• BMB Reports
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• v.39 no.1
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• pp.9-15
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• 2006

To prove whether error catastrophe /lethal mutagenesis is the primary antiviral mechanism of action of ribavirin against foot-and-mouth disease virus (FMDV). Ribavirin passage experiments were performed and supernatants of $Rp_1$ to $Rp_5$ were harvested. Morphological alterations as well as the levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected using the supernatants of $Rp_1$ to $Rp_5$ and control were measured by microscope, real-time RT-PCR, western-blotting and plaque assays, respectively. The mutation frequency was measured by sequencing the complete P1- and 3D-encoding region of FMDV after a single round of virus infection from ribavirin-treated or untreated FMDV-infected cells. Ribavirin treatment for FMDV caused dramatically inhibition of multiplication in cell cultures. The levels of viral RNAs, proteins, and infectious particles in the BHK-21 cells infected were more greatly reduced along with the passage from $Rp_1$ to $Rp_5$, moreover, nucleocapsid protein could not be detected and no recovery of infectious virus in the supernatant or detection of intracellular viral RNA was observed at the $Rp_5$-infected cells. A high mutation rate, giving rise to an 8-and 11-fold increase in mutagenesis and resulting in some amino acid substitutions, was found in viral RNA synthesized at a single round of virus infection in the presence of ribavirin of $1000\;{\mu}M$ and caused a 99.7% loss in viral infectivity in contrast with parallel untreated control virus. These results suggest that the antiviral molecular mechanism of ribavirin is based on the lethal mutagenesis/error catastrophe, that is, the ribavirin is not merely an antiviral reagent but also an effective mutagen.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.880-884
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• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.

• Journal of Korean Neurosurgical Society
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• v.37 no.6
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• pp.443-448
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• 2005

Objective: A variety of genetic alterations in human glioblastoma comprises signal transduction and cell cycle arrest control of cellular processes. Subtractive hybridization is potentially a faster method for identifying differentially expressed genes associated with a particular disease state. Using the technique of subtraction, we isolated novel genes that are overexpressed in glioblastoma tissue as compared to normal brain tissue. Methods: We evaluated the differential expression of genes in each of hybridizing tester and driver cDNAs to digested 130 clones. After sequencing of 130 clones and homology search, this study performed to determine mRNA expression of the unknown gene, "clone 47", in brain tissue, glioblasoma, and several cancer cell lines by reverse transcription-polymerase chain reaction (RT-PCR). To test the time course for Go-phase arrest, serum stimulation and expression at various times for RT-PCR performed. Results: We identified 23 novel genes by BLAST of the digested 130 clones. The expressions of "clone 47" mRNA of glioblastoma and several cancer lines were significantly higher than normal brain tissues and several normal cell lines. We confirmed the mRNA expression of "clone 47" was up-regulation for $0.5{\sim}1hr$ of WI-38 cell differentiation. Conclusion: The novel gene, "Clone 47" is upregulated in glioblastoma tissue and several cancer cell lines. This gene is time dependent activation during time course of serum stimulation. This result suggests that "clone 47" playa role in brain tumorigenesis and the activation of this "clone 47" may be necessary for the development of cancer.

• Journal of Chest Surgery
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• v.42 no.2
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• pp.141-147
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• 2009

Background: We performed this study to identify the tumor suppressor genes located in the long arm of chromosome 21 in non-small cell lung cancer. Material and Method: The genes of USP25 in 21q11.2, NCAM2, ADAMTS1 in 21q21.2, and Claudin-8 (CLDN8), Claudin-17 (CLDN17) and TIAM1 in 21q22.1 were investigated for their gene expressions, genetic alterations and promoter methylation. Result: The expressions of CLDN8 and CLDN17 were significantly decreased in 7 (L132, H157, H358, H522, H1299, H1703 and HCC2108) of 13 cell lines, and the expression of ADAMTS1 was also significantly reduced in 6 cell lines (A549, SW900, H1299, H1373, H1703 and H1793). There were no genetic alterations by PCR-SSCP and cDNA cloning in the cell lines with a decreased gene. In the cell lines with a decreased gene expression, the mRNA expression was increased significantly with treatment of 5-Aza-CdR. Conclusion: These results suggest that the ADMTS1, CLDN8 and CLDN17 may act as tumor suppressor genes.

• Journal of Gastric Cancer
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• v.3 no.1
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• pp.26-32
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• 2003

Purpose: Genomic alterations and abnormal expression of the fragile histidine triad (FHIT) gene in gastric cancer were examined to determine whether the FHIT gene is actually a frequent target for alteration during gastric carcinogenesis. Materials and Methods: To correlate DNA and RNA lesions of the FHIT gene with the effect on FHIT protein expression, in 40 gastric cancers, we investigated the FHIT gene for loss of heterozygisity (LOH), aberrant transcripts, and protein expression. Results: Allelic loss at D3S1300 was detected in 7 of 38 ($19\%$) informative cases. Aberrant transcripts were observed in 20 of 40 ($50\%$) cases. Significant reduction of FHIT protein expression was observed in 22 of 40 ($55\%$) cases. Aberrant FHIT transcription was shown to be associated with loss of FHIT protein expression. However, aberrent FHIT transcripts themselves were not associated with any clinicopathological parameters, such as age, sex, tumor site, or clinical stage. Moreover, there was no association between the presence of LOH at D3S1300 and the expression of aberrant FHIT transcripts. Conclusion: The high frequency of aberrant FHIT transcripts, the significant rate of LOH at D3S1300, and the altered expression of the FHIT protein indicate that alterations of the FHIT gene can play an important role in gastric carcinogenesis.

