• Journal of Animal Science and Technology
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• v.64 no.6
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• pp.1024-1034
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• 2022

Identifying effective biomarkers for the diagnosis of male fertility is crucial for improving animal production and treating male infertility in humans. Ras-related proteins (Rab) are associated with morphological and motion kinematic functions in spermatozoa. Moreover, Rab2A, a Rab protein, is a possible male fertility-related biomarker. The present study was designed to identify additional fertility-related biomarkers among the various Rab proteins. First, the expression of Rab proteins (Rab3A, 4, 5, 8A, 9, 14, 25, 27A, and 34A) from 31 duroc boar spermatozoa was measured before and after capacitation; correlation between Rab protein expression and litter size was evaluated by statistical analysis. The results showed that the expression of Rab3A, 4, 5, 8A, 9, and 25 before capacitation and Rab3A, 4, 5, 8A, 9, and 14 after capacitation were negatively correlated with litter size. Moreover, depending on the cutoff values calculated by receiver operating curves, an increase in litter size was observed when evaluating the ability of the Rab proteins to forecast litter size. Therefore, we suggest that Rab proteins may be potential fertility-related biomarkers that could help select superior sires in the livestock industry.

• Journal of Integrative Natural Science
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• v.16 no.2
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• pp.47-51
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• 2023

Ras small GTPases are involved in regulating various cellular signaling pathways including cell migration, proliferation, and differentiation. Ras GTPase subfamily is comprised of 15 proteins; 11 Ras, 3 Rap, and one Rheb related protein. Some Ras proteins, such as RasC and RasG, have been identified for their major functions, but there are proteins whose functions have not been studied yet, such as RasU and RasX. Here, we investigated the roles of RasU in cell motility and development. RasU shows the highest homology with RasX. To investigate the functions of RasU, rasU null cells were used to observe the phenotype. Cells lacking RasU were larger and more spread than wild-type cells. These results indicate that RasU plays a negative role in cell spreading. In addition, we investigated the roles of RasU in cell motility and development of Dictyostelium cells and found that rasU null cells exhibited decreased random migration speed and delayed developmental process. These results suggest that RasU plays an important role in cell motility and development.

• Molecules and Cells
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• v.20 no.2
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• pp.196-200
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• 2005

The neurofibromatosis type2 (NF2) tumor suppressor gene product, merlin, is structurally related to the ezrin-radixin-moesin (ERM) family of proteins that anchor the actin cytoskeleton to specific membrane proteins and participate in cell signaling. However, the basis of the tumor suppressing activity of merlin is not well understood. Previously, we identified a role of merlin as an inhibitor of the Ras-ERK signaling pathway. Recent studies have suggested that phosphorylation of merlin, as of other ERM proteins, may regulate its function. To determine whether phosphorylation of merlin affects its suppression of Ras-ERK signaling, we generated plasmids expressing full-length merlin with substitutions of serine 518, a potential phosphorylation site. A substitution that mimics constitutive phosphorylation (S518D) abrogated the ability of merlin to suppress effects of the Ras-ERK signaling pathway such as Ras-induced SRE transactivation, Elk-mediated SRE transactivation, Ras-induced ERK phosphorylation and Ras-induced focus formation. On the other hand, an S518A mutant, which mimics nonphosphorylated merlin, acted like wild type merlin. These observations show that mimicking merlin phosphorylation impairs not only growth suppression by merlin but also its inhibitory action on the Ras-ERK signaling pathway.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.12-20
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• 2020

Cell adhesion plays an important role in the differentiation of the morphogenesis and the trophectoderm epithelium of the blastocyst. In the porcine embryo, CDH1 mediated adhesion initiates at compaction before blastocyst formation, regulated post-translationally via protein kinase C and other signaling molecules. Here we focus on muscle RAS oncogene homolog (M-RAS), which is the closest relative to the RAS related proteins and shares most regulatory and effector interactions. To characterize the effects of M-RAS on embryo compaction, we used gain- and loss-of-function strategies in porcine embryos, in which M-RAS gene structure and protein sequence are conserved. We showed that knockdown of M-RAS in zygotes reduced embryo development abilities and CDH1 expression. Moreover, the phosphorylation of ERK was also decreased in M-RAS KD embryos. Overexpression of M-RAS allows M-RAS KD embryos to rescue the embryo compaction and blastocyst formation. Collectively, these results highlight novel conserved and multiple effects of M-RAS during porcine embryo development.

