Semi-quantitative RT-PCR Assay Research Articles

Microarray-based detection and expression analysis of drug resistance in an animal model of peritoneal metastasis from colon cancer

Chemotherapy drugs efficiently eradicate rapidly dividing differentiated cells by inducing cell death, but poorly target slowly dividing cells, including cancer stem cells and dormant cancer cells, in the later course of treatment. Prolonged exposure to chemotherapy results in a decrease in the proportion of apoptotic cells in the tumour mass. To investigate and characterize the molecular basis of this phenomenon, microarray-based expression analysis was performed to compare tHcred2-DEVD-EGFP-caspase 3-sensor transfected C-26 tumour cells that were harvested after engraftment into mice treated with or without 5-FU. Peritoneal metastasis was induced by intraperitoneal injection of C-26 cells, which were subsequently reisolated from omental metastatic tumours after the mice were sacrificed by the end of the 10th day after tumour injection. The purity of reisolated tHcred2-DEVD-EGFP-caspase 3-sensor-expressing C-26 cells was confirmed using FLIM, and total RNA was extracted for gene expression profiling. The validation of relative transcript levels was carried out via real-time semiquantitative RT‒PCR assays. Our results demonstrated that chemotherapy induced the differential expression of mediators of cancer cell dormancy and cell survival-related genes and downregulation of both intrinsic and extrinsic apoptotic signalling pathways. Despite the fact that some differentially expressed genes, such as BMP7 and Prss11, have not been thoroughly studied in the context of chemoresistance thus far, they might be potential candidates for future studies on overcoming drug resistance.

Deletion of the OsLA1 Gene Leads to Multi-Tillering and Lazy Phenotypes in Rice

Plant architecture, one of the key factors that determine grain yield in rice, is mainly affected by components such as plant height, tiller number, and panicle morphology. For this paper, we obtained a multi-tillering and lazy mutant from a japonica rice cultivar, Wuyunjing 7 (WYJ7), via treatment with a heavy ion beam. Compared to WYJ7, the mutant showed a significant increase in tiller angle, tiller number, number of primary and secondary branches, and number of grains; however, the plant height and grain thickness of the mutant was significantly decreased. Phenotypic analysis of the F1 hybrids revealed that the multi-tillering and lazy mutant phenotypes were regulated by a recessive gene. The segregation ratio of 1׃3 of the mutant phenotype and the wild-type plant in the F2 population indicated that the former was controlled by a single gene named Multi-Tillering and Lazy 1 (MTL1). Bulked segregant analysis was performed using the individual plants with extremely typical tiller angles in the F2 population. The MTL1 gene was initially mapped within a region of 5.58–17.64 Mb on chromosome 11. By using the F2 segregated population for fine mapping, the MTL1 gene was ultimately fine mapped within the range of 66.67 kb on chromosome 11. The analysis of genes in this region revealed the presence of the previously identified LAZY1 (LA1) gene. Genomic PCR amplification and semi-quantitative RT-PCR assays showed that the LA1 gene could not be amplified and was not expressed, thus indicating that the MTL1 gene might be identical to the LA1 gene. This study suggests that the multi-tillering and lazy mutant phenotypes might be caused by the deletion of LA1 function. This finding can guide further investigations on the functional mechanisms of the LA1 gene, thus enriching the theoretical knowledge of plant architecture in relation to rice.

Effects of natural compounds and commercial antibiotics on Pseudomonas aeruginosa quorum sensing

Pseudomonas aeruginosa is a Gram-negative bacterium designated by the WHO as a critical priority microorganism due to its virulence, controlled by a quorum sensing (QS) system. QS is regulated} through specific subsystems: LasI/LasR, RhlI/RhlR, and PQS/MvfR. Several natural compounds can inhibit these QS mechanisms. In this study, we determined the effect of curcumin, reserpine, and their mixtures with two commercial antibiotics (gentamicin and azithromycin) on P. aeruginosa QS mechanisms: mvfR gene expression and the production of pyocyanin and rhamnolipids. Antibiotic and natural compounds’ minimal inhibitory concentrations (MICs) were determined via microdilution assays. Gentamicin, azithromycin, curcumin, reserpine, and their mixtures exerted variable effects on mvfR gene expression, as assessed via semi-quantitative RT-PCR assays. Curcumin, reserpine, and gentamicin inhibited mvfR gene expression better than azithromycin, and the mixtures curcumin-gentamicin and reserpine-gentamicin outperformed gentamicin alone in inhibiting mvfR gene expression and decreasing pyocyanin and rhamnolipids production, revealing the synergistic effect of these mixture components. The mixtures of curcumin and gentamicin and reserpine and gentamicin may become alternatives to complement or enhance conventional methods currently used to treat P. aeruginosa infections.

