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1.  In Silico Identification and Characterization of N-Terminal Acetyltransferase Genes of Poplar (Populus trichocarpa) 
N-terminal acetyltransferase (Nats) complex is responsible for protein N-terminal acetylation (Nα-acetylation), which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS) and auxiliary subunits (AS) have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A–F), being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.
doi:10.3390/ijms15021852
PMCID: PMC3958825  PMID: 24473137
acetyltransferase; Nα-acetylation; genome identification; woody plants; phylogenetic analysis
2.  Amino-Terminated Generation 2 Poly(amidoamine) Dendrimer as a Potential Broad-Spectrum, Nonresistance-Inducing Antibacterial Agent 
The AAPS Journal  2012;15(1):132-142.
The treatment of septicemia caused by antibiotic-resistant bacteria is a great challenge in the clinic. Because traditional antibiotics inevitably induce bacterial resistance, which is responsible for many treatment failures, there is an urgent need to develop novel antibiotic drugs. Amino-terminated Poly(amidoamine) dendrimers (PAMAM-NH2) are reported to have antibacterial activities. However, previous studies focused on high generations of PAMAM-NH2, which have been found to exhibit high toxicities. The present study aimed to clarify whether low generations of PAMAM-NH2 could be used as novel antibacterial agents. We found that generation 2 (G2.0) PAMAM-NH2 showed significant antibacterial effects against antibiotic-sensitive and antibiotic-resistant strains but exhibited little toxicity to human gastric epithelial cells and did not induce antibiotic resistance in bacteria. Scanning and transmission electron microscopy analyses suggested that G2.0 PAMAM-NH2 might inhibit the growth of bacteria by destroying their cell membranes. The administration of G2.0 PAMAM-NH2 dose-dependently improved the animal survival rate of mice infected with extended-spectrum beta lactamase-producing Escherichia coli (ESBL-EC) and of animals infected with a combination of ESBL-EC and methicillin-resistant Staphylococcus aureus. A treatment regimen of 10 mg/kg of G2.0 PAMAM-NH2 starting 12 h before inoculation followed by 10 mg/kg at 0.5 h after inoculation rescued 100% of singly infected mice and 60% of multiply infected mice. The protective effects were associated with the reduction of the bacterial titers in the blood and with the morphological amelioration of infected tissues. These findings demonstrate that the G2.0 PAMAM-NH2 is a potential broad-spectrum and nonresistance-inducing antibiotic agent with relatively low toxicity.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9416-8) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-012-9416-8
PMCID: PMC3535096  PMID: 23135925
antibacterial activity; antibiotic resistance; extended-spectrum beta lactamase-producing Escherichia coli; methicillin-resistant Staphylococcus aureus; PAMAM dendrimers
3.  Resveratrol Protects Mouse Oocytes from Methylglyoxal-Induced Oxidative Damage 
PLoS ONE  2013;8(10):e77960.
Methylglyoxal, a reactive dicarbonyl compound, is mainly formed from glycolysis. Methylglyoxal can lead to the dysfunction of mitochondria, the depletion of cellular anti-oxidation enzymes and the formation of advanced glycation ends. Previous studies showed that the accumulation of methylglyoxal and advanced glycation ends can impair the oocyte maturation and reduce the oocyte quality in aged and diabetic females. In this study, we showed that resveratrol, a kind of phytoalexin found in the skin of grapes, red wine and other botanical extracts, can alleviate the adverse effects caused by methylglyoxal, such as inhibition of oocyte maturation and disruption of spindle assembly. Besides, methylglyoxal-treated oocytes displayed more DNA double strands breaks and this can also be decreased by treatment of resveratrol. Further investigation of these processes revealed that methylglyoxal may affect the oocyte quality by resulting in excessive reactive oxygen species production, aberrant mitochondrial distribution and high level lipid peroxidation, and resveratrol can block these cytotoxic changes. Collectively, our results showed that resveratrol can protect the oocytes from methylglyoxal-induced cytotoxicity and this was mainly through the correction of the abnormity of cellular reactive oxygen species metabolism.
doi:10.1371/journal.pone.0077960
PMCID: PMC3806792  PMID: 24194906
4.  Identifying the Genome-Wide Sequence Variations and Developing New Molecular Markers for Genetics Research by Re-Sequencing a Landrace Cultivar of Foxtail Millet 
PLoS ONE  2013;8(9):e73514.
