Showing papers in "Acta Biochimica et Biophysica Sinica in 2007"
TL;DR: This review introduces the recent developments in Fourier transform infrared (FTIR) spectroscopy technique and its applications to protein structural studies.
Abstract: Infrared spectroscopy is one of the oldest and well established experimental techniques for the analysis of secondary structure of polypeptides and proteins. It is convenient, non-destructive, requires less sample preparation, and can be used under a wide variety of conditions. This review introduces the recent developments in Fourier transform infrared (FTIR) spectroscopy technique and its applications to protein structural studies. The experimental skills, data analysis, and correlations between the FTIR spectroscopic bands and protein secondary structure components are discussed. The applications of FTIR to the secondary structure analysis, conformational changes, structural dynamics and stability studies of proteins are also discussed.
2,685 citations
TL;DR: The zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies, and the EF1α, Rpl13α and 18S rRNA genes are more suitable as a reference gene panel for zebra fish tissue analysis.
Abstract: The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or house- keeping genes. However, there is emerging evidence that even reference genes can be regulated under differ- ent conditions. qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The β-actin, EF1α and Rpl13α genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1α, Rpl13α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.
582 citations
TL;DR: Special emphasis is given to the importance of that type of enzyme and their related phenolic ferulic acid compound in biotechnological processes, and industrial and medicinal applications.
Abstract: Feruloyl esterases represent a diverse group of hydrolases catalyzing the cleavage and formation of ester bonds between plant cell wall polysaccharide and phenolic acid. They are widely distributed in plants and microorganisms. Besides lipases, a considerable number of microbial feruloyl esterases have also been discovered and overexpressed. This review summarizes the latest research on their classification, production, and biophysicochemical properties. Special emphasis is given to the importance of that type of enzyme and their related phenolic ferulic acid compound in biotechnological processes, and industrial and medicinal applications.
146 citations
TL;DR: Some strategies derived from tetracycline-inducible system alone, as well as the combined use of Tet-based systems and Cre/lox P switching gene expression system, have been newly developed to allow more flexibility for exploring gene functions in health and disease, and produce credible transgenic animal models for various human diseases.
Abstract: To accurately analyze the function of transgene(s) of interest in transgenic mice, and to generate credible transgenic animal models for multifarious human diseases to precisely mimic human disease states, it is critical to tightly regulate gene expression in the animals in a conditional manner. The ability to turn gene expression on or off in the restricted cells or tissues at specific time permits unprecedented flexibility in dissecting gene functions in health and disease. Pioneering studies in conditional transgene expression have brought about the development of a wide variety of controlled gene expression systems, which meet this criterion. Among them, the tetracycline-controlled expression systems (e.g. Tet-off system and Tet-on system) have been used extensively in vitro and in vivo. In recent years, some strategies derived from tetracycline-inducible system alone, as well as the combined use of Tet-based systems and Cre/lox P switching gene expression system, have been newly developed to allow more flexibility for exploring gene functions in health and disease, and produce credible transgenic animal models for various human diseases. In this review these newly developed strategies are discussed.
84 citations
TL;DR: Results suggest that OA acts as the effective component of FLL by exerting its cytotoxicity towards target tumor cells through activation of caspases and cleavage of PARP.
Abstract: It has been shown that Fructus Ligustri Lucidi (FLL), a promising traditional Chinese medicine, can inhibit the growth of tumors. However, the effective component and molecular mechanism of FLL act to inhibit tumor proliferation are unclear. In this study, we demonstrated that oleanolic acid (OA), a principal chemical component of FLL, inhibited the proliferation of human leukemia HL60 cells in culture. MTT assay showed that treatment of HL60 cells with FLL crude extracts or OA dramatically blocked the growth of target tumor cell in a time- and dose-dependent manner. Morphological changes of the nuclei and DNA fragmentation showed that apoptotic cell death occurred in the HL60 cells after treating with FLL extracts (20 mg/ml) or OA (3.65×10 −2 mg/ml). Furthermore, flow cytometry assay showed that treatment of HL60 cells with FLL or OA caused an increased accumulation of G1 and sub-G1 subpopulations. Western blot analysis showed that caspase-9 and caspase-3 were activated, accompanied by the cleavage of poly (ADP-ribose) polymerase (PARP) in the target cells during FLL- or OA-induced apoptosis. These results suggest that OA acts as the effective component of FLL by exerting its cytotoxicity towards target tumor cells through activation of caspases and cleavage of PARP.
79 citations
TL;DR: The results indicated that increasing levels of HHcy significantly increased genome hypomethylation in B1 repetitive elements, and showed that the varied detrimental effects ofHHcy could be attributed to different concentrations through different mechanisms.