Neuropeptide FF-related gene in fish (Larimichthys polyactis): identification, characterization, and potential anti-inflammatory function.

Neuropeptide FF (NPFF), an octapeptide of the RFamide-related peptides (FaRPs), is involved in regulatory function in various biological processes. The regulatory role of NPFF in the immune and inflammatory response was currently being revealed. Neuropeptide FF-related gene (termed LpNPFF) and its two receptors, NPFF receptor 1 (LpNPFFR1) and NPFF receptor 2 (LpNPFFR2) were identified by PCR and Semi-quantitative RT-PCR assay. Effect of LpNPFF on the production of nitric oxide (NO) in macrophage RAW264.7 cell was divided into PBS group, lipopolysaccharide (LPS) group, LPS treated with LpNPFF group, and LPS treated with LpNPFF and receptor antagonist RF9 group. Then specimens were measured by color reaction at 570nm absorbance value. Sequence analysis showed that LpNPFF cDNA consists of 835 nucleotides with a 5'- untranslated region (UTR) of 150 base pair (bp), an open reading frame (ORF) of 384bp and a 3'-UTR of 300bp (Accession No. MT012894). The ORF encodes 127 amino acid (aa) residues with a hydrophobic signal peptide at N-terminus and two presumptive peptides with -PQRFa structure, LpNPFF (1) and LpNPFF (2). LpNPFFR1 and LpNPFFR2 encode 427 and 444 aa residues respectively, which both have seven hydrophobic TMDs and identified as G protein coupled receptors (GPCRs). Results of tissue distribution showed that LpNPFF and receptors were highly expressed in the brain and gonad. Furtherly, in vitro assay found LpNPFF could inhibit NO production in RAW 264.7 macrophages under inflammatory stress with LPS, while its receptor antagonist RF9 caused the evoke of NO generation. These results contribute to the further study of neuropeptide evolution in marine organisms, and also provide a new research idea for exploring the related functions of NPFF gene.

Retraction Note to: Lupeol enhances inhibitory effect of 5-fluorouracil on human gastric carcinoma cells.

Lupeol, a dietary triterpene present in many fruits and medicinal plants, has been reported to possess many pharmacological properties including cancer-preventive and anti-cancer effects in vitro and in vivo. Here, we investigated the anti-cancer efficacy and adjuvant chemotherapy action of lupeol in gastric cancer (GC) cells (SGC7901 and BGC823) and explored the underlying mechanisms. Cells were treated with lupeol and/or 5-fluorouracil (5-Fu) and subjected to cell viability, colony formation, apoptosis, western blot, semiquantitative RT-PCR, and xenograft tumorigenicity assay. Our results showed that lupeol and 5-Fu inhibited the proliferation of SGC7901 and BGC823 cells, and combination treatment with lupeol and 5-Fu resulted in a combination index < 1, indicating a synergistic effect. Co-treatment with lupeol and 5-Fu induced apoptosis through up-regulating the expressions of Bax and p53 and down-regulating the expressions of survivin and Bcl-2. Furthermore, co-treatment displayed more efficient inhibition of tumor weight and volume on BGC823 xenograft mouse model than single-agent treatment with 5-Fu or lupeol. Taken together, our findings highlight that lupeol sensitizes GC to 5-Fu treatment, and combination treatment with lupeol and 5-Fu would be a promising therapeutic strategy for human GC treatment.

Changes in transcript levels of cassava superoxide dismutase and catalase during interaction with Phytopythium sp.