Foxtail millet (Setariaitalica) is a drought-resistant, barren-tolerant grain crop and forage. Currently, it has become a new model plant for cereal crops and biofuel grasses. Although two reference genome sequences were released recently, comparative genomics research on foxtail millet is still in its infancy. Using the Solexa sequencing technology, we performed genome re-sequencing on one important foxtail millet Landrace, Shi-Li-Xiang (SLX). Compared with the two reference genome sequences, the following genetic variation patterns were identified: 762,082 SNPs, 26,802 insertion/deletion polymorphisms of 1 to 5 bp in length (indels), and 10,109 structural variations (SVs) between SLX and Yugu1 genomes; 915,434 SNPs, 28,546 indels and 12,968 SVs between SLX and Zhang gu genomes. Furthermore, based on the Yugu1 genome annotation, we found out that ~ 40% SNPs resided in genes containing NB-ARC domain, protein kinase or leucine-rich repeats, which had higher non-synonymous to synonymous SNPs ratios than average, suggesting that the diversification of plant disease resistance proteins might be caused by pathogen pressure. In addition, out of the polymorphisms identified between SLX and Yugu1, 465 SNPs and 146 SVs were validated with more than 90% accuracy, which could be used as DNA markers for whole-genome genotyping and marker-assisted breeding. Here, we also represented an example of fine mapping and identifying a waxy locus in SLX using these newly developed DNA markers. This work provided important information that will allow a deeper understanding of the foxtail millet genome and will be helpful for dissecting the genetic basis of important traits in foxtail millet.
doi:10.1371/journal.pone.0073514
PMCID: PMC3769310  PMID: 24039970
5.  Systemic administration of low dosage of tetanus toxin decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus 
Laboratory Animal Research  2013;29(3):148-155.
In the present study, we investigated the effect of Tetaus toxin (TeT) on cell proliferation and neuroblast differentiation using specific markers: 5-bromo-2-deoxyuridine (BrdU) as an exogenous marker for cell proliferation, Ki-67 as an endogenous marker for cell proliferation and doublecortin (DCX) as a marker for neuroblasts in the mouse hippocampal dentate gyrus (DG) after TeT treatment. Mice were intraperitoneally administered 2.5 and 10 ng/kg TeT and sacrificed 15 days after the treatment. In both the TeT-treated groups, no neuronal death occurred in any layers of the DG using neuronal nuclei (NeuN, a neuron nuclei maker) and Fluoro-Jade B (F-J B, a high-affinity fluorescent marker for the localization of neuronal degeneration). In addition, no significant change in glial activation in both the 2.5 and 10 ng/kg TeT-treated-groups was found by GFAP (a marker for astrocytes) and Iba-1 (a marker for microglia) immunohistochemistry. However, in the 2.5 ng/kg TeT-treated-group, the mean number of BrdU, Ki-67 and DCX immunoreactive cells, respectively, were apparently decreased compared to the control group, and the mean number of each in the 10 ng/kg TeT-treated-group was much more decreased. In addition, processes of DCX-immunoreactive cells, which projected into the molecular layer, were short compared to those in the control group. In brief, our present results show that low dosage (10 ng/kg) TeT treatment apparently decreased cell proliferation and neuroblast differentiation in the mouse hippocampal DG without distinct gliosis as well as any loss of adult neurons.
doi:10.5625/lar.2013.29.3.148
PMCID: PMC3791348  PMID: 24106509
Exotoxin; neuronal damage; neurogenesis; sub-granular zone; granule cell
6.  Genetic variants of the class A scavenger receptor gene are associated with coronary artery disease in Chinese 
Journal of Biomedical Research  2012;26(6):418-424.
