Cytotoxicity,genotoxicity and mechanism of action (via gene expression analysis) of the indole alkaloid aspidospermine (antiparasitic) extracted from Aspidosperma polyneuron in HepG2 cells

Aspidospermine is an indole alkaloid with biological properties associated with combating parasites included in the genera Plasmodium, Leishmania and Trypanossoma. The present study evaluated the cytotoxicity (resazurin test), genotoxicity (comet assay) and mechanism of action (gene expression analysis via qRT-PCR) of this alkaloid in human HepG2 cells. The results demonstrated that treatment with aspidospermine was both cytotoxic (starting at 75 μM) and genotoxic (starting at 50 μM). There was no significant modulation of the expression of the following genes: GSTP1 and GPX1 (xenobiotic metabolism); CAT (oxidative stress); TP53 and CCNA2 (cell cycle); HSPA5, ERN1, EIF2AK3 and TRAF2 (endoplasmic reticulum stress); CASP8, CASP9, CASP3, CASP7, BCL-2, BCL-XL BAX and BAX (apoptosis); and PCBP4, ERCC4, OGG1, RAD21 and MLH1 (DNA repair). At a concentration of 50 μM (non-cytotoxic, but genotoxic), there was a significant increase in the expression of CYP1A1 (xenobiotic metabolism) and APC (cell cycle), and at a concentration of 100 μM, a significant increase in the expression of CYP1A1 (xenobiotic metabolism), GADD153 (endoplasmic reticulum stress) and SOD (oxidative stress) was detected, with repression of the expression of GR (xenobiotic metabolism and oxidative stress). The results of treatment with aspidospermine at a 100 μM concentration (the dose indicated in the literature to achieve 89 % reduction of the growth of L. amazonensis) suggest that increased oxidative stress and an unfolded protein response (UPR) occurred in HepG2 cells. For the therapeutic use of aspidospermine (antiparasitic), chemical alteration of the molecule to achieve a lower cytotoxicity/genotoxicity in host cells is recommended.     

Preferential pattern of mouse neutrophil cell death in response to various stimulants

Neutrophils undergo cell death processes once their physiological function has been fulfilled. Apoptosis, necrosis, pyroptosis, or NETosis, a unique form of cell death, could occur, depending on the type of stimulant or inhibitory intervention. We investigated whether phorbol myristate acetate (PMA) and Klebsiella pneumoniae (KP), serving as stimulants, or whether an inhibitor (cytochalasin B, CytB) could alter the morphology and gene expression pattern associated with mouse neutrophil cell death. Fluorescence microscopy, flow cytometry, and real-time PCR approaches were used to identify morphological changes, percentages of cell death, and gene expression patterns, respectively. The results showed an increase in the percentage of cell death in PMA and KP groups, whereas CytB exerted inhibitory effects. Fluorescently stained cell nuclei revealed significantly different percentages of cell death among treatments. Moreover, there was a stepwise increase in cell death from 90 to 150 min after stimulation. Quantification of the release of neutrophil extracellular traps (NETs) demonstrated clearly elevated amounts in cells stimulated with KP, while the amount of NETs was slightly increased or unchanged with PMA or CytB treatments. Analysis of the genes involved in cell death revealed that mRNA expression of CASP1, IL1β, IL18, MCL1, NLRP3, and PYCARD was down-regulated in the PMA group, with the exception of AIM2 and CASP3. The expression of AIM2, IL1β, MCL1, and NLRP3 was up-regulated in KP-stimulated neutrophils, while CASP1, CASP3, IL18, and PYCARD genes were down-regulated. In summary, a spectrum of specific cell stimulants and exposure durations accounted for different outcomes in mouse neutrophil cell death.     

Type 1 diabetes mellitus induces structural changes and molecular remodelling in the rat kidney

Previous studies indicated that chlorogenic acid, a compound present in many fruits and vegetables, has anti-cancer activities. We report that chlorogenic acid regulates the expression of apoptosis-related genes and self-renewal-related stem cell markers in cancer cells. The lung cancer cell line A549 was cultured with or without chlorogenic acid. The presence of chlorogenic acid decreased cell proliferation as measured by MTT activity. Polymerase chain reaction (PCR) showed that treatment of cells with chlorogenic acid reduced the expression of BCL2 but increased that of both BAX and CASP3. Chlorogenic acid enhanced annexin V expression as measured using fluorescently labeled annexin V. Chlorogenic acid also induced p38 MAPK and JNK gene expression. Meanwhile, several agents, including SB203580 (p38 MAP kinase inhibitor), N-acetylcysteine (antioxidant inhibitor), dipyridamole (phosphodiesterase inhibitor), and apocynin (NADPH-oxidase inhibitor) blocked chlorogenic acid-induced BAX gene expression. Chlorogenic acid reduced gene expression levels of stem cell-associated markers NANOG, POU5F1, and SOX2. Together these results indicate that chlorogenic acid affects the expression of apoptosis-related genes that are part of oxidative stress and p38 MAP-dependent pathways, as well as genes encoding stem cell markers. In conclusion, chlorogenic acid may contribute to the polyphenolic anti-cancer effect associated with consumption of vegetables and fruits.     