• BMB Reports
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• v.35 no.2
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• pp.172-177
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• 2002

We previously reported that cholesteryl ester transfer protein (CETP) inhibitory peptides (designated $P_{28}$ and $P_{10})$ have anti-atherogenic effects in hypercholesterolemic rabbits (Biochim. Biophys. Acta (1998) 1391, 133-144). To further investigate those effects, we studied rabbit plasma that was collected after 30 h of a $P_{28}$ or $P_{10}$ injection. We found that there is a strong correlation between the in vivo CETP inhibition effects and alterations of lipoprotein particle size distribution in rabbit plasma, as determined on an agarose gel electrophoresis and gel filtration column chromatography. In vivo effects of the peptide were observed again in C57BL/6 mice that expressed simian CETP. The $P_{28}$ or $P_{10}$ peptide ($7\;{\mu}g/g$ of body weight) that was dissolved in saline was injected subcutaneously into the mice. The $P_{28}$ injection caused the partial inhibition of plasma CETP activity up to 50%, decreasing the total plasma cholesterol concentration by 30%, and increasing the ratio of HD/total-cholesterol concentration by 150% in the CETP-transgenic (tg) mice. The CETP inhibition by the $P_{28}$ or $P_{10}$ made alterations that modulated the size re-distribution of the lipoproteins in the blood stream. Particle size of the very low (VLDL) and low density lipoproteins (LDL) from the peptide-injected group was highly decreased compared to the saline-injected group (determined on the gel filtration column chromatography). In contrast, The HDL particle size of the $P_{28}$-injected group increased compared to the control group (saline-injected). The expression level of the CETP mRNA of the $P_{28}$-injected CETP-tg mouse appeared lower than the saline-injected CETP-tg mouse. These results suggest that the injection of the CETP inhibitory peptide could affect the CETP expression level in the liver by influencing lipoprotein metabolism.

• Tuberculosis and Respiratory Diseases
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• v.49 no.6
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• pp.703-714
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• 2000

Background : Surfactant protein A (SP-A) is important in the regulation of surfactant secretion, synthesis and recycling. SP-A has important roles in regulating surfactant metabolism as well as in determining surfactant's physical properties. Since systemic sepsis is one of the common causes of acute respiratory distress syndrome (ARDS) and abnormalities in surfactant function have been described in ARDS, the authors investigated the effects of endotoxemia on the accumulation of mRNA encoding SP-A and SP-A protein content. Methods : Adult rats were given various doses of intraperitoneal endotoxin from Salmonella enteritidis and sacrificed at different times. SP-A mRNA was measured by filter hybridization method. Lung SP-A protein content was determined by double sandwich ELISA assay using a polyclonal antiserum raised in rabbits against purified rat SP-A. Results : 1) The accumulation of SP-A mRNA in the endotoxin treated group 24 hours after 2mg/kg and 5mg/kg endotoxin treatments was significantly increased 50.9% and 27.3%, respectively, compared to the control group (P<0.001, P<0.025). 2) The accumulation of SP-A mRNA 24 hours in the 5mg/kg endotoxin treated group was significantly increased by 26.5% compared to the control group (P<0.01). 3) Total amount of lung SP-A was not altered at 24 hours by various doses of treatment. Total lung SP-A content 144 hours after endotoxin administration was significantly decreased by 51.4% compared to the control group (P<0.01). Conclusions : The specific regulation of SP-A by various time course in vivo is evident. The late decline in SP-A protein content was unexpected and suggests that SP-A may be differentially regulated during lung inflammation. The functional significance of these alterations remains to be clarified.

• Journal of Nutrition and Health
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• v.51 no.6
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• pp.489-497
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• 2018

Purpose: Obesity is often associated with disturbances in the mineral metabolism. The purpose of this study was to investigate the effects of high-fat diet-induced obesity on tissue zinc concentrations and zinc transporter expressions in mice. Methods: C57BL/6J male mice were fed either a control diet (10% energy from fat, control group) or a high-fat diet (45% energy from fat, obese group) for 15 weeks. The zinc concentrations in the serum, stool, and various tissues were measured by inductively coupled plasma (ICP)-atomic emission spectrophotometry or ICP-mass spectrophotometry. The levels of zinc transporter mRNAs in the liver, duodenum, and pancreas were measured by real-time RT-PCR. The levels of serum adipokines, such as leptin and IL-6, were determined. Results: The total body weight, adipose tissue weight, and hepatic TG and cholesterol concentrations were significantly higher in the obese group, as compared to the control group. The obese group had significantly higher levels of serum leptin and pro-inflammatory IL-6 concentrations, and had significantly lower levels of serum alkaline phosphatase activity. The zinc concentrations of the liver, kidney, duodenum, and pancreas were all significantly lower in the obese group than in the control group. On the other hand, the fecal zinc concentrations were significantly higher in the obese group than in the control group. The serum zinc concentrations were not significantly different between the two groups. The ZnT1 mRNA levels of the liver and the pancreas were significantly higher in the obese group, as compared to the control group. Hepatic Zip10 mRNA was also increased in the obese group. Conclusion: Our study findings suggest that obesity increases fecal zinc excretion and lowers the tissue zinc concentrations, which may be associated with alterations in the zinc transporter expressions.