Among the biotic factors affecting the production of cassava (Manihot esculenta Crantz), root rot is one of the most severe diseases worldwide. In northern Brazil, the oomycete Phytopythium sp. causes cassava soft root rot disease in the Amazon region. Seeking to understand this plant-pathogen interaction, our main aim was to evaluate changes in expression levels of superoxide dismutase (SOD) and catalase (CAT) genes in susceptible cassava infected by Phytopythium sp., using semi-quantitative RT-PCR assays. Thus, four SOD (three CuZnSOD and one MnSOD) and three CAT genes were selected from the cassava genome available in the Phytozome Database. Comparative sequence analysis revealed high identity between the deduced MeSOD and MeCAT proteins with SODs and CATs from GenBank Database. In addition, putative conserved motifs and metal-binding domains were found within MeCuZnSOD1-3 and MeMnSOD sequences. The deduced MeCAT proteins contain putative domains and conserved motifs, such as a peroxisomal targeting signal. Furthermore, phylogenetic analysis showed that MeCuZnSOD1 and MeCuZnSOD3 proteins were related to SOD [Cu–Zn] 2 (XP_021686055.1) from Hevea brasiliensis, while MeCuZnSOD2 was close to SOD [Cu–Zn] (XP_012089157.1) from Jatropha curcas. MeMnSOD showed a phylogenetic relationship with mitochondrial SOD [Mn] (XP_021684793.1) from H. brasiliensis. The results also revealed phylogenetic proximity of MeCAT2 and MeCAT3 with CAT isozyme 2 (XP_021675148.1), while MeCAT1 was close to CAT isozyme 1 (XP_021675149.1), both from H. brasiliensis. Semi-quantitative RT-PCR assays showed that Phytopythium sp. was able to suppress the expression of MeSOD genes at early stages of infection, while at 48 h after inoculation (HAI) cassava responded by increasing the expression of the MeCuZnSOD3 gene. For the CAT genes, the MeCAT1 gene showed an expression pattern similar to MeCuZnSOD3, while the expression of the MeCAT3 gene was increased at 24 and 48 HAI. The results revealed that cassava responded to Phytopythium sp. infection through changes in transcript levels of different SOD and CAT isoforms, which are known enzymes with crucial functions regarding the oxidative burst produced during plant-pathogen interactions.

Transcription factor ZmPLATZ2 positively regulate the starch synthesis in maize

Maize is one of the three major crops worldwide based on its yield and quality. Starch is crucial to both the yield and quality of maize as it accounts more than 60% of the seed weight, and its structure influences the quality of the crop. Starch synthase I (SSI) contributes to the majority of the starch synthase activity in the maize endosperm. An in-depth understanding of the starch synthesis regulatory mechanism would provide opportunities for improving the yield and quality of maize. In this study, ZmPLATZ2, a plant AT-rich sequence and zinc-binding protein (PLATZ) transcription factor related to starch synthesis, was selected based on co-expression analysis. The semiquantitative RT-PCR and qRT-PCR assays revealed that ZmPLATZ2 had a high expression in the endosperm, and reached the peak at 12 days after pollination (DAP). Different treatments demonstrated that ZmPLATZ2 was downregulated by the presence of sucrose. Subsequent transactivation and subcellular localization analyses showed that ZmPLATZ2 was localized in the nuclei without transactivation. Yeast one-hybrid and transient expression in maize endosperm indicated that ZmPLATZ2 could bind to the promoters of ZmSSI, ZmISA1, and ZmISA2 and increase their gene expression. After ZmPLATZ2 overexpression in rice, four starch synthesis genes were significantly upregulated in the transgenic plant, including the OsSSI gene. In vitro DAP-seq data showed that ZmPLATZ2 could bind to the CAAAAAAA element. In conclusion, our data support that ZmPLATZ2 binds to the CAAAAAAA element in the ZmSSI promoter and mediates the Glu signal pathway.

A Cassava CPRF-2-like bZIP Transcription Factor Showed Increased Transcript Levels during Light Treatment

