• Korean Journal of Veterinary Research
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• v.43 no.4
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• pp.667-675
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• 2003

The safety of Brucella abortus strain RB51(SRB51) was investigated in dairy cows and Korean native cattle of 4~7th month of gestation. From experimentally inoculated cattle, 18 of 25 (72.0%) dairy cows, and 3 of 10 (30.0%) Korean native cattle were aborted or delivered premature fetus. There were no significant differences in the incidence of abortion depending on the inoculation route (intramuscular and subcutaneous) and dosages ($1{\times}10^9$, $2.8{\times}10^9$, and $4.0{\times}10^9$ CFU). The antibodies to the SRB51 were measured by a dot blot enzyme-linked immunosorbent assay. The highest titers to SRB51 were detected between 5~7 weeks after inoculation and the specitic antibody could be detected up to 28 weeks after inoculation. The SRB51 was isolated from amnio-allantoic fluid, bronchial lymph node, mammary gland, and supramammary lymph node in 5 of 25 dairy cows during 4 weeks after either abortion or delivery. Although SRB51 was isolated from 4 of 24 aborted fetus or normally delivered calves at parturition time, it was not isolated during 4 weeks afterward. Eleven of twentyfive dairy cows showed the endometritis and/or necrosis until 6 weeks after delivery, no lesions were seen at 8 weeks after delivery and uterus from control dairy cows. The results of present study revealed that SRB51 might induce the clinical signs of brucellosis in the pregnant cattle at 4~7th month of gestation.

• The Plant Pathology Journal
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• v.29 no.4
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• pp.410-417
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• 2013

In 2011-2012, sixty nine samples were collected from alfalfa plants showing viral infection symptoms in Riyadh region. Mechanical inoculation with sap prepared from two collected samples out of twenty five possitive for Alfalfa mosaic virus (AMV) by ELISA were produced systemic mosaic on Vigna unguiculata and Nicotiana tabacum, local lesion on Chenopodium amaranticolor and C. quinoa. Vicia faba indicator plants that induce mosaic and mottle with AMV-Sagir isolate and no infection with AMV-Wadi aldawasser isolate. Approximately 700-bp was formed by RT-PCR using AMV coat protein specific primer. Samples from infected alfalfa gave positive results, while healthy plant gave negative result using dot blot hybridization assay. The nucleotide sequences of the Saudi isolates were compared with corresponding viral nucleotide sequences reported in GenBank. The obtained results showed that the AMV from Australia, Brazil, Puglia and China had the highest similarity with AMV-Sajer isolate. While, the AMV from Spain and New Zealaland had the lowest similarity with AMV-Sajer and Wadi aldawasser isolates. The data obtained in this study has been deposited in the GenBank under the accession numbers KC434083 and KC434084 for AMV-Sajer and AMV-Wadialdawasser respectively. This is the first report regarding the gnetic make up of AMV in Saudi Arabia.

• Journal of Plant Biotechnology
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• v.35 no.1
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• pp.87-94
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• 2008

Porcine epidemic diarrhea virus (PEDV) is an infectious and highly contagious virus of swine. In order to develop the transgenic tobacco culture cells producing PEDV antigen protein, four vectors expressing PEDV spike protein (SP) gene under the control of a CaMV 35S promoter were constructed. Four fragments of the SP region of PEDV, SP1 (444 bp, 1487-1930 bp), SP2 (1.7 kb, 2300-3987 bp), SP3 (1.4 kb, 1559-2950 bp), and SP4 (2.6 kb, 9-2643 bp) were amplified by PCR and then C-MYC tag was fused to the end of each SP gene, respectively. These cassettes are inserted into the pCAMBIA2300 (named as 35S::SP1-M, 35S::SP2-M 35S::SP3-M, and 35S::SP4-M, respectively). Tobacco (cv. BY-2) cultured cells were transformed by co-cultivation with Agrobacterium tumefaciens harboring expression vector. We selected kanamycin-resistant calli and checked for the presence of the introduced SP gene using PCR, resulting 70% of them showed the foreign gene. We selected the lines with high-level expression of PEDV antigen protein based on dot blot analysis. Southern blot analysis confirmed that the PEDV SP gene was integrated into the genome of the tobacco cultured cells. Northern blot analysis showed that the introduced gene was highly expressed in transgenic cultured cells. Transgenic tobacco cultured cells-derived antigen induced immunogenicity in mice as determined by a plaque reduction neutralization assay. These results suggest that the vectors expressing PEDV spike protein gene in this study will be useful for the development of transgenic plants and cultured cells producing PEDV antigene protein.

• Proceedings of the Korea Society of Environmental Toocicology Conference
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• 2003.05a
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• pp.187-187
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• 2003

Methylmercury (MeHg), one of the heavy metal compounds, can cause severe damage to the central nervous system in humans. Many reports have shown that MeHg is poisonous to human body through contaminated foods and has released into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established so far. In this study, two methods, cDNA Microarray and SSH, were performed to assess the expression profile against MeHg and to identify differentially expressed genes by MeHg in neuroblastoma cell line. TwinChip Human-8K (Digital Genomics) was used with total RNA from SH-SY5Y (human neuroblastoma cell line) treated with solvent (DMSO) and 6.25 uM (IC50) MeHg. And we performed forward and reverse SSH method on mRNA derived from SH-SY5Y treated with DMSO and MeHg (6.25 uM). Differentially expressed cDNA clones were sequenced and were screened by dot blot and ribonuclease protection assay to confirm that individual clones indeed represent differentially expressed genes. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences may provide an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as environmental pollutants.

• International Journal of Industrial Entomology and Biomaterials
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• v.7 no.2
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• pp.165-173
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• 2003

Cytokines represent an essential part of the innate immune response in mammals. Recently, several studies have reported the presence of cytokine-like activities and molecules in the invertebrates such as echinoderms, tunicates, mollusks and insects. In our serial study, we investigated presence of cytokines in the silkworm, Bombyx mori, infected with several immune inducers. Western blotting analysis using rabbit anti-human cytokines showed the presence of IL-6-like molecule in the hemolymph collected at 8 and 24 hrs after infection with peptidoglycan and oligodeoxynucleotide, and the molecular weight of the proteins was ∼45 kDa. We attempted to isolate the molecule by gel permeation HPLC, anion exchange chromatography, ultra centrifugation, and immuno-dot-blot assay, but until now the effort was not much successful yet. It, however, does not appear that the IL-6-like molecule in the silkworm larvae is a mere experimental artifact happened by Western blotting analysis. Instead, further experiment on this subject probably will provide us more fruitful result as detected in other invertebrates including insects.

• Korean Journal of Veterinary Research
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• v.41 no.1
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• pp.51-57
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• 2001

A procedure for extraction and purification of 8 kDa antigen of Brucella abortus RB51 was developed. Bacteria heat inactivated at $60^{\circ}C$, 30 min was extracted by 1% sarcosine and followed by fluid pressure liquid gel filtration chromatography of 2 series, Superose 12 HR 10/30 and Sephacryl S-100. There was produced $71.46{\mu}g/g$(wet) of 8 kDa antigen, and it resisted 1% trypsin, solved 1% triton X-100 higher than distilled water and inactivated 0.1% proteinase K. These results show that 8 kDa antigen may be a lipoprotein existed cell surface of B. abortus RB51. Also, we developed ELISA using purified 8 kDa surface antigen of Brucella abortus RB51 strain, its specificity and sensitivity was 95.0%, 98.6%, respectively. As compared with dot-blot assay using whole cell and ELISA using 8 kDa antigen, its correlation was 93.5%.

• BMB Reports
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• v.37 no.6
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• pp.720-725
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• 2004

A number of signaling molecules contain small pleckstrin homology (PH) domains capable of binding phosphoinositides or proteins. Phospholipase C (PLC)-${\gamma}1$ has two putative PH domains, an $NH_2$-terminal (PH1) and a split PH domain ($nPH_2$ and $cPH_2$). We previously reported that the split PH domain of PLC-${\gamma}1$ binds to phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)$P_2$) (Chang et al., 2002). To identify the amino acid residues responsible for binding with PI(4)P and PI(4,5)$P_2$, we used site-directed mutagenesis to replace each amino acid in the variable loop-1 (VL-1) region of the PLC-${\gamma}1$ $nPH_2$ domain with alanine (a neutral amino acid). The phosphoinositide-binding affinity of these mutant molecules was analyzed by Dot-blot assay followed by ECL detection. We found that two PLC-${\gamma}1$ nPH2 domain mutants, P500A and H503A, showed reduced affinities for phosphoinositide binding. Furthermore, these mutant PLC-${\gamma}1$ molecules showed reduced PI(4,5)$P_2$ hydrolysis. Using green fluorescent protein (GFP) fusion protein system, we showed that both $PH_1$ and $nPH_2$ domains are responsible for membrane-targeted translocation of PLC-${\gamma}1$ upon serum stimulation. Together, our data reveal that the amino acid residues $Pro^{500}$ and $His^{503}$ are critical for binding of PLC-${\gamma}1$ to one of its substrates, PI(4,5)$P_2$ in the membrane.