Suppression of Wnt/ß-catenin Signaling in PDAC via METTL16-mediated N6-methyladenosine Modification of DVL2.

Pancreatic cancer is a formidable cause of cancer-related deaths worldwide and has witnessed a more than twofold increase in incidence over the last 25 years. The most frequently occurring form of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), accounting for the majority of pancreatic cancer cases. N6-methyladenosine (m6A), the most abundant transcript modification, has been implicated in the pathogenesis of numerous human cancers, including pancreatic cancer. Despite this, the functional role of methyltransferase-like 16 (METTL16), a critical m6A methyltransferase, in PDAC remains elusive. In this study, we demonstrate that METTL16 expression is significantly diminished in PDAC, rendering it a promising prognostic indicator. Strikingly, both in vitro and in vivo assays revealed accelerated metastasis and invasion of PDAC cells upon METTL16 knockdown, while overexpression of METTL16 exerted an opposite effect. Mechanistically, METTL16 regulates DVL2 expression by suppressing its translation via m6A modification, thereby regulating Wnt/ß-catenin signaling., Our results unveil the downregulation of METTL16 as a concomitant increase in DVL2 levels via m6A modification promoting the progression of PDAC. Thus, we propose METTL16 as a novel therapeutic candidate for targeted PDAC treatment.

The Wet Suction Technique Enhances the Diagnostic Efficacy and Aspirate Quality of EUS-FNA for Solid Lesions: A Multicenter Retrospective Study in China.

GOALS: To comprehensively compare the wet suction technique with the conventional dry suction technique for endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in solid lesions. BACKGROUND: Optimal suction techniques for EUS-FNA remain uncertain when approaching solid lesions. STUDY: We performed a retrospective study of EUS-FNA at 3 medical centers in China. A total of 203 patients were enrolled who received 2 passes of EUS-FNA with 22-G needles. If the first pass underwent dry suction, the second pass was wet suction. Otherwise, the order of suction technique is opposite. Diagnostic accuracy, sample quality (including cellularity and blood contamination), and sample quantity (including specimen adequacy, the maximum intact specimen length, and the total specimen length) were compared between wet-suction and dry-suction techniques. RESULTS: The patients included 143 pancreatic lesions and 60 nonpancreatic lesions. Compared with the dry suction technique, the wet suction technique yielded a significantly higher diagnostic accuracy (85.22% vs. 72.41%, P =0.002), better specimen adequacy score and cellularity score ( P <0.0001), and lower blood contamination score ( P <0.0001). In the subgroup analysis, wet suction provided significantly higher diagnostic accuracy in pancreatic cancer without chronic pancreatitis ( P <0.05), and better cellularity score and specimen adequacy score, lower blood contamination score, and longer maximum intact specimen length and total specimen length in various lesions than that in dry suction. CONCLUSIONS: The wet suction technique resulted in significantly higher diagnostic accuracy in pancreatic cancer without chronic pancreatitis, and better cellularity and histologic specimen in most of solid lesions.

RELA-induced MiR-21 Exerts Oncogenic Effects on PDAC via Targeting of ARHGAP24.

Inflammation is one of the inducing factors of pancreatic ductal adenocarcinoma (PDAC), and microRNAs have been confirmed to be involved in the occurrence and development of PDAC. However, whether RELA, an inflammatory regulator, is involved in the regulation of PDAC by miRNA remains to be further studied. In the present study miR-21 was characterized and its upstream regulatory mechanism was investigated, as well as its functional effects and target genes in pancreatic ductal adenocarcinoma (PDAC). In situ hybridization analysis confirmed increased miR-21 expression levels in PDAC tissues. The results of the chromatin immunoprecipitation and dual-luciferase reporter assays demonstrated that transcription factor RELA modulated miR-21 transcription in the PDAC, PANC-1 and MIA PaCa-2 cell lines. Subsequently, a cell viability assay, EdU staining assay and flow cytometry analysis, demonstrated that miR-21 promoted cell proliferation and cell cycle progression, but inhibited cell apoptosis in vitro. Furthermore, a xenograft assay demonstrated that miR-21 accelerated tumor growth in vivo. Mechanistically, miR-21 directly regulated the expression of Rho GTPase activating protein 24 (ARHGAP24), which was indicated to be a tumor suppressor gene. Moreover, both miR-21 and ARHGAP24 were strongly associated with clinical features and may therefore serve as valuable biomarkers in PDAC prognosis.

