Functional Mechanism of Antimicrobial Peptide Bomidin and Its Safety for Macrobrachium rosenbergii.

Macrobrachium rosenbergii is an economically important source of crustacean seafood worldwide. Vibrio parahaemolyticus is an important aquatic pathogen that causes epidemics of acute hepatopancreatic necrosis in shrimp populations, which results in significant economic losses to aquaculture farmers. To prevent the antibiotics abuse, which has become a serious threat to human health, novel anti-infective strategies are urgently required to control V. parahaemolyticus. Antimicrobial peptides, which exhibit favourable germicidal activity compared to traditional antibiotics, can be used as a key method to prevent and treat bacterial diseases. Herein, an antimicrobial peptide, bomidin, was expressed through genetic engineering technology. The minimum inhibitory concentration (MIC) of bomidin showed a significant inhibitory effect on V. parahaemolyticus that was equivalent to that of ampicillin. Subsequently, the mechanism of action of recombinant bomidin was explored using PNP and ONPG assays to investigate the effects on membrane permeability. These assays indicated that bomidin penetrated the germ membrane and induced the release of cytoplasmic contents and ultimately interacted with DNA to form a bomidin-DNA complex that inhibits bacterial survival. Transmission electron microscopy and scanning electron microscopy revealed that bomidin could cause damage and dysfunction to the cell wall and membrane. Bomidin was nontoxic to mouse red blood cells within a concentration range that was much larger than the MIC. Toxicity assays revealed that 0.02 mg/mL bomidin was safe for use with juvenile freshwater prawns of M. rosenbergii and significantly inhibited the growth of V. parahaemolyticus in cultured water. These results demonstrated that synthetic peptide bomidin had great antibacterial effect against V. parahaemolyticus and therefore a therapeutic potential in aquaculture.

Antibacterial activity and its mechanisms of a recombinant Funme peptide against Cronobacter sakazakii in powdered infant formula.

Cronobacter sakazakii (Cs) is a typical foodborne bacterium that infect powdered infant formula (PIF) worldwide. In this study, a recombinant antimicrobial peptide, branded as Funme peptide (FP)was applied to protect PIF from Cs contamination. The result from the antimicrobial activity assay showed that the minimum inhibitory concentration (MIC) of BMAP-27 peptide, FP and Ampicillin against Cs were 250.0, 125.0 and 15.6 µg/mL, respectively, indicating FP possessed higher MIC than that of Ampicillin, and lower MIC than that of BMAP-27. The minimum biofilm eradication concentration (MBEC) assay showed that FP at 2 × MIC (250.0 µg/mL) could completely eradicated Cs biofilms. The antibacterial activity of FP might be due to the increasing permeability and the release of cytoplasmic ß-galactosidase of Cs. The results acquired from transmission electron microscopy and scanning electron microscopy indicated that FP induced the disruption and dysfunction of cell walls and membranes. Moreover, safety assay showed that FP had low cytotoxicity to human erythrocytes. The present study investigated the antibacterial effects and mechanisms of FP against Cs, providing promising evidence to apply this novel antimicrobial agent against Cs contamination in foods and food processing facilities.

Interruption of intrachromosomal looping by CCCTC binding factor decoy proteins abrogates genomic imprinting of human insulin-like growth factor II.

Monoallelic expression of IGF2 is regulated by CCCTC binding factor (CTCF) binding to the imprinting control region (ICR) on the maternal allele, with subsequent formation of an intrachromosomal loop to the promoter region. The N-terminal domain of CTCF interacts with SUZ12, part of the polycomb repressive complex-2 (PRC2), to silence the maternal allele. We synthesized decoy CTCF proteins, fusing the CTCF deoxyribonucleic acid-binding zinc finger domain to CpG methyltransferase Sss1 or to enhanced green fluorescent protein. In normal human fibroblasts and breast cancer MCF7 cell lines, the CTCF decoy proteins bound to the unmethylated ICR and to the IGF2 promoter region but did not interact with SUZ12. EZH2, another part of PRC2, was unable to methylate histone H3-K27 in the IGF2 promoter region, resulting in reactivation of the imprinted allele. The intrachromosomal loop between the maternal ICR and the IGF2 promoters was not observed when IGF2 imprinting was lost. CTCF epigenetically governs allelic gene expression of IGF2 by orchestrating chromatin loop structures involving PRC2.

Associated chromosome trap for identifying long-range DNA interactions.

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.

CTCF regulates allelic expression of Igf2 by orchestrating a promoter-polycomb repressive complex 2 intrachromosomal loop.

CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allele-specific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes.

IVF results in de novo DNA methylation and histone methylation at an Igf2-H19 imprinting epigenetic switch.