bZIP proteins participate in the regulation of gene expression, playing crucial roles in various biological processes in plants, including response to environmental changes. Luminosity is an environmental factor of extreme importance for plant metabolism, acting as a regulator of its growth and development. Despite advances in the identification of bZIP proteins in several plant species, studies on these transcription factors in cassava are lacking. Cassava (Manihot esculenta Crantz) is one of the most important food crops in tropical and subtropical regions, mainly in developing countries, where its storage root is a major source of calories for low-income people. Our main aim was the isolation of a cDNA sequence encoding a bZIP protein from cassava (MebZIP) as well as the in silico characterization of its nucleotide and deduced amino acid sequences. In addition, we evaluated the expression pattern of the MebZIP gene in response to light, and its possible relationship with regulation of the chalcone synthase (MeCHS) gene. RT-PCR and 3' and 5' RACE assays were used to isolate the full-length cDNA sequence of MebZIP. Bioinformatics tools were used to characterize the nucleotide and amino acid sequences of MebZIP. Semiquantitative RT-PCR assays were used to evaluate the expression levels of MebZIP and MeCHS genes. We isolated the full-length cDNA sequence of MebZIP with a 1320-bp ORF encoding a deduced protein with a predicted molecular weight and isoelectric point of 47 kDa and 5.85, respectively. Comparative analyses with GenBank sequences showed high identity of MebZIP with bZIP CPRF-2 of Hevea brasiliensis (XP_021650934) and Petroselinum crispum (Q99090.2). Besides the basic region and leucine zipper domains, MebZIP contains putative conserved domains (D1- D4), found in parsley CPRF-2 and bZIP proteins closely related to this protein. Since CPRF proteins are known for their function in regulation of the CHS gene by light, we evaluated the expression levels of the MebZIP gene and the possible target gene to be regulated by MebZIP (the MeCHS gene) in cassava under light conditions. Semi-quantitative RT-PCR assays revealed that MebZIP transcription increased in response to white light, with maximum expression levels at 6 h of light exposure. On the other hand, the expression levels of the MeCHS gene were statistically constant in all samples, indicating that they were not influenced by the experimental conditions used here. The putative MebZIP protein identified in this work contains the conserved domains (bZIP, D1-D4) that indicate its functionality, thus allowing it to be considered a new member of the bZIP transcription factor CPRF-2 family. The expression levels of the MebZIP gene increased during white light exposure, indicating a potential function in light-response in cassava.

Identification and Expression Analyses of Olfactory Gene Families in the Rice Grasshopper, Oxya chinensis, From Antennal Transcriptomes.

The rice grasshopper Oxya chinensis is an important agricultural pest of rice and other gramineous plants. Chemosensory genes are crucial factors in direct interactions with odorants in the olfactory process. Here we identified genes encoding 18 odorant-binding proteins (OBPs), 13 chemosensory proteins (CSPs), 94 olfactory receptors (ORs), 12 ionotropic receptors (IRs), and two sensory neuron membrane proteins (SNMPs) from O. chinensis using an transcriptomic approach. Semi-quantitative RT-PCR assays revealed that six OBP-encoding genes (OchiOBP4, 5, 8, 9, 10, and 14), one CSP gene (OchiOBP10) and two IR genes (OchiIR28 and 29) were exclusively expressed in antennae, suggesting their roles in olfaction. Real-time quantitative PCR analyses revealed that genes expressed exclusively or predominantly in antennae also displayed significant differences in expression levels between males and females. Among the differentially expressed genes, 17 OR-encoding genes, one CSP- and one SNMP-gene showed female-biased expression, suggesting that they may be involved in some female-specific behaviors such as seeking oviposition site; whereas the three remaining OR-encoding genes showed male-biased expression, indicating their possible roles in sensing female sex pheromones. Our results laid a solid foundation for future studies to reveal olfactory mechanisms as well as designing strategies for controlling this rice pest.

Candidate genes involved in spiroacetal biosynthesis in the oriental fruit fly, Bactrocera dorsalis

Spiroacetals are widespread in nature as components of volatile semiochemical secretions from many insect species. The general pathway for spiroacetal biosynthesis in Bactrocera sp. is preliminarily established, but many genes involved in this pathway remain to be characterized. By analyzing transcriptomes of the rectal glands (RGs) from immature and mature females of the oriental fruit fly, Bactrocera dorsalis, a set of genes encoding two acetyl-CoA carboxylases (ACCs), two fatty acid synthases (FASs), eight desaturases (DESs), twelve fatty acyl-CoA reductases (FARs), seventy-two cytochrome P450 enzymes (CYPs), and twenty-three odorant binding proteins (OBPs) were identified. We investigated the expression of candidate genes in immature and mature stages based on the RNA-seq data and Real-time quantitative PCR. Expression profiling revealed that some of these genes were primarily expressed in female rectal glands among different tissues, and were up-regulated in mature females. Semi-quantitative RT-PCR assays were also adapted to examine tissue-specific expression of selected candidate genes. Additionally, their putative functions in spiroacetal synthesis and transportation are proposed. Our study provided large-scale sequence information for further functional studies on spiroacetal biosynthetic pathways.

