Characterization of DNA-binding activity of Zα domains from poxviruses and the importance of the β-wing regions in converting B-DNA to Z-DNA

The E3L gene is essential for pathogenesis in vaccinia virus. The E3L gene product consists of an N-terminal Zα domain and a C-terminal double-stranded RNA (dsRNA) binding domain; the left-handed Z-DNA-binding activity of the Zα domain of E3L is required for viral pathogenicity in mice. E3L is highly conserved among poxviruses, including the smallpox virus, and it is likely that the orthologous Zα domains play similar roles. To better understand the biological function of E3L proteins, we have investigated the Z-DNA-binding behavior of five representative Zα domains from poxviruses. Using surface plasmon resonance (SPR), we have demonstrated that these viral Zα domains bind Z-DNA tightly. Ability of Zα[subscript E3L] converting B-DNA to Z-DNA was measured by circular dichroism (CD). The extents to which these Zαs can stabilize Z-DNA vary considerably. Mutational studies demonstrate that residues in the loop of the β-wing play an important role in this stabilization. Notably the Zα domain of vaccinia E3L acquires ability to convert B-DNA to Z-DNA by mutating amino acid residues in this region. Differences in the host cells of the various poxviruses may require different abilities to stabilize Z-DNA; this may be reflected in the observed differences in behavior in these Zα proteins.Korean Science and Engineering Foundation (National Research Laboratory Program (NRL-2006-02287))Korean Science and Engineering Foundation (Ubiquitome Research Program (M10533010002-06N3301-00210))Korean Science and Engineering Foundation (21C Frontier Functional Proteomics Program (FPR06B2-120))National Institutes of Health (U.S.)Ellison Medical FoundationKorea (South). Ministry of Science and Technology (National Laboratory program (NRL-2006-02287)

A peptide with alternating lysines can act as a highly specific Z-DNA binding domain

Many nucleic acid binding proteins use short peptide sequences to provide specificity in recognizing their targets, which may be either a specific sequence or a conformation. Peptides containing alternating lysine have been shown to bind to poly(dG–d5meC) in the Z conformation, and stabilize the higher energy form [H. Takeuchi, N. Hanamura, H. Hayasaka and I. Harada (1991) FEBS Lett., 279, 253–255 and H. Takeuchi, N. Hanamura and I. Harada (1994) J. Mol. Biol., 236, 610–617.]. Here we report the construction of a Z-DNA specific binding protein, with the peptide KGKGKGK as a functional domain and a leucine zipper as a dimerization domain. The resultant protein, KGZIP, induces the Z conformation in poly(dG–d5meC) and binds to Z-DNA stabilized by bromination with high affinity and specificity. The binding of KGZIP is sufficient to convert poly(dG–d5meC) from the B to the Z form, as shown by circular dichroism. The sequence KGKGKGK is found in many proteins, although no functional role has been established. KGZIP also has potential for engineering other Z-DNA specific proteins for future studies of Z-DNA in vitro and in vivo

De Novo Design and Synthesis of Ultra-Short Peptidomimetic Antibiotics Having Dual Antimicrobial and Anti-Inflammatory Activities

Ravichandran N. Murugan, Mija Ahn, Eunha Hwang, Ji-Hyung Seo, Chaejoon Cheong, Jeong Kyu Bang, Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk, Republic of KoreaBinu Jacob, Song Yub Shin, Department of Bio-Materials, Graduate School and Department of Cellular and Molecular Medicine, School of Medicine, Chosun University, Gwangju, Republic of KoreaHoik Sohn, Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, United States of AmericaHyo-Nam Park, Jae-Kyung Hyun, Division of Electron Microscopic Research, Korea Basic Science Institute, Daejeon, Republic of KoreaEunjung Lee, Ki-Woong Jeong, Yangmee Kim, Department of Bioscience and Biotechnology, Institute of SMART Biotechnology, Konkuk University, Seoul, Republic of KoreaKy-Youb Nam, Bioinformatics and Molecular Design Research Center, Yonsei University Research Complex, Seoul, Republic of KoreaBackground: Much attention has been focused on the design and synthesis of potent, cationic antimicrobial peptides (AMPs) that possess both antimicrobial and anti-inflammatory activities. However, their development into therapeutic agents has been limited mainly due to their large size (12 to 50 residues in length) and poor protease stability.-- Methodology/Principal Findings: In an attempt to overcome the issues described above, a set of ultra-short, His-derived antimicrobial peptides (HDAMPs) has been developed for the first time. Through systematic tuning of pendant hydrophobic alkyl tails at the N(π)- and N(τ)-positions on His, and the positive charge of Arg, much higher prokaryotic selectivity was achieved, compared to human AMP LL-37. Additionally, the most potent HDAMPs showed promising dual antimicrobial and anti-inflammatory activities, as well as anti–methicillin-resistant Staphylococcus aureus (MRSA) activity and proteolytic resistance. Our results from transmission electron microscopy, membrane depolarization, confocal laser-scanning microscopy, and calcein-dye leakage experiments propose that HDAMP-1 kills microbial cells via dissipation of the membrane potential by forming pore/ion channels on bacterial cell membranes. -- Conclusion/Significance: The combination of the ultra-short size, high-prokaryotic selectivity, potent anti-MRSA activity, anti-inflammatory activity, and proteolytic resistance of the designed HDAMP-1, -3, -5, and -6 makes these molecules promising candidates for future antimicrobial therapeutics.This work was supported in part by the Korea Basic Science Institute's research program grants T33418 (J.K.B) and T33518 (J-k.H.), and the Korea Research Foundation, funded by the Korean Government (KRF-2011-0009039 to S.Y.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.ChemistryBiochemistryEmail: [email protected] (JKB)Email: [email protected] (SYS

