Improving Energy Metabolism of Deproteinized Extract of Calf Blood Through Regulation of Hmgcs2, Cpt1a,Angptl4, Cyp8b1, and Ehhadh Genes in Mice

Herein, we described the physicochemical properties of deproteinized extract of calf blood(DECB) and established a hypoxia model treated with or without DECB to detect the sugar, lactic acid, protein, and ATP contents of mice and then identified and analyzed the differentially expressed genes between two groups using mRNA expression chip. According to the results of the airtight hypoxia experiment, mice in the model+DECB group had a signifi? cantly prolonged time of hypoxia tolerance compared with the model group. The biochemical test indicated that DECB could significantly increase the level of sugar, ATP and proteins and reduce the amount of lactic acid in mice. It also revealed that Hmgcs2, Cptla, Angptl4, Cyp8b], and Ehhadh genes were involved in mice energy metabolism, and were closely associated witli metabolic signaling pathway. These results suggest that DECB might be a potential drug to treat metabolic diseases. Among the genes with differential expression under hypoxia, Angptl4, Cyp8bl, and Ehhadh were critical factors for sugar metabolism. Hmgcs2 provided energy directly, and Cptla regulated cellular inflammatory responses, promoting energy metabolism.     

Identification of differentially expressed proteins in the heart of translationally controlled tumor protein over-expressing transgenic mice

We previously reported that transgenic (TG) mice over-expressing translationally controlled tumor protein (TCTP) developed systemic arterial hypertension at about 6 weeks after birth. In the present study, we identified, using proteomics technologies, 24 other proteins that were differentially expressed in the heart of TCTP over-expressing TG mice. These 24 proteins are involved in a variety of biological processes such as reactive oxygen species metabolism, cytoskeleton organization, fatty acid metabolism, amino acid metabolism and energy metabolism. We determined protein expression levels of the peroxiredoxin (Prx)2, Prx3, myosin light chain 1, stress protein (heat shock protein) 25K, and T-complex protein 1 alpha subunit by western blot analysis. Over-expression of TCTP probably regulates the expression of other proteins which play a pivotal role in a variety of cellular functions in TCTP over-expressing TG mice.     

Nuclear protein profiling of Jurkat cells during heat stress-induced apoptosis by 2-DE and MS/MS

Heat stress causes severe constraints on numerous physiological functions of cells, such as the repression of splicing of mRNA precursors. In this study, we performed proteomic profiling of a nuclear fraction of Jurkat cells during heat stress using 2-DE and LC-MS/MS. We found 10 protein spots whose expression had changed after heat stress at 43 degrees C for 30 min. Seven of those protein spots, periodic tryptophan protein 1 homolog (PWP1), importin beta-1 subunit, sumoylated protein, splicing factor 3a subunit 3 (SF3a3), TAR DNA-binding protein 43, U2 small nuclear ribonucleoprotein auxiliary factor 35 kDa subunit (U2AF35) and small ubiquitin-related modifier-1 (SUMO-1) were downregulated, while three other protein spots, Protein SET, 40S ribosomal protein SA and 60S acidic ribosomal protein P0 were upregulated by the heat stress. We focused on the downregulation of two splicing factors, which might participate in the repression of pre-mRNA processing by heat stress, leading to cell apoptosis.     

Comparative proteomic analysis of grain development in two spring wheat varieties under drought stress