Identification of genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig

Suppression subtractive hybridization was used to identify genes showing differential expression profile associated with growth rate in skeletal muscle tissue of Landrace weanling pig. Two subtracted cDNA populations were generated from musculus longissimus muscle tissues of selected pigs with extreme expected breeding values at the age of 100 kg. Three upregulated genes (EEF1A2, TSG101 and TTN) and six downregulated genes (ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7) in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clones selected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis confirmed difference in expression profiles of the identified genes in musculus longissimus muscle tissues between the two Landrace weanling pig groups with divergent genetic propensity for growth rate. Further, differential expression of the identified genes except for the TTN was validated by Western blot analysis. Additionally, the eight genes other than the ATP5C1 co-localized with the same chromosomal positions as QTLs that have been previously identified for growth rate traits. Finally, the changes of expression predicted from gene function suggested association of upregulation of expression of the EEF1A2, TSG101 and TTN genes and downregulation of the ATP5B, ATP5C1, COQ3, HADHA, MYH1 and MYH7 gene expression with increased growth rate. The identified genes will provide an important insight in understanding of the molecular mechanism underlying growth rate in Landrace pig breed.     

SkQ1 Controls <Emphasis Type="Italic">CASP3</Emphasis> Gene Expression and Caspase-3-Like Activity in the Brain of Rats under Oxidative Stress

Here, we studied the effect of the mitochondria-targeted antioxidant SkQ1 (plastoquinone cationic derivative) on the CASP3 gene expression and caspase-3 activity in rat cerebral cortex and brain mitochondria under normal conditions and in oxidative stress induced by hyperbaric oxygenation (HBO). Under physiological conditions, SkQ1 administration (50 nmol/kg, 5 days) did not affect the CASP3 gene expression and caspase-3-like activity in the cortical cells, as well as caspase-3-like activity in brain mitochondria, but caused a moderate decrease in the content of primary products of lipid peroxidation (LPO) and an increase in the reduced glutathione (GSH) level. HBO-induced oxidative stress (0.5 MPa, 90 min) was accompanied by significant upregulation of CASP3 mRNA and caspase-3-like activity in the cerebral cortex, activation of the mitochondrial enzyme with simultaneous decrease in the GSH content, increase in the glutathione reductase activity, and stimulation of LPO. Administration of SkQ1 before the HBO session maintained the basal levels of the CASP3 gene expression and enzyme activity in the cerebral cortex cells and led to the normalization of caspase-3-like activity and redox parameters in brain mitochondria. We hypothesize that SkQ1 protects brain cells from the HBO-induced oxidative stress due to its antioxidant activity and stimulation of antiapoptotic mechanisms.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.     

Light quality modifies the expression of photosynthetic genes in maize seedlings

Although maize (Zea mays L.) plants utilize light efficiently, the expression of high light-efficient genes and stomatal factors is regulated by light conditions and affects photosynthesis of plants. In this study, we investigated the effects of different light qualities on the expression of the photosynthetic genes, such as pep1, pdk1, ZmSTOMAGEN, and psad1, and on stomatal function in maize seedlings. For both maize genotypes, Zhengdan 958 and Xianyu 335, light with wavelengths shorter than 490 nm enhanced the expression of pdk1 and ZmSTOMAGEN, whereas the expression of pdk1 positively correlated with ZmSTOMAGEN. Light with wavelengths longer than 630 nm or shorter than 490 nm (band pass filter) increased the expression of pep1 and psad1. Although the expression of four genes in Zhengdan 958 was significantly higher than that of Xianyu 335, changes in the expression of ZmSTOMAGEN, pdk1, or pep1 exerted no significant influence on stomatal function and photosynthetic rate. Our results suggest that light with wavelengths shorter than 490 nm promoted the expression of stomatal proteins and pdk1, facilitated the absorption of inorganic elements, and contributed to stomatal function in photosynthesis. Meanwhile, light with wavelengths longer than 630 nm inhibited the expression of pep1 and pdk1. Light with wavelengths longer than 630 nm or shorter than 490 nm promoted the expression of pep1, pdk1, and psad1.     