Patterns of cardiometabolic multimorbidity and the risk of depressive symptoms in a longitudinal cohort of middle-aged and older Chinese.

BACKGROUND: Cardiometabolic diseases (CMDs) are associated with depression. However, it is unclear whether coexisting CMDs may increase the risk of depression. We examined associations between cardiometabolic multimorbidity and depressive symptoms among middle-aged and older Chinese. METHODS: Participants aged ≥45 years were enrolled from the China Health and Retirement Longitudinal Study 2011-2018 (N = 18,002). Cardiometabolic multimorbidity was defined as the coexistence of ≥2 CMDs, including stroke, heart disease, diabetes, hypertension, and dyslipidemia. Depressive symptoms were assessed by the Center for Epidemiological Studies Depression Scale. We used generalized estimating equation models to examine associations between cardiometabolic multimorbidity and depressive symptoms, including the dose effect of disease count and prevalent disease combinations, as well as individual and additive effects of specific CMDs. RESULTS: The prevalence of cardiometabolic multimorbidity was 24.5%. A higher number of CMDs had an additive dose effect on depressive symptoms that persisted consistently in specific CMDs. Stroke only, heart disease only, and diabetes only were each associated with a higher risk of depressive symptoms compared with no CMDs. CMD combinations involving stroke, heart disease, or diabetes were each associated with an increased risk of depressive symptoms compared with the absence of stroke, heart disease, or diabetes. LIMITATION: Self-reported chronic conditions. CONCLUSION: Stroke, heart disease, and diabetes showed individual and additive effects on CMD combinations, whereas hypertension and dyslipidemia only showed associations with depressive symptoms in combinations with other CMDs. These results suggest person-centered healthcare of mental health prevention and treatment for middle-aged and older adults with individual or multiple CMDs.

Prediction of the oxidation potential of PM2.5 exposures from pollutant composition and sources.

The inherent oxidation potential (OP) of atmospheric particulate matter has been shown to be an important metric in assessing the biological activity of inhaled particulate matter and is associated with the composition of PM2.5. The current study examined the chemical composition of 388 personal PM2.5 samples collected from students and guards living in urban and suburban areas of Beijing, and assessed the ability to predict OP from the calculated metrics of carcinogenic risk, represented by ELCR (excess lifetime cancer risk), non-carcinogenic risk represented by HI (hazard index), and the composition and sources of the particulate matter using multiple linear regression methods. The correlations between calculated ELCR and HI and the measured OP were 0.37 and 0.7, respectively. HI was a better predictor of OP than ELCR. The prediction models based on pollutants (Model_1) and pollution sources (Model_2) were constructed by multiple linear regression method, and Pearson correlation coefficients between the predicted results of Model_1 and Model_2 with the measured volume normalized OP are 0.81 and 0.80, showing good prediction ability. Previous investigations in Europe and North America have developed location-specific relationships between the chemical composition of particulate matter and OP using regression methods. We also examined the ability of relationships between OP and composition, sources, developed in Europe and North America, to predict the OP of particulate matter in Beijing from the composition and sources determined in Beijing. The relationships developed in Europe and North America provided good predictive ability in Beijing and it suggests that these relationships can be used to predict OP from the chemical composition measured in other regions of the world.

Value of Crystal Vue technique in detecting the placenta accreta spectrum located in c-section scar area.