• Applied Microscopy
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• v.43 no.1
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• pp.27-33
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• 2013

Inflammatory cells express the inflammatory cytokines and growth factors induced by lipopolysaccharide (LPS). Odontoblasts are located at the pulp-dentin interface and extend their cell processes far into the dentin where they are the first cells to encounter microorganisms or their products. Therefore, this study examined the expression of some growth factors related to the signal pathway, such as growth factor receptor binding protein 2 (Grb2)-Ras in odontoblast-like dental pulp cells, after a treatment with LPS. After 60 minutes, the mRNA and protein expression levels of Grb2 and Ras were higher in the LPS-treated cells than in the control cells. The level of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression was increased significantly to a level similar to that of Grb2 and Ras at 60 minutes. The platelet-derived growth factor-AA (PDGF-AA) mRNA level was expressed strongly in the odontoblast like dental pulp cells without an association with LPS stimulation. Scanning electron microscopy revealed many extensions of the cytoplasmic processes and the number of processes increased gradually at 30, 60 and 90 minutes after LPS stimulation. From these results VEGF and bFGF expression might be induced through the Grb2-Ras signal transduction pathway in LPS treated odontoblasts.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.778-783
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• 2008

Anthrax is an infectious disease caused by toxigenic strains of the Gram-positive bacterium Bacillus anthracis. To identify the mitochondrial proteins that are expressed differently in murine macrophages infected with spores of B. anthracis Sterne, proteomic and MALDI-TOF/MS analyses of uninfected and infected macrophages were conducted. As a result, 13 mitochondrial proteins with different expression patterns were discovered in the infected murine macrophages, and some were identified as ATP5b, NIAP-5, ras-related GTP binding protein B isoform CRAa, along with several unnamed proteins. Among these proteins, ATP5b is related to energy production and cytoskeletal rearrangement, whereas NIAP-5 causes apoptosis of host cells due to binding with caspase-9. Therefore, this paper focused on ATP5b, which was found to be down regulated following infection. The downregulated ATP5b also reduced ATP production in the murine macrophages infected with B. anthracis spores. Consequently, this study represents the first mitochondrial proteome analysis of infected macrophages.

• Radiation Oncology Journal
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• v.35 no.3
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• pp.281-288
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• 2017

Purpose: The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. Materials and Methods: CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. Results: CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. Conclusion: CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

• Journal of Pharmacopuncture
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• v.9 no.2
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• pp.17-37
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• 2006

