Identification and functional validation of HLA-C as a potential gene involved in colorectal cancer in the Korean population.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide and is influenced by environmental and genetic factors. Although numerous genetic loci for CRC have been identified, the overall understanding of the genetic factors is yet to be elucidated. We sought to discover new genes involved in CRC applying genetic association analysis and functional study. RESULTS: We conducted exome array analysis on 194 CRC and 600 control subjects for discovering new candidate CRC genes. Fisher's exact test detected one exome-wide significant functional locus for CRC on SMCO1 (P < 10-6) and two suggestive functional loci on HLA-C and NUTM1 (10-6 ≤ P < 10-4). To evaluate the biological role of three candidate CRC genes, the differential expression of these genes between CRC and non-cancer colorectal cells was analyzed using qRT-PCR and publicly available gene expression data. Of three genes, HLA-C consistently revealed the significant down-regulation in CRC cells. In addition, we detected a reduction in cell viability in the HLA-C overexpression CRC cell line, implying the functional relevance of HLA-C in CRC. To understand the underlying mechanism exerted by HLA-C in CRC development, we conducted RNA sequencing analyses of HLA-C overexpression CRC cells and non-cancer colorectal cells. Pathway analysis detected that significantly down-regulated genes in HLA-C overexpression CRC cells were highly enriched in cancer-related signaling pathways such as JAK/STAT, ErbB, and Hedgehog signaling pathways. CONCLUSIONS: Exome array CRC case-control analysis followed by functional validation demonstrated that HLA-C likely exerts its influence on CRC development via cancer-related signaling pathways.

Ca2+ Regulation of Cav3.3 T-type Ca2+ Channel Is Mediated by Calmodulin.

Calcium-dependent inactivation of high voltage-activated Ca2+ channels plays a crucial role in limiting rises in intracellular calcium (Ca2+i). A key mediator of these effects is calmodulin, which has been found to bind the C-terminus of the pore-forming α subunit. In contrast, little is known about how Ca2+i can regulate low voltage-activated T-type Ca2+ channels. Using whole cell patch clamp, we examined the biophysical properties of Ca2+ current through the three T-type Ca2+ channel isoforms, Cav3.1, Cav3.2, or Cav3.3, comparing internal solutions containing 27 nM and l µM free Ca2+ Both activation and inactivation kinetics of Cav3.3 current in l µM Ca2+i solution were more rapid than those in 27 nM Ca2+i solution. In addition, both activation and steady-state inactivation curves of Cav3.3 were negatively shifted in the higher Ca2+i solution. In contrast, the biophysical properties of Cav3.1 and Cav3.2 isoforms were not significantly different between the two internal solutions. Overexpression of CaM1234 (a calmodulin mutant that doesn't bind Ca2+) occluded the effects of l µM Ca2+i on Cav3.3, implying that CaM is involved in the Ca2+i regulation effects on Cav3.3. Yeast two-hybrid screening and co-immunoprecipitation experiments revealed a direct interaction of CaM with the carboxyl terminus of Cav3.3. Taken together, our results suggest that Cav3.3 T-type channel is potently regulated by Ca2+i via interaction of Ca2+/CaM with the carboxyl terminus of Cav3.3.

Development and evaluation of a semifield test for repellent efficacy testing.

Estimation of the efficacy of mosquito repellents requires both laboratory and field tests. The results of field tests are more meaningful, but the safety of volunteers in such tests may be a significant concern. In the current study, we compared tests of mosquito repellent efficacy under semifield conditions in an outdoor enclosure with those under laboratory and field conditions. In this study, we assessed the efficacy of N,N-diethyl-meta-toluamide under laboratory conditions with human volunteers and under semifield and field conditions with Centers for Disease Control and Prevention traps and experimental mice. A semifield test may be a suitable replacement for the more difficult field test for assessment of mosquito repellent efficacy. Semifield tests should be considered when developing new guidelines for testing.

Repression of porcine endogenous retrovirus infection by human APOBEC3 proteins.

It has been shown that porcine endogenous retrovirus (PERV) can infect human cells, indicating that PERV transmission poses a serious concern in pig-to-human xenotransplantation. A number of recent studies have reported on retrovirus interference by antiviral proteins. The most potent antiviral proteins are members of the APOBEC family of cytidine deaminases, which are involved in defense against retroviral attack. These proteins are present in the cytoplasm of mammalian cells and inhibit retroviral replication. To evaluate the inhibition of PERV transmission by human APOBEC3 proteins, we co-transfected 293T cells with a PERV molecular clone and human APOBEC3F or APOBEC3G expression vectors, and monitored PERV replication competency using a quantitative analysis of PERV pol genes. The replication of PERVs in cells co-expressing human APOBEC3s was reduced by 60-90% compared with PERV-only control. These results suggest that human APOBEC3G and APOBEC3F might serve a potential barrier function against PERV transmission in xenotransplantation.