Evaluating the Sensitivity of Mycobacterium tuberculosis to Biotin Deprivation Using Regulated Gene Expression

In the search for new drug targets, we evaluated the biotin synthetic pathway of Mycobacterium tuberculosis (Mtb) and constructed an Mtb mutant lacking the biotin biosynthetic enzyme 7,8-diaminopelargonic acid synthase, BioA. In biotin-free synthetic media, ΔbioA did not produce wild-type levels of biotinylated proteins, and therefore did not grow and lost viability. ΔbioA was also unable to establish infection in mice. Conditionally-regulated knockdown strains of Mtb similarly exhibited impaired bacterial growth and viability in vitro and in mice, irrespective of the timing of transcriptional silencing. Biochemical studies further showed that BioA activity has to be reduced by approximately 99% to prevent growth. These studies thus establish that de novo biotin synthesis is essential for Mtb to establish and maintain a chronic infection in a murine model of TB. Moreover, these studies provide an experimental strategy to systematically rank the in vivo value of potential drug targets in Mtb and other pathogens

Oncogenic CagA Promotes Gastric Cancer Risk via Activating ERK Signaling Pathways: A Nested Case-Control Study

Background: CagA cellular interaction via activation of the ERK signaling pathway may be a starting point in the development of gastric cancer. This study aimed to evaluate whether genes involved in ERK downstream signaling pathways activated by CagA are susceptible genetic markers for gastric cancer. Methods: In the discovery phase, a total of 580 SNPs within +/-5 kbp of 30 candidate genes were genotyped to examine an association with gastric cancer risk in the Korean Multi-center Cancer Cohort (100 incident gastric cancer case-control sets). The most significant SNPs (raw or permutated p value??0.02) identified in the discovery analysis were re-evaluated in the extension phase using unconditional logistic regression model (400 gastric cancer case-control sets). Combined analyses including pooled-and meta-analysis were conducted to summarize all the results. Results: 24 SNPs in eight genes (ERK, Dock180, C3G, Rap1, Src, CrkL, Mek and Crk) were significantly associated with gastric cancer risk in the individual SNP analyses in the discovery phase (p??0.05). In the extension analyses, ERK rs5999749, Dock180 rs4635002 and C3G rs7853122 showed marginally significant gene-dose effects for gastric cancer. Consistently, final combined analysis presented the SNPs as significantly associated with gastric cancer risk (OR = 1.56, [95% CI: 1.19-2.06], OR = 0.61, [95% CI: 0.43-0.87], OR = 0.59, [95% CI: 0.54-0.76], respectively). Conclusions: Our findings suggest that ERK rs5999749, Dock180 rs4635002 and C3G rs7853122 are genetic determinants in gastric carcinogenesis

Performance of Papanicolaou Testing and Detection of Cervical Carcinoma In Situ in Participants of Organized Cervical Cancer Screening in South Korea