• Journal of Life Science
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• v.33 no.10
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• pp.828-834
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• 2023

The cDNA that encodes transmembrane protein 258 (Tmem258) was cloned from Gryllus bimaculatus and named GbTmem258. This protein comprises 80 amino acids, has no N-glycosylation site, and contains five potential phosphorylation sites at two serines, two threonines, and one tyrosine. The predicted molecular mass of GbTmem258 is 9.06 kDa, and its theoretical isoelectric point is 5.5. The tertiary structure of GbTmem258 was predicted using the available secondary structure information, which suggests the presence of alpha helices (52.5%), random coils (22.5%), extended strands (16.25%), and beta turns (8.75%). Homology analysis revealed that GbTmem258 exhibits high similarity at the amino-acid level to Tmem258 found in other species. The effect of starvation and refeeding on GbTmem258 mRNA expression was also examined in this study. It was found that GbTmem258 mRNA expression in the hindgut progressively increased throughout the starvation period, peaking at almost 1.5 times the control level after six days of starvation. However, refeeding for one to two days after the six-day starvation period restored GbTmem258 mRNA expression to the control level. In fat body, GbTmem258 mRNA expression was almost 3-fold higher during starvation compared to the control level. Refeeding for one to two days after the six-day fast resulted in a decline in the expression to about a 2.5-fold increase over the control level. Throughout the starving and refeeding periods, no other tissues showed any discernible alterations in GbTmem258 mRNA expression.

• Development and Reproduction
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• v.13 no.4
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• pp.257-264
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• 2009

A neurotoxin, 6-hydroxydopamine (6-OHDA) has been widely used to create animal model for Parkinson's disease (PD) due to its specific toxicity against dopaminergic (DA) neurons. Since DA signals modulate a broad spectrum of CNS physiology, one can expect profound alterations in neuroendocrine activities of both PD patients and 6-OHDA treated animals. Limited applications of 6-OHDA injection model, however, have been made on the studies of hypothalamuspituitary neuroendocrine circuits. The present study was performed to examine whether blockade of brain catecholamine (CA) biosynthesis with 6-OHDA can make any alteration in the transcriptional activities of hypothalamus-pituitary hormone genes in adult male rats. Three-month-old male rats (SD strain) were received 6-OHDA ($200{\mu}g$ in $10{\mu}\ell$ of saline/animal) by intracerebroventricular (icv) injection, and sacrificed after two weeks. To determine the mRNA levels of hypothalamuspituitary hormone genes, total RNAs were extracted and applied to the semi-quantitative RT-PCRs. The mRNA levels of tyrosine hydroxylase (TH), the rate-limiting enzyme for the catecholamine biosynthesis, were significantly lower than those from the control group (control:6-OHDA=1:0.72${\pm}$0.02AU, p<0.001), confirming the efficacy of 6-OHDA injection. The mRNA levels of gonadotropin-releasing hormone (GnRH) and corticotropin releasing hormone (CRH) in the hypothalami from 6-OHDA group were significantly lower than those from the control group (GnRH, control:6-OHDA=1:0.39${\pm}$0.03AU, p<0.001; CRH, control:6-OHDA=1:0.76${\pm}$0.07AU, p<0.01). There were significant decreases in the mRNA levels of common alpha subunit of glycoprotein homones (Cg$\alpha$), LH beta subunit (LH-$\beta$), and FSH beta subunit (FSH-$\beta$) in pituitaries from 6-OHDA group compared to control values (Cg$\alpha$, control:6-OHDA=1:0.81${\pm}$0.02AU, p<0.001; LH-$\beta$, control:6-OHDA=1:0.68${\pm}$0.04AU, p<0.001; FSH-$\beta$, control:6-OHDA=1:0.84${\pm}$0.05AU, p<0.001). Similarly, the level of adrenocorticotrophic hormone (ACTH) transcripts from 6-OHDA group was significantly lower than that from the control group (control: 6-OHDA=1:0.86${\pm}$0.04AU, p<0.01). The present study demonstrated that centrally injected DA neurotoxin could downregulate the transcriptional activities of the two hypothalamus-pituitary neuroendocrine circuits, i.e., GnRH-gonadotropins and CRH-ACTH systems. These results suggested that hypothalamic CA input might affect on the activities of gonad and adrenal through modulation of hypothalamus-pituitary function, providing plausible explanation for frequent occurrence of sexual dysfunction and poor stress-response in PD patients.