A Simple Mouse Model for the Study of Human Immunodeficiency Virus.

Humanized mouse models derived from immune-deficient mice have been the primary tool for studies of human infectious viruses, such as human immunodeficiency virus (HIV). However, the current protocol for constructing humanized mice requires elaborate procedures and complicated techniques, limiting the supply of such mice for viral studies. Here, we report a convenient method for constructing a simple HIV-1 mouse model. Without prior irradiation, NOD/SCID/IL2Rγ-null (NSG) mice were intraperitoneally injected with 1 × 10(7) adult human peripheral blood mononuclear cells (hu-PBMCs). Four weeks after PBMC inoculation, human CD45(+) cells, and CD3(+)CD4(+) and CD3(+)CD8(+) T cells were detected in peripheral blood, lymph nodes, spleen, and liver, whereas human CD19(+) cells were observed in lymph nodes and spleen. To examine the usefulness of hu-PBMC-inoculated NSG (hu-PBMC-NSG) mice as an HIV-1 infection model, we intravenously injected these mice with dual-tropic HIV-1DH12 and X4-tropic HIV-1NL4-3 strains. HIV-1-infected hu-PBMC-NSG mice showed significantly lower human CD4(+) T cell counts and high HIV viral loads in the peripheral blood compared with noninfected hu-PBMC-NSG mice. Following highly active antiretroviral therapy (HAART) and neutralizing antibody treatment, HIV-1 replication was significantly suppressed in HIV-1-infected hu-PBMC-NSG mice without detectable viremia or CD4(+) T cell depletion. Moreover, the numbers of human T cells were maintained in hu-PBMC-NSG mice for at least 10 weeks. Taken together, our results suggest that hu-PBMC-NSG mice may serve as a relevant HIV-1 infection and pathogenesis model that could facilitate in vivo studies of HIV-1 infection and candidate HIV-1 protective drugs.

Identification of Porcine Endogenous Retrovirus (PERV) packaging sequence and development of PERV packaging viral vector system.

Studies of the retroviruses have focused on the specific interaction of the nucleocapsid protein with a packaging signal in the viral RNA as important for this selectivity, but the packaging signal in porcine endogenous retrovirus (PERV) has not been defined. Herein, we identified and analyzed this packaging signal in PERV and found hairpin structures with conserved tetranucleotides in their loops and nucleocapsid recognition sequences; both of which are key elements in the viral packaging signal of MLV. We evaluated packaging efficiency of sequence variants isolated from viral and proviral integrated genomes. All viral packaging sequences (Ψ) were identical, while five distinct packaging sequences were identified from proviral sources. One proviral sequence (Ψ1) was identical to that of the viral Ψ and had the highest packaging efficiency. Three variants (Ψ2, Ψ3, Ψ4) maintained key elements of the viral packaging signal, but had nucleotide replacements and consequently demonstrated reduced packaging efficiency. Despite of the same overall hairpin structure, the proviral variant (Ψ5) had only one GACG sequence in the hairpin loop and showed the lowest packaging efficiency other than ∆Ψ, in which the essential packaging sequence was removed. This result, thus, defined the packaging sequences in PERV and emphasized the importance of nucleotide sequence and RNA structure in the determination of packaging efficiency. In addition, we demonstrate efficient infection and gene expression from the PERV based viral vector, which may serve as a novel alternative to current retroviral expression systems.

Enhanced Anti-inflammatory Effects of γ-irradiated Pig Placenta Extracts.

