[Show abstract][Hide abstract] ABSTRACT: SERBP1 (SERPINE1 mRNA binding protein 1) is an arginine methylated RNA binding protein and the modification affects protein interaction and intracellular localization. In the present study, we show that under normal growth condition without stress, SERBP1 interacts with some arginine methylated and stress granule-associated proteins such as hnRNPA1, FMRP, FXR1 in an RNA-dependent manner. We also show that after arsenite treatment, a fraction of full-length SERBP1 protein co-localizes with the typical stress granule marker TIA-1in the cytoplasmic SGs. Truncated SERBP1 with the N-terminal, middle RG or C-terminal deletions, or else any single domain segment containing the N-terminal, middle or C-terminal region, was recruited to SGs upon arsenite treatment but with reduced efficiency. Besides, upon arsenite treatment, diffuse cytoplasmic localization of SERBP1 was shifted to nuclear-dominant and concentrated nucleolar distribution. Similar distribution was observed when cells were treated with a methylation inhibitor adenosine periodate (AdOx) and was also detected in the N- or C-terminal domain deletions and all three single domain fragments even without stress induction. We further demonstrate that AdOx treatment delays the association/dissociation of SERBP1 with SGs. Hypomethylation retains SERBP1 in the nucleus/nucleolus regardless of arsenite treatment. Our study indicates that arginine methylation is correlated with the recruitment of SERBP to as well as its retention in stress granules and nucleoli. Our study is the first report of an RNA binding protein that can be shifted simultaneously to cytoplasmic SGs and nucleoli, two RNP-concentrated subcellular compartments, upon stress. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Increased expression of cellular membrane bound glucose-regulated protein 78 (GRP78) is considered to be one of the biomarkers for gastric cancers. Therefore, peptides or molecules with specific recognition to GRP78 can act as a guiding probe to direct conjugated imaging agents to localized cancers. Based on this rationale, GRP78-guided polymeric micelles were designed and manufactured for nuclear imaging detection of tumors. Thiolated GRP78 binding peptide (GRP78BP) was first labeled with maleimide-terminated poly(ethylene glycol)- poly(ɛ-caprolactone) and then mixed with diethylenetriaminepentaacetic acid (DTPA)-linked poly(ethylene glycol)-poly(ɛ-caprolactone) to form DTPA/GRP78BP-conjugated micelles. The coupling efficiency of micelles with radioisotope indium-111 ((111)In) was measured and analyzed by instant thin layer chromatography. The coupling efficiency of DTPA-conjugated micelles and DTPA/GRP78BP-conjugated micelles with (111)In was 85% and 93%, respectively. For characterization and trace imaging, the radioisotope (111)In-targeting tumors were detected and imaged in a xenograft murine model using nano single photon emission computed tomography/computed tomography. The results revealed that the radioactive intensity measured in the animals administered with GRP78BP-guided (111)In-labeled micelles was statistically higher than that in animals administered with (111)In-labeled micelles, demonstrating that GRP78BP more than doubled the accumulation of micelles to the tumor tissue (P < 0.05). The results indicate that the gastric cancer biomarker GRP78 is a probing target in the application of nuclear imaging for tumor diagnosis. This novel GRP78BP-guided micelle agent may be applied in clinical practice to complement the histological diagnosis.
Full-text · Article · Apr 2013 · International Journal of Nanomedicine
[Show abstract][Hide abstract] ABSTRACT: Protein arginine methylation regulates a broad array of cellular processes. SERBP1 implicated in tumor progression through its putative involvement in the plaminogen activator protease cascade, is an RNA-binding protein containing an RG-rich domain and an RGG box domain that might be methylated by protein arginine N-methyltransferases (PRMTs). Asymmetric dimethylarginine (aDMA) was detected in SERBP1 and an indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) significantly reduced the methylation signals. Arginines in the middle RG and C-terminal RGG region of SERBP1 are methylated based on the analyses of different deletion constructs. The predominant type I protein arginine methyltransferase PRMT1 co-immunoprecipitated with SERBP1 and the level of bound PRMT1 decreased upon the addition of AdOx. Recombinant PRMT1 methylated SERBP1 and knockdown of PRMT1 significantly reduced the aDMA level of SERBP1, indicating that SERBP1 is specifically methylated by PRMT1. Immunofluorescent analyses of endogenous SERBP1 showed predominant cytoplasmic localization of SERBP1. Treatment of AdOx or PRMT1 siRNA increased the nuclear localization of SERBP1. Analyses of different deletions indicated that the middle RG region is important for the nuclear localization while both N- and C- terminus are required for nuclear export. Low methylation of the C-terminal RGG region also favors nuclear localization. In conclusion, the RG-rich and RGG box of SERBP1 is asymmetrically dimethylated by PRMT1 and the modification affects protein interaction and intracellular localization of the protein. These findings provide the basis for dissecting the roles of SERBP1.