AIMS: Excessive placental invasion is a life-threatening obstetric disease. Determining the extent of placental villi invasion prenatally is crucial for formulating a surgical plan for pregnant women. The objective of this study was to explore the diagnostic accuracy of the Crystal Vue technique combined with two-dimensional (2D) ultrasound in detecting the degree of placenta accreta spectrum (PAS) located in the C-section scar area. MATERIALS AND METHODS: Twenty-seven pregnant women with a strong suspicion of PAS underwent 2D ultrasound combined with a Crystal Vue examination. The diagnosis of 2D ultrasound alone and Crystal Vue combined with 2D ultrasound was statistically calculated, respectively. Cohen's kappa (k) was used to measure the consistency between these two ultrasound diagnosis and the postoperative diagnosis. RESULTS: The postoperative diagnosis of 27 pregnant women was as follows: 6 cases of placental accreta, 11 cases of placental increta, 2 cases of placental percreta, 2 cases of placental accrete and placental increta, 2 cases of placental accreta and placental percreta, and 4 cases without PAS. Compared with the postoperative diagnosis, 20 cases (74.07%) were correctly diagnosed by 2D ultrasound alone, 6 cases were misdiagnosed, and one case the diagnosis was incomplete, which were substantially consistent with the postoperative diagnosis (k=0.612, p<0.01). Twenty-six cases (96.30%) were correctly diagnosed by Crystal Vue combined with 2D ultrasound; only one case was incomplete diagnosed which was  almost perfectly consistent with the postoperative diagnosis (k=0.934, p<0.01). CONCLUSIONS: Combining the Crystal Vue technique with 2D ultrasound can improve the diagnostic accuracy of ultrasound for detecting all types of PAS located in C-section scar area.

Comparative assessment of the biocompatibility and degradation behavior of Zn-3Cu and JDBM alloys used for biliary surgery.

This study was designed to investigate the biocompatibility and the degradation behavior of a Zn-3Cu alloy, a Zn-3Cu coating alloy, a Mg-Nd-Zn-Zr (denoted as JDBM) alloy and a JDBM coating alloy to choose the optimal alloy for common bile duct (CBD) surgery. In the in vitro degradation experiments, we observed the surface morphology of the samples and determined the elements of the corrosion products. In the in vitro cytotoxicity experiments, the cell morphology and cytotoxicity were observed and tested. In the in vivo experiments, in addition to analyzing the samples, we also analyzed the variations in serum magnesium, serum creatinine (CREA), blood urea nitrogen (BUN), total bilirubin (TB) and glutamic pyruvic transaminase (GPT). Moreover, important tissue samples from the CBD, liver, kidney and spleen were taken for histological evaluation. The in vitro degradation experiments revealed that the surface corrosion of the JDBM and JDBM coating alloys were more obvious than that of Zn-3Cu and Zn-3Cu coating alloys, and the degradation rate of the JDBM coating alloy was the slowest. The in vitro cytotoxicity assessment showed that the JDBM alloy and JDBM coating alloy extracts were biologically safe for L-929 cells, while the Zn-3Cu alloy and Zn-3Cu coating alloy extracts were harmful to L-929 cells. In the in vivo experiments, neither the JDBM alloy nor the JDBM coating alloy affected the function or morphology of the bile duct, liver, kidney or spleen. Similar to the in vitro degradation behavior, the surface corrosion of the JDBM alloy was more significant than that of the JDBM coating alloy. Our data suggested that the JDBM coating alloy is a safe, biodegradable material for CBD surgery.

Combined Real-Time Three-Dimensional Hysterosalpingo-Contrast Sonography with B Mode Hysterosalpingo-Contrast Sonography in the Evaluation of Fallopian Tube Patency in Patients Undergoing Infertility Investigations.