Porcine placenta extract (PPE) is known to possess anti-inflammatory properties owing to its high concentration of bioactive substances. However, the need to eliminate blood-borne infectious agents while maintaining biological efficacy raises concerns about the optimal method for sterilizing PPE. Therefore, the objective of this study was to compare the effects of the standard pressurized heat (autoclaving) method of sterilization with γ-irradiation on the anti-inflammatory effects of PPE. The anti-inflammatory actions of these two preparations of PPE were evaluated by measuring their inhibitory effects on the production of NO, the expression of iNOS protein, and the expression of iNOS, COX2, TNF-α, IL-1ß, and IL-6 mRNA in lipopolysaccharide-stimulated RAW 264.7 cells. Compared with autoclaved PPE, γ-irradiated PPE showed significantly greater inhibition of NO production and iNOS protein expression, and produced a greater reduction in the expression of iNOS, COX2, TNF-α, IL-1ß, and IL-6 mRNA. These results provide evidence that the sterilization process is crucial in determining the biological activity of PPE, especially its anti-inflammatory activity. Collectively, our data suggest that γ-irradiated PPE acts at the transcriptional level to effectively and potently suppresses the production of NO and the expression of pro-inflammatory cytokines.

Effect of AcHERV-GmCSF as an Influenza Virus Vaccine Adjuvant.

INTRODUCTION: The first identification of swine-originated influenza A/CA/04/2009 (pH1N1) as the cause of an outbreak of human influenza accelerated efforts to develop vaccines to prevent and control influenza viruses. The current norm in many countries is to prepare influenza vaccines using cell-based or egg-based killed vaccines, but it is difficult to elicit a sufficient immune response using this approach. To improve immune responses, researchers have examined the use of cytokines as vaccine adjuvants, and extensively investigated their functions as chemoattractants of immune cells and boosters of vaccine-mediated protection. Here, we evaluated the effect of Granulocyte-macrophage Colony-Stimulating Factor (GmCSF) as an influenza vaccine adjuvant in BALB/c mice. METHOD AND RESULTS: Female BALB/c mice were immunized with killed vaccine together with a murine GmCSF gene delivered by human endogenous retrovirus (HERV) envelope coated baculovirus (1 × 10(7) FFU AcHERV-GmCSF, i.m.) and were compared with mice immunized with the killed vaccine alone. On day 14, immunized mice were challenged with 10 median lethal dose of mouse adapted pH1N1 virus. The vaccination together with GmCSF treatment exerted a strong adjuvant effect on humoral and cellular immune responses. In addition, the vaccinated mice together with GmCSF were fully protected against infection by the lethal influenza pH1N1 virus. CONCLUSION: Thus, these results indicate that AcHERV-GmCSF is an effective molecular adjuvant that augments immune responses against influenza virus.

Comparison of Repellency Effect of Mosquito Repellents for DEET, Citronella, and Fennel Oil.

To confirm that Korean Food and Drug Administration (KFDA) guidelines are applicable to test the efficacy of mosquito repellents, these guidelines were used to test the efficacy and complete protection times (CPTs) of three representative mosquito repellents: N,N-diethyl-3-methylbenzamide (DEET), citronella, and fennel oil. The repellency of citronella oil decreased over time, from 97.9% at 0 h to 71.4% at 1 h and 57.7% at 2 h, as did the repellency of fennel oil, from 88.6% at 0 h to 61.2% at 1 h and 47.4% at 2 h. In contrast, the repellency of DEET remained over 90% for 6 h. The CPT of DEET (360 min) was much longer than the CPTs of citronella (10.5 min) and fennel oil (8.4 min). These results did not differ significantly from previous findings, and hence confirm that the KFDA guidelines are applicable for testing the efficacy of mosquito repellents.

Therapeutic potential of an AcHERV-HPV L1 DNA vaccine.

Cervical cancer is strongly associated with chronic human papillomavirus infections, among which HPV16 is the most common. Two commercial HPV vaccines, Gardasil and Cervarix are effective for preventing HPV infection, but cannot be used to treat existing HPV infections. Previously, we developed a human endogenous retrovirus (HERV)-enveloped recombinant baculovirus capable of delivering the L1 genes of HPV types 16, 18, and 58 (AcHERV-HP16/18/58L1, AcHERV-HPV). Intramuscular administration of AcHERVHPV vaccines induced a strong cellular immune response as well as a humoral immune response. In this study, to examine the therapeutic effect of AcHERV-HPV in a mouse model, we established an HPV16 L1 expressing tumor cell line. Compared to Cervarix, immunization with AcHERVHPV greatly enhanced HPV16 L1-specific cytotoxic T lymphocytes (CTL) in C57BL/6 mice. Although vaccination could not remove preexisting tumors, strong CTL activity retarded the growth of inoculated tumor cells. These results indicate that AcHERV-HPV could serve as a potential therapeutic DNA vaccine against concurrent infection with HPV 16, 18, and 58.

Isolation of a gene, PnFL-1, expressed in Pharbitis cotyledons during floral induction.