No preview · Article · Aug 2012 · Journal of Cellular Biochemistry
[Show abstract][Hide abstract] ABSTRACT: The survivals of gastric cancer (GC) patients are associated with early diagnosis and effective treatments. Therefore, it is urgent for the discovery of early GC biomarkers and tumor-targeting therapeutics. The aim of this study was to uncover putative tissue biomarkers of GC using 2D DIGE and then apply one of these specific markers in GC treatment. We found three putative biomarkers of GC with significant differences in expression level compared to adjacent normal tissue, including glucose-regulated protein 78 (GRP78) and glutathione s-transferase pi (GSTpi) with increased expression level, and alpha-1 antitrypsin (A1AT) with reduced expression level. The overexpressed GRP78 was used as a targeted protein for guiding the drugs to tumor cells, leading to more effective treatment for GC xenografts. Our results demonstrated that the designated GRP78-binding peptide based on the sequence, WIFPWIQL, was selectively prone to recognize and bind to GC MKN45 cells in vitro, and also improve the delivery efficiency of polymeric micelles-encapsulated drugs into tumor cells and displayed better therapeutic outcome in experimental animals. This strategy of GRP78-mediated drug targeting system may bring chemotherapeutic drugs with more precise targeting to tumor cells, leading to minimize side effects on patients after chemotherapy.
[Show abstract][Hide abstract] ABSTRACT: Human neutrophil peptides (HNPs) -1, -2 and -3 are significantly upregulated and were reported as biomarkers in gastric cancer (GC). However, the tissue location and function of HNPs 1-3 are still unclear in GC, and the spatial distribution of the triad needs to be disclosed. The aims of this study were to investigate the distribution and relationships among HNPs-1, -2 and -3, and assess whether infiltrated neutrophils accumulate in gastric tumor.
In this study, paired samples (n=33) of the GC tissues and adjacent normal tissues from the same patients were obtained from surgery. Expression of HNPs 1-3 were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The distributions of the HNPs 1-3 in GC tissues were investigated. After verification of HNPs-1 by immunohistochemistry, infiltrated neutrophils were also detected. Then, an in vitro assay was used to observe the binding capacity and measure the cytotoxic effect of HNPs-1 against AGS cells.
Comparing to neighboring normal tissue, expressional level of HNPs 1-3 were significantly higher and their distributions overlapped in cancerous tissues of GC patients with high abundance in the lamina propria, whereas HNPs-1 was identified as the highest major peak. Moreover, HNPs-1, -2 and -3 correlated with each other. Besides, we also observed that increased infiltrated neutrophils accumulating in GC tissues, indicating that a strong positive correlation between HNPs 1-3 and infiltrated neutrophils. In addition, the further investigated demonstrated that the major peptide, HNPs-1, was statistically increased with the advance of tumor development from the early to advanced stage of GC (p< 0.05). Moreover, we also noticed that HNPs-1 with a great binding capacity to GC AGS cells in vitro can inhibit tumor cell growth.
Our results suggest that neutrophil secreted peptides, HNPs 1-3, increased in the GC tissues and could be used as potential biomarkers detected using MALDI-TOF MS, implying that elevated neutrophils may be used as a tumor target for tumor treatment. The binding capacity of HNPs-1 with GC cells implies that tracking molecules conjugated with HNPs-1 could be applied as a specific probe for GC diagnoses.
