A combined approach for locating box H/ACA snoRNAs in the human genome

A novel combined method for locating box H/ACA small nucleolar RNAs (snoRNAs) is described, together with a software tool. The method adopts both a probabilistic hidden Markov model (HMM) and a minimum free energy (MFE) rule, and filters possible candidate box H/ACA snoRNAs obtained from genomic DNA sequences. With our novel method 12 known box H/ACA snoRNAs, and one strong candidate were identified in 30 nucleolar protein genomic sequences.     

Modulation of immunity against herpes simplex virus infection via mucosal genetic transfer of plasmid DNA encoding chemokines

In this study, we examined the effects of murine chemokine DNA, as genetic adjuvants given mucosally, on the systemic and distal mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV) by using the mouse model. The CC chemokines macrophage inflammatory protein 1beta (MIP-1beta) and monocyte chemotactic protein 1 (MCP-1) biased the immunity to the Th2-type pattern as judged by the ratio of immunoglobulin isotypes and interleukin-4 cytokine levels produced by CD4(+) T cells. The CXC chemokine MIP-2 and the CC chemokine MIP-1alpha, however, mounted immune responses of the Th1-type pattern, and such a response rendered recipients more resistant to HSV vaginal infection. In addition, MIP-1alpha appeared to act via the upregulation of antigen-presenting cell (APC) function and the expression of costimulatory molecules (B7-1 and B7-2), whereas MIP-2 enhanced Th1-type CD4(+) T-cell-mediated adaptive immunity by increasing gamma interferon secretion from activated NK cells. Our results emphasize the value of using the mucosal route to administer DNA modulators such as chemokines that function as adjuvants by regulating the activity of innate immunity. Our findings provide new insight into the value of CXC and CC chemokines, which act on different innate cellular components as the linkage signals between innate and adaptive immunity in mucosal DNA vaccination.     

Exploring the Genetic Signature of Body Size in Yucatan Miniature Pig

Since being domesticated about 10,000–12,000 years ago, domestic pigs (Sus scrofa domesticus) have been selected for traits of economic importance, in particular large body size. However, Yucatan miniature pigs have been selected for small body size to withstand high temperature environment and for laboratory use. This renders the Yucatan miniature pig a valuable model for understanding the evolution of body size. We investigate the genetic signature for selection of body size in the Yucatan miniature pig. Phylogenetic distance of Yucatan miniature pig was compared to other large swine breeds (Yorkshire, Landrace, Duroc and wild boar). By estimating the XP-EHH statistic using re-sequencing data derived from 70 pigs, we were able to unravel the signatures of selection of body size. We found that both selections at the level of organism, and at the cellular level have occurred. Selection at the higher levels include feed intake, regulation of body weight and increase in mass while selection at the molecular level includes cell cycle and cell proliferation. Positively selected genes probed by XP-EHH may provide insight into the docile character and innate immunity as well as body size of Yucatan miniature pig.     

Genetic structure and variability of the working dog inferred from microsatellite marker analysis

Working dogs serve as military watch dogs, search dogs, rescue dogs, and guide dogs with un-come-at-able character. They are drafted by in-training examination including concentration, capacity for locomotion, boldness and earthly desires. In this study, genetic diversity and relationships among two groups of working dogs (pass and fail group in-training examination) were assessed based on 15 microsatellite markers in 25 individuals of working dogs (military watch dogs and Korean search dogs). For the 15 microsatellite markers, the values of allelic richness (A _R ) ranged from 2.21 (pass group) to 1.60 (fail group) in military watch dogs, while A _R ranged from 2.79 (pass group) to 2.72 (fail group) in Korean search dogs. Among 52 different alleles of military watch dogs, 22 alleles were detected in pass group only, while 8 alleles in fail group only. In case of Korean search dogs, 3 alleles were observed in pass group only, while 13 alleles in fail group only. These group-specific unique alleles reflect good biomarker for selecting working dogs (military watch dogs and Korean search dogs), indicating that those group specific microsatellite alleles could separate working dogs to be pass or fail group in out-training dog population. Taken together, this study demonstrates the feasibility of microsatellite analyses for the selection of superior working dogs objectively. Furthermore, this approach could be used for the proper selection of working dogs in combination with in-training examination.     