Expression profiles of defense genes in cassava storage roots upon exposure to Phytopythium sp., causal agent of soft root rot disease

Cassava is one of the most important crops in Africa, Asia and Latin America, where millions of people depend on its starchy storage roots as their main source of energy. In Brazil, cassava is cultivated in all regions; however, soft root rot disease has hampered its production mainly in the Amazon region. The identification of Phytopythium sp., as the causal agent of soft root rot disease in cassava plants collected in Brazil, was recently reported. Here, our main aim was to evaluate changes in expression levels of genes possibly involved in compatible cassava- Phytopythium sp. interaction. Semi-quantitative RT-PCR assays were performed to evaluate the expression levels of genes coding for proteins with known functions in plant defense, such as enzymes involved in oxidative burst and the phenylpropanoid pathway, in detached cassava storage roots inoculated and non-inoculated with pathogen. Our results showed that cassava responded to pathogen infection through up-regulation of genes coding for superoxide dismutase, phenylalanine ammonia-lyase, chalcone synthase, cysteine proteinase inhibitor and RING zinc finger protein. Furthermore, the expression pattern of the allergenic-related glutamic acid-rich protein and translationally controlled tumor protein genes in detached roots used here validated the absence of post-harvest physiological deterioration, a known endogenous disorder affecting the storage roots of cassava at 24–72 h after harvest. This is the first study of interaction between cassava and Phytopythium sp. at the molecular level contributing to understanding how cassava responds to pathogen infection, as well as to future strategies of molecular breeding regarding the tolerance to soft root rot disease.

A novel FADS2 isoform identified in human milk fat globule suppresses FADS2 mediated Δ6-desaturation of omega-3 fatty acids

IntroductionThe only known non-pharmacological means to alter long chain polyunsaturated fatty acid (LCPUFA) abundance in mammalian tissue is by altering substrate fatty acid ratios. Alternative mRNA splicing is increasingly recognized as a modulator of protein structure and function. Here we report identification of a novel alternative transcript (AT) of fatty acid desaturase 2 (FADS2) that inhibits production of omega-3 but not omega-6 LCPUFA, discovered during study of ATs in human milk fat globules (MFG). MethodsHuman breastmilk collected from a single donor was used to isolate MFG. An mRNA-sequencing library was constructed from the total RNA isolated from the MFG. The constructed library was sequenced using an Illumina HiSeq instrument operating in high output mode. Expression levels of evolutionary conserved FADSAT were measured using cDNA from MFG by semi-quantitative RT-PCR assay. ResultsRNA sequencing revealed >15,000 transcripts, including moderate expression of the FADS2 classical transcript (CS). A novel FADS2 alternative transcript (FADS2AT2) with 386 amino acids was discovered. When FADS2AT2 was transiently transfected into MCF7 cells stably expressing FADS2, delta-6 desaturation (D6D) of alpha-linolenic acid 18:3n−3 → 18:4n−3 was suppressed as were downstream products 20:4n−3 and 20:5n−3. In contrast, no significant effect on D6D of linoleic acid 18:2n−6 → 18:3n−6 or downstream products was observed. FADS2, FADS2AT1 and 5 out of 8 known FADS3AT were expressed in MFG. FADS1, FADS3AT3, and FADS3AT5 are undetectable. ConclusionThe novel, noncatalytic FADS2AT2 regulates FADS2CS-mediated Δ6-desaturation of omega-3 but not omega-6 PUFA biosynthesis. This spliced isoform mediated interaction is the first molecular mechanism by which desaturation of one PUFA family but not the other is modulated.