Abstract: Hyperhomocysteinemia (HHcy), which is an independent risk factor for atherosclerosis, might cause dysregulation of gene expression, but the characteristics and key links involved in its pathogenic mechanisms are still poorly understood. The objective of the present study was to investigate the effect of HHcy on DNA methylation and the underlying mechanism of homocysteine (Hcy)-induced DNA methylation. HHcy was induced in Sprague-Dawley rats after 4 weeks of a low, medium or high methionine diet. The levels of total homocysteine, S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) were detected by high-performance liquid chromatography. The expression levels of genes and proteins of S-adenosylhomocysteine hydrolase, DNA methyltransferase and methyl-CpG-binding domain 2 were detected by real-time reverse transcription-polymerase chain reaction and Western blot analysis. A high-throughput quantitative methylation assay using fluorescence-based real-time polymerase chain reaction was employed to determine the levels of DNA methylation. The results indicated that HHcy induced the elevation of AdoHcy concentration, the decline of AdoMet concentration, the ratios of AdoMet/AdoHcy and the RNA and protein expression of S-adenosylhomocysteine hydrolase and methyl-CpG-binding domain 2, as well as an increase of DNA methyltransferase activity. With different methylation-dependent restriction endonucleases, the aberrant demethylation was found to prefer CCGG sequences to CpG islands. Increasing levels of HHcy significantly increased genome hypomethylation in B1 repetitive elements. The impacts of different levels of HHcy showed that the varied detrimental effects of HHcy could be attributed to different concentrations through different mechanisms. In mild and moderate HHcy, the Hcy might primarily influence the epigenetic regulation of gene expression through the interference of transferring methyl-group metabolism. However, at high Hcy concentrations, the impacts might be more injurious through oxidative stress, apoptosis and inflammation.
74 citations
TL;DR: Current knowledge on the structure and function of the NS1A protein is outlined, a multifunctional protein that plays a significant role in resisting the host antiviral response during the influenza infection.
Abstract: The avian influenza A virus currently prevailing in Asia causes fatal pneumonia and multiple organ failure in birds and humans. Despite intensive research, understanding of the characteristics of influenza A virus that determine its virulence is incomplete. NS1A protein, a non-structural protein of influenza A virus, was reported to contribute to its pathogenicity and virulence. NS1A protein is a multifunctional protein that plays a significant role in resisting the host antiviral response during the influenza infection. This review briefly outlines the current knowledge on the structure and function of the NS1A protein.
73 citations
TL;DR: Results indicated that JcPIP2 probably played a role in drought resistance in J. curcas, and indicated that this protein is ubiquitously located in all tested tissues of the plant.
Abstract: Water channel proteins, aquaporins, play fundamental roles in transmembrane water movements in plants. A new full-length cDNA encoding aquaporin was isolated from the seedlings of Jatropha curcas. The gene of the plasma membrane intrinsic protein (PIP) from J. curcas (JcPIP2) contained an 843 bp open reading frame encoding a protein of 280 amino acids. The amino acid sequence showed 94% identity with Ricinus communis PIP. Injection of JcPIP2 complementary RNA into Xenopus oocytes increased 10-fold the osmotic water permeability of the oocytes. Immunodetection of JcPIP2 with anti-JcPIP2 antibody indicated that this protein is ubiquitously located in all tested tissues of the plant. To investigate the relationship between aquaporins and drought resistance in J. curcas, the abundance of JcPIP2 was examined in seedlings of two J. curcas populations, GaoYou CSC63 and YanBian S1, under water deficit with PEG6000. Under field conditions, those two populations, GaoYou CSC63 was resistant to water deficit, but YanBian S1 was sensitive to water deprivation. With the increasing degree of drought stress, JcPIP2 level increased in seedlings of GaoYou CSC63, whereas there was no significant change in seedlings of YanBian S1. Compared with YanBian S1, GaoYou CSC63 also showed higher root hydraulic conductivity and lower decreasing trend in the seedlings under water deficit. These results indicated that JcPIP2 probably played a role in drought resistance in J. curcas.
68 citations
TL;DR: The molecular mechanism of ethylenediaminetetraacetic acid (EDTA)-induced membrane destabilization has been studied using a combination of four biophysical techniques on artificial lipid membranes and growth in size and shape of the membrane protrusion was found to be time-dependent upon exposure to EDTA.
