• Asian-Australasian Journal of Animal Sciences
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• v.15 no.10
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• pp.1458-1468
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• 2002

There is increasing interest in exploiting natural products as feed additives to solve problems in animal nutrition and livestock production. Essential oils and saponins are two types of plant secondary compounds that hold promise as natural feed additives for ruminants. This paper describes recent advances in research into these additives. The research has generally concentrated on protein metabolism. Dietary essential oils caused rates of NH$_3$ production from amino acids in ruminal fluid taken from sheep and cattle receiving the oils to decrease, yet proteinase and peptidase activities were unchanged. Hyper-ammonia-producing (HAP) bacteria were the most sensitive of ruminal bacteria to essential oils in pure culture. Essential oils also slowed colonisation and digestion of some feedstuffs. Ruminobacter amylophilus may be a key organism in mediating these effects. Saponin-containing plants and their extracts appear to be useful as a means of suppressing the bacteriolytic activity of rumen ciliate protozoa and thereby enhancing total microbial protein flow from the rumen. The effects of some saponins seems to be transient, which may stem from the hydrolysis of saponins to their corresponding sapogenin aglycones, which are much less toxic to protozoa. Saponins also have selective antibacterial effects which may prove useful in, for example, controlling starch digestion. These studies illustrate that plant secondary compounds, of which essential oils and saponins comprise a small proportion, have great potential as 'natural' manipulators of rumen fermentation, to the potential benefit of the farmer and the environment.

• The Plant Pathology Journal
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• v.33 no.3
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• pp.213-228
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• 2017

Plasmodesmata (PDs) are specialized intercellular channels that facilitate the exchange of various molecules, including sugars, ribonucleoprotein complexes, transcription factors, and mRNA. Their diameters, estimated to be 2.5 nm in the neck region, are too small to transfer viruses or viral genomes. Tobacco mosaic virus and Potexviruses are the most extensively studied viruses. In viruses, the movement protein (MP) is responsible for the PD gating that allows the intercellular movement of viral genomes. Various host factors interact with MP to regulate complicated mechanisms related to PD gating. Virus replication and assembly occur in viral replication complex (VRC) with membrane association, especially in the endoplasmic reticulum. VRC have a highly organized structure and are highly regulated by interactions among the various host factors, proteins encoded by the viral genome, and the viral genome. Virus trafficking requires host machineries, such as the cytoskeleton and the secretory systems. MP facilitates the virus replication and movement process. Despite the current level of understanding of virus movement, there are still many unknown and complex interactions between virus replication and virus movement. While numerous studies have been conducted to understand plant viruses with regards to cell-to-cell movement and replication, there are still many knowledge gaps. To study these interactions, adequate research tools must be used such as molecular, and biochemical techniques. Without such tools, virologists will not be able to gain an accurate or detailed understanding of the virus infection process.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.890-894
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• 2001

The isolation of genomic DNA from mammalian cells is usually performed by cell lysis followed by protein digestion, extraction, and finally, ethanol precipitation of the chromosomal DNA. However, in the case of large sample numbers or when only small amounts of starting materials are available, such conventional methods are not efficient and are cumbersome to be applied. Some alternative methods have been described as well as having commercial DNA isolation kits to be available, nevertheless, there is room left for much improvement. In the present study, a novel method is introduced, where it simplifies conventional protocols by omitting some time-consuming steps such as protease incubation or DNA precipitation and its resuspension. Using paramagnetic silica resins, the genomic DNA was purified over a magnetic field, and the bound DNA was eluted with a low-salt buffer. The fidelity and effectiveness of this novel method was determined by using normal and apoptotic cells as a starting material and then compared to other protocols. The high speed and convenience along with its high efficiency in detecting apoptotic chromosomal DNA will prove this method to be an improved alternative in the isolation of genomic DNA from mammalian cells.