Suppression of lung inflammation by the methanol extract of Spilanthes acmella Murray is related to differential regulation of NF-κB and Nrf2

Ethnopharmacological relevanceAlthough Spilanthes acmella has been used to relieve inflammation, fever, pain, or infection in traditional Asian medicine, experimental evidence supporting these functions is scarce. Here, we examined an anti-inflammatory function and a possible underlying mechanism of S. acmella Murray (SAM). Materials and methodThe methanol extract of SAM was fingerprinted by HPLC. C57BL/6 mice were administered with a single intratracheal (i.t.) LPS and 2 h later with a single i.t. SAM. The effect of SAM on lung inflammation was assessed by histology, semi-quantitative RT-PCR, and MPO assay of lung tissue. The effects of SAM on a pro-inflammatory factor NF-κB and an anti-inflammatory factor Nrf2 were analyzed by immunoblotting of nuclear proteins and by semi-quantitative RT-PCR analysis of mRNA of the genes governed by these transcription factors. V5-Nrf2 was precipitated by an anti-V5 antibody and the ubiquitinated V5-Nrf2 was revealed by immunoblotting of HA-tagged ubiquitin. ResultsThe i.t. SAM robustly diminished a neutrophilic lung inflammation induced by i.t. LPS treatment of mice. In RAW 264.7 cells, SAM suppressed the nuclear localization of NF-κB and the expression of NF-κB-dependent cytokine genes. SAM increased the level of Nrf2 in the nucleus and the expression of Nrf2-dependent genes while suppressing ubiquitination of Nrf2. ConclusionOur results suggest that SAM can suppress a neutrophilic inflammation in mouse lungs, which is associated with suppressed NF-κB and activated Nrf2. Our results provide experimental evidence supporting the anti-inflammatory function of S. acmella.

Transcript and protein expression analysis of proteases in the blood stages of Plasmodium falciparum

Proteases are crucial enzymes with varying roles in living organisms. In the malaria parasite Plasmodium falciparum, the role of proteases has been deciphered mainly in the asexual blood stages and shown to represent promising drug targets. However, little is known about their functions in the sexual blood stages, which are important for transmission of the disease from the human to the mosquito vector. Determination of their stage-specific expression during the malaria life-cycle is crucial for the effective design of multi-stage anti-malaria drugs aimed at eradicating the disease. In this study, we screened the P. falciparum genome database for putative proteases and determined the transcript and protein expression profiles of selected proteases in the plasmodial blood stages using semi-quantitative RT-PCR and indirect immunofluorescence assay. Database mining identified a total of 148 putative proteases, out of which 18 were demonstrated to be expressed in the blood stages on the transcript level; for 12 of these proteins synthesis was confirmed. While three of these proteases exhibit gametocyte-specific expression, two are restricted to the asexual blood stages and seven are found in both stages, making them interesting multi-stage drug targets.

Isolation and characterization of LcSAP, a Leymus chinensis gene which enhances the salinity tolerance of Saccharomyces cerevisiae.

A number of members of the SAP ("stress-associated protein") gene family have been implicated in the plant stress response. Here, a SAP gene has been isolated using PCR RACE from the perennial grass Leymus chinensis, a species which has reputation for ecological adaptability. The 17.6kDa LcSAP product comprised 161 residues, including both an A20 domain and an AN1 domain, a feature of type I SAPs. Using a semi-quantitative RT-PCR assay to profile its transcription, it was shown that LcSAP was more strongly transcribed in the leaf than in the root under control conditions. The level of LcSAP transcription began to rise 6h after the plant's exposure to 400mM NaCl, and the abundance of transcript remained stable for at least 24h. Exposing the plant to 100mM Na2CO3 also induced LcSAP transcription, but the abundance of SAP transcript faded after 6h. When LcSAP was introduced into yeast cells, the transgenic cells grew better than wild type ones when the medium contained 1.4M NaCl. The ability of LcSAP to respond to salinity stress in yeast suggests that it also makes a contribution to the stress tolerance shown by L. chinensis.

Transgenic sugarcane overexpressing CaneCPI-1 negatively affects the growth and development of the sugarcane weevil Sphenophorus levis.

