Sini Decoction (SNT) is a traditional formula recognized for its efficacy in warming the spleen and stomach and dispersing cold. However, elucidating the mechanism of action of SNT remains challenging due to its complex multiple components. This study utilized a synergistic approach combining two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE)-based drug affinity responsive target stability (DARTS) with label-free quantitative proteomics techniques to identify the direct and indirect protein targets of SNT in myocardial infarction. The analysis identified 590 proteins, with 30 proteins showing significant upregulation and 51 proteins showing downregulation when comparing the SNT group with the model group. Through the integration of 2D-DIGE DARTS with proteomics data and pharmacological assessments, the findings indicate that protein disulfide-isomerase A3 (PDIA3) may serve as a potential protein target through which SNT provides protective effects on myocardial cells during myocardial infarction.
Myocardial Infarction/genetics* ; Proteomics/methods* ; Drugs, Chinese Herbal/chemistry* ; Animals ; Protein Disulfide-Isomerases/genetics* ; Male ; Two-Dimensional Difference Gel Electrophoresis/methods* ; Humans ; Rats ; Rats, Sprague-Dawley ; Electrophoresis, Gel, Two-Dimensional

To construct a high-quality Panax ginseng cDNA library, transcription factors binding to the P. ginseng PgD14 gene promoter were screened by yeast one-hybrid, and proteins interacting with the P. ginseng Pgpht2-1 gene-encoded protein were screened by yeast two-hybrid. In this study, root tissues of P. ginseng were used as materials. Gateway technology was used to construct the P. ginseng yeast one-hybrid library, and duplex-specific nuclease(DSN) homogenization technology was used to construct the P. ginseng yeast two-hybrid library. The pAbAi-PgD14-Pro961 vector was used as bait to screen candidate transcription factors that might bind to the PgD14 gene promoter from the yeast one-hybrid library, and the pGBKT7-Pgpht2-1 vector was used as bait to screen candidate proteins that might interact with the Pgpht2-1 gene-encoded protein from the yeast two-hybrid library. The yeast one-hybrid library had a size of 1.20×10~7 CFU, a recombination rate of 100%, and an average inserted fragment length of more than 1 000 bp. The yeast two-hybrid library had a size of 1.832×10~5 CFU, a recombination rate of 100%, and an average inserted fragment length of about 1 000 bp. The recombinant vectors pAbAi-PgD14-Pro961 and pGBKT7-Pgpht2-1 were transformed into Y1HGold and AH109 strains, respectively, and interacting proteins were screened by yeast one-hybrid and yeast two-hybrid. As a result, 54 transcription factors that could bind to the PgD14 gene promoter of P. ginseng and 42 proteins that may interact with the protein encoded by the Pgpht2-1 gene were identified. This study successfully constructed the P. ginseng yeast one-hybrid and yeast two-hybrid cDNA libraries, laying a foundation for subsequent studies on the functions of the P. ginseng PgD14, Pgpht2-1, and other genes.
Panax/metabolism* ; Two-Hybrid System Techniques ; Plant Proteins/metabolism* ; Gene Library ; Plant Roots/chemistry* ; Protein Binding ; Transcription Factors/metabolism* ; Promoter Regions, Genetic

Animals ; Electrophoresis, Gel, Two-Dimensional ; Male ; Muscles ; Physical Conditioning, Animal ; Proteome ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To screen proteins interacting with ring finger protein 216(RNF216) through yeast two hybrid experiment, and further clarify the role of RNF216 in the pathogenesis of gonadotropin-releasing hormone deficiency. METHODS: A recombinant expression vector pGBKT7-RNF216 was constructed and transformed into yeast Y2HGold, which was hybridized with a human cDNA library in order to screen proteins interacting with RNF216. The interaction was verified in yeast Y2HGold. RESULTS: A recombinant expression vector pGBKT7-RNF216 was successfully constructed and expressed in yeast Y2HGold. Filamin B (FLNB) was identified by yeast two hybrid experiment, and their interaction was verified in yeast Y2HGold. CONCLUSION An interaction between FLNB and RNF216 was identified through yeast two hybrid experiment. RNF216 may affect the proliferation and migration of GnRH neurons by regulating FLNB or FLNB/FLNA heterodimers.
Gene Library ; Gonadotropin-Releasing Hormone/genetics* ; Humans ; Proteins ; Two-Hybrid System Techniques ; Ubiquitin-Protein Ligases/genetics*

