• Journal of Veterinary Science
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• v.24 no.6
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• pp.82.1-82.12
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• 2023

Background: The current conventional serotyping based on antigen-antisera agglutination could not provide a better understanding of the potential pathogenicity of Salmonella enterica subsp. enterica serovar Brancaster. Surveillance data from Malaysian poultry farms indicated an increase in its presence over the years. Objective: This study aims to investigate the virulence determinants and antimicrobial resistance in S. Brancaster isolated from chickens in Malaysia. Methods: One hundred strains of archived S. Brancaster isolated from chicken cloacal swabs and raw chicken meat from 2017 to 2022 were studied. Two sets of multiplex polymerase chain reaction (PCR) were conducted to identify eight virulence genes associated with pathogenicity in Salmonella (invasion protein gene [invA], Salmonella invasion protein gene [sipB], Salmonella-induced filament gene [sifA], cytolethal-distending toxin B gene [cdtB], Salmonella iron transporter gene [sitC], Salmonella pathogenicity islands gene [spiA], Salmonella plasmid virulence gene [spvB], and inositol phosphate phosphatase gene [sopB]). Antimicrobial susceptibility assessment was conducted by disc diffusion method on nine selected antibiotics for the S. Brancaster isolates. S. Brancaster, with the phenotypic ACSSuT-resistance pattern (ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline), was subjected to PCR to detect the corresponding resistance gene(s). Results: Virulence genes detected in S. Brancaster in this study were invA, sitC, spiA, sipB, sopB, sifA, cdtB, and spvB. A total of 36 antibiogram patterns of S. Brancaster with a high level of multidrug resistance were observed, with ampicillin exhibiting the highest resistance. Over a third of the isolates displayed ACSSuT-resistance, and seven resistance genes (β-lactamase temoneira [bla_TEM], florfenicol/chloramphenicol resistance gene [floR], streptomycin resistance gene [strA], aminoglycoside nucleotidyltransferase gene [ant(3")-Ia], sulfonamides resistance gene [sul-1, sul-2], and tetracycline resistance gene [tetA]) were detected. Conclusion: Multidrug-resistant S. Brancaster from chickens harbored an array of virulence-associated genes similar to other clinically significant and invasive non-typhoidal Salmonella serovars, placing it as another significant foodborne zoonosis.

• Molecules and Cells
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• v.46 no.12
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• pp.764-777
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• 2023

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC₅₀ values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

• Biomedical Science Letters
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• v.19 no.2
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• pp.142-146
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• 2013

Enterotoxigenic Bacteroides fragilis (ETBF) causes inflammatory diarrhea in humans and animals and is also implicated in colorectal cancer. ETBF-infected mice exhibit a prominent large intestinal inflammation characterized by neutrophil infiltration and induction of the Th17 response. In this study, we examined differences in the secreted cytokine profile of the cecum and proximal colon of ETBF-infected mice using an antibody array. Of the cytokines examined, we found that the cecal tissues from ETBF-infected mice secreted elevated levels of G-CSF, IL-6, IL-17 and LIX compared to non-toxigenic Bacteroides fragilis (NTBF) and Mock infected mice. The proximal colon tissues from ETBF-infected mice secreted higher levels of G-CSF, IL-6, KC, LIX, MIP-1g and MCP-1. This study demonstrates that the cecum and colon should be considered separately when assays are used to determine immune responsiveness to enteric infections.

• The Plant Pathology Journal
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• v.34 no.1
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• pp.35-43
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• 2018

Root-knot nematodes (Meloidogyne spp.) are parasites that attack many field crops and orchard trees, and affect both the quantity and quality of the products. A root-colonizing bacterium, Pseudomonas chlororaphis O6, possesses beneficial traits including strong nematicidal activity. To determine the molecular mechanisms involved in the nematicidal activity of P. chlororaphis O6, we constructed two mutants; one lacking hydrogen cyanide production, and a second lacking an insecticidal toxin, FitD. Root drenching with wild-type P. chlororaphis O6 cells caused juvenile mortality in vitro and in planta. Efficacy was not altered in the fitD mutant compared to the wild-type but was reduced in both bioassays for the mutant lacking hydrogen cyanide production. The reduced number of galls on tomato plants caused by the wild-type strain was comparable to that of a standard chemical nematicide. These findings suggest that hydrogen cyanide-producing root colonizers, such as P. chlororaphis O6, could be formulated as "green" nematicides that are compatible with many crops and offer agricultural sustainability.

• BMB Reports
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• v.30 no.5
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• pp.337-341
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• 1997

In order to investigate the role of a small GTP-binding protein RhoA in lysophosphatidic acid (LPA)-induced stress fiber formation, C3 ADP-ribosyltransferase was prepared by expressing in E. coli and then applied to Rat-2 fibroblasts. C3 transferase isolated from E. coli was as effective as the toxin from Clostridium botulinum in ADP-ribosylation of RhoA. Incubation of the cells with C3 transferase for 2 days induced ADP-ribosylation of RhoA by a dose-dependent manner, with a sub-maximal induction at $25\;{\mu}g/ml$. As expected, LPA-induced stress fiber formation was completely blocked by pre-incubation with C3 transferase for 2 days. However, exogenously added C3 transferase had no significant effect on the formation of phosphatidylethanol by LPA. These results suggested that phospholipase D was not activated by RhoA in the LPA-induced stress fiber formation.