Transgenic sugarcane expressing CaneCPI-1 exhibits resistance to Sphenophorus levis larvae. Transgenic plants have widely been used to improve resistance against insect attack. Sugarcane is an economically important crop; however, great losses are caused by insect attack. Sphenophorus levis is a sugarcane weevil that digs tunnels in the stem base, leading to the destruction of the crop. This insect is controlled inefficiently by chemical insecticides. Transgenic plants expressing peptidase inhibitors represent an important strategy for impairing insect growth and development. Knowledge of the major peptidase group present in the insect gut is critical when choosing the most effective inhibitor. S. levis larvae use cysteine peptidases as their major digestive enzymes, primarily cathepsin L-like activity. In this study, we developed transgenic sugarcane plants that overexpress sugarcane cysteine peptidase inhibitor 1 (CaneCPI-1) and assessed their potential through feeding bioassays with S. levis larvae. Cystatin overexpression in the transgenic plants was evaluated using semi-quantitative RT-PCR, RT-qPCR, and immunoblot assays. A 50% reduction of the average weight was observed in larvae that fed on transgenic plants in comparison to larvae that fed on non-transgenic plants. In addition, transgenic sugarcane exhibited less damage caused by larval attack than the controls. Our results suggest that the overexpression of CaneCPI-1 in sugarcane is a promising strategy for improving resistance against this insect.

PS 01-18] MECHANISM OF HYPERTENSIVE CARDIAC HYPERTROPHY IN CULTURED NEONATAL RAT VENTRICULAR CARDIAC MYOCYTES

Objective: hypertrophy and impaired cardiac function are commonly observed in some patients with hypertension. Angiotensin II is a critical growth factor to mediate cardiac hypertrophy. MicroRNAs (miRs) have been identified and associated with several cardiovascular diseases, including ischemic cardiomyopathy and cardiac hypertrophy. MiR-21 was reported to be upregulated during adult rat cardiac remodeling. The importance of miR-21* in cardiomyocyte hypertrophy has also been demonstrated. Design and Method: Wistar rats (weight, 120–150 g) were purchased. Ventricular cardiomyocytes were isolated from 1 to 2-day-old neonatal wistar rat as described previously with minor modification. After 24 h of serum starvation, the cardiomyocytes were stimulated with or without 1 umol/L Ang II for 48 h. The hypertrophy cardiomyocytes were identified by semi-quantitative RT-PCR assay. Total miRNAs were extracted using mirVANA miRNA isolation kit. The miRNAs quality and quantity were determined by a NanoDrop 1000 Spectrophotometer. The miRNAs were reverse transcribed by TaqMan MicroRNA Reverse Transcription Kit. qRT-PCR was performed using TaqMan Universal PCR Master Mix II on the ABI 7500 System according to the manufacturer's recommendations. Total proteins isolated from cultured cardiomyocytes, with or without 1 umlo/L Ang II for 48 h, were prepared by M-PER mammalian protein extraction reagent. SDS-polyacrylamide gel electrophoresis was performed using standard procedures. Results: We demonstrated that miR-21* was markedly upregualted in Ang II-induced neonatal rat cardiac myocytes, while miR-21 was reduced. We might speculate that the miRNA expression profile is different between neonatal rat and adult rat cardiac myocytes. Our results further showed that Ang II led to a significant reduction of PTEN expression level and a significant promotion of phosphorylation level of PI3K. In contrast, treatment with prohypertrophic molecule Ang II reduced the phosphorylation level of Akt. Conclusions: We therefore speculate that miR-21* might have two opposite effects on Ang II-induced cardiac hypertrophy through various signaling pathways.

A R2R3-MYB transcription factor gene in common wheat (namely TaMYBsm1) involved in enhancement of drought tolerance in transgenic Arabidopsis

We isolated the TaMYBsm1 genes, encoding R2R3-type MYB proteins in common wheat, aimed to uncover the possible molecular mechanisms related to drought response. The TaMYBsm1 genes, TaMYBsm1-A, TaMYBsm1-B and TaMYBsm1-D, were isolated and analyzed from the common wheat cultivar Shimai 15. Their expression patterns under PEG 6000 and mannitol were monitored by semi-quantitative RT-PCR and β-glucuronidase (Gus) assay. The function of TaMYBsm1-D under drought stress in transgenic Arabidopsis plants was investigated, and the germination rate, water loss rate, as well as the proline and malondialdehyde (MDA) content were compared with that in wild type (WT) plants. The expression of three downstream genes (DREB2A, P5CS1 and RD29A) in TaMYBsm1-D transgenic plants was analyzed. The R2R3-MYB domains of the MYBsm1 proteins were highly conserved in plants. In addition, the TaMYBsm1 proteins were targeted to the nucleus and contained transcriptional activation domains (TADs). Gus assay and semi-quantitative RT-PCR analysis demonstrated that the TaMYBsm1 genes were up-regulated when the wheat was treated by PEG and mannitol. Compared with WT plants, the germination rates were much higher, but the water loss rates were much lower in TaMYBsm1-D overexpression plants. TaMYBsm1-D transgenic plants showed distinct higher proline contents but a lower MDA content than the WT plants. The three downstream genes were highly expressed in TaMYBsm1-D transgenic plants. We concluded from these results that TaMYBsm1 genes play an important role in plant drought stress tolerance through up-regulation of DREB2A, P5CS1 and RD29A. The increase of proline content and decrease of MDA content may also be involved in the drought response.

