic and apoptotic cell death induced by Captan on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Captan is one of the most widely used broad-spectrum fungicide applied to control several early and late diseases of grapes, apples, and other fruits and vegetables, and as other phthalimide fungicides is defined as a multisite compound with thiol-reactivity. Captan can affect non-target organisms as yeasts, modifying microbial populations and fermentation processes. In this study, we asked whether Captan thiol-reactivity and other mechanisms are involved in acute Captan-induced cell death on aerobic growing Saccharomyces cerevisiae. Thus for, we analyze cellular protein and non-protein thiols, cell membrane integrity, reactive oxygen species accumulation, phosphatidylserine externalization, and apoptotic mutants behavior. The results showed that when submitted to acute Captan treatment most cells lost their membrane integrity and died by necrosis due to Captan reaction with thiols. However, part of the cells, even maintaining their membrane integrity, lost their culture ability. These cells showed an apoptotic behavior that may be the result of non-protein thiol depletion and consequent increase of reactive oxygen species (ROS). ROS accumulation triggers a metacaspase-dependent apoptotic cascade, as shown by the higher viability of the yca1-deleted mutant. Together, necrosis and apoptosis are responsible for the high mortality detected after acute Captan treatment of aerobically growing cells of S. cerevisiae.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Selection of optimal flocculant for effective harvesting of the fucoxanthin-rich marine microalga <Emphasis Type="Italic">Isochrysis galbana</Emphasis>

Flocculation harvesting of the fucoxanthin-rich marine microalga Isochrysis galbana has received little attention. Therefore, we attempted to screen for an optimal chemical flocculant and optimize flocculation conditions from five chemical flocculants—ferric chloride (FC), aluminum sulfate (AS), polyaluminum chloride (PAC), aluminum potassium sulfate (APS), and zinc sulfate (ZS)—for effective flocculation of I. galbana. The growth rate, photosynthetic performance, and fucoxanthin content were determined in re-suspended flocculated algal cells and in the flocculation supernatant cultured algal cells. The results showed that high growth rate and fucoxanthin accumulation were observed when FC was used as the flocculant in I. galbana cultures, which indicated that FC may cause less harm to I. galbana than the other aluminum-based flocculants. Furthermore, satisfactory flocculation efficiency was also observed when FC was used to flocculate I. galbana, and the FC dosage was less than that required for flocculation of I. galbana using PAC, APS, and AS. Thus, we selected FC as the optimal flocculant for harvesting I. galbana based on its flocculation efficiency together with algal physiological performance, growth rate, and fucoxanthin content.     

Cytotoxic and apoptotic activities of extract of <Emphasis Type="Italic">Amaranthus</Emphasis><Emphasis Type="Italic">spinosus</Emphasis> L. in <Emphasis Type="Italic">Allium cepa</Emphasis> and human erythrocytes

The present study examined the apoptosis inducing effects of Amaranthus spinosus L. aqueous extract in Allium cepa root meristematic cells and human erythrocytes. Cytogenetic assay revealed many apoptosis inducing cytogenetic aberrations viz., cytoplasmic breakage, cytoplasmic disintegration, cytoplasmic shrinkage, receding of cytoplasm, cytoplasmic vacuolation, enucleated cell, ghost cell, nuclear vacuolation, nuclear fragmentation and nuclear disintegration. A remarkable modification of red blood cell surface morphology was observed in the result of RBC assay. The treated RBCs show membrane blebbing and shrinkage, features typical for apoptosis in nucleated cells. Significant induction of cell death was observed in treated Allium root tip cells after Evans blue staining, disclosing the membrane damage potential of the plant extract. TTC assay results in reduced mitochondrial/metabolic activity in Allium root tip cells after treatment, designating the adverse effect of plant extract on mitochondrial respiratory chain. These results confirm the apoptosis inducing potential of A. spinosus extract. Confirming the present results by further in vitro studies, it can be effectively targeted against cell proliferation during cancer treatment by inducing apoptosis. Thus from the present investigation it can be concluded that the aqueous extract of A. spinosus exhibited apoptosis induction and cytotoxic activities.     