BACKGROUND: The present study measured the performance of the Papanicolaou (Pap) test and detection of cervical carcinoma in situ (CIS) and cancer in participants of organized cervical cancer screening in South Korea, and examined differences in the proportion of CIS according to socio-demographic factors. METHODS: Data were obtained from the National Cancer Screening Program and National Health Insurance Cancer Screening Program databases. We analyzed data from 4,072,997 screenings of women aged 30 years or older who underwent cervical cancer screening by Pap test between January 1, 2005 and December 31, 2006. We calculated the performances of the Pap test and compared that according to socio-demographic factors. RESULTS: The positivity rate for all screenings was 6.6%. The cancer detection rate (CDR) and interval cancer rate (ICR) were 0.32 per 1,000 screenings, and 0.13 per 1,000 negative screenings, respectively. About 63.4% of screen-detected CIS+ cases (CIS or invasive cervical cancer) were CIS. The CDR and ICR, and percentage of CIS among all CIS+ were significantly different by age group and health insurance status. The odds ratios of CDR and ICR were higher for Medical Aid Program (MAP) recipients compared with National Health Insurance (NHI) beneficiaries. The likelihood of a detected CIS+ case to be CIS was significantly lower among MAP recipients than among NHI beneficiaries. CONCLUSIONS: The difference in performance of cervical cancer screening among different socio-demographic groups may indicate an important influence of socio-demographic factors on preventive behavior. The findings of the study support the critical need for increasing efforts to raise awareness and provide more screening in at-risk populations, specifically low-income groups

Directed Induction of Functional Motor Neuron-Like Cells from Genetically Engineered Human Mesenchymal Stem Cells

Cell replacement using stem cells is a promising therapeutic approach to treat degenerative motor neuron (MN) disorders, such as amyotrophic lateral sclerosis and spinal cord injury. Human bone marrow-derived mesenchymal stem cells (hMSCs) are a desirable cell source for autologous cell replacement therapy to treat nervous system injury due to their plasticity, low immunogenicity, and a lower risk of tumor formation than embryonic stem cells. However, hMSCs are inefficient with regards to differentiating into MN-like cells. To solve this limitation, we genetically engineered hMSCs to express MN-associated transcription factors, Olig2 and Hb9, and then treat the hMSCs expressing Olig2 and Hb9 with optimal MN induction medium (MNIM). This method of induction led to higher expression (>30% of total cells) of MN markers. Electrophysiological data revealed that the induced hMSCs had the excitable properties of neurons and were able to form functional connections with muscle fibers in vitro. Furthermore, when the induced hMSCs were transplanted into an injured organotypic rat spinal cord slice culture, an ex vivo model of spinal cord injury, they exhibited characteristics of MNs. The data strongly suggest that induced Olig2/Hb9-expressing hMSCs were clearly reprogrammed and directed toward a MN-like lineage. We propose that methods to induce Olig2 and Hb9, followed by further induction with MNIM have therapeutic potential for autologous cell replacement therapy to treat degenerative MN disorders

Genetic Susceptibility on CagA-Interacting Molecules and Gene-Environment Interaction with Phytoestrogens: A Putative Risk Factor for Gastric Cancer

OBJECTIVES: To evaluate whether genes that encode CagA-interacting molecules (SRC, PTPN11, CRK, CRKL, CSK, c-MET and GRB2) are associated with gastric cancer risk and whether an interaction between these genes and phytoestrogens modify gastric cancer risk. METHODS: In the discovery phase, 137 candidate SNPs in seven genes were analyzed in 76 incident gastric cancer cases and 322 matched controls from the Korean Multi-Center Cancer Cohort. Five significant SNPs in three genes (SRC, c-MET and CRK) were re-evaluated in 386 cases and 348 controls in the extension phase. Odds ratios (ORs) for gastric cancer risk were estimated adjusted for age, smoking, H. pylori seropositivity and CagA strain positivity. Summarized ORs in the total study population (462 cases and 670 controls) were presented using pooled- and meta-analysis. Plasma concentrations of phytoestrogens (genistein, daidzein, equol and enterolactone) were measured using the time-resolved fluoroimmunoassay. RESULTS: SRC rs6122566, rs6124914, c-MET rs41739, and CRK rs7208768 showed significant genetic effects for gastric cancer in both the pooled and meta-analysis without heterogeneity (pooled OR = 3.96 [95% CI 2.05-7.65], 1.24 [95% CI = 1.01-1.53], 1.19 [95% CI = 1.01-1.41], and 1.37 [95% CI = 1.15-1.62], respectively; meta OR = 4.59 [95% CI 2.74-7.70], 1.36 [95% CI = 1.09-1.70], 1.20 [95% CI = 1.00-1.44], and 1.32 [95% CI = 1.10-1.57], respectively). Risk allele of CRK rs7208768 had a significantly increased risk for gastric cancer at low phytoestrogen levels (p interaction<0.05). CONCLUSIONS: Our findings suggest that SRC, c-MET and CRK play a key role in gastric carcinogenesis by modulating CagA signal transductions and interaction between CRK gene and phytoestrogens modify gastric cancer risk

Transcriptional recapitulation and subversion of embryonic colon development by mouse colon tumor models and human colon cancer

Colon tumors from four independent mouse models and 100 human colorectal cancers all exhibited striking recapitulation of embryonic colon gene expression from embryonic days 13.5-18.5