Recent studies suggest that IVF and assisted reproduction technologies (ART) may result in abnormal genomic imprinting, leading to an increased frequency of Angelman syndrome (AS) and Beckwith-Weidemann syndrome (BWS) in IVF children. To learn how ART might alter the epigenome, we examined morulas and blastocysts derived from C57BL/6J X M. spretus F1 mice conceived in vivo and in vitro and determined the allelic expression of four imprinted genes: Igf2, H19, Cdkn1c and Slc221L. IVF-derived mouse embryos that were cultured in human tubal fluid (HTF) (Quinn's advantage) media displayed a high frequency of aberrant H19 imprinting, whereas in vivo and IVF embryos showed normal maternal expression of Cdkn1c and normal biallelic expression of Igf2 and Slc221L. Embryonic stem (ES) cells derived from IVF blastocysts also showed abnormal Igf2/H19 imprinting. Allele-specific bisulphite PCR reveals abnormal DNA methylation at a CCCTC-binding factor (CTCF) site in the imprinting control region (ICR), as the normally unmethylated maternal allele acquired a paternal methylation pattern. Chromatin immunoprecipitation (ChIP) assays indicate an increase of lysine 4 methylation (dimethyl Lys4-H3) on the paternal chromatin and a gain in lysine 9 methylation (trimethyl Lys9-H3) on the maternal chromatin at the same CTCF-binding site. Our results indicate that de novo DNA methylation on the maternal allele and allele-specific acquisition of histone methylation lead to aberrant Igf2/H19 imprinting in IVF-derived ES cells. We suggest that ART, which includes IVF and various culture media, might cause imprinting errors that involve both aberrant DNA methylation and histone methylation at an epigenetic switch of the Igf2-H19 gene region.

Identification and cloning of a submergence-induced gene OsGGT (glycogenin glucosyltransferase) from rice (Oryza sativa L.) by suppression subtractive hybridization.

A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5'- and 3'-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17-1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970-20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293-51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses.

Overexpression of the AP2/EREBP transcription factor OPBP1 enhances disease resistance and salt tolerance in tobacco.

Osmotin promoter binding protein 1 (OPBP1), an AP2/EREBP-like transcription factor of tobacco (Nicotiana tabacum), was isolated using a yeast one-hybrid system. RNA gel blot analysis indicated that expression of the OPBP1 gene was induced by elicitor cryptogein, NaCl, ethephon, methyl jasmonate, as well as cycloheximide. Transient expression analysis using an OPBP1-eGFP fusion gene in onion epidermal cells revealed that the OPBP1 protein was targeted to the nuclear. Further, electrophoretic mobility shift assays demonstrated that the recombinant OPBP1 protein could bind to an oligonucleotide containing the GCC-box cis element. Transgenic tobacco plants with an over expression of the OPBP1 gene accumulated high levels of PR-1a and PR-5d genes and exhibited enhanced resistance to infection by Pseudomonas syringae pv tabaci and Phytophthora parasitica var nicotianae pathogens. They also exhibited increased tolerance to salt stress. These results suggest that OPBP1 might be a transcriptional regulator capable of regulating expression in sets of stress-related genes.

Cloning of a putative monogalactosyldiacylglycerol synthase gene from rice (Oryza sativa L.) plants and its expression in response to submergence and other stresses.

Suppression subtractive hybridization was used to construct a subtractive cDNA library from plants of non-submerged and 7-day-submerged rice (Oryza sativa L., FR13A, a submergence-tolerant cultivar). One clone of the subtractive cDNA library, S23, was expressed abundantly during submergence. The full length of S23 was amplified using 5'- and 3'-rapid amplification of cDNA ends, and found to consist of 1,671 bp with an open reading frame of 1,077 bp (181-1257) encoding 358 amino acids. Its deduced amino acid sequence showed a high homology with monogalactosyldiacylglycerol synthase (UDPgalactose: 1,2-diacylglycerol 3-beta-D-galactosyl transferase; EC 2.4.1.46, MGDG synthase) from Arabidopsis thaliana; therefore, we named the gene OsMGD. Time-course studies showed that the expression of OsMGD in the rice cultivars FR13A and IR42 (submergence-susceptive cultivar) during submergence was gradually increased and that expression in FR13A was higher than in IR42. The expression of OsMGD in FR13A was influenced by benzyladenine and illumination. The accumulation of OsMGD mRNA in both FR13A and IR42 was also increased by ethephon, gibberellin, drought and salt treatment, but cold stress had no effect on the expression of the gene. These results suggest that the expression of OsMGD mRNA requires benzyladenine or illumination, and that the process is also mediated by ethephon and gibberellin. Salt and drought stress have an effect similar to that of submergence. Furthermore, the enhanced expression of OsMGD may relate to photosynthesis, and play an important role during submergence.

Cloning of two cysteine proteinase genes, CysP1 and CysP2, from soybean cotyledons by cDNA representational difference analysis.

By cDNA representational difference analysis (cDNA RDA) and rapid amplification of cDNA ends (RACE), we isolated two cDNAs, CysP1 and CysP2, from the cotyledons of growing soybean (Glycine max (L.) Merr.) seedlings. CysP1 cDNA is 1265 bp in size with a 1089-bp open reading frame (ORF), and CysP2 cDNA is 1270 bp in size with a 1089-bp ORF. Either CysP1 or CysP2 encodes a cysteine proteinase (CPR) with a C-terminal KDEL motif. The similarities between CysP1 and CysP2 are 93.5% in nucleotide sequences and 93.6% in deduced amino acid sequences. Furthermore, we determined the nucleotide sequences of CysP1 genomic DNA (1846 bp) and CysP2 genomic DNA (1831 bp). Both consisted of four exons and three introns. RNA-blot analysis revealed that both CysP1 and CysP2 were expressed from 6 days after germination (DAG) to 13 or 14 DAG in the cotyledons of growing seedlings and did so in a short period (9-12 DAG) in rejuvenated cotyledons. The transcripts of CysP1 and CysP2 were also detected in the root, flower and pod of soybean plants. Their physiological roles in the cotyledons of growing seedlings are discussed.