Protective immunity induced by systemic and mucosal delivery of DNA vaccine expressing glycoprotein B of pseudorabies virus

A murine model immunized by systemic and mucosal delivery of plasmid DNA vaccine expressing glycoprotein B (pCIgB) of pseudorabies virus (PrV) was used to evaluate both the nature of the induced immunity and protection against a virulent virus. With regard to systemic delivery, the intramuscular (i.m.) immunization with pCIgB induced strong PrV-specific IgG responses in serum but was inefficient in generating a mucosal IgA response. Mucosal delivery through intranasal (i.n.) immunization of pCIgB induced both systemic and mucosal immunity at the distal mucosal site. However, the levels of systemic immunity induced by i.n. immunization were less than those induced by i.m. immunization. Moreover, i.n. genetic transfer of pCIgB appeared to induce Th2-biased immunity compared with systemic delivery, as judged by the ratio of PrV-specific IgG isotypes and Th1- and Th2-type cytokines produced by stimulated T cells. Moreover, the immunity induced by i.n. immunization did not provide effective protection against i.n. challenge of a virulent PrV strain, whereas i.m. immunization produced resistance to viral infection. Therefore, although i.n. immunization was a useful route for inducing mucosal immunity at the virus entry site, i.n. immunization did not provide effective protection against the lethal infection of PrV.     

Removal and inactivation of human immunodeficiency virus (HIV-1) by cold ethanol fractionation and pasteurization during the manufacturing of albumin and immunoglobulins from human plasma

Viral safety is a prerequisite for manufacturing clinical albumin and immunoglobulins from human plasma pools. This study was designed to evaluate the efficacy of cold ethanol fractionation and pasteurization (60°C heat treatment for 10 h) for the removal inactivation of human immunodeficiency virus type 1 (HIV-1) during the manufacturing of albumin and immunoglobulins. Samples from the relevant stages of the production process were spiked with HIV-1, and the amount of virus in each fraction was quantified by the 50% tissue culture infectious dose (TCID₅₀). Both fraction IV fractionation and pasteurization steps during albumin processing were robust and effective in inactivating HIV-1, titers of which were reduced from an initial 8.5 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved were ≥4.5 and ≥6.5, respectively. In addition, fraction III fractionation and pasteurization during immunoglobulins processing were robust and effective in eliminating HIV-1. HIV-1 titers were reduced from an initial 7.3 log₁₀ TCID₅₀ to undetectable levels. The log reduction factors achieved in this case were ≥4.9 and ≥5.3, respectively. These results indicate that the process investigated for the production of albumin and immunoglobulins have sufficient HIV-1 reducing capacity to achieve a high margin of safety.     

Long interspersed element-1 open reading frame 1 protein expression profiles in ovarian cancers

Long interspersed element-1 (LINE-1) retrotransposons are autonomous mobile DNA elements with unique activity that account for about one-fifth of the human genome. Recently, it has been reported that the expression of LINE-1 is closely related to cancer prognosis, and LINE-1 hypomethylation might contribute to the acquisition of aggressive tumor behavior. Despite the importance of LINE-1 expression in cancers, research on the expression of LINE-1 open reading frame (ORF) proteins is very limited. Here, we investigated the expression profiles of LINE-1 ORF1p in ovarian cancer tissue microarrays containing 100 surgical specimens including adjacent normal ovary tissue, primary ovarian cancers, and metastatic ovarian cancers in lymph node. The tissue microarray was stained with mouse monoclonal antibody to LINE-1 ORFp1 for immunofluorescence analysis, and expression levels were evaluated by image analysis. LINE-1 ORFp was significantly overexpressed in ovarian cancers compared with normal tissues and especially upregulated in metastatic ovarian cancers. In addition, the expression of LINE-1 ORF1p was significantly higher in older ovarian cancer patients compared with young patients. These results indicate that expression of LINE-1 ORF1p is related to the progression of ovarian cancers and, in particular, to the age of the patient and the metastatic potential of the cancer.     

Determination of free acetaldehyde in total blood for investigating the effect of aspartate on metabolism of alcohol in mice

To explore the effect of sodium l-aspartate monohydrate (aspartate) as a NAD⁺ regenerating agent for acetaldehyde in alcohol metabolism, a simple HPLC method has been developed for the measurement of free acetaldehyde in total mice blood digested with alcohol and aspartate. The blood samples were collected in EDTA Vacutainer tubes, and treated with 2,4-dinitrophenylhydrazine (DNP hydrazine) reagent in total blood. Acetaldehyde DNP hydrazone was extracted from total blood and analyzed by HPLC using an Ultrasphere ODS column. The compounds were separated using acetonitrile–water (50:50, v/v) as mobile phase and detected at 356 nm. The detection limit for acetaldehyde DNP hydrazone was 0.1 ppm. A blank determination was carried out for each analysis and subtracted from the results. The amount of acetaldehyde in blood has been determined as a function of time lapse after sole alcohol administration and aspartate ingestion followed by alcohol administration, respectively. This comparative analysis demonstrates that the ingestion of aspartate before the administration of alcohol dramatically decreases the aldehyde level in blood, and aspartate may be utilized as a prospective antagonist for acceleration of ethanol metabolism and prevention of acetaldehyde toxicity.