[Show abstract][Hide abstract] ABSTRACT: Excessive consumption of alcohol contributes to alcoholic liver disease. Fatty liver is the early stage of alcohol-related liver disease. The aim of this study was to search for specific serological biomarkers of alcoholic fatty liver (AFL) compared to healthy controls, non-alcoholic fatty liver (NAFL) and liver fibrosis in a rodent model.
Serum samples derived from animals with AFL, NAFL, or liver fibrosis were characterized and compared using two-dimensional differential gel electrophoresis. A matrix-assisted laser desorption ionization-time of flight tandem mass spectrometer in conjunction with mascot software was used for protein identification. Subsequently, Western blotting and flexible multi-analyte profiling were used to measure the expressions of the putative biomarkers present in the serum of animals and clinical patients.
Eight differential putative biomarkers were identified, and the two most differentiated proteins, including upregulated C-reactive protein (CRP) and downregulated haptoglobin (Hp), were further investigated. Western blotting validated that CRP was dramatically higher in the serum of AFL compared to healthy controls and other animals with liver disease of NAFL or liver fibrosis (p < 0.05). Moreover, we found that CRP and Hp were both lower in liver fibrosis of TAA-induced rats and clinical hepatitis C virus-infected patients.
The results suggest that increased levels of CRP are an early sign of AFL in rats. The abnormally elevated CRP induced by ethanol can be used as a biomarker to distinguish AFL from normal or otherwise diseased livers.
Full-text · Article · Aug 2011 · Journal of Biomedical Science
[Show abstract][Hide abstract] ABSTRACT: The aim of this study is to compare efficacy and complications between fundus-down and conventional laparoscopic cholecystectomy (LC) in treating contracted gallbladders with gallstones.
Between January 1999 and May 2008, 64 patients with contracted gallbladders and gallstones were included in the study. Main outcome measures included conversion rate, complication rate, bile duct injury rate, operation time, and postoperative stay.
The average postoperative hospital stay for fundus-down technique was 5 ± 3 days, and 7 ± 3 days for conventional technique (P = 0.003). The conversion rate and complication rate were 0% (0/33) and 3.00% (1/33) for fundus-down technique, and 32.3% (10/31) and 22.6% (7/31) for conventional technique (P = 0.0009 and 0.02, respectively). In subgroup analysis, fundus-down LC seemed to lower the bile duct injury rate from 2/31 (6.5%) to 0/33 (0%) compared with 6/1,468 (0.4%) (P = 0.01 between 6.5% and 0.4% vs. P = 1.00 between 0% and 0.4%).
It appears that fundus-down laparoscopic cholecystectomy is associated with lower conversion and complication rates and shorter postoperative hospital stay as compared with conventional laparoscopic cholecystectomy when used to treat patients with contracted gallbladders and gallstones.
No preview · Article · Jan 2011 · Surgical Endoscopy
[Show abstract][Hide abstract] ABSTRACT: We analyzed autoantibodies against tumor-associated antigens (TAAs) in the serum of patients with endometrioma and healthy controls to determine whether autoantibodies can be accurate biomarkers for the diagnosis of ovarian endometrioma.
Serum samples were obtained from 56 patients with endometriosis and 66 healthy women who served as normal controls. The titers of antibodies against a panel of eight TAAs were analyzed using enzyme-linked immunosorbent assay.
We found that the serum IGFII mRNA-binding protein 1 (IMP1) autoantibody and cyclin B1 autoantibody could discriminate between healthy controls and endometriosis patients (AUC-ROC 0.777; 95% confidence interval [CI] 0.694-0.860, P<0.0005, and AUC-ROC 0.614; 95%confidence interval [CI] 0.513-0.714, P=0.031, respectively). Using 0.073 and 0.007 as the cutoff values for IMP1 and Cyclin B1 autoantibody, respectively, the sensitivity and specificity of IMP1 were 85.7 and 63.6%, respectively. When cylcin B1 was combined with IMP1, the specificity increased to 72.7% and the sensitivity slightly decreased to 83.9%.
Our data suggest that IMP1 alone or combined with cyclin B1 seems to fulfill the requirements of sensitivity and specificity to become a useful clinical biomarker of endometrioma. However, further studies will be required to establish the predictive value and to support the clinical use of IMP1/cyclin B1 in the diagnosis and/or screening of endometriosis.