Novel Pectate Lyase Genes of Heterodera glycines Play Key Roles in the Early Stage of Parasitism.

Pectate lyases are known to play a key role in pectin degradation by catalyzing the random cleavage of internal polymer linkages (endo-pectinases). In this paper, four novel cDNAs, designated Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7, that encode pectate lyases were cloned and characterized from the soybean cyst nematode, Heterodera glycines. The predicted protein sequences of HG-PEL-3, HG-PEL-4 and HG-PEL-6 differed significantly in both their amino acid sequences and their genomic structures from other pectate lyases of H. glycines (HG-PEL-1, HG-PEL-2 and HG-PEL-7). A phylogenetic study revealed that the pectate lyase proteins of H. glycines are clustered into distinct clades and have distinct numbers and positioning of introns, which suggests that the pectate lyase genes of H. glycines may have evolved from at least two ancestral genes. A Southern blot analysis revealed that multiple Hg-pel-6-like genes were present in the H. glycines genome. In situ hybridization showed that four novel pectate lyases (Hg-pel-3, Hg-pel-4, Hg-pel-6 and Hg-pel-7) were actively transcribed in the subventral esophageal gland cells. A semi-quantitative RT-PCR assay supported the finding that the expression of these genes was strong in the egg, pre-parasitic second-stage juvenile (J2) and early parasitic J2 stages and that it declined in further developmental stages of the nematode. This expression pattern suggests that these proteins play a role in the migratory phase of the nematode life cycle. Knocking down Hg-pel-6 using in vitro RNA interference resulted in a 46.9% reduction of the number of nematodes that invaded the plants and a 61.5% suppression of the development of H. glycines females within roots compared to the GFP-dsRNA control. Plant host-derived RNAi induced the silencing of the Hg-pel-6gene, which significantly reduced the nematode infection levels at 7 Days post inoculation (dpi). Similarly, this procedure reduced the number of female adults at 40 dpi, which suggests the important roles of this gene in the early stages of parasitism. Our combined data suggest that two types of pectate lyases are present in the H. glycines genome and may have different roles during infection.

Lupeol enhances inhibitory effect of 5-fluorouracil on human gastric carcinoma cells.

Lupeol, a dietary triterpene present in many fruits and medicinal plants, has been reported to possess many pharmacological properties including cancer-preventive and anti-cancer effects in vitro and in vivo. Here, we investigated the anti-cancer efficacy and adjuvant chemotherapy action of lupeol in gastric cancer (GC) cells (SGC7901 and BGC823) and explored the underlying mechanisms. Cells were treated with lupeol and/or 5-fluorouracil (5-Fu) and subjected to cell viability, colony formation, apoptosis, western blot, semiquantitative RT-PCR, and xenograft tumorigenicity assay. Our results showed that lupeol and 5-Fu inhibited the proliferation of SGC7901 and BGC823 cells, and combination treatment with lupeol and 5-Fu resulted in a combination index < 1, indicating a synergistic effect. Co-treatment with lupeol and 5-Fu induced apoptosis through up-regulating the expressions of Bax and p53 and down-regulating the expressions of survivin and Bcl-2. Furthermore, co-treatment displayed more efficient inhibition of tumor weight and volume on BGC823 xenograft mouse model than single-agent treatment with 5-Fu or lupeol. Taken together, our findings highlight that lupeol sensitizes GC to 5-Fu treatment, and combination treatment with lupeol and 5-Fu would be a promising therapeutic strategy for human GC treatment.