1.ISOLATION AND PURIFICATION OF IMMUNE MODULATING LACTOFERRIN FROM MONGOL BOVINE COLOSTRUM
Chingunjav E ; Jambal B ; Amarsaikhan B ; Gerelmaa T ; Narantsetseg L ; Sarantuya R ; Bilegtsaikhan Ts ; Purevjargal N ; Tengis A ; Javkhlan B ; Tsendmaa Ts ; Galindev B ; Munkhtulga L ; Nyambayar D ; Munkhbat B ; Baigalmaa B
Innovation 2017;11(1):30-33
BACKGROUND
Bovine colostrums is the milk secreted by cows during the first few days after parturition. It
contains many essential nutrients and bioactive components, including growth factors,
immunoglobulins, lactoperoxidase, lactoferrin and cytokines ets. Lactoferrin has been reported
for its multifunctional properties such as antifungal, antibacterial, antiviral antioxidant and
anticancer activities. The aims of this study focused on the isolation and purification of lactoferrin
from Mongolian bovine colostrums. Lactoferrin purified using HiTrap DEAE an ion exchange
chromatography. Lactoferrin purification efficiency was about 60.5%. The single band of purified
lactoferrin has been observed in SDS-PAGE electrophoresis.
METHODS
Bovine colostrum was collected at a cow farm in the Darkhan province of Mongolia. At first
the cream was separated by centrifugation (10000 xg 20 min at 4oC). In order to separate the
whey, the samples were precipitated with 1mol/l to pH 4.6 and centrifuged at 10000 g 20 min
again. The samples of whey were stored at -18oC to the analysis. Lactoferrin was purified by
HiTrap DEAE an ion exchange chromatography using 0.005 M phosphate buffer (pH 7.7) and
linear gradient NaCl from 0.25M, 0.5M, 1M. During chromatography, protein in the eluents was
monitored by ultraviolet absorbation at 280 nm with the instrument. Purity test done by using
polyacrylamide gel electrophoresis under denaturated condition (SDS-PAGE) method by Laemmli
(1970). For HPLC determination of the lactoferrin by Shimadzu Nexera X2 HPLC system with UV/
VIS detector were used. Detection was carried out at the wavelength 280 nm. Separation was
performed on a chromatographic column Protein R C18 ,2.2 x 150 mm, 5 μm particle size. Linear
gradient and flow rate 0.2 ml/min were used. Mobile phase a consisted of water / acetonitrile/
trifluoroacetic acid ( 95:5:0.1). The column temperature was set at 40oC and injection volume
was 10 μl. Data were collected and evaluated by software Lab Solution. An external standard
method for quantification analytes was used.
RESULTS
Purified lactoferrin in the present study had a good concentration and purification efficiency
was about 60.5 %. Protein fraction from 1M NaCl gradient delivers sharp and clean peak to
HPLC chromatogram that fits intensity and retention time of standard bovine lactoferrin.
Ammount of lactoferrin in bovine colostrums was 0.6 mg/ml and it`s molecular weight 80 kDa as
a standard sample. The retention time of lactoferrin fraction which is purified by SDS-PAGE gel
electrophoresis. The peak of fraction same compared to the standard lactoferrin 5.8 minutes
by HPLC analysis.
CONCLUSION
Ion exchange chromatography shows reliable and easy isolation of lactoferrin from Mongol
bovine colostrum.
2.EFFECT OF TLR7 LIGAND ON SIGNAL TRANSDUCTION OF INTERFERON GAMMA
Baasansuren E ; Javkhlan B ; Baljinnyam T ; Erkhembayar Sh ; Batkhishig M ; Dolgorsuren S ; Enkhsaikhan L ; Ulziisaikhan J ; Khongorzul B ; Baigalmaa B ; Galindev B ; Sodnomtsogt L ; Nyambayar D ; Nyamdorj D ; Munkhtuvshin N ; Munkhbat B ; Bilegtsaikhan Ts
Innovation 2017;11(4):14-17
BACKGROUND: Toll like receptors (TLRs) are a class of proteins that key role in the innate immune system. TLR7 is expressed on monocytes, macrophages and dendritic cells, T cell, B cell and eosinophiles. TLR7, originally identified as recognizing imidaquinoline, loxibrine, broprimine and ssRNA, ssRNA viruses such as vesicular stomatitis virus, influenza A virus and human immunodefiency virus. It is known that virus ssRNA affects signaling molecule of IFN-y. Objective: To determine gene and protein activation of IFN-y signal transduction by TLR7 ligand in the endothelial cells.