Aims: Acinetobacter baumannii has been identified as one of the six most pathogenic bacteria that is the cause of most hospital bacterial infections according to Infectious Disease Society of America (IDSA). These nosocomial pathogens are notorious worldwide due to its ability in causing lethal infections among immunocompromised patients and its resistance to many strong antibiotics. This study aims to compare the expressed proteins of two A. baumannii strain, ATCC 19606 and a pathogenic clinically isolated strain known as AB-13. Methodology and results: AB-13 clinically strain was isolated from the lower respiratory tract of a patient with pneumonia. In this study, the proteomic profile of both ATCC 19606 and AB-13 are produced using 2-dimensional gel electrophoresis. The total protein contents were extracted, quantified and separated using 2-DE with a pH range of 4-7 to acquire the proteomic profile for comparison. The final analytical gel was analysed using Delta2D software and among the 324 protein spots successfully resolved, 10 spots exhibited signs of differential expression with 7 spots found to be downregulated and 3 spots upregulated (p< 0.01). These differences could signify the evolution AB-13 has undergone as it acquires traits ultimately aiding in its survivability, antimicrobial resistance and pathogenicity within varied environments especially during infections. Conclusion, significance and impact of study These findings support the presence of variation in AB-13 from a proteomic perspective, highlighting the pathogen’s evolution improving survivability and pathogenicity, warranting in-depth exploration towards understanding A. baumannii virulence and pathogenicity.
Acinetobacter baumannii--pathogenicity ; Two-Dimensional Difference Gel Electrophoresis ; Proteomics

two or more consecutive unexplained miscarriages and with no female factor abnormality as revealed by gynaecologic investigation including karyotyping and age matched fertile healthy volunteers (n=20). All samples were collected during 2013 to 2015 after getting institutional ethical approval and written consent from the participants. Seminal ejaculates were collected by masturbation after 2 to 3 days of sexual abstinence and analyzed according to World Health Organization 5th criteria 2010. Two-dimensional difference gel electrophoresis followed by mass spectrophotometric analysis was used to identify differentially expressed proteins (DEPs). Western blotting was used for validation of the key proteins.RESULTS: The data identified 36 protein spots to be differentially expressed by more than 2-fold change with p<0.05 considered as significant. Matrix-assisted laser desorption/ionization time of flight/mass spectrometry identified GPx4, JIP4, ZN248 to be overexpressed while HSPA2, GSTM5, TF3C1, CC74A was underexpressed in RPL group. Western blot analysis confirmed the differential expression of key redox associated proteins GPx4 and HSPA2 in the RPL group. Functional analysis revealed the involvement of key biological processes that includes spermatogenesis, response to oxidative stress, protein folding and metabolic process.CONCLUSIONS: The present study provides a snapshot of the altered protein expression levels consistent with the potential involvement of the sperm chromatin landscape in early embryonic development.]]>
Abortion, Spontaneous ; Biological Processes ; Blotting, Western ; Chromatin ; Embryo Loss ; Embryonic Development ; Female ; Healthy Volunteers ; Humans ; Karyotyping ; Male ; Masturbation ; Metabolism ; Oxidation-Reduction ; Oxidative Stress ; Pregnancy ; Prospective Studies ; Protein Folding ; Proteomics ; Sexual Abstinence ; Spectrum Analysis ; Spermatogenesis ; Spermatozoa ; Two-Dimensional Difference Gel Electrophoresis ; World Health Organization

BACKGROUND: Irritable bowel syndrome (IBS) is one of the most common functional intestinal diseases, but its pathogenesis is still unknown. The present study aimed to screen the differentially expressed proteins in the mucosa of colon between IBS with diarrhea (IBS-D) patients and the healthy controls. METHODS: Forty-two IBS-D patients meeting the Rome III diagnostic criteria and 40 control subjects from July 2007 to June 2009 in Chinese PLA General Hospital were enrolled in the present study. We examined the protein expression profiles in mucosa of colon corresponding to IBS-D patients (n = 5) and controls (n = 5) using 2-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Secondly, Western blot and immunohistochemical analysis were carried out to validate the screened proteins in 27 IBS-D patients and 27 controls. Thirdly, high-performance liquid chromatography (HPLC) was further carried out to determine ATP concentration in the mucosa of colon between 10 IBS-D patients and 8 controls. Comparisons between 2 groups were performed by Student's t-test or Mann-Whitney U-test. RESULTS: Twelve differentially expressed proteins were screened out. The α-enolase (ENOA) in the sigmoid colon (0.917 ± 0.007 vs. 1.310 ± 0.100, t = 2.643, P = 0.017) and caecum (0.765 ± 0.060 vs. 1.212 ± 0.122, t = 2.225, P = 0.023), Isobutyryl-CoA dehydrogenase (ACAD8) in the sigmoid colon (1.127 ± 0.201 vs. 1.497 ± 0.392, t = 7.093, P = 0.008) of the IBS-D group were significantly lower while acetyl-CoA acetyltransferase (CT) in the caecum (2.453 ± 0.422 vs. 0.931 ± 0.652, t = 8.363, P = 0.015) and ATP synthase subunit d (ATP5H) in the sigmoid (0.843 ± 0.042 vs. 0.631 ± 0.042, t = 8.613,P = 0.007) of the IBS-D group was significantly higher, compared with the controls. The ATP concentration in the mucosa of the sigmoid colon in IBS-D group was significantly lower than that of control group (0.470 [0.180, 1.360] vs. 5.350 [2.230, 7.900], U = 55, P < 0.001). CONCLUSIONS Many proteins related to energy metabolism presented differential expression patterns in the mucosa of colon of the IBS-D patients. The abnormalities in energy metabolism may be involved in the pathogenesis of IBS which deserves more studies to elucidate.
Adenosine Triphosphate ; metabolism ; Adult ; Blotting, Western ; Colon ; metabolism ; pathology ; Diarrhea ; enzymology ; metabolism ; pathology ; Electrophoresis, Gel, Two-Dimensional ; Energy Metabolism ; genetics ; physiology ; Female ; Humans ; Immunohistochemistry ; Intestinal Mucosa ; enzymology ; metabolism ; pathology ; Irritable Bowel Syndrome ; enzymology ; metabolism ; pathology ; Male ; Mass Spectrometry ; Middle Aged ; Proteome ; metabolism

