• Journal of Microbiology and Biotechnology
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• v.34 no.3
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• pp.525-537
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• 2024

Pilins are protein subunits of pili. The pilins of type IV pili (T4P) in pathogenic bacteria are well characterized, but anything is known about the T4P proteins in acidophilic chemolithoautotrophic microorganisms such as the genus Acidithiobacillus. The interest in T4P of A. thiooxidans is because of their possible role in cell recruitment and bacterial aggregation on the surface of minerals during biooxidation of sulfide minerals. In this study we present a successful ad hoc methodology for the heterologous expression and purification of extracellular proteins such as the minor pilin PilV of the T4P of A. thiooxidans, a pilin exposed to extreme conditions of acidity and high oxidation-reduction potentials, and that interact with metal sulfides in an environment rich in dissolved minerals. Once obtained, the model structure of A. thiooxidans PilV revealed the core basic architecture of T4P pilins. Because of the acidophilic condition, we carried out in silico characterization of the protonation status of acidic and basic residues of PilV in order to calculate the ionization state at specific pH values and evaluated their pH stability. Further biophysical characterization was done using UV-visible and fluorescence spectroscopy and the results showed that PilV remains soluble and stable even after exposure to significant changes of pH. PilV has a unique amino acid composition that exhibits acid stability, with significant biotechnology implications such as biooxidation of sulfide minerals. The biophysics profiles of PilV open new paradigms about resilient proteins and stimulate the study of other pilins from extremophiles.

• Journal of Plant Biotechnology
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• v.42 no.2
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• pp.123-128
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• 2015

Pregermination treatment of cocoa beans either with the testa, group PCB (+T), or without the testa, group PCB (-T), was studied here to determine whether this treatment (incubation up to 120 h at $25^{\circ}C$) has any effect on the levels of allergenic proteins or on chemical composition. Our proximate analysis included carbohydrates, proteins, and lipids. We used western blotting to measure changes in the amounts of allergenic proteins in the cocoa beans during the pregermination treatment. The protein and carbohydrate content of both groups (with or without the testa) decreased slightly during this treatment, whereas lipid content increased. Group PCB (-T) showed more rapid metabolic processes than did group PCB (+T) during the pregermination treatment. Western blot analysis showed that the cocoa beans contained an allergenic protein of ~28 kDa. Removal of the testa strongly reduced the amount of this allergenic protein after 72 h of the pregermination treatment. Generally, the pregermination treatment increased antioxidant activity in both groups. Significant differences in the antioxidant activity were observed between groups PCB (-T) and PCB (+T). Particularly, group PCB (-T) showed high antioxidant activity at 72 h of the pregermination treatment. Thus, the combination of cocoa beans without the testa and pregermination treatment (72 h) seems to be the optimal method for production of hypoallergenic cocoa beans rich in antioxidants for patients with allergic disorders.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.16
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• pp.6977-6982
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• 2014

Background: The aim of this study was to investigate the effect of a Lipofectamine2000 (Life2000) Transfection Reagent transfected psiRNA-STAT3 plasmid on 4T1 breast cancer cells. Materials and Methods: MTT was used to detect the cell proliferation of breast cancer 4T1 cells at different periods (0h, 6h, 8h, 10h); the cell cycle was assessed by flow cytometry; variation of apoptosis and mitochondrial membrane potential was observed under a fluorescence microscope; immunohistochemical staining was used to determine the expression of caspase-3 and cyclin-D1 protein. Results: An obvious effect of inhibition to 4T1 cancer cells could be observed at 8h after the psiRNA-STAT3 was transfected. Typical alterations of apoptotic morphological features were visible in the psiRNA-STAT3 treatment group. Mitochondrial membrane potential decreased significantly, the number of cells was increased in G0/G1 phase, and the number of cells was decreased in S phase, and the data were statistically significant (p<0.05), compared with the Scramble and Mock groups. Expression of caspase-3 protein was increased significantly, while that of cyclin D1 was significantly decreased. Conclusions: Life2000 transfected psiRNA-STAT3 plasmid can inhibit 4T1 tumor cell proliferation and promote apoptosis of 4T1 tumor cells, which process depends on the regulation of expression of cyclin D1 and caspase-3 protein.

• Parasites, Hosts and Diseases
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• v.49 no.1
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• pp.79-83
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• 2011

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage ${\lambda}$ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, ${\alpha$-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.