Abstract: The molecular mechanism of ethylenediaminetetraacetic acid (EDTA)-induced membrane destabilization has been studied using a combination of four biophysical techniques on artificial lipid membranes. Data from Langmuir film balance and epifluorescence microscopy revealed the fluidization and expansion effect of EDTA on phase behavior of monolayers of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixtures of DPPC and metal-chelating lipids, such as N(alpha),N(alpha)-Bis[carboxymethyl]-N(epsilon)-[(dioctadecylamino)succinyl]-L-lysine or 1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl]. A plausible explanation could be drawn from the electrostatic interaction between negatively charged groups of EDTA and the positively charged choline head group of DPPC. Intercalation of EDTA into the lipid membrane induced membrane curvature as elucidated by atomic force microscopy. Growth in size and shape of the membrane protrusion was found to be time-dependent upon exposure to EDTA. Further loss of material from the lipid membrane surface was monitored in real time using a quartz crystal microbalance. This indicates membrane restabilization by exclusion of the protrusions from the surface. Loss of lipid components facilitates membrane instability, leading to membrane permeabilization and lysis.
65 citations
TL;DR: The expression and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H1299 and SPC-A-1 are detected, thus providing a potential choline-starvation strategy of cancer interference through targeting choline transporting.
Abstract: Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential "choline-starvation" strategy of cancer interference through targeting choline transporters, especially CTL1.
61 citations
TL;DR: The posttranslational modification of histones plays an important role in chromatin regulation, and the imbalance of histone methylation has been linked to cancers.
Abstract: The posttranslational modification of histones plays an important role in chromatin regulation. Histone methylation influences constitutive heterochromatin, genomic imprinting, X-chromosome inactivation and gene transcription. Histone demethylase catalyzes the removal of methyl groups on lysine or arginine residues of histones. Two kinds of histone lysine demethylases have been identified, including lysine specific demethylase 1 and Jumonji C (JmjC) domain family proteins. These histone demethylases are involved in the regulation of gene expression. Histone modification is a dynamic process, and the imbalance of histone methylation has been linked to cancers. Therefore, histone demethylases may represent a new target for anti-cancer therapy.
TL;DR: Integrons were prevalent and played an important role in multidrug resistance in ESBL-producing K. pneumoniae.
Abstract: This study is concerned with the level of antibiotic resistance of extended-spectrum β- lactamase (ESBL)-producing Klebsiella pneumoniae, isolated in Shantou, China, and its mechanism. Seventy- four non-repetitive clinical isolates of K. pneumoniae producing ESBLs were isolated over a period of 2 years. Antibiotic susceptibility, carried out by Epsilometer test, showed that most of the isolates were multiresistant. Polymerase chain reaction showed that, among the several types of β-lactamases, SHV was the most prevalent, TEM was the second most prevalent, and CTX-M was the least prevalent. Sixty-nine isolates were positive for integrase gene IntI1, but no IntI2 or IntI3 genes were found. The variable region of class 1 integrons were amplified and further identified by sequencing. Thirteen different gene cassettes and 11 different cassette combinations were detected. Dfr and aadA cassettes were predominant and cassette combinations dfrA12, orfF and aadA2 were most frequently found. No gene cassettes encoding ESBLs were found. Integrons were prevalent and played an important role in multidrug resistance in ESBL-producing K. pneumoniae.
TL;DR: These breakthrough findings shed light on the current understanding of the structure and function of the various F-box proteins and some protein-protein interaction motifs at their carboxy-terminus, such as Trp-Asp repeats or leucine rich repeats.
Abstract: The F-box protein is an important component of the E3 ubiquitin ligase Skp1-Cullin-F-box protein complex. It binds specific substrates for ubiquitin-mediated proteolysis. The F-box proteins contain a signature F-box motif at their amino-terminus and some protein-protein interaction motifs at their carboxy- terminus, such as Trp-Asp repeats or leucine rich repeats. Many F-box proteins have been identified to be involved in plant hormone response as receptors or important medial components. These breakthrough findings shed light on our current understanding of the structure and function of the various F-box proteins, their related plant hormone signaling pathways, and their roles in regulating plant development.
TL;DR: It was found that mean pulmonary arterial pressure (mPAP) increased significantly after 7 d of hypoxia, and linear correlation analysis showed that HIF-1α mRNA, TGF-β1 mRNA,TGF-α protein were positively correlated with mPAP, vessel morphometry and right ventricular hypertrophy index.