The Effect of Sodium Fluoride on Cell Apoptosis and the Mechanism of Human Lung BEAS-2B Cells In Vitro

Sodium fluoride (NaF) is a source of fluoride ions used in many applications. Previous studies found that NaF suppressed the proliferation of osteoblast MC3T3 E1 cells and induced the apoptosis of chondrocytes. However, little is known about the effects of NaF on human lung BEAS-2B cells. Therefore, we investigated the mode of cell death induced by NaF and its underlying molecular mechanisms. BEAS-2B cells were treated with NaF at concentrations of 0, 0.25, 0.5, 1.0, 2.0, and 4.0 mmol/L. Cell viability decreased and apoptotic cells significantly increased as concentrations of NaF increased over specific periods of time. The IC₅₀ of NaF was 1.9 and 0.9 mM after 24 and 48 h, respectively. The rates of apoptosis increased from 4.8 to 37.7% after NaF exposure. HE staining, electron microscopy, and single cell gel electrophoresis revealed that morphological changes of apoptosis increased with exposure concentrations. RT-PCR and Western blotting were used to detect the apoptotic pathways. The expressions of bax, caspase-3, caspase-9, p53, and the cytoplasmic CytC of the NaF groups increased, while bcl-2 and mitochondrial CytC decreased compared with that of the control group (P < 0.05). Further, the fluorescence intensities of ROS in the NaF groups were higher than those in the control group, and the membrane potential of mitochondria in the NaF group was significantly lower than that of the control group (P < 0.05). These findings suggested that NaF induced apoptosis in the BEAS-2B cells through mitochondria-mediated signal pathways. Our study provides the theoretical foundation and experimental basis for exploring the mechanisms of human lung epithelial cell damage and cytotoxicity induced by fluorine.     

Measurement of individual cell strength of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> in cell culture

Botryococcus braunii is a microalga considered for biofuel production and may require physical disruption of cells/colonies for efficient hydrocarbon extraction. In this study, the strength of individual cells of B. braunii was measured using a nanoindenter. From the load and cell size, the pressure for bursting the cell was calculated to be 56.9 MPa. This value is 2.3–10 times those of Saccharomyces cerevisiae and Chlorella vulgaris found in another research, because B. braunii has two types of cell walls with different thicknesses. The energy required to disrupt 1 g of dry B. braunii cells, estimated by load-displacement curves, is 3.19 J g^?1 which is 0.19–1.2 times higher than those of S. cerevisiae and C. vulgaris. When using a high-pressure homogenizer for disrupting B. braunii cells, the cell disruption degree increased with the treatment pressure at above 30 MPa, and 70% of cells were disrupted at 80 MPa.     

Induction of Apoptotic Cell Death on Human Cervix Cancer HeLa cells by Extract from <Emphasis Type="Italic">Loranthus yadoriki</Emphasis>

Loranthus yadoriki, one of the Korean mistletoe species, has been already known for anti-viral effects, but the molecular basis that it caused apoptosis in cancer cells was not definitely revealed yet. The aim of this study was to estimate the mechanisms of apoptotic cell death of the extract from Loranthus yadoriki (named as ELY) in human cervix HeLa cells. We identified that ELY prevented the proliferation of HeLa cells between 50 and 300 μg/mL which did not affect non-cancerous HaCaT cells. In addition, ELY induced a morphological change and nucleus disruption as well as an accumulation of sub-G1 phase in HeLa cells. The mechanism study, by using western blot analysis, showed that the phosphorylation of Fas-associated death domain (FADD), Bim and Bak was up-regulated by ELY treatment. Furthermore, the expression of cytochrome c and Apaf-1 was increased by ELY treatment. In immunofluorescence staining, the increased intensity of cleaved caspase-3 and cleaved PARP was also observed under ELY treatment. Sequentially, the caspase cascade was activated by ELY from caspase-8 to caspase-3 and from caspase-9 to caspase-3, in both extrinsic and intrinsic pathways. The results of this study demonstrate that ELY has anti-cancer effects on human cervix cancer HeLa cells via caspase cascade in apoptotic signaling pathways.     

Effect of ATP and Bax on the apoptosis of <Emphasis Type="Italic">Eimeria tenella</Emphasis> host cells

Background

Eimeria tenella (E. tenella) is a species of Eimeria that causes haemorrhagic caecal coccidiosis, resulting in major economic losses in the global poultry industry. After E. tenella infection, the amount of ATP and Bax in host cells showed highly significant changes. Therefore, it is necessary to investigate the effects of ATP and Bax on the apoptosis of E. tenella host cells.