MATERIAL: In study we used mouse aortic linear endothelial cell which is cultured (END-D) in 5% heat- inactivated fetal calf serum (FCS), medium (DMEM) containing antibiotic mix(penicillin G, streptomycin, amphotericin B) at 37°C (5% CO2). Endothelial cells treated with synthetic IFN-γ and imiquimodligands, then the NO (nitric oxide) concentration in the supernatant is determined by Griess reagent. Endothelial cells are cultured in 6 well cell culture plate and in each well 2*104cells are expected to be grown for 24 hours of culture. Then, the cells are treated with synthetic IFN-γ and имиквимод ligand for 6 hours and the NO signaling gene activation iNOS mRNA expression which is induced by IFN-γ is determined by RT-qPCR. Endothelial cells are cultured in 12 well cell culture plate and in each well 2*104 cells are expected to be grown for 18 hours of culture. Then, the cells are treated with synthetic IFN-γ and imiquimodligands for 24 hours and the NO signaling protein iNOS expression which is induced by IFN-γ is determined by western blotting. The experiment was conducted as representation mean of at least three test results. The difference between statistical probabilities is determined by the “Students” t test. The p<0.01 value is assumed to be statistically different.
RESULTS: TLR7 ligand imiquimodaugmented interferon gamma induced nitric oxide production TLR7 ligand imiquimodincreased interferon gamma induced iNOS mRNA gene expression. TLR7 ilgand imiquimodup-regulated interferon gamma induced iNOS protein expression.
CONCLUSIONS: TLR7 ligand imiquimod augments IFN-γ signaling in the endothelial cells. This synergistic effect has revealed in the levels of gene and protein expression.
3.Study on influence of the CpG DNA on activation of IFN-γ signaling transduction regulatory proteins
Baljinnyam T ; Khulan U ; Erkhembayar Sh ; Baasansuren E ; Javkhlan B ; Batkhishig M ; Enkhsaikhan L ; Ulziisaikhan J ; Baigalmaa B ; Galindev B ; Tsevelmaa N ; Khongorzul B ; Sodnomtsogt L ; Munkhbat B ; Munkhtuvshin N ; Bilegtsaikhan Ts
Mongolian Medical Sciences 2018;186(4):10-13
Introduction:
When human body encounters external pathogens primary/innate immunity cells are activated by
recognizing them and secondary/adaptive immunity is activated consecutively. In our previous study,
we revealed that there is a synergistic action between TLR9 and IFN-γ signaling in the endothelial cells.
Purpose:
To determine the role of negative and positive regulator proteins on the IFN-γ/TLR9 signaling pathway.
Methods:
In this study, murine endothelial cell (END-D) culture was used. END-D cells pre-treated with TLR9
ligand CpG DNA and then stimulated with IFN-γ. The negative (SHP-2, SOCS1, PIAS1) and positive
(p38) regulator protein expression was detected by Western blotting.
Results and Conclusion
Treatment by TLR9 ligand CpG DNA and IFN-γ increased positive regulator p38 phosphorylation in 0.5
hour. CpG DNA inhibited IFN-γ negative regulator PIAS1 protein expression in 6 hour and SOCS1 and
SHP-2 expression could not affect in 4 hour.
4.Role of negative regulators on the TLR7 ligand/IFN-γ signaling in the endothelial cells
Baasansuren E ; Javkhlan B ; Baljinnyam T ; Khulan U ; Batkhishig M ; Enkhsaikhan L ; Ulziisaikhan J ; Khongorzul B ; Baigalmaa B ; Galindev B ; Tsevelmaa N ; Sodnomtsogt L ; Nyambayar D ; Munkhtuvshin N ; Munkhbat B ; Bilegtsaikhan Ts
Health Laboratory 2018;8(1):14-18
Introduction:
Toll like receptors (TLRs) are a class of proteins that key role in the innate immune system. The SOCS1 and SHP2 proteins are negative-feed loop inhibitors of signaling of JAK/STAT and TLRs pathways.
Purpose:
To determine negative regulator protein activation which is activated through TLR7 ligand/IFN-γ signal transduction in endothelial cells.
Methods:
We used mouse aortic linear endothelial cell (END-D); protein expressio was detected by western blotting
Results:
We analyzed a time dependent stimulation effects of negative regulator proteins stimulated by TLR7 ligand/IFN-γ in endothelial cell cultures. Imiquimod of 10 μg/ml treatment of 1 hr was followed by 100 ng/ml IFN-γ stimulation for 1-8hr to analysis of negative regulator SOCS1 and SHP2 protein expression.