OBJECTIVE: This prospective study aimed to investigate the use of real-time three-dimensional hysterosalpingo-contrast sonography (4D-HyCoSy), using contrast agent SonoVue, with B mode hysterosalpingo-contrast sonography (B mode-HyCoSy), to evaluate tubal patency and the wall of the Fallopian tubes in infertility patients. METHOD: In total, we recruited 739 women with fertility requirements from the First Affiliated Hospital of Shantou Medical College between January 2017 and July 2018. All cases received 4D-HyCoSy using contrast agent SonoVue, immediately followed by the B mode-HyCoSy. Of these patients, 145 showed pathological findings in the Fallopian tubes during HyCoSy; 34 of these (62 Fallopian tubes) were verified by laparoscopy and the dye test against routine reference standards. Sonographic findings, along with laparoscopic findings and dye test results, were used to compare the two techniques using the Cohen kappa coefficient. We also investigated the duration of examination and pain score. RESULTS: Compared with laparoscopy and the dye test, the tubal occlusion diagnostic accordance rates for 4D-HyCoSy were 88.7% (32+23)/62, with a kappa coefficient of 0.769 and a 76.9% agreement rate. Distal occlusion diagnostic accordance rates for 4D-HyCoSy were 100% (8/8) with a k coefficient of 1.000 and a 100% agreement rate. CONCLUSIONS: The use of 4D-HyCoSy, with B mode-HyCoSy, for the diagnosis of tubal patency is safe, feasible, noninvasive, and highly accurate. B mode-HyCoSy allowed us to observe tubal walls in an intuitive manner.

Applying WCACG modified process is beneficial on reduced door-to-balloon time of acute STEMI patients.

BACKGROUND: Various systems have employed with the objective to reduce the time from emergency medical services contact to balloon inflammation for ST-elevation myocardial infraction (STEMI) patients. The WCACG message system was used to an alternative communication platform to improve confirmation of the diagnosis and movement to treatment, resulted in shorten the door-to-balloon (D-to-B) time for STEMI patients. METHODS: We collected 366 STEMI patients admitted at the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, Department of Cardiology, during the period from June 2013 to October 2015. The patients were divided into two groups one underwent the current GC processes and the other group was handled using WCACG system. We compared between two groups with several indicators including D-to-B time, duration of hospitalization, associated costs, and incidence of adverse cardiovascular events. RESULTS: The results show that the new method with WCACG system significantly reduced the average D-to-B time (from 100.42 ± 25.14 mins to 79.81 ± 20.51 mins, P < 0.05) compared to the GC processes, and also reduced the duration, costs and undesirable cardiac incidence during hospitalization. CONCLUSIONS: The modified WCACG process is an applicable system to save pieces of time and efficiently integrate the opinions of experts in emergency.

miR-21 promotes EGF-induced pancreatic cancer cell proliferation by targeting Spry2.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant cancer that lacks effective targets for therapy. Alteration of epidermal growth factor (EGF) expression has been recognized as an essential molecular event in pancreatic carcinogenesis. Accumulating studies have demonstrated that miRNAs play critical roles in EGF signaling regulation, tumor initiation, cell proliferation and apoptosis. Here, we demonstrated that miR-21 expression was induced by EGF in pancreatic cancer cells. miR-21 promoted EGF-induced proliferation, inhibited cell apoptosis and accelerated cell cycle progression. In vivo experiments confirmed the influence of miR-21 on tumor growth. Mechanistic studies revealed that miR-21 targeted MAPK/ERK and PI3K/AKT signaling pathways to modulate cell proliferation. In addition, Spry2 was proven to be a target of miR-21. Furthermore, miR-21 and Spry2 were significantly related to clinical features and may be valuable predictors of PDAC patient prognosis.

The efficacy of temporary placement of nasobiliary drainage following endoscopic metal stenting to prevent post-ERCP cholangitis in patients with cholangiocarcinoma.