Objectives : To observe the changes in the serum proteins after intravenous injection of cultivated wild ginseng pharmacopuncture. Methods : Blood was collected before and after the administration of cultivated wild ginseng pharmacopuncture and only the serum was taken. Then differences in the spots on the scanned image after carrying out 2-Dimensional electrophoresis were located and conducted mass analysis and protein identification. Results : Following results were obtained from the comparative analysis of serum proteins before and after the administration of cultivated wild ginseng pharmacopuncture. 1. 28 spots were identified before and after the administration. 2. In confirming manifestation degree, spots with more than two-times increase were 204, 1302, 2205, 3105, 7104, 8006, spots with more than one-time increase were 1101, 1505, 2013, 2403, 3009, 3010, 4002, 4009, 6704, 8101, and spots with decrease were 205, 801, 803, 3205, 5202, 6105, 6106, 7103, 9001, 9003. 3. After conducting protein identification, proteins 205, 804, 1302, 4009, 6105, 6106 are unidentified yet, and 1l01 is unnamed protein. Protein 204 is identified as complement receptor CR2-C3d, 801 as YAPl protein, 803 as antitrypsin polymer, 1505 as PRO0684, 2013 and 3010 as proapolipoprotein, 2205 as USP48, 2403 as vitamin D binding protein, 3009 as complement component 4A preprotein, 3105 as immunoglobulin lambda chain, 3205 as transthyretin, 4002 as Ras-related protein Ral-A, 4204 as beta actin, 5202 and 7104 as apolipoprotein Ll, 6704 as alpha 2 macroglobulin precursor, 7103 as complement component 3 precursor, 8006 as testis-specific protein Y, 8101 as transferrin, 9001 as (Alpha-Oxy, Beta-(Cl12g)deoxy) T-State Human Hemoglobin, and 9003 as human hemoglobin. 4. Immune protein CR2-C3d(204), which acts against microbes and pathogenic organisms, was increased by more than two-times after the administration of pharmacopuncture. 5. Antitrypsin(803), which is secreted with inflammatory response in the lungs, was reduced after the administration of pharmacopuncture. 6. Proapolipoprotein(2013, 3010) and apolipoprotein(7104), key components of the HDL-cholesterol which plays an important role in preventing arteriosclerosis, were increased after the administration of pharmacopuncture. 7. Vitamin D binding protein(DBP, 2403), protecting the lung at the time of inflammatory response, was increased after the administration of pharmacopuncture. 8. Transthyretin(TTR, 3205), which is the main protein causing familial amyloid polyneuropathy(FAP), was decreased after the administration of pharmacopuncture. 9. Ras-related protein Ral-A(4002) that controls phospholipid metabolism, cytoskeletal formation, and membrane traffic, was increased after the administration of pharmacopuncture. 10. Testis-specific protein Y(8006), which takes part in determination of the gender, was increased by more than two-times after the administration of pharmacopuncture. 11. Transferrin(8101), which balances the iron level in the body, was increased after the administration of pharmacopuncture. Conclusion : Above results support the notion that intravenous injection of cultivated wild ginseng pharmacopuncture induce changes in serum proteins and this research can be a pioneer work in finding biomarkers.

• Journal of Plant Biology
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• v.39 no.2
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• pp.85-92
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• 1996

Small GTP-binding proteins are divided into three major group: Ras, Rho and Ypt/Rab. They have the conserved regions designed G1 to G5 that are critical in GDP/GTP exchange, GTP-induced conformational change and GTP hydrolysis. We isolated and characterized genomic DNA or cDNAfragments encoding G1 to G3 domains of small GTP-binding protein Rab and Rho from several plant species using two different PCR-based cloning strategies. Seven rab DNA fragments were isolated from 4 different plants, mung-bean, tobacco, rice and pepper using two degenerate primers corresponding to the GTP-binding domain G1 and G3 in small GTP-binding proteins. The amino acid sequences among these rab DNA fragments and other known small GTP-binding proteins shows that they belong to the Ypt/Rab family. Six rho DNA fragments were isolated from 5 different plants, mung-bean, rice, Arabidopsis, Allium and Gonyaulax using the nested PCR method that involves four degenerate primers corresponding to the GTP-binding domain G1, G3 and G4. The rho DNA fragments cloned show more than 90% homology to each other. Sequence comparison between plant and other known Rho family genes suggests that they are closely related (67 to 82% amino acid identity). Sequence analysis and southern blot analysis of rab and rho in mung-bean suggest than thses genes are encoded by multigene family in mung-bean.

• Journal of Applied Biological Chemistry
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• v.50 no.3
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• pp.109-114
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• 2007

Sar1p is a ras-related GTP-binding protein that functions in intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi complex. The effector domain of Ras family proteins is highly conserved and this domain is functionally interchangeable in plant, yeast and mammalian Sar1. Using a recombinant Brassica sar1 protein (Bsar1p) harboring point mutations in its effector domain, we here investigated the ability of Sar1p to bind and hydrolyze GTP and to interact with the two sar1-specific regulators, GTPase activating protein (GAP) and guanine exchange factor (GEF). The T51A and T55A mutations impaired Bsar1p intrinsic GTP-binding and GDP-dissociation activity. In contrast, mutations in the switch domain of Bsar1 did not affect its intrinsic GTPase activity. Moreover, the P50A, P54A, and S56A mutations affected the interaction between Bsar1p and GAP. P54A mutant protein did not interact with two regulating proteins, GEF and GAP, even though the mutation didn't affect the intrinsic GTP-binding, nucleotide exchange or GTPase activity of Bsar1p.