Abstract: The present study was undertaken to investigate the dynamic expression of hypoxia inducible factor-1α (HIF-1α) and transforming growth factor-β1 (TGF-β1) in hypoxia-induced pulmonary hypertension of rats. It was found that mean pulmonary arterial pressure (mPAP) increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d of hypoxia. HIF-1α mRNA staining was less positive in the control, hypoxia for 3 d and hypoxia for 7 d, but began to enhance significantly after 14 d of hypoxia, then remained stable. Expression of HIF-1α protein in the control was less positive, but was up-regulated in pulmonary arterial tunica intima of all hypoxic rats. TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-β1 protein staining was less positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia, and then weakening after 14 and 21 d of hypoxia. Western blotting showed that HIF-1α protein levels increased significantly after 7 d of hypoxia and then remained at a high level. TGF-β1 protein level was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia, and then decreasing after 14 and 21 d of hypoxia. Linear correlation analysis showed that HIF-1α mRNA, TGF-β1 mRNA, TGF-β1 protein were positively correlated with mPAP, vessel morphometry and right ventricular hypertrophy index. TGF-β1 protein (tunica adventitia) was negatively correlated with HIF-1α mRNA. Taken together, our results suggest that changes in HIF-1α and TGF-β1 expression after hypoxia play an important role in hypoxia-induced pulmonary hypertension of rats.
TL;DR: Six new miRNAs from rat hippocampus were cloned and expressed at significantly higher levels in the hippocampus than in other tissues, including cerebral cortex, heart, liver, lung and kidney.
Abstract: MicroRNAs (miRNAs) are small regulatory molecules post-transcriptionally suppressing mRNA activity. Many miRNAs in various organisms have been cloned but many unknown miRNAs remain to be identified. Here we describe the cloning of six new miRNAs from rat hippocampus. Among them, four were not found in the rat miRBase, but were identical to their human and/or mouse homolog, therefore they were designated as rno-miR-92b, rno-miR-146b, rno-let-7g, and rno-miR-551b. The other two were derived from the other arms of the known miRNA precursors of rno-miR-330 and rno-miR-384, and were not found in miRBase of all organisms. They were designated as rno-miR-330* and rno-miR-384*. The expression of these miRNAs was confirmed by RNA-tailing and primer-extension real-time reverse transcription-polymerase chain reaction. These six miRNAs were expressed at significantly higher levels in the hippocampus than in other tissues, including cerebral cortex, heart, liver, lung and kidney. miR-384* was 10 times more abundant than miR-384 in rat hippocampus, but little difference was found between miR-330* and miR-330 expression in the same tissue.
TL;DR: The results show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of cal protectin in the gut.
Abstract: Calprotectin, a heterodimeric complex belonging to the S100 protein family, has been found predominantly in the cytosolic fraction of neutrophils. In the present study, human calprotectin was purified from neutrophils using two-step ion exchange chromatography. The purified protein was used for circular dichroism study and fluorescence analysis in the presence of calcium and zinc at physiological concentrations, as well as for assessment of its inhibitory activity on the K562 leukemia cell line. The thermal stability of the protein at pH 7.0 (physiological pH) and 8.0 (similar to intestinal pH) was also compared. The results of cell proliferation analysis revealed that human calprotectin initiated growth inhibition of the tumor cells in a dose-dependent manner. The intrinsic fluorescence emission spectra of human calprotectin (50 microg/ml) in the presence of calcium and zinc ions show a reduction in fluorescence intensity, reflecting a conformational change within the protein with exposure of aromatic residues to the protein surface that is important for the biological function of calprotectin. The far ultraviolet-circular dichroism spectra of human calprotectin in the presence of calcium and zinc ions at physiological concentrations show a decrease in the alpha-helical content of the protein and an increase in beta- and other structures. Our results also show that increasing the pH level from 7.0 to 8.0 leads to a marked elevation in the thermal stability of human calprotectin, indicating a significant role for pH in the stability of calprotectin in the gut.
TL;DR: It is suggested that the conserved OmpW could be an effective vaccine candidate against infection by V. alginolyticus.
Abstract: Vibrio alginolyticus is one of the Vibrio pathogens common to humans and marine animals. During infection and induction of the host immune response, outer membrane proteins of bacteria play an important role. In this study, an outer membrane protein gene (ompW) was cloned from V. alginolyticus and expressed in Escherichia coli. The 645 bp open reading frame (ORF) encodes a protein of 214 amino acid residues with a predicted molecular weight of 23.3 kDa. The amino acid sequence showed a high identity with that of Photobacterium damselae (96.2%) and Vibrio parahaemolyticus (94.4%). The alignment analysis indicated that OmpW was highly conserved. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the gene was over-expressed in E. coli BL21(DE3). Western blot analysis revealed that the expressed protein had immunoreactivity. The recombinant protein was purified by affinity chromatography on Ni-NTA Superflow resin. Large yellow croaker vaccinated with the purified OmpW showed significantly increased antibody to OmpW, which could resist the infection by V. alginolyticus. A specific antibody was detected by enzyme-linked immunosorbent assay. This study suggested that the conserved OmpW could be an effective vaccine candidate against infection by V. alginolyticus.