Results

The ATP-treated group and the V5-treated group had higher E. tenella infection rates than the untreated group at 24, 48, 72, 96, and 120 h after infection with E. tenella. The results of flow cytometry showed that compared with the control group, the mitochondrial permeability transition pore (MPTP) opening in the untreated group was highly significantly increased (P?<?0.01) at 4, 24, 48, 72, 96, and 120 h. Moreover, results from Hoechst-Annexin V-PI staining and flow cytometry showed that the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were significantly lower (P?<?0.05) or highly significantly lower (P?<?0.01) than those of the control group at 4 h, while the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were higher at varying degrees than those in the control group at 24–120 h (P?<?0.05 or P?<?0.01). After treatment with ATP and Bax inhibitors, the rates of early apoptosis, late apoptosis, and necrosis, in addition to the MPTP opening in both the ATP-treated and V5-treated groups, were significantly lower (P?<?0.05) or highly significantly lower (P?<?0.01) than those in the untreated group.

Conclusions

ATP and Bax play important roles in regulating the apoptosis of E. tenella host cells.

     

Identification and characterization of a complex chromosomal replication origin in<Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

In the budding yeast,S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (±300 bp). In contrast, in mammalian cells andDrosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just likeS. cerevisiae origins. Because the fission yeast,Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than doesS. cerevisiae, we thought thatS. pombe replication origins might prove to resemble — and thus be helpful models for — animal cell origins. An attempt to test this possibility using 2D gel techiques resulted in identification of a replication origin near theura4 gene on chromosome III ofS. pombe. The 2D gel patterns produced by thisS. pombe origin indeed resemble the patterns produced by animal cell origins and show that theS. pombe origin cannot be precisely located. The data suggest an initiation zone of 3–5 kbp. Some aspects of the 2D gel patterns detected at theS. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.     

Genome sequence of <Emphasis Type="Italic">Candida versatilis</Emphasis> and comparative analysis with other yeast

The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae.     

Heavy metal accumulation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells armed with metal binding hexapeptides targeted to the inner face of the plasma membrane

Accumulation of heavy metals without developing toxicity symptoms is a phenotype restricted to a small group of plants called hyperaccumulators, whose metal-related characteristics suggested the high potential in biotechnologies such as bioremediation and bioextraction. In an attempt to extrapolate the heavy metal hyperaccumulating phenotype to yeast, we obtained Saccharomyces cerevisiae cells armed with non-natural metal-binding hexapeptides targeted to the inner face of the plasma membrane, expected to sequester the metal ions once they penetrated the cell. We describe the construction of S. cerevisiae strains overexpressing metal-binding hexapeptides (MeBHxP) fused to the carboxy-terminus of a myristoylated green fluorescent protein (myrGFP). Three non-toxic myrGFP-MeBHxP (myrGFP-H6, myrGFP-C6, and myrGFP-(DE)3) were investigated against an array of heavy metals in terms of their effect on S. cerevisiae growth, heavy metal (hyper) accumulation, and capacity to remove heavy metal from contaminated environments.     

Functional analysis of recombinant human and <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> <Emphasis Type="Italic">O</Emphasis>-GlcNAc transferases expressed in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.     

Changes in intracellular metabolism underlying the adaptation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to ethanol stress

Saccharomyces cerevisiae is often stressed by the ethanol which accumulates during the production of bioethanol by the fermentation process. The study of ethanol-adapted S. cerevisiae strains provide an opportunity to clarify the molecular mechanism underlying the adaptation or tolerance of S. cerevisiae to ethanol stress. The aim of this study was to clarify this molecular mechanism by investigating the ethanol adaptation-associated intracellular metabolic changes in S. cerevisiae using a gas chromatography–mass spectrometry-based metabolomics strategy. A partial least-squares-discriminant analysis between the parental strain and ethanol-adapted strains identified 12 differential metabolites of variable importance with a projection value of >1. The ethanol-adapted strains had a more activated glycolysis pathway and higher energy production than the parental strain, suggesting the possibility that an increased energy production and energy requirement might be partly responsible for an increased ethanol tolerance. An increased glycine content also partly contributed to the higher ethanol tolerance of the ethanol-adapted strains. The decreased oleic acid content may be a self-protection mechanism of ethanol-adapted strains to maintain membrane integrity through decreasing membrane fluidity. We suggest that while being exposed to ethanol stress, ethanol-adapted S. cerevisiae cells may remodel their metabolic phenotype and the composition of their cell membrane to adapt to ethanol stress and acquire higher ethanol tolerance.