A novel gene, designated PnFL-1, was isolated from flower-induced cotyledons in a short-day plant, Pharbitis nil, using messenger RNA differential display. PnFL-1 has no similarity to genes with known functions in databases, but the deduced amino acid sequence of the gene has 58% homology with a hypothetical protein of Arabidopsis including a phosphatidic acid phosphatase 2 domain. PnFL-1 is a single-copy gene that is expressed during the inductive dark period. Expression of PnFL-1 increased gradually from the 6th to 16th h of a 16-h dark period, and expression was extinguished by a brief exposure to light at the 8th hour of the dark period (night break treatment). PnFL-1 mRNA was found in cotyledons and leaves but not in stems and roots. These results indicate that the transcription of PnFL-1 in Pharbitis may be photoperiodically regulated and associated with photoperiodic events.

Cadmium activates Arabidopsis MPK3 and MPK6 via accumulation of reactive oxygen species.

Cadmium (Cd) is a non-essential toxic heavy metal that influences normal growth and development of plants. However, the molecular mechanisms by which plants recognize and respond to Cd remain poorly understood. We show that, in Arabidopsis, Cd activates the mitogen-activated protein kinases, MPK3 and MPK6, in a dose-dependent manner. Following treatment with Cd, these two MAPKs exhibited much higher activity in the roots than in the leaves, and pre-treatment with the reactive oxygen species (ROS) scavenger, glutathione, effectively inhibited their activation. These results suggest that the Cd sensing signaling pathway uses a build-up of ROS to trigger activation of Arabidopsis MPK3 and MPK6.

Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense.

Arabidopsis thaliana WRKY38 and WRKY62, encoding two structurally similar type III WRKY transcription factors, are induced in a Nonexpressor of PR Gene1 (NPR1)-dependent manner by salicylic acid (SA) or by virulent Pseudomonas syringae. Disease resistance and SA-regulated Pathogenesis-Related1 (PR1) gene expression are enhanced in the wrky38 and wrky62 single mutants and, to a greater extent, in the double mutants. Overexpression of WRKY38 or WRKY62 reduces disease resistance and PR1 expression. Thus, WRKY38 and WRKY62 function additively as negative regulators of plant basal defense. WRKY38 and WRKY62 interact with Histone Deacetylase 19 (HDA19). Expression of HDA19 is also induced by P. syringae, and the stability of its induced transcripts depends on SA and NPR1 in infected plants. Disruption of HDA19 leads to compromised resistance, whereas its overexpression results in enhanced resistance to P. syringae. Thus, HDA19 has a role opposite from those of WRKY38 and WRKY62 in basal resistance to the bacterial pathogen. Both WRKY38 and WRKY62 are transcriptional activators in plant cells, but their activation activities are abolished by overexpressed HDA19. Interaction of WRKY38 and WRKY62 with HDA19 may act to fine-tune plant basal defense responses.

Pathogen-induced Arabidopsis WRKY7 is a transcriptional repressor and enhances plant susceptibility to Pseudomonas syringae.

The Arabidopsis (Arabidopsis thaliana) WRKY7 gene is induced by pathogen infection and salicylic acid (SA) treatment and may therefore play a role in plant defense responses. Here, we show that WRKY7 is localized in the nucleus, recognizes DNA molecules with the W-box (TTGAC) elements, and functions as a transcriptional repressor in plant cells. To study its biological functions directly, we have characterized both loss-of-function T-DNA insertion and RNAi mutants and gain-of-function transgenic overexpression plants for WRKY7 in Arabidopsis. The T-DNA insertion and RNAi mutant plants displayed enhanced resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae as measured by significant decrease in both bacterial growth and symptom development as compared to those in wild-type plants. The enhanced resistance in the loss-of-function mutants was associated with increased induction of SA-regulated Pathogenesis-Related 1 (PR1) by the bacterial pathogen. Transgenic plants that constitutively overexpress WRKY7 have altered leaf growth and morphology strikingly similar to those observed in the previously isolated eds8 mutant plants. Like eds8 mutant plants, WRKY7-overexpressing plants supported more growth of P. syringae and developed more severe disease symptoms than wild-type plants. The enhanced susceptibility of both the WRKY7-overexpressing plants and the eds8 mutant correlated with reduced expression of defense-related genes, including PR1, but significantly increased accumulation of SA after pathogen infection, probably due to reduced negative feedback of SA synthesis. Thus, pathogen-induced WRKY7 transcription factor play a negative role in defense responses to P. syringae.