Two spring wheat varieties Ningchun 4 and Chinese Spring with good and poor resistance to abiotic stress, respectively, were used to investigate proteomic changes in the developing grains under drought stress by a comparative proteomics approach. A total of 152 protein spots showed at least twofold differences in abundance on two-dimensional electrophoresis (2-DE) maps, of which 28 and 68 protein spots were identified by MALDI-TOF and MALDI-TOF/TOF mass spectrometry, respectively. Of the 96 identified protein spots, six different expression patterns were found and they were involved in stress/defense/detoxification, carbohydrate metabolism, photosynthesis, nitrogen metabolism, storage proteins and some other important functions. Comparative proteomic analysis revealed that under the drought conditions the decreased degree of ascorbate peroxidases was more significant in Chinese Spring than in Ningchun 4 during grain development whereas translationally controlled tumor protein, which was significantly upregulated at 14 DAF, was present in Ningchun 4 and absent in Chinese Spring. The Rubisco large subunit displayed an upregulated expression pattern in Ningchun 4. In addition, two drought-tolerant proteins, triosephosphate isomerase and oxygen-evolving complex showed B and F type expression patterns in Chinese Spring, but D and B types in Ningchun 4, respectively. These differentially expressed proteins might be responsible for the stronger drought resistance of Ningchun 4 compared to Chinese Spring.     

Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.     

Expression Profiling of Bioactive Genes from Moringa oleifera

Plants are under constant assault by biotic and abiotic agents. When an elicitor is prologued, an immense reprogramming of plant gene expression and defense responses are initiated, which could be a natural source for potential drug development and insertional mutagenesis. In this regard, differential expression analysis of a medicinal plant Moringa oleifera was performed for bioactive genes at seedling stage, using differential display-RT-PCR technique. Infected seedlings with a fungus Fusarium solani collected at different time intervals, showed a massive change in their gene expression profile. The data analysis revealed that at least 150 pathogen-induced and about 60 suppressed genes were differentially expressed at 8-h postinoculation of the biotic stress. Fifty-five selective genes were disunited and reamplified. Sequence analysis of these potential genes illustrated that these genes had properties of some induced peroxidase mRNA, cell proliferation, others were mitogen activated protein kinases, ribosomal protein genes, defense regulating genes, and a few also had structural properties. Further studies about the utility of these genes in plant metabolism could assist to develop improved transgenic breeds with enhanced value of infection tolerance not only of M. oleifera but of other cultivars also.     

Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.     

Pre-incubated with BSA-complexed free fatty acids alters ER stress/autophagic gene expression by carboxylated multi-walled carbon nanotube exposure in THP-1 macrophages

Pre-incubation with BSA-complexed free fatty acids influenced the colloidal aspects of carboxylated multiwalled carbon nanotubes (c-MWCNTs), and altered macrophage foam cell formation through the modulation of ER stress/autophagic gene expression.     

Downregulation of two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 correlates with high metastatic potentials of human SN12C renal cell carcinoma cell clones

Proteomic differential display analysis was performed on human renal cell carcinoma cell SN12C clones having different metastatic potentials by using 2-DE and LC-MS/MS. The SN12C cell clones were SN12C parent cell line, SN12C-clone 2, SN12C-clone 4, and SN12C-PM6. The SN12C parent cell line was established from an HRCC surgical specimen. SN12C-clone 4 has lower, and SN12C-clone 2 and SN12C-PM6 have higher metastatic potential than SN12C parent cells. We found eight protein spots whose expression level was different between low metastatic clones and high metastatic clones. The protein expression of three appeared to be higher in high metastatic clones than low metastatic clones, and that of other five protein spots appeared to be lower in high metastatic clones than low metastatic clones. These spots were selected, digested and analyzed by LC-MS/MS analysis, and they were identified by peptide sequencing tag. In high metastatic potential clones, two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) were downregulated. These results suggest that UCH-L1 expression seems to be associated with the metastatic potential of HRCC SN12C cell clones.     

Invertase production is related to the nitrogen source in Hansenula anomala

Differences in invertase accumulation of Hansenula anomala cultivated in ammonium and nitrate are reported. Media supplemented with sucrose and ammonium sulfateas the solecarbon and nitrogen source, respectively, show strong media acidification (pH 3.0 or lower), and vigorous cell growth. Invertase activity was not detected under such conditions. A cell-free imitation experiment suggests that, under such low pH, extensive chemical break-down of sucrose (>22%) occurs. Thus, H. anomala is able togrow under strong acidic conditions that permit sucrose metabolism by the uptake of monosaccharides generated from chemical hydrolysis. In addition, invertase activity is not present in cells grown in nitrate as nitrogen source at pH 5.0, but at pH 7.0 activity is detected. If ammonium is supplied instead of nitrate, cells grown at pH 5.0 show invertase activity and at pH 7.0 high levels of activity are detected. These results indicate a specific physiological response of the sucrose metabolism to the presence of alternate nitrogen source.     