Differential expression of two <Emphasis Type="Italic">CONSTANS-LIKE1</Emphasis> genes in potato

Although the CONSTANS gene and its CONSTANS-LIKE1 (COL1) orthologs are known to control the photoperiod-dependent floral transition in many plant species, the role of these genes in Solanum development has not been sufficiently elucidated. Previously we characterized two forms of CONSTANS-LIKE1 genes, sCOL1 and lCOL1, in potato (Solanum tuberosum ssp. tuberosum). To prove that these genes were functional, we followed their expression in potato cv. Early Rose with the real-time PCR technique. Both sCOL1 and lCOL1 displayed characteristic day-night patterns of expression under long-day and short-day conditions. The profiles and amplitudes of expression dramatically differed in two genes, with the maximum sCOL1 expression exceeding that of lCOL1 by an order of magnitude.     

Selection of reference genes for quantitative real-time PCR in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> under salt stress

Real time quantitative PCR (qPCR) is widely used in gene expression analysis for its accuracy and sensitivity. Reference genes serving as endogenous controls are necessary for gene normalization. In order to select an appropriate reference gene to normalize gene expression in Casuarina equisetifolia under salt stress, 10 potential reference genes were evaluated using real time qPCR in the leaves and roots of plants grown under different NaCl concentrations and treatment durations. GeNorm, NormFinder, and BestKeeper analyses reveal that elongation factor 1-alpha (EF1α) and ubiquitin-conjugating enzyme E2 (UBC) were the most appropriate reference genes for real time qPCR under salt stress. However, β-tubulin (βTUB) and actin 7, which were widely used as reference genes in other plant species, were not always stably expressed. The combination of EF1α, UBC, uncharacterized protein 2, DNAJ homolog subfamily A member 2, and glyceraldehyde-3-phosphate dehydrogenase should be ideal reference genes for normalizing gene expression data in all samples under salt stress. It indicates the need for reference gene selection for normalizing gene expression in C. equisetifolia. In addition, the suitability of reference genes selected was confirmed by validating the expression of WRKY29-like and expansin-like B1. The results enable analysis of salt response mechanism and gene expression in C. equisetifolia.     

Plant DNA methyltransferase genes: Multiplicity,expression, methylation patterns

Expression and methylation patterns of genes encoding DNA methyltransferases and their functionally related proteins were studied in organs of Arabidopsis thaliana plants. Genes coding for the major maintenance-type DNA methyltransferases, MET1 and CMT3, and the major de novo-type DNA methyltransferase, DRM2, are actively expressed in all organs. Similar constitutively active expression was observed for genes encoding their functionally related proteins, a histone H3K9 methyltransferase KYP and a catalytically non-active protein DRM3. Expression of the MET1 and CMT3 genes is significantly lower in developing endosperm compared with embryo. Vice versa, expression of the MET2a, MET2b, MET3, and CMT2 genes in endosperm is much more active compared with embryo. A special maintenance DNA methylation system seems to operate in endosperm. The DNMT2 and N6AMT genes encoding putative methyltransferases are constitutively expressed at low levels. CMT1 and DRM1 genes are expressed rather weakly in all investigated organs. Most of the studied genes have methylation patterns conforming to the “body-methylated gene” prototype. A peculiar feature of the MET family genes is methylation at all three possible site types (CG, CHG, and CHH). The most weakly expressed among genes of their respective families, CMT1 and DRM1, are practically unmethylated. The MET3 and N6AMT genes have unusual methylation patterns, promoter region, and most of the gene body devoid of any methylation, and the 3'-end proximal part of the gene body is highly methylated.     

Gene expression profiles of the thermotolerant yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain KKU-VN8 during high-temperature ethanol fermentation using sweet sorghum juice

Objectives

To investigate gene expression profiles of the thermotolerant yeast Saccharomyces cerevisiae strain KKU-VN8, a potential high-ethanol producer, in response to various stresses during high-temperature ethanol fermentation using sweet sorghum juice (SSJ) under optimal conditions.

Results

The maximal ethanol concentration obtained by S. cerevisiae KKU-VN8 using SSJ at 40 °C was 66.6 g/l, with a productivity of 1.39 g/l/h and a theoretical ethanol yield of 81%. Quantitative RT-PCR assays were performed to investigate the gene expression profiles of S. cerevisiae KKU-VN8. Differential expression of genes encoding heat-shock proteins (HSP82, HSP104, SSA4), genes involved in trehalose metabolism (TPS1, TPS2, NTH1) and genes involved the glycolytic pathway (ADH1, ADH2, CDC19) at various time points during fermentation was observed. The expression levels of HSP82, HSP104, SSA4, ADH1 and CDC19 were significantly higher than those of the controls (10.2-, 4-, 8-, 8.9- and 5.9-fold higher, respectively). In contrast, the expression levels of TPS1, TPS2, NTH1 and ADH2 were approx. 2-fold less than those of the controls.

Conclusions

The highly expressed genes encoding heat-shock proteins, HSP82 and SSA4, potentially play an important role in helping S. cerevisiae KKU-VN8 cope with various stresses that occur during high-temperature fermentation, leading to higher ethanol production efficiency.