• Journal of Microbiology and Biotechnology
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• v.25 no.1
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• pp.119-126
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• 2015

Among the various human growth factors, epidermal growth factor (hEGF, consisting of 53 amino acids) has various effects on cell regeneration, stimulation of proliferation, migration of keratinocytes, formation of granulation tissues, and stimulation of fibroblast motility, which are important for wound healing. Owing to their multiple activities, EGFs are used as pharmaceutical and cosmetic agents. However, their low productivity, limited target specificity, and short half-life inhibit their application as therapeutic agents. To overcome these obstacles, we fused the collagen-binding domain (CBD) of Vibrio mimicus metalloprotease to EGF protein. About 18 or 12 amino acids (aa) (of the 33 total amino acids), which were essential for collagen-binding activity, were combined with the N- and C-termini of EGF. We constructed, expressed, and purified EGF (53 aa)-CBD (18 aa), EGF (53 aa)-CBD (12 aa), CBD (18 aa)-EGF (53 aa), and CBD (12 aa)-EGF (53 aa). These purified recombinant proteins increased the numbers of cells in treated specimens compared with non-treated specimens and control hEGF samples. The collagen-binding activities were also evaluated. Furthermore, CBD-hybridized hEGF induced phosphorylation of the EGF receptor. These results suggested that these fusion proteins could be applicable as small therapeutic agents in wound tissue healing.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.579-586
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• 2010

A genomic DNA fragment encoding a putative maltooligosyltrehalose synthase (NfMTS) for trehalose biosynthesis was cloned by the degenerate primer-PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTS was 2,799 bp in length and encoded 933 amino acid residues constituting a 106.6 kDa protein. The deduced amino acid sequence of NfMTS contained 4 regions highly conserved for MTSs. By expression of NfMTS in E. coli, it was demonstrated that the recombinant protein catalyzed the conversion of maltohexaose to maltooligosyl trehalose. The $K_m$ of the recombinant enzyme for maltohexaose was 1.87 mM and the optimal temperature and pH of the recombinant enzyme was at $50^{\circ}C$ and 7.0, respectively. The expression of MTS of N. flagelliforme was upregulated, and both trehalose and sucrose contents increased significantly in N. flagelliforme during drought stress. However, trehalose accumulated in small quantities (about 0.36 mg/g DW), whereas sucrose accumulated in high quantities (about 0.90 mg/g DW), indicating both trehalose and sucrose were involved in dehydration stress response in N. flagelliforme and sucrose might act as a chemical chaperone rather than trehalose did during dehydration stress.

• Toxicological Research
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• v.33 no.3
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• pp.219-224
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• 2017

Lipin1 was identified as a phosphatidate phosphatase enzyme, and it plays a key role in lipid metabolism. Since free radicals contribute to metabolic diseases in the liver, this study investigated the effects of free radicals on the regulation of Lipin1 expression in Huh7 and AML12 cells. Hydrogen peroxide induced mRNA and protein expression of Lipin1 in Huh7 cells, which was assayed by quantitative RT-PCR and immunoblotting, respectively. Induction of Lipin1 by hydrogen peroxide was confirmed in AML12 cells. Hydrogen peroxide treatment significantly increased expression of sterol regulatory element-binding protein (SREBP)-2, but not SREBP-1. Moreover, nuclear translocation of SREBP-2 was detected after hydrogen peroxide treatment. Hydrogen peroxide-induced Lipin1 or SREBP-2 expression was significantly reduced by N-acetyl-$\small{L}$-cysteine treatment, indicating that reactive oxygen species (ROS) were implicated in Lipin1 expression. Next, we investigated whether the hypoxic environments that cause endogenous ROS production in mitochondria in metabolic diseases affect the expression of Lipin1. Exposure to hypoxia also increased Lipin1 expression. In contrast, pretreatment with antioxidants attenuated hypoxia-induced Lipin1 expression. Collectively, our results show that ROS activate SREBP-2, which induces Lipin1 expression.