Age-dependent variations of cell response to oxidative stress: proteomic approach to protein expression and phosphorylation

We investigated the protein profiles of variously aged rat astrocytes in response to oxidative stress. After H2O2-exposure of cells at 100 microM for 30 min, the relative intensity of ten protein spots changed on two-dimensional (2-D) gels compared with control gels after silver staining. Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis after in-gel digestion revealed that six of these spots corresponded to three kinds of proteins, each of which was composed of a protein and its modified form with a different isoelectric point (pI). These three proteins were identified as peroxiredoxins (PRDXs) II and III, and calpactin I light chain (p11). H2O2-exposure increased the intensity of the spot with lower pI and simultaneously decreased that of the spot with higher pI for both PRDXs II and III. In addition, the expression of annexin VII, S-adenosyl-L-homocysteine hydrolase, elongation factor II fragment (EF-II), and adenosine deaminase was increased by H2O2-exposure in astrocytes from variously aged rats. Using the Pro-Q Diamond staining, heat shock protein 60 kDa (Hsp 60) and alpha-tubulin were observed to be phosphorylated upon H2O2-exposure. While phosphorylation of alpha-tubulin was correlated positively with age, the changes in abundance of ten protein spots as described above were independent of age. These results suggest that aging does not suppress the responses aimed at limiting injury and promoting repair brought about by severe oxidative stress, and might affect cell dynamics including the formation of microtubules.     

2-D PAGE analysis of pesticide-induced stress proteins of E. coli

Logarithmically growing batch cultures of Escherichia coli were exposed to sublethal concentrations of pyrethroid and carbamate pesticides of four different technical grades. This induced 17–20 stress proteins, as observed using two-dimensional polyacrylamide gel electrophoresis. An E. coli culture growing exponentially in Luria Bertani medium (cell density ~2.3×10⁹ cells/ml) was exposed to predetermined sublethal doses of individual pesticides. The cells were harvested after 30 minutes of induction and the stress response was developed in fresh LB medium for three hours under the same growth conditions. Cell pellets were obtained and stored in sonication buffer. Two-dimensional polyacrylamide gel electrophoresis was performed to resolve the proteins. Visualization of the protein spots by rapid silver staining showed 17–20 stress proteins which were absent in the standard protein profile of E. coli. On average 29% of these stress proteins were unique to the pollutant, while the remaining stress proteins overlapped with those of other pesticides. The iso-electric points (PIs) and molecular weights of the proteins were determined by comparing with protein markers with known PIs and molecular weights. Furthermore, upon comparing the pesticide-induced proteins within the same class and between the two different classes (pyrethroid and carbamate), it was apparent that the general nature of the stress remained the same throughout, which indirectly proved that the gene or set of genes responsible for stress expression are also the same, irrespective of the chemical nature of the substituents of the pesticides.     

Involvement of Heat-Shock Protein 70 and P53 Proteins in Attenuation of UVC-lnduced Apoptosis by Thermal Stress in Hepatocellular Carcinoma Cells

Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. In this study, we found that a prior exposure to mild stress (42 degrees C) can protect HepG2 (p53+/+) cells from a subsequent UVC-induced apoptosis determined by DNA fragmentation and ratio of sub-G1 peak, but no heat-enhanced protection was found in Hep3B (p53-/-) cells. Although a similar inductive pattern of HSP70 protein and mRNA was detected in the two cell lines under thermal stress, the effect of thermal stress on UVC-induced apoptosis in HepG2 and Hep3B cells was obviously different. Overexpression of HSP70 by transient transfection of HSP70 expression vector in HepG2 cells significantly inhibited UVC-induced cell death; however, this inhibitory effect did not occur in transfected-Hep3B cells. Treatment of HepG2 cells with p53-specific antisense oligonucleotide could effectively block the antiapoptotic effect of thermal stress on UVC-induced apoptosis and increase of intracellular wild-type p53 protein by transfecting wtp53 expression plasmid into Hep3B cells yielded more resistance to UVC irradiation after prior thermal stress exposure. The results reveal an involvement of p53 in the antiapoptotic effect of thermal stress on UVC irradiation. Finally, a p53 protein increase was detected in UVC-treated HepG2 cells and could be coimmunoprecipitated with HSP70 after a thermal stress treatment. Prolonged p53 binding activity and enhanced expression of p53-controlled genes such as G1 arrest and DNA damage 45 and wild-type p53 activation factor 1/Cdk-interacting protein 1 by thermal stress are also observed in UVC-irradiated HepG2 cells. Based on these results, we propose that the antiapoptotic effect of thermal stress is mediated by increasing HSP70 and modulating intracellular p53 function.     

The mode of action of hormones

Higher organisms having anatomically and functionally differentiated organ systems must be able to coordinate biochemical processes within and between the various organs or cells, even under rapidly changing environmental conditions. Hormones play an important part in the regulation processes: they promote numerous metabolic reactions and differentiation processes, and show considerable differences in the speed with which they act. Some hormones act almost instantaneously by stimulating adenyl cyclase and thus initiating the production of adenosine 3′,5′-monophosphate (cyclic AMP), which passes on the hormone message (“second messenger”) to the intracellular metabolism by modifying enzyme activities. Delayed-action hormones, mainly morphogenetic hormones, act primarily on the cell nucleus where they induce the synthesis of certain enzymes by activation of genes.     

Deoxyhypusine synthase is phosphorylated by protein kinase C in vivo as well as in vitro

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. We earlier observed that yeast recombinant deoxyhypusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold following treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca(2+), suggesting a possible involvement of PKC. We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in different cell lines in order to define its role on the regulation of eIF5A in the cell. Deoxyhypusine synthase was found to be phosphorylated by endogenous kinases in CHO, NIH3T3, and chicken embryonic cells. The highest degree of phosphorylation was found in CHO cells. Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cells was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly diminished by diacyl ethylene glycol (DAEG), a PKC inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca(2+)/DAG. Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca(2+) plus DAG. Close association of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune coprecipitation and was PMA-, and Ca(2+)/phospholipid dependent. These results suggest that phosphorylation of deoxyhypusine synthase was PKC-dependent cellular event and open a path for possible regulation in the interaction with eIF5A precursor for hypusine synthesis.     

Proteomic Analysis of Differentially Expressed Proteins of Arabidopsis thaliana Response to Specialist Herbivore Plutella xylostella

The changes of the proteome in Arabidopsis thaliana leaves were examined by specialist Plutella xylostella.Analysis of about 1100 protein spots on each 2DE gel revealed 38 differentially expressed protein spots in abun-dance of which 34 proteins were identified by MALDI-TOF/TOF MS.Among the insect feeding responsive proteins,a few proteins involved in carbon metabolism were identified including proteins associated with the Calvin cycle in the chloroplast and TCA cycle in the mitochondria,indicating carbon metabolism related proteins may play crucial roles in induced defense response in plants under insect infestation.The analysis elucidates the subcellular location of proteins demonstrates that about 50% of proteins are in the chloroplast,which shows the chloroplast has a key role in the insect feeding response for plant.Gene expression analysis of 10 different proteins by quantitative real-time PCR shows that four proteins of the mRNA level were correlated well with the protein level.This study further dissected the nature of insect infestation as a stress signal and some novel insect feeding responsive proteins identified may play an important role in induced defence machanism for plant.