• Korean Journal of Plant Resources
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• v.28 no.5
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• pp.582-590
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• 2015

In the present study, we investigated the effect of 70% EtOH extract from Hippophae Rhamnoides L. leaves (HRL) on the anti-obesity effect in 3T3-L1 cells. The effects of HRL on lipid accumulation in 3T3-L1 cells were examined using Oil Red O staining. In addition, we examined the gene expression levels by using RT-PCR and western blot. The results of this analysis showed that 100 ㎍/㎖ HRL significantly increased the inhibition of lipid accumulation by 82.25%; significantly decreased the mRNA expression of sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and fatty acid synthase (FAS) in 3T3-L1 cells as well as the stimulated protein expression of AMP-activated protein kinase (AMPK); and suppressed the expression level of PPARγ. These results suggest that HRL can prevent adipogenesis through activation of AMPKα and inhibition of adipogenesis transcription factors.

• Journal of Microbiology and Biotechnology
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• v.32 no.11
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• pp.1471-1478
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• 2022

2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (Kan^R) should be dependent on FucT2 solubility. Kan^R was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

• Food Science of Animal Resources
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• v.30 no.2
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• pp.190-197
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• 2010

The present study was conducted to determine the effect of tannery waste protein concentrate (TWPC) on fattening of cattle and the carcass and meat quality, with the aim of replacing the costly commercial protein concentrate (Jasoprot) with a more economical and effective alternative. Twelve young cattle (six male and six female) were fed during the study period on a control diet (T1) with 10% Jasoprot and on two test diets: 5% TWPC + 5% Jasoprot (T2) and 10% TWPC (T3). The test diets significantly affected (p<0.05) live weight gain and profitability compared to the control diet, perhaps due to the increased protein and essential amino acid content, relative to Jasoprot. TWPC was free of aflatoxin. Sensory-evaluated organoleptic scores did not differ among the groups. Chemical composition was normal as other beef and was non toxic especially within recommended chromium level ($1.90{\pm}0.6{\mu}g$) Total lipid contents were higher (p<0.05) in T3, and moisture, ash and crude protein contents were almost similar (p>0.05) among the three groups. It is concluded that TWPC or an equal mixture of TWPC and Jasoprot may be an economic and efficient alternative protein source to Jasoprot in the cattle industry, which minimizes adverse effects on carcass and sensory meat quality.

• Journal of International Academy of Physical Therapy Research
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• v.4 no.1
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• pp.479-487
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• 2013

This study is intended to examine the tDCS and Morris Water maze training in Alzheimer's disease(AD) rats on Tau protein expression. Experiment groups were divided into four groups and assigned 16 rats to each group. Group I was a control group(AD induced by scopolamine); Group II was a experimental control group(AD injured by scopolamine and treatment tacrine); Group III was a group of tDCS application after AD injured by scopolamine; Group IV was a group of morris water maze training after AD injured by scopolamine. In cognition test, the outcome of group II was significantly lower than the groups(p<.001). and group III, IV were significantly low result at 14 days(p<.05). In histological finding, the experimental groups were destroy of micro vessels and finding of cell atropy and swelling. Group III, IV were decreased in degeneration of liver and kidney cells. In immuno- histochemistric response of BDNF and tau protein in hippocampus, BDNF expression of Group II was more increase than the other groups. and increase of BDNF expression was III, IV were higher than group I at 21 days. Tau protein expression of Group II was more decrease than the other groups. and decrease of Tau protein expression was III, IV were lower than group I at 21 days. These result suggest that improved tDCS and morris water maze training after scopolamine induced is associated with dynamically altered expression of BDNF and Tau protein in hippocampus and that is related with cognitive function.

• Korean Journal of Microbiology
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• v.47 no.4
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• pp.348-355
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• 2011

Staphylococcus aureus is a major human pathogen that is associated with various types of local and systemic infection. Staphylococcal protin A (SPA), a highly expressed surface component of S. aureus, may have a role in virulence such as activating inflammation and interfering with immune clearance. We examined the effect of recombinant SPA on inflammatory response in human HaCaT keratinocytes. The recombinant SPA protein was prepared using the pET-28a Vector System in Escherichia coli. The expression of pro-inflammatory related adhesion molecules and cytokines in HaCaT cells incubated for 6, 12, and 24 h with SPA (2 ${\mu}g$/ml) was analyzed by comparative RT-PCR or ELISA. The expression of E-selectin, ICAM-1, MCP-1, IL-6 and IL-8 was significantly increased in HaCaT from 6 to 24 h after treatment with SPA. SPA showed the effect on the adhesion-promoting ability of U937 monocytes to HaCaT cells. Our data demonstrate that SPA stimulates inflammatory response of HaCaT cells, implicating an important factor for exacerbation of skin inflammation of immunologic disease.