No preview · Article · Sep 2010 · Journal of Clinical Laboratory Analysis
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to compare the efficacy and safety of laparoscopic primary closure of the common bile duct (CBD) combined with percutaneous transhepatic cholangiographic drainage (PTCD) and laparoscopic choledocholithotomy with T-tube placement for the treatment of CBD stones. Between January 1991 and July 2002, 50 patients with choledocholithiasis and a CBD diameter larger than or equal to 1 cm underwent laparoscopic CBD explorations. The study group consisted of 10 patients undergoing laparoscopic primary closure of the CBD combined with PTCD. The control group consisted of 40 patients undergoing laparoscopic choledocholithotomy with T-tube placement. Parameters were compared statistically. The study group showed higher female/male ratio (6/4 vs. 8/32, P = 0.02), less stone numbers (1.90 +/- 0.88 vs. 3.40 +/- 1.65, P = 0.0078), shorter operation time (138 +/- 37 minutes vs. 191 +/- 75 minutes, P = 0.014), and shorter postoperative stays (7 +/- 3 days vs. 10 +/- 3 days, P = 0.0013). It seems that laparoscopic primary closure of the CBD combined with PTCD can shorten the operation time and postoperative stays as compared with laparoscopic choledocholithotomy with T-tube placement for the treatment of CBD stones.
No preview · Article · May 2010 · The American surgeon
[Show abstract][Hide abstract] ABSTRACT: The goal of our study was to evaluate the influence of genetic polymorphisms of matrix metalloproteinases (MMP)-2, MMP-3 and MMP-7 on susceptibility to endometrial cancer.
In the present study, we enrolled a total of 118 patients with endometrial cancer confirmed by histopathology, and 229 unrelated healthy individuals. Polymorphism for the MMP-2 (rs2285053), MMP-3 (rs3025058) and MMP-7 (rs11568818) genes was genotyped by polymerase chain reaction-restriction enzyme length polymorphism.
The frequencies of MMP-7 -181 G/G and A/G genotypes were found to be significantly higher in cancer patients compared with healthy controls (p = 0.017). Stratification showed that individuals with MMP-7 -181 G allele were at increased risk for endometrial cancer when >50 years of age [odds ratios (OR) = 2.03; 95% confidence interval (CI) 1.21-3.39], endometrioid (OR = 1.80; 95% CI 1.11-2.92), low (stage I-II) (OR = 1.73; 95% CI 1.05-2.83) or high stage (stage III-IV) (OR = 2.69; 95% CI 1.16-6.24). Compared with the A/A genotype, the A/G + G/G genotype modified the risk of developing endometrial carcinoma and significance was detected in patients over 50 years old, and those with endometrioid type and high stage endometrial cancer. However, no significant difference in MMP-2 (-735 C/T) and MMP-3 (6A/5A) genotypes was observed between endometrial carcinoma cases and controls.
This is the first report on the association of MMP-2, MMP-3 and MMP-7 gene polymorphisms in endometrial cancer. Our results suggest that individuals with the MMP-7 -181 G/G and A/G genotype may have an increased risk of developing endometrial cancer.
No preview · Article · Mar 2010 · Clinical Chemistry and Laboratory Medicine
[Show abstract][Hide abstract] ABSTRACT: Tissue inhibitors of metalloproteinases (TIMPs) are a family of multifunctional proteins known to possess a broad range of biological activities involved in tumorgenesis and mRNA expression of TIMP family members has been shown to be upregulated in numerous cancers and correlates with clinical outcomes. We investigated the association of TIMP-1 and TIMP-2 gene polymorphism with risk of endometrial cancer.
In the present case-control study, we enrolled a total of 118 endometrial cancer patients confirmed by histopathology and 229 unrelated healthy individuals. Polymorphism for TIMP-1 (_372C>T) and TIMP-2 (_418G>C and _303C>T) genes was genotyped by PCR-RFLP.