TL;DR: Results show that tetrandrine can induce apoptosis and abrogate radiation-induced G2 arrest in CNE cells.
Abstract: Tetrandrine is known to exert antitumor effect, however, little is known about its effect on nasopharyngeal carcinoma cells. In this study, we tested tetrandrine-induced apoptosis and radiosensitivity in nasopharyngeal carcinoma cell line CNE and investigated the possible mechanisms. Using flow cytometry and DNA electrophoresis, we found that tetrandrine could induce cell apoptosis. Further, it was shown that the level of Bcl-2 mRNA decreased and Bax mRNA increased after addition of tetrandrine by using reverse transcription-polymerase chain reaction. X-ray-induced G2 arrest was abrogated by treatment with tetrandrine, as detected by flow cytometry and mitotic index. The accumulation of cyclinB1 protein and the suppression of Cdc2 tyrosine-15 and Cdc25C serine-216 phosphorylation were detected in irradiated cells treated with tetrandrine using Western blot analysis. Taken together, these results show that tetrandrine can induce apoptosis and abrogate radiation-induced G2 arrest in CNE cells.
TL;DR: It is shown that A3F was up-regulated by interferon (IFN)-alpha in primary hepatocytes and multiple liver cell lines as well as macrophages, and expression levels were comparable to those in CD4+ T lymphocytes in some individuals.
Abstract: Human cytidine deaminase APOBEC3F (A3F) has broad anti-viral activity against hepatitis B virus and retroviruses including human immunodeficiency virus type 1. However, its regulation in viral natural target cells such CD4 + T lymphocytes, macrophages, and primary liver cells has not been well studied. Here we showed that A3F was up-regulated by interferon (IFN)-α in primary hepatocytes and multiple liver cell lines as well as macrophages. Although the IFN-α signaling pathway was active in T lymphoid cells and induction of other IFN stimulated genes such as PKR was detected, A3F and APOBEC3G (A3G) were not induced by IFN-α in these cells. Thus, additional factors other than known IFN-stimulated genes also regulated IFN-α-induced A3F expression distinctly. A3F and A3G expression levels in primary hepatocytes, especially after IFN-α stimulation, were comparable to those in CD4 + T lymphocytes in some individuals. Significant variations of A3F and A3G expression in primary hepatocytes from various subjects were observed. Individual variations in A3F and/or A3G regulation and expression might influence the clinical outcomes of hepatitis B infection.
TL;DR: It is concluded that p26 might be involved in protecting the embryo from physiological stress during embryonic development by preventing irreversible protein damage during embryonicDevelopment.
Abstract: The protein p26 is a small heat shock protein that functions as a molecular chaperone to protect embryos by preventing irreversible protein damage during embryonic development. A 542 bp fragment of the p26 gene was cloned and sequenced. The fragment encoded 174 amino acid residues and the amino acid sequence contained the α-crystallin domain. Phylogenetic analysis showed that eight Artemia populations were divided into four major groups. Artemia sinica (YC) belonged to the East Asia bisexual group. Expres sion of the p26 gene at different developmental stages of A. sinica was quantified using real-time quantitative polymerase chain reaction followed by cloning and sequencing. The relationship between the quantity of p26 gene expression and embryonic development was analyzed. The results indicated that massive amounts of p26 were expressed during the development of A. sinica. At the developmental stage of 0 h, A. sinica expressed the highest level of p26. As development proceeded, expression levels of the p26 gene reduced significantly. There was a small quantity of p26 gene expression at the developmental stages of 16 h and 24 h. We concluded that p26 might be involved in protecting the embryo from physiological stress during embryonic development.
TL;DR: The results showed that Bcl-XL siRNA contributed to an increase of DDP-induced cell death in non-small-cell lung cancer and sensitized cells to DDP, leading to increased the effectiveness of the drug in treating non- small- cell lung cancer.