In untreated cells, there was low activations of negative regulator SOCS1 and SHP2 proteins. IFN-γ stimulation alone had increased SOCS1 and SHP2 protein expressions, also imiquimod treatment highly elevated SOCS1 and SHP2 expressions. However imiquimod and IFN-γ doubled treatment have decreased activation of negative regulator SOCS1 and SHP2 proteins. These findings suggest SOCS1 and SHP2 proteins are inhibitors in the TLR7 ligand/IFN-γ signaling.
Conclusion
Negative regulators, SOCS1 and SHP2 strongly suppressed activations of TLR7 ligand/IFN-γ signaling
5.The effect of TLR9 ligand on IFN-ү signaling
Erkhembayar Sh ; Battsetseg Ts ; Baljinnyam T ; Altai E ; Baasansuren E ; Javkhlan B ; Batkhishig M ; Dolgorsuren S ; Ulziisaikhan J ; Enkhsaikhan L ; Tsendmaa Ts ; Galindev B ; Khongorzul B ; Baigalmaa B ; Nyambayar D ; Munkhbat B ; Bilegtsaikhan Ts
Health Laboratory 2017;6(1):15-23
Introduction:
The aim of this research project is to elucidate the crosstalk of innate and adaptive immune reactions against the DNA containing bacteria.
:
This study held in the Core laboratory, Science Technology Center, Mongolian National University of
Medical Sciences (MNUMS). Murine aortal endothelial cells, END-D cultured and the cell viability checked by MTT assay. In addition, the NO production, protein and gene expression studied by Griess Reagent
assay, R.T-PCR and immunoblotting, respectively.
Results:
0.1µM, 1µM and 10µM of TLR9 ligand exhibited no cytotoxic action against the cells by MTT assay. IFN-ү alone induced NO production in END-D cells. In the other hand, TLR9 ligand at 0.1µM, 1µM and 10µM up-regulated IFN-ү induced NO production in dose dependent manner. RTPCR results exhibit that TLR9 ligand up regulates iNOS mRNA. Immunoblotting analysis showed the enhanced iNOS protein expression and phosphorylation of STAT1 in cells pre-treated with TLR9 ligand.
Discussion:
We have demonstrated CpG DNA, TLR9 ligand, up-regulates IFN-ү induced NO via enhanced IFN-ү signaling. The result of Western Blotting and RT-PCR support the up-regulation of NO. CpG DNA can be used as agent against virus and bacteria. Further research need to be conducted.
Conclusion
TLR9 ligand, CpG DNA up-regulates IFN-ү induced NO production in time and dose dependent manner. TLR9 ligand augments the expression of iNOS mRNA and STAT1 phosphorylation in response to IFN-ү.
6.Inhibitory action of Lipopolysaccharide-induced signal transductions by Valproic acid
Ulziisaikhan J ; Tsogtsaikhan S ; Yokochi T ; Enkhsaikhan L ; Jambaldorj J ; Javkhlan B ; Baigalmaa B ; Tsevelmaa N ; Galindev B ; Sodnomtsogt L ; Munkhtuvshin N ; Munkhbat B ; Bilegtsaikhan Ts
Health Laboratory 2019;9(1):12-20
Introduction:
Valproic acid (VPA) has been used in the treatment of seizures and bipolar disorders. In the present
study, we examined how VPA affected PI3K-Akt pathway in response to LPS by using mouse
RAW 264.7 macrophage cells.
Material and methods:
Mouse RAW 264.7 macrophage-like cells cultured and the cell viability
checked by MTT and TUNEL assay. In addition, protein expression and protein interaction were
detected by immune blotting and immune precipitation, respectively. TLR4 expression on cell
surface studied by FACS analysis.
Results:
The MTT and TUNEL assays demonstrated no significant difference between VPA at 2
mM treated and untreated control cells. VPA attenuated LPS-induced phosphorylation of
phosphatidylinositol 3-kinase (PI3K) and Akt, but not nuclear factor (NF)-κB and mitogen activated protein kinases (MAPKs). There was no significant difference in the TLR4 expression on
the cell surface between cells treated with or without VPA. VPA inhibited LPS-induced PI3K/Akt
signal transduction in a dose dependent manner.
Conclusion
VPA at 2mM exhibits nontoxic effect in the RAW 264.7 cells. VPA down regulates
LPS-induced phosphorylation of Akt via inhibition of PI3K activation.