• Molecules and Cells
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• v.21 no.3
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• pp.330-336
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• 2006

Since its identification in 1989, hepatitis C virus has been the subject of extensive research. The biology of the virus and the development of antiviral drugs are closely related. The RNA polymerase activity of nonstructural protein 5B was first demonstrated in 1996. NS5B is believed to localize to the perinuclear region, forming a replicase complex with other viral proteins. It has a typical polymerase structure with thumb, palm, and finger domains encircling the active site. A de novo replication initiation mechanism has been suggested. To date, many small molecule inhibitors are known including nucleoside analogues, non-nucleoside analogues, and pyrophosphate mimics. NS5B interacts with other viral proteins such as core, NS3, 4A, 4B, and 5A. The helicase activity of NS3 seems necessary for RNA strand unwinding during replication, with other nonstructural proteins performing modulatory roles. Cellular proteins interacting with NS5B include VAMP-associated proteins, heIF4AII, hPLIC1, nucleolin, PRK2, ${\alpha}$-actinin, and p68 helicase. The interactions of NS5B with these proteins might play roles in cellular trafficking, signal transduction, and RNA polymerization, as well as the regulation of replication/translation processes.

• The Plant Pathology Journal
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• v.19 no.1
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• pp.19-23
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• 2003

Turnip crinkle vims (TCV) inoculation onto resistant Arabidopsis ecotype Dijon（Di-17) leads to a hypersensitive response (HR) on the inoculated leaves. A dominant gene, HRT, which confers an HR to TCV, has been cloned from Di-17 plants by map-based cloning. HRT is a LZ-NBS-LRR class resistance gene and it belongs to a small gene family that includes RPP8, which confers resistance to Peronospora parasitica Emco5. Outside of the LRR region, HRT and RPP8 proteins share 98% amino acid identity while their LRR regions are less conserved (87% identity). HRT-transformed Arabidopsis plants developed an HR but generally remained susceptible to TCV due to a dominant RRT allele, which is not compatible with resistance. However, several transgenic plants that over-expressed HRT much higher than Di-l7 showed micro-HR or no HR when inoculated with TCV and were resistant to infection. Both the HR and resistance are dependent on salicylic acid but independent of NPRI, ethylene, or jasmonic acid. Arabidopsis plants containing both TCV coat protein gene and HRT developed massive necrosis and death in seedlings, indicating that the TCV coat protein is an avirulence factor detected by the HRT.

• Ocean and Polar Research
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• v.40 no.1
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• pp.15-22
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• 2018

In the present study, we investigated temporal variation in the reproductive effort and biochemical contents in adult Manila clam Ruditapes philippinarum at Shi-Heung-Ri beach on the east coast of Jeju Island. Gonad-somatic index (GSI), a ratio of the egg mass to the total biomass determined using ELISA increased dramatically from late May to June (7.1% to 19.5%). In mid-July, GSI dropped to 15.1%, indicating spawning on a small scale during this period. GSI also declined dramatically from late August (15.5%) to September (4.3%), suggesting that massive spawning occurred during this period. A positive correlation was observed between clam size and potential fecundity, while potential fecundity ranged between 1.19-8.40 million eggs/clam. Total proteins and lipids in the tissue increased from late spring to mid-summer, coinciding with an increase in GSI. Protein, lipid and carbohydrate contents in the tissue were lowest in January, indicating that clams were suffering from poor nutritional circumstances, which may be associated with poor food supply from the environment. Monthly increase and/or decrease in the body weight were closely related to gonad maturation and subsequent spawning, which was also linked to temporal changes in the contents of protein, lipid and carbohydrates in the tissue.

• Journal of Applied Biological Chemistry
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• v.43 no.3
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• pp.147-151
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• 2000

A gene, nprM, from Bacillus megaterium ATCC 14945 was obtained by PCR using primers synthesized based on two nprM sequences from two different strains, and cloned into Escherichia coli. The gene nprM encoded an extracellular neutral protease, and the molecular mass of the expressed enzyme was estimated to be approximately 36kDa on a denaturating gel. The enzyme was activated by $Ca^{2+}$, and the optimum concentration of $Ca^{2+}$ was 5 mM. The enzyme was inhibited by EDTA but not by PMSF. The optimal pH and temperature of the cloned enzyme were $50^{\circ}C and pH 7.5-8.0, respectively, and were similar to those of the enzyme from the gene gonor cell. The cloned NprM caused internal cleavage of the native endoglucanase of B. subtilis BSE616 as a model foregin protein, and resulted in a small truncated but still active endoglucanase.