Frequency of TIMP-1_372C/C genotype and 372-C allele differed significantly between patients with endometrial cancer (38.1% and 56.4% respectively) and healthy individuals (22.7% and 44.1% respectively). Individuals with TIMP-1_372 C/C genotype were at higher risk of endometrial cancer (OR=2.37; 95%CI: 1.33-4.22). Stratification analysis showed that individuals with TIMP-1_372 C/C genotype were at increased risk for endometrioid (OR=2.46; 95% CI 1.34-4.53) and low stage (stages I-II) endometrial cancer (OR=3.24; 95% CI 1.22-4.13). However, no significant differences in TIMP-2_418G>C and TIMP-2_303C>T genotypes were observed between endometrial carcinoma cases and controls.
Individuals with TIMP-1_372C/C genotype were at significantly higher risk of endometrial cancer.
No preview · Article · Sep 2009 · Clinica chimica acta; international journal of clinical chemistry
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine whether cytokine expression (interleukin [IL]-1beta, IL-6, IL-8, IL-10, and tumor necrosis factor [TNF]-alpha), C-reactive protein, and endotoxins on the first day of intensive care unit (ICU) admission are associated with hospital mortality in severe community-acquired pneumonia (CAP).
This was a prospective study with bronchoalveolar lavage (BAL) and blood sampling.
This study was carried out in a 44-bed medical ICU of a 1700-bed university hospital.
Participants included 112 mechanically ventilated patients with severe CAP.
Serum and BAL fluid IL-1beta, IL-6, IL-8, IL-10, TNF-alpha, C-reactive protein, and endotoxins on the first day of ICU admission were obtained.
The concentrations of TNF-alpha in BALF and IL-6, IL-8, IL-10, and TNF-alpha in serum were higher in nonsurvivors than in survivor patients with CAP. Of these 112 patients with severe CAP (39%), 44 developed acute respiratory distress syndrome (ARDS); these patients seemed to have higher serum IL-6, IL-8, and IL-10 levels than did the non-ARDS group. Furthermore, in the ARDS population, we found that the endotoxin levels in the BAL fluid were higher in the survival than in the nonsurvival group and BAL fluid concentrations of IL-6, IL-8, and IL-1beta and sera levels of IL-6 and IL-10 were lower in the survival than in the nonsurvival group, and they were associated with a high negative predictive value.
Serum and BAL fluid levels of the studied cytokines on admission may provide valuable prognostic information for patients with severe CAP.
No preview · Article · Aug 2009 · Journal of critical care
[Show abstract][Hide abstract] ABSTRACT: The human endometrium manifests different and distinct morphologies and physiologies during the different phases of the menstrual cycle. We aimed to determine which candidate genes demonstrate differential expression patterns in the endometrium during different phases of the menstrual cycle.
Using differential display reverse transcription polymerase chain reaction to compare day 5 and day 18 human glandular endometrium obtained by laser capture microdissection, we identified a specific gene, NUDT9 (nucleoside diphosphate-linked moiety X motif 9). NUDT9 is known to function as a highly specific adenosine diphosphate ribose pyrophosphatase and has been mapped to chromosome 4q22.1. It gives rise to two alternatively spliced messenger RNAs, NUDT9alpha and NUDT9beta, encoding a member of the Nudix hydrolase family. In this study, we purified NUDT9 protein and produced an antibody, which we then used for immunohistochemical studies.
Using this anti-NUDT9 antibody, we successfully demonstrated that NUDT9 protein was differentially expressed in endometrial glandular cells at different phases of the menstrual cycle. NUDT9 was also found to be expressed more prominently in the epithelial glandular component than in the stromal component of human endometrial carcinomas.
We suggest that NUDT9 may be involved in the regulation of the menstrual cycle and may be related to the proliferation of glandular cells in the human endometrium.