Abstract: Bcl-XL is overexpressed in a variety of human tumors and is involved in tumorigenesis and chemoresistance. This study investigated the inhibitory effect of the hairpin Bcl-XL small interfering RNA (siRNA) on the expression of the Bcl-XL gene in the cisplatin (DDP)-resistant human lung adenocarcinoma cell line A549/DDP, and the effect of Bcl-XL siRNA on drug sensitization in A549/DDP cells. Bcl-XL siRNA and negative siRNA plasmids were constructed and stably transfected into A549/DDP cells. Reverse transcription-polymerase chain reaction and Western blot analysis were used to detect the target gene expression. Spontaneous apoptosis of cells was detected by acridine orange and ethidium bromide staining. Drug sensitivity of the cells to DDP was analyzed with dimethylthiazol-diphenyltetrazolium bromide (MTT) and flow cytometry. Expression levels of Bcl-XL mRNA and protein in siRNA stable transfectants were clearly reduced as compared with negative siRNA transfectants and untreated cells. MTT results indicated that Bcl-XL transfectants had a higher cell inhibition rate than the negative vector or untreated cells after treatment with 0.2-200 micarog/ml DDP. Flow cytometry revealed increased apoptosis in Bcl-XL siRNA cells. After the addition of 20 microg/ml DDP, siRNA targeting of the Bcl-XL gene specifically down-regulated gene expression in A549/DDP cells, increased spontaneous apoptosis, and sensitized cells to DDP. The results showed that Bcl-XL siRNA contributed to an increase of DDP-induced cell death in non-small-cell lung cancer and sensitized cells to DDP, leading to increased the effectiveness of the drug in treating non-small-cell lung cancer.
TL;DR: SiRNA targeting of the Bcl-2 gene specifically down-regulated gene expression in A549/DDP cells, increased spontaneous apoptosis, and sensitized cells to DDP and DADS.
Abstract: Bcl-2 is overexpressed in a variety of human tumors and is involved in tumorigenesis and chemoresistance. In this study, we investigated the inhibitory effect of the hairpin Bcl-2 small interfering (si)RNA on the expression of the Bcl-2 gene in the cisplatin (DDP)-resistant human lung adenocarcinoma cell line A549/DDP, and the effect of Bcl-2 siRNA on drug sensitization in A549/DDP cells. Bcl-2 siRNA and negative siRNA plasmids were constructed and stably transfected into A549/DDP cells. Reverse transcription- polymerase chain reaction, immunofluorescence microscopy and Western blot analysis were used to detect the target gene expression. Spontaneous cell apoptosis was detected by acridine orange and ethidium bromide staining. Drug sensitivity of the cells to DDP and diallyl disulfide (DADS) was analyzed by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Expression levels of Bcl-2 mRNA and protein in siRNA stable transfectants were clearly reduced compared with negative siRNA transfectants and untreated cells. MTT results indicated that Bcl-2 transfectants had a higher cell inhibition rate after treatment with 0.2−200 µg/ml DDP or 50−200 µM DADS. Flow cytometry revealed increased apoptosis in Bcl-2 siRNA cells. After the addition of 20 µg/ml DDP or 100 µM DADS, siRNA targeting of the Bcl-2 gene specifically down-regulated gene expression in A549/DDP cells, increased spontaneous apoptosis, and sensitized cells to DDP and DADS.
TL;DR: The MTT and cytometry analyses showed that recombinant BTI could specifically inhibit the proliferation of IM-9 human B lymphoblastoid cells (from patient with multiple myeloma) in a dose-dependent manner.
Abstract: The gene of buckwheat trypsin inhibitor (BTI) has been cloned and expressed in Escherichia coli. The yield of this recombinant inhibitor was over 12 mg/L by using one-step purification on a Ni 2+ -NTA Sepharose column. Its molecular weight was 9322.1 Da, determined by mass spectrum analysis. The MTT and cytometry analyses showed that recombinant BTI could specifically inhibit the proliferation of IM-9 human B lymphoblastoid cells (from patient with multiple myeloma) in a dose-dependent manner. The test of recombinant BTI-induced apoptosis in IM-9 cells implied that the inhibitor might have potential application in the treatment of cancer.
TL;DR: Two pentadecamer peptides are synthesized based on cDNA sequences of alpha 3/5 conotoxins from C. achatinus and experiments indicate that both toxins strongly prefer thealpha(1)-delta subunit interface instead of the alpha(1-gamma binding site on the mouse muscle nicotinic acetylcholine receptor.