BACKGROUND/AIMS: Although endoscopic metal biliary endoprosthesis (EMBE) is widely accepted as the most suitable drainage method for patients with unresectable malignant obstruction, uncontrolled post-procedural cholangitis is still a problem. We aimed to validate a new treatment modality to prevent post-ERCP cholangitis in patients with unresectable cholangiocarcinoma. PATIENTS AND METHODS: A total of 378 patients who were diagnosed with unresectable malignant biliary obstruction and underwent EMBE or temporary endoscopic nasobiliary drainage (ENBD) following EMBE placement, from January 2010 to July 2016, were enrolled in this retrospective study. Incidence of cholangitis, related infectious indicators, success rate of biliary drainage, and occurrence of complications were evaluated. RESULTS: The risk of overall cholangitis and related infectious indicators was significantly lower in EMBE plus ENBD group than that in EMBE group. The occurrence of cholangitis was 2.4% versus 11.9% (P = 0.004). On further analysis of subgroups, although no difference was detected in nonhilar cholangiocarcinoma subgroup, the incidence of cholangitis and related infectious indicators in hilar cholangiocarcinoma subgroup with EMBE modality were distinctly higher than that with EMBE plus ENBD modality (type I + II was 18.5% vs 0%, P < 0.05; type III + IV was 19.8% vs 3.8%, P < 0.05). No significant difference was found in successful biliary drainage rate and procedure-related complications when all subgroups were compared. CONCLUSIONS: The temporary placement of ENBD following EMBE is a simple and effective treatment modality to prevent post-ERCP cholangitis, especially in patients with hilar cholangiocarcinoma.

Dilation catheter-guided mini-forceps biopsy improves the diagnostic accuracy of malignant biliary strictures.

BACKGROUND: Tissue sampling for biliary stricture is important for differential diagnosis and further treatment. The aim of this study was to assess a novel dilation catheter-guided mini-forceps biopsy (DCMB) method in the diagnosis of malignant biliary strictures. METHODS: 42 patients with malignant biliary stricture who underwent both brush cytology and DCMB during endoscopic retrograde cholangiopancreatography between October 2014 and November 2015 were retrospectively included. During DCMB, the mini biopsy forceps was introduced into the biliary stricture through the dilation catheter, and then the position and direction of the forceps were adjusted to obtain tissue samples. RESULTS: The positive rate of DCMB was significantly higher than that of brush cytology (81.0 % [34/42] vs. 38.1 % [16/42]; P < 0.001). No severe complications occurred; three patients (7.1 %) experienced mild procedure-related acute pancreatitis. CONCLUSIONS: The novel DCMB technique was a practical, safe, efficient, and low-costing method for diagnosing malignant biliary stricture with a high accuracy rate.

Endoscopic ultrasonography-guided drainage combined with trans-duodenoscope cyclic irrigation technique for walled-off pancreatic necrosis.

BACKGROUND: Endoscopic ultrasonography-guided drainage has been established as a good treatment modality in the management of walled-off pancreatic necrosis, but the unmanageable infection of postoperation is still a thorny problem due to the poor drainage ability for solid necrotic debris only through transmural stent and nasocystic catheter. AIMS: Introduce a novel therapeutic method, namely endoscopic ultrasonography-guided drainage combined with cyclic irrigation technique in managing patients with walled-off pancreatic necrosis. METHODS: 18 patients with severe acute pancreatitis complicated with walled-off pancreatic necrosis received treatment with endoscopic ultrasonography-guided drainage combined with cyclic irrigation were involved in this retrospective study. RESULTS: 17 of 18 patients with walled-off pancreatic necrosis were treated by this new therapeutic method. Subsequent surgery was performed in 1 case due to uncontrolled infection, complications such as perforation, bleeding or multiple organ failure were not observed. Treatment success rate was high (16 in 17, 94.12%). CONCLUSION: Endoscopic ultrasonography-guided drainage combined with cyclic irrigation is an effective treatment option for symptomatic walled-off pancreatic necrosis to facilitate drainage and obviate the need for subsequent surgery or endoscopic necrosectomy.

Molecular cloning and functional characterization of a mouse gene upregulated by lipopolysaccharide treatment reveals alternative splicing.

Treatment of mouse cells with lipopolysaccharide (LPS) potently initiates an inflammatory response, but the underlying mechanisms are unclear. We therefore sought to characterize cDNA sequences of a new mouse LPS-responsive gene, and to evaluate the effects of MLrg. Full-length cDNAs were obtained from LPS-treated NIH3T3 cells. We report that the MLrg gene produces two alternative splice products (GenBank Accession Nos. DQ316984 and DQ320011), respectively, encoding MLrgW and MLrgS polypeptides. Both proteins contain zinc