The class A scavenger receptor, encoded by the macrophage scavenger receptor 1 (MSR1) gene, is a pattern recognition receptor (PPR) primarily expressed in macrophages. It has been reported that genetic polymorphisms of MSR1 are significantly associated with the number of diseased vessels and coronary artery narrowing greater than 20% in Caucasians. However, whether it links genetically to coronary artery disease (CAD) in Chinese is not defined. Here, we performed an independent case-control study in a Chinese population consisting of 402 CAD cases and 400 controls by genotyping ten single nucleotide polymorphisms (SNPs) of MSR1. We found that rs416748 and rs13306541 were significantly associated with an increased risk of CAD with per allele odds ratio (OR) of 1.56 [95% confidence interval (CI) = 1.28-1.90; P < 0.001] and 1.70 (95% CI = 1.27-2.27; P < 0.001), respectively. Our results indicate that genetic variants of MSR1 may serve as predictive markers for the risk of CAD in combination with traditional risk factors of CAD in Chinese population.
doi:10.7555/JBR.26.20110116
PMCID: PMC3597046  PMID: 23554780
class A scavenger receptor gene; single nucleotide polymorphisms (SNPs); coronary artery disease; Chinese population
7.  Underlying Mechanism of In vivo and In vitro Activity of C-terminal–amidated Thanatin Against Clinical Isolates of Extended-Spectrum β-lactamase–Producing Escherichia coli 
The Journal of Infectious Diseases  2011;203(2):273-282.
Background. Infections with extended-spectrum β-lactamase–producing Escherichia coli (ESBL-EC) have developed resistance to current therapies. Therefore, the underlying mechanisms of in vivo and in vitro activity of C-terminal–amidated thanatin (A-thanatin) against clinical isolates of ESBL-EC were studied in an attempt to resolve this problem.
Methods. A-thanatin was synthesized to determine its minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and kill curve for ESBL-EC. The hemolytic toxicity, stability, and resistance induction of A-thanatin were determined. ESBL-EC–infected mice were used to determine the in vivo activity of A-thanatin. Scanning and transmission electron microscopy and fluorescence microscopy were used to study the underlying mechanism of A-thanatin.
Results. A-thanatin is highly effective against ESBL-EC in vitro, with MIC values ≤4 μg/mL. It has been confirmed that A-thanatin has little hemolysis and relative high stability in plasma. Excellent in vivo therapeutic effects were also observed in a septicemic animal model, with survival rates of 50.0%, 66.7%, and 91.7% in the low-dose, middle-dose, and high-dose groups, respectively. Membrane permeabilization may be a major biological action of A-thanatin.
Conclusions. Because the development of multidrug resistance limits the available therapeutic options, A-thanatin may provide a novel strategy for treating ESBL-EC infection and other infections due to multidrug-resistant bacteria.
doi:10.1093/infdis/jiq029
PMCID: PMC3071056  PMID: 21288828
8.  Targeting RNA Polymerase Primary σ70 as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid 
PLoS ONE  2012;7(1):e29886.
Background
Methicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to “druggable” target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ70 (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition.
Methodology/Principal Findings
By synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)3K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ70 in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ70 were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM.
Conclusions
The present result suggested that RNAP primary σ70 is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections.
doi:10.1371/journal.pone.0029886
PMCID: PMC3254626  PMID: 22253815
9.  Co-blockade of mecR1/blaR1 signal pathway to restore antibiotic susceptibility in clinical isolates of methicillin-resistant Staphylococcus aureus 
Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is caused by the production of low-affinity penicillin-binding protein 2a and β-lactamases, which are encoded by mecA and blaZ, respectively. Expressions of the two key genes are mutually regulated by MecI and BlaI. The aim of this study was to design specific anti-mecR1 and anti-blaR1 deoxyribozymes and identify the restoration of susceptibility in MRSA isolates with mecI or blaI or no deletions by interfering with the mutual regulation of mecA and blaZ.
Material and methods
Specific deoxyribozymes were designed by using the program RNA structure 4.6. RNA substrates were obtained by transcription in vitro and used to assess the target cleavage of DNAzymes. Transcription of mecR1-mecA and blaR1-blaZ was analysed by real time RT-PCR. The susceptibility of MRSA was tested.
Results
Specific deoxyribozymes showed efficient catalytic activity to each own substrate mecR1 or blaR1 in vitro and caused the reduction of mecR1 and blaR1 transcription in vivo. Furthermore, simultaneous administration of two DNAzymes to knockdown mecR1 and blaR1 resulted in increased susceptibility of all MRSA strains tested in this study.
Conclusions
These results demonstrated that combined use of the two specific phosphorothioate deoxyribozymes could be a viable and promising strategy to restore the susceptibility of almost all MRSA clinical isolates.
doi:10.5114/aoms.2011.23404
PMCID: PMC3258742  PMID: 22295022
methicillin-resistant Staphylococcus aureus; mecR1; blaR1; phosphorothioate deoxyribozyme
10.  Identification and validation of rice reference proteins for western blotting 
Journal of Experimental Botany  2011;62(14):4763-4772.