Abstract: Every cone snail produces a mixture of different conotoxins and secretes them to immobilize their prey and predators. α3/5 Conotoxins, isolated from fish-hunting cone snails, target muscle nicotinic acetylcholine receptors. The structure and function of α3/5 conotoxin from the piscivorous Conus achatinus have not been studied. We synthesized two pentadecamer peptides, Ac1.1a and Ac1.1b, with appropriate disulfide bonding, based on cDNA sequences of α3/5 conotoxins from C. achatinus. Ac1.1a and Ac1.1b differ by only one amino acid residue. They have similar potency on blocking recombinant mouse muscle acetylcholine receptor expressed in Xenopus laevis oocytes, with IC50 values of 36 nM and 26 nM, respectively. For Ac1.1b, deletion of the first three N-terminal amino acids did not change its activity, indicating that the N-terminus is not involved in the interaction with its receptor. Furthermore, our experiments indicate that both toxins strongly prefer the α1–δ subunit interface instead of the α1–γ binding site on the mouse muscle nicotinic acetylcholine receptor. These peptides provide additional tools for the study of the structure and function of nicotinic receptor. FullText for HTML: https://doi.org/10.1111/j.1745-7270.2007.00301.x
TL;DR: Findings indicate that copper binding to Abeta(1-40) can give rise to greater production of H(2)O(2), which leads to a breakdown in the integrity of the plasma membrane and subsequent neuronal death.
Abstract: β-amyloid peptide (Aβ) is considered to be responsible for the formation of senile plaques, which is the hallmark of Alzheimer's disease (AD). Oxidative stress, manifested by protein oxidation and lipid peroxidation, among other alterations, is a characteristic of AD brain. A growing body of evidence has been presented in support of Aβ1−40 forming an oligomeric complex that binds copper at a CuZn superoxide dismutase-like binding site. Aβ1−40Cu(II) complexes generate neurotoxic hydrogen peroxide (H2O2) from O2 via Cu 2+ reduction, though the precise reaction mechanism is unclear. The toxicity of Aβ1−40 or the Aβ1−40Cu(II) complexes to cultured primary cortical neurons was partially attenuated when (+)-α-tocopherol (vitamin E) as free radical antioxidant was added at a concentration of 100 μM. The data derived from lactate dehydro- genase (LDH) release and the formation of H2O2 confirmed the results from the MTT assay. These findings indicate that copper binding to Aβ1−40 can give rise to greater production of H2O2, which leads to a break- down in the integrity of the plasma membrane and subsequent neuronal death. Groups treated with vitamin E exhibited much slighter damage, suggesting that vitamin E plays a key role in protecting neuronal cells from dysfunction or death.
TL;DR: The deduced amino acid sequence revealed that CBHI has a modular structure with a predicted molecular mass of 56 kDa and consists of a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region.
Abstract: A cellobiohydrolase 1 gene (cbh1) was cloned from Penicillium chrysogenum FS010 by a modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). DNA sequencing shows that cbh1 has an open reading frame of 1590 bp, encoding a putative protein of 529 amino acid residues. The deduced amino acid sequence revealed that CBHI has a modular structure with a predicted molecular mass of 56 kDa and consists of a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region. The putative gene product is homologous to fungal cellobiohydrolases in Family 7 of the glycosyl hydrolases. A novel cbh1 promoter (1.3 kb) was also cloned and sequenced, which contains seven putative binding sites (5'-SYGGRG-3') for the carbon catabolite repressor CRE1. Effect of various carbon sources to the cbh1 transcription of P. chrysogenum was examined by Northern analysis, suggesting that the expression of cbh1 is regulated at transcriptional level. The cbh1 gene in cold-adaptive fungus P. chysogenum was expressed as an active enzyme in Saccharomyces cerevisiae H158. The recombinant CBHI accumulated intracellularly and could not be secreted into the medium.
TL;DR: A lectin with a novel N-terminal amino acid sequence was purified from the rhizomes of Aspidistra elatior Blume and chemical modification and spectrum analysis indicated that tryptophan, arginine, cysteine and carboxyl group residues were essential for its hemagglutinating activity.
Abstract: A lectin with a novel N-terminal amino acid sequence was purified from the rhizomes of Aspidistra elatior Blume by ammonium sulphate precipitation, ion exchange chromatography on diethylaminoethyl-Sepharose and carboxymethyl-Sepharose and gel filtration chromatography on Sephacryl S-100. The A. elatior Blume lectin (AEL) is a heterotetramer with a molecular mass of 56 kDa and composed of two homodimers consisting of two different polypeptides of 13.5 kDa and 14.5 kDa held together by noncovalent interactions. Hapten inhibition assay indicated that hemagglutinating activity of AEL towards rabbit erythrocytes could be inhibited by D-mannose, mannan, thyroglobulin and ovomucoid. The lectin was stable up to 70 oC, and showed maximum activity in a narrow pH range of 7.0−8.0. Chemical modification and spectrum analysis indicated that tryptophan, arginine, cysteine and carboxyl group residues were essential for its hemagglutinating activity. However, they might not be present in the active center, except some carboxyl group residues. AEL also showed significant in vitro antiproliferative activity towards Bre-04 (66%), Lu-04 (60%) and HepG2 (56%) of human cancer cell lines.