No preview · Article · Jul 2009 · Taiwanese journal of obstetrics & gynecology
[Show abstract][Hide abstract] ABSTRACT: Our previous studies found that insulin-like growth factor-I receptor (IGF1R) signaling blockade caused cardiac hypertrophy, and that apoptosis is required for upregulating the IGF-II and the IGF-II/ mannose 6-phosphate receptor (IGF2R) gene. However, the role of IGF-II in the regulation of cell apoptosis through IGF2R is little known. In this study, we hypothesized that IGF-II may induce cell apoptosis through IGF2R but is dependent on IGF1R activity. Western blots and TUNEL assay revealed that in the presence of IGF1R, exogenous IGF-II acts, like IGF-I, would increase phospho-Akt through IGF1R, but does not affect the caspase 3 activation and apoptotic induction in H9c2 cardiomyoblast cells. Conversely, AG1024, an inhibitor of IGF1R activity, causes cell apoptosis, and the treatment with IGF-II further enhances this process, implying that it occurs through IGF2R. Moreover, immunoprecipitation assay revealed that treatment with IGF-II could enhance the interaction of IGF2R with Galphai and Galphaq but reduce its binding with Galphas, resulting in the reduction of phospho-PKA and the activation of PLC-beta. Taken together, these data provide new insight into the dual role of IGF-II in the control of IGF1R dependent cell apoptosis and involved activation of IGF2R signaling. Improving IGF1R activity and suppressing IGF2R may be a good strategy to prevent the progression of heart disease with cardiomyocyte apoptosis.
No preview · Article · Feb 2009 · The Chinese journal of physiology
[Show abstract][Hide abstract] ABSTRACT: The role played by IGF-II in signal transduction through the IGF-II/mannose-6-phosphate receptor (IGF2R) in heart tissue has been poorly understood. In our previous studies, we detected an increased expression of IGF-II and IGF2R in cardiomyocytes that had undergone pathological hypertrophy. We hypothesized that after binding with IGF-II, IGF2R may trigger intracellular signaling cascades involved in the progression of pathologically cardiac hypertrophy. In this study, we used immunohistochemical analysis of the human cardiovascular tissue array to detect expression of IGF2R. In our study of H9c2 cardiomyoblast cell cultures, we used the rhodamine phalloidin staining to measure the cell hypertrophy and western blot to measure the expression of cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cells treated with IGF-II. We found that a significant association between IGF2R overexpression and myocardial infarction. The treatment of H9c2 cardiomyoblast cells with IGF-II not only induced cell hypertrophy but also increased the protein level of ANP and BNP. Using Leu27IGF-II, an analog of IGF-II which interacts selectively with the IGF2R, to specifically activate IGF2R signaling cascades, we found that binding of Leu27IGF-II to IGF2R led to an increase in the phosphorylation of protein Kinase C (PKC)-alpha and calcium/calmodulin-dependent protein kinase II (CaMKII) in a Galphaq-dependent manner. By the inhibition of PKC-alpha/CaMKII activity, we found that IGF-II and Leu27IGF-II-induced cell hypertrophy and upregulation of ANP and BNP were significantly suppressed. Taken together, this study provides a new insight into the effects of the IGF2R and its downstream signaling in cardiac hypertrophy. The suppression of IGF2R signaling pathways may be a good strategy to prevent the progression of pathological hypertrophy.
Preview · Article · Jun 2008 · Journal of Endocrinology
[Show abstract][Hide abstract] ABSTRACT: The IGF-II/mannose 6-phosphate receptor (IGF2R) function in extracellular matrix (ECM) remodeling is known to occur as a result of transforming growth factor-beta (TGF-beta) activation and plasmin in the proteolytic cleavage level caused by the interaction between latent TGF-beta and urokinase plasminogen activator receptor (uPAR) respectively. In one of our previous studies, we found IGF-II and IGF2R dose-dependently correlated with the progression of pathological hypertrophy remodeling following complete abdominal aorta ligation. However, how this IGF2R signaling pathway responds specifically to IGF-II and regulates the myocardial ECM remodeling process is unclear. We found that IGF2R was aberrantly expressed in myocardial infarction scars. The matrix metalloproteinase-9 (MMP-9) zymographic activity was elevated in H9c2 cardiomyoblast cells treated with IGF-II, but not IGF-I. Treatment with Leu27IGF-II, an IGF2R specifically binding IGF-II analog, resulted in significant time-dependent increases in the MMP-9, tissue-type plasminogen activator (tPA), and urokinase plasminogen activator (uPA); and a reduction in the tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) protein expression. Furthermore, IGF2R expression inhibition by siRNA blocked the IGF-II-induced MMP-9 activity. We hypothesize that after IGF-II is bound with IGF2R, the resulting signal disrupts the balance in the MMP-9/TIMP-2 expression level and increases plasminogen activator (PAs) expression involved in the development of myocardial remodeling. If so, IGF2R signaling inhibition may have potential use in the development of therapies preventing heart fibrosis progression.