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.
Cordyceps ; chemistry ; Desiccation ; methods ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; analysis ; Molecular Weight

5-HT₆ receptor (5-HT₆R) is implicated in cognitive dysfunction, mood disorder, psychosis, and eating disorders. However, despite its significant role in regulating the brain functions, regulation of 5-HT₆R at the molecular level is poorly understood. Here, using yeast two-hybrid assay, we found that human 5-HT₆R directly binds to neuro-oncological ventral antigen 1 (Nova-1), a brain-enriched splicing regulator. The interaction between 5-HT₆R and Nova-1 was confirmed using GST pull-down and co-immunoprecipitation assays in cell lines and rat brain. The splicing activity of Nova-1 was decreased upon overexpression of 5-HT₆R, which was examined by detecting the spliced intermediates of gonadotropin-releasing hormone (GnRH), a known pre-mRNA target of Nova-1, using RT-PCR. In addition, overexpression of 5-HT₆R induced the translocation of Nova-1 from the nucleus to cytoplasm, resulting in the reduced splicing activity of Nova-1. In contrast, overexpression of Nova-1 reduced the activity and the total protein levels of 5-HT₆R. Taken together, these results indicate that when the expression levels of 5-HT₆R or Nova-1 protein are not properly regulated, it may also deteriorate the function of the other.
Animals ; Brain ; Cell Line ; Cytoplasm ; Eating ; Gonadotropin-Releasing Hormone ; Humans ; Immunoprecipitation ; Mood Disorders ; Psychotic Disorders ; Rats ; RNA Precursors ; RNA-Binding Proteins ; Serotonin ; Two-Hybrid System Techniques

To establish a two-dimensional gel electrophoresis (2-DE) map for comparative proteomic analysis of rat spinal cord with chronic morphine tolerance, and to detect differentially expression proteins that are associated with chronic morphine tolerance.
 Methods: Sixteen male SD rats received the intrathecal catheterization operation and they were randomly divided into a morphine tolerance group (MT group, n=8) and a saline group (NS group, n=8). The lumbar enlargement segments of the MT group and the NS group spinal cord were harvested and proteins were separated by 2-DE. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The 2-DE maps were visualized after coomassie blue staining and analyzed using PDQuest analysis software. Identification of differential protein spots was conducted by MALDI-TOF-MS, and the Mascot query software was used to search Swiss-Prot database for bioinformatics analysis. Western blotting was used to verify the expression of some differentially expressed proteins.
 Results: A total of 1 000 spots were identified in 2-DE maps of rat spinal cord tissues from the MT group and the NS group, and 36 proteins were significantly differentially expressed in the MT group compared with the NS group. Identification was conducted by MALDI-TOF-MS and Swiss-Prot database through Mascot query software, and a total of 14 proteins were obtained. Among them, 2 protein spots were down-regulated in the MT group compared with that in the NS group, and 12 protein spots were up-regulated in the MT group compared with that in the NS group. Two kinds of proteins (NUDAA, ENOG) were verified by Western blotting and the results were consistent with proteomics data.
 Conclusion: The optimized 2-DE profiles for the proteome of spinal cord tissue in rats with chronic morphine tolerance is established preliminarily, which showed that morphine tolerance can cause changes in the expression of various proteins in the spinal cord.
Animals ; Electrophoresis, Gel, Two-Dimensional ; Male ; Morphine ; Proteome ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Spinal Cord