TL;DR: Results showed that H2O2 induced AChE activity, a functional marker in apoptosis, increases in neuronal-like PC12 cells, and Glutathione inhibited the increase of A cholinesterase activity, suggesting that reactive oxygen species (ROS) play a key role in this process.
Abstract: Acetylcholinesterase (AChE) is thought to play an important role during apoptosis. Our results showed that H2O2 induced AChE activity, a functional marker in apoptosis, increases in neuronal-like PC12 cells. Glutathione, which is involved in cellular redox homeostasis, inhibited the increase of AChE activity, suggesting that reactive oxygen species (ROS) play a key role in this process. Further investigation showed that the elevation of AChE was observed after the degradation of Akt, release of cytochrome c from mitochondria into the cytosol, and activation of caspase family members. When nerve growth factor (NGF) was present, with the maintenance of Akt level, the elevation of AChE, the cytochrome c diffusion, as well as apoptosis were markedly attenuated in H2O2-treated PC12 cells. However, wortmannin, an inhibitor of the PI3K/Akt pathway, accelerated the apoptosis and increased the AChE activity. The overexpression of constitutively activated Akt, which is a downstream signalling element of the NGF receptor TrkA, delayed mitochondrial collapse and inhibited elevation of AChE activity. Thus, NGF prevented apoptosis and elevation of AChE activity by activating the Akt pathway and stabilizing the function of mitochondria.
TL;DR: Generally, the accumulation levels of CMV-Fny ORFs in the co-infection were higher than those in the single infection, whereas the accumulation of ZYMV-SD CP ORF showed a reverse result.
Abstract: Cucumber mosaic virus (CMV) and zucchini yellow mosaic virus (ZYMV) are two principal viruses infecting cucurbitaceous crops, and their synergy has been repeatedly observed. In our present work, a real-time reverse transcription-polymerase chain reaction procedure was established to study the accumulation kinetics of these two viruses in single and combined infections at the molecular level. The accumulations of open reading frames (ORFs) for 1a, 2a, 3a and coat protein (CP) of CMV and CP of ZYMV were tested. In the single infection, CMV-Fny ORFs accumulated to their maxima in cucumber or bottle gourd at 14 d post-inoculation (dpi), and gradually declined thereafter. ZYMV-SD CP ORF reached maximal accumulation at 14 and 28 dpi on cucumber and bottle gourd, respectively. However, when co- infected with CMV-Fny and ZYMV-SD, the maximal accumulation levels of all viral ORFs were delayed. CMV-Fny ORFs reached their maxima at 21 dpi on both hosts, and ZYMV-SD CP ORF reached maximal accumulation at 21 and 28 dpi on cucumber and bottle gourd, respectively. Generally, the accumulation levels of CMV-Fny ORFs in the co-infection were higher than those in the single infection, whereas the accumulation of ZYMV-SD CP ORF showed a reverse result.
TL;DR: The results indicated that the attenuation of CMV, by adding satRs or deleting the 2b gene, was due to the low accumulation ofCMV genomic RNAs, and that satRNA-mediated reduction of CMv genomicRNAs accumulation in N. tabacum was possibly related to the 2a gene.
Abstract: Satellite RNAs (satRNAs) are molecular parasites that interfere with the pathogenesis of the helper viruses. In this study, the relative accumulation of cucumber mosaic virus (CMV)-Fny genomic RNAs with or without satRNAs were quantitatively analyzed by real-time RT-PCR. The results showed that satRs apparently attenuated the symptoms of CMV-Fny on Nicotiana tabacum by depressing the accumulation of CMV-Fny genomic RNAs, tested as open reading frames. The accumulation of CMV-Fny 1a, 2a, 2b, 3a, and CP genes was much higher than that of CMV-Fny with satRs added (CMV-Fsat), at different inoculation times. CMV-FnyDelta2b, in which the complete 2b gene and 41 amino acids at the C-terminal of the 2a gene were deleted, caused only a slight mosaic effect on N. tabacum seedlings, similar to that of CMV-Fsat, but the addition of satRs to CMV-FnyDelta2b showed further decrease in the accumulation of CMV-FnyDelta2b genomic RNAs. Our results indicated that the attenuation of CMV, by adding satRs or deleting the 2b gene, was due to the low accumulation of CMV genomic RNAs, and that satRNA-mediated reduction of CMV genomic RNAs accumulation in N. tabacum was possibly related to the 2b gene.