Studies of rice protein expression have increased considerably with the development of rice functional genomics. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. Comparison among the profiling of translation and transcription [expressed sequence tags (EST) and massively parallel signature sequencing (MPSS)] revealed that a correlation existed. Based on the standard curves derived from the antigen–antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.
doi:10.1093/jxb/err084
PMCID: PMC3192993  PMID: 21705388
Antibody-based proteomics; rice (Oryza sativa L.); reference gene; reference protein; western blotting
11.  Tetra­aqua­(5,5′-dimethyl-2,2′-bipyridine-κ2 N,N′)zinc(II) sulfate 
The asymmetric unit of the title compound, [Zn(C12H12N2)(H2O)4]SO4, consists of a ZnII complex cation, a sulfate anion and four mol­ecules of water coordinated to the ZnII atom. The ZnII complex cation, with approximate twofold symmetry, displays a slightly distorted octa­hedral geometry around the ZnII atom, which is coordinated by two N atoms from a 5,5′-dimethyl-2,2′-bipyridine ligand and by the O atoms of four water mol­ecules. In the crystal, O—H⋯O hydrogen bonds help to establish the packing.
doi:10.1107/S160053680902488X
PMCID: PMC2977444  PMID: 21583333
12.  RFXB and its splice variant RFXBSV mediate the antagonism between IFNγ and TGFβ on COL1A2 transcription in vascular smooth muscle cells 
Nucleic Acids Research  2009;37(13):4393-4406.
Cytokines secreted by infiltrating immune cells during atherogenesis modulate vascular remodeling. One exemplary event is the antagonism between transformed growth factor (TGF-β) and interferon gamma (IFN-γ) on the transcriptional control of type I collagen gene (COL1A2). Previously we have reported that IFN-γ up-regulates regulatory factor for X-box B (RFXB) to repress collagen transcription while down-regulates the expression of RFXBSV, a splice variant of RFXB that blocks collagen repression in fibroblasts. Here we demonstrate that TGF-β abrogated COL1A2 repression by IFN-γ through altering the relative expression of RFXB and RFXBSV. Unlike RFXB, RFXBSV did not bind to the collagen promoter and competed with RFXB for the co-repressor histone deacetylase 2 (HDAC2), limiting HDAC2 recruitment to the collagen transcription start site as evidenced by chromatin immunoprecipitation assays. Over-expression of RFXB by lentiviral infection in HASMCs enhanced HDAC2 enlistment, promoted histone deacetylation surrounding the collagen site by IFN-γ, and blocked the TGF-β antagonism, a pattern reversed by RFXBSV infection. On the contrary, silencing of RFXB, but not both RFXB and RFXBSV, expression promoted the TGF-β antagonism. Thus, we have identified a novel mechanism whereby TGF-β antagonizes the IFN-γ repression of collagen transcription in HASMCs and as such provided new insights into antiatherogenic strategies.
doi:10.1093/nar/gkp398
PMCID: PMC2715248  PMID: 19465385
13.  catena-Poly[[[diaqua­terbium(III)]-μ-6-carboxy­nicotinato-μ-pyridine-2,5-di­carboxyl­ato] dihydrate] 
The title compound, {[Tb(C7H3NO4)(C7H4NO4)(H2O)2]·2H2O}n, is isotypic with the analogous TmIII compound [Li, Zhang, Wang & Bai (2009). Acta Cryst. E65, m411]. The TbIII atom is octa­coordinated by two water mol­ecules and by four carboxyl­ate O atoms and two pyridyl N atoms from two pyridine-2,5-dicarboxyl­ate (2,5-pydc) and two 6-carboxy­nicotinate (2,5-Hpydc) ligands. The 2,5-pydc and 2,5-Hpydc ligands bridge TbIII atoms, generating helical coordination polymers along [001]. An extensive network of O—H⋯O hydrogen bonds is formed between the coordination polymers and the uncoordinated water mol­ecules. The refined Flack parameter of 0.54 (2) suggests inversion twinning.
doi:10.1107/S1600536809008824
PMCID: PMC2968984  PMID: 21582353

Results 1-13 (13)