Preview · Article · Jun 2008 · Journal of Molecular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: Human dihydrolipoamide dehydrogenase (hE3) is a common component of alpha-ketoacid dehydrogenase complexes. Mutations of this homodimeric protein cause E3 deficiency and are always fatal. To investigate its reaction mechanism, we first performed multiple sequence alignment with other 17 eukaryotic E3s. According to hE3 structure and the result of multiple sequence alignment, two amino acids, T148 and R281, were subjected to mutagenesis and four hE3 mutants, T148G, T148S, R281N, and R281K, were expressed and assayed. The specific activities of T148G, T148S, R281N, and R281K are 76.34%, 88.62%, 12.50%, and 11.93% to that of wild-type E3, respectively. The FAD content analysis indicated that the FAD content of these mutant E3s were about 71.0%, 92%, 96%, and 93% that of wild-type E3, respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic data demonstrated that the K(cat) of forward reaction of all mutants, except T148 mutants, were decreased dramatically. The results of kinetic study suggest that T148 is not important to E3 catalytic function and R281 play a role in the catalytic function of the E3.
Full-text · Article · Feb 2008 · Journal of Biomedical Science
[Show abstract][Hide abstract] ABSTRACT: According to the multiple alignment of various dihydrolipoamide dehydrogenases (E3s) sequences, three human mutant E3s of the conserved residues in the center domain, N286D, N286Q, and D320N were created, over-expressed and purified. We characterized these mutants to investigate the reaction mechanism of human dihydrolipoamide dehydrogenases. The specific activities of N286D, N286Q, and D320N are 30.84%, 24.57% and 48.60% to that of the wild-type E3 respectively. The FAD content analysis indicated that these mutant E3s about 96.0%, 99.4% and 82.7% of FAD content compared to that of wild-type E3 respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic's data demonstrated that the K(cat) of both forward and reverse reactions of these mutant proteins were decreased. These results suggest that N286 and D320 play a role in the catalytic function of the E3.
[Show abstract][Hide abstract] ABSTRACT: Due to their similarity to type IV pilus (Tfp) subunits, the pseudopilins, XpsG, -H, -I, -J and -K, have been predicted to form a pilus-like structure in the type II secretion (T2S) pathway. While overexpression of GspG can result in the formation of bundle structures, the functions of other pseudopilin are not known yet. In this study, we investigate the mutual interaction among the pseudopilins and characterize the specialized minor pseudopilin, XpsJ. By using gel filtration and Ni-NTA affinity chromatography, a linearly ordered interactive relationship is revealed among the four pseudopilins, XpsG-XpsI-XpsH-XpsJ. Notably, unlike the mutant XpsJ194 staying in the inner membrane, wild type XpsJ stayed in the outer membrane and blocked the extension of overexpressed XpsG to outside of the cell. By analogy with the Type I pilus structures, we hypothesize that the XpsH and XpsI might act as an adaptor to connect XpsJ with the major pseudopilin XpsG, and XpsJ might act as a tip to restrict the out-growth of XpsG in the pilus-like structure of the T2S pathway.
Full-text · Article · Feb 2005 · Journal of Biomedical Science
[Show abstract][Hide abstract] ABSTRACT: p73 is one of the p53 family members which are transcription factors involved in the regulation of cell proliferation, apoptosis, and differentiation. In this study, we cloned the p73 cDNA from zebrafish ovary RNA. The consensus open reading frame (1923bp) encodes a polypeptide of 640 amino acids which shares 70-95% identity to the p73 of other vertebrates. Expression of zebrafish p73 mRNA is restricted to tissues such as skin, fin, brain, ovary, and testis, in contrast to the ubiquitous expression of zebrafish p53 and p63. During embryonic development, p73 transcripts are detected from the zygote period to the early larva stage. Whole-mount in situ hybridization reveals that p73 expression is in the brain, including olfactory bulbs, telencephalon, and hypothalamus, as well as in the pharyngeal arches and the nose. Moreover, p73 protein is found in the ovary and testis sections by immunohistochemical staining.
No preview · Article · Aug 2003 · Biochemical and Biophysical Research Communications