• Journal of Microbiology and Biotechnology
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• v.30 no.10
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• pp.1552-1558
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• 2020

With an increase in the consumption of non-heated fresh food, foodborne shiga toxin-producing Escherichia coli (STEC) has emerged as one of the most problematic pathogens worldwide. Endolysin, a bacteriophage-derived lysis protein, is able to lyse the target bacteria without any special resistance, and thus has been garnering interest as a powerful antimicrobial agent. In this study, rV5-like phage endolysin targeting E. coli O157:H7, named as LysECP26, was identified and purified. This endolysin had a lysozyme-like catalytic domain, but differed markedly from the sequence of lambda phage endolysin. LysECP26 exhibited strong activity with a broad lytic spectrum against various gram-negative strains (29/29) and was relatively stable at a broad temperature range (4℃-55℃). The optimum temperature and pH ranges of LysECP26 were identified at 37℃-42℃ and pH 7-8, respectively. NaCl supplementation did not affect the lytic activity. Although LysECP26 was limited in that it could not pass the outer membrane, E. coli O157: H7 could be effectively controlled by adding ethylenediaminetetraacetic acid (EDTA) and citric acid (1.44 and 1.14 log CFU/ml) within 30 min. Therefore, LysECP26 may serve as an effective biocontrol agent for gram-negative pathogens, including E. coli O157:H7.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.7
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• pp.1044-1051
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• 2016

The present study compared the differential functions of two groups of adjuvants, Montanide incomplete Seppic adjuvant (ISA) series and Quil A, cholesterol, dimethyl dioctadecyl ammonium bromide, and Carbopol (QCDC) formulations, in chicken by analyzing published microarray data associated with each type of vaccine adjuvants. In the biological function analysis for differentially expressed genes altered by two different adjuvant groups, ISA series and QCDC formulations showed differential effects when chickens were immunized with a recombinant immunogenic protein of Eimeria. Among the biological functions, six categories were modified in both adjuvant types. However, with respect to "Response to stimulus", no biological process was modified by the two adjuvant groups at the same time. The QCDC adjuvants showed effects on the biological processes (BPs) including the innate immune response and the immune response to the external stimulus such as toxin and bacterium, while the ISA adjuvants modified the BPs to regulate cell movement and the response to stress. In pathway analysis, ISA adjuvants altered the genes involved in the functions related with cell junctions and the elimination of exogenous and endogenous macromolecules. The analysis in the present study could contribute to the development of precise adjuvants based on molecular signatures related with their immunological functions.

• Journal of Life Science
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• v.14 no.2
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• pp.301-308
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• 2004

The morphology of the apical pore complex, the first apical plate and the posterior sulcal plates in a new isolate of Alexandrium tamarense (Lebour) Balech from the Bay of Chinhae was compared with other that of toxic strains of A. tamarense previously isolated from Korean waters. Although this isolate was morphologically identical to these toxic strains, high performance liquid chromatography and mouse bioassay showed no evidence of toxin production. The nontoxic A. tamarense strain showed a strong positive binding activity with PNA lectin, indicating a high density of lactose and galactose residues on the cell surface, and in SDS-PAGE and Western blot analysis a unique protein of about 21-kDa molecular sizes was observed. These findings demonstrate that the use of PNA and immunobioassay could be used to discriminate between toxic and nontoxic strains of A. tamarense.

• Animal cells and systems
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• v.7 no.3
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• pp.173-186
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• 2003

The transition metal cadmium is a serious occupational and environmental toxin. To inhibit cadmium-induced damage, cells respond by increasing the expression of genes that encode stress-responsive proteins. The metal-regulatory transcription factor 1 (MTF-1) is a key regulator of heavy-metal induced transcription of metallothionein-I and II and other genes in mammals and other metazoans. Transcriptional activation of genes by MTF-1 is mediated through binding to metal-responsive elements in the target gene promoters. Phosphorylation of MTF-1 plays a critical role in the cadmium-inducible transcriptional activation of metallothionein and other responses. Studies using inhibitors indicate that multiple kinases and signal transduction cascades, including those mediated by protein kinase C, tyrosine kinase and casein kinase II, are essential for cadmium-mediated transcriptional activation. In addition, calcium signaling is also involved in regulating metal-activated transcription. In several species, cadmium induces heat shock genes. Recently much progress has been made in elucidating the cellular machinery that regulates this metal-inducible gene expression. This review summarizes these recent advances in understanding the role of some known cadmium-responsive genes and the molecular mechanisms that activate metal-responsive transcription factor, MTF-1.

• Animal cells and systems
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• v.14 no.3
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• pp.155-160
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• 2010

Extracellular signal-regulated kinases 1/2 (ERK1/2) play important roles in a variety of biological processes including cell growth and differentiation. We have previously reported that GAR-3 activates ERK1/2 via phospholipase C and protein kinase C, presumably through pertussis toxin (PTX)-insensitive Gq proteins, in Chinese hamster ovary (CHO) cells. Here we provide evidence that GAR-3 also activates ERK1/2 through PTX-sensitive G proteins, phosphatidylinositol 3-kinase (PI 3-kinase), and Src family kinases in CHO cells. We further show that in human embryonic kidney (HEK293) cells, epidermal growth factor receptor and Ras are required for efficient ERK1/2 activation by GAR-3. Taken together, our data indicate that GAR-3 evokes ERK1/2 activation through multiple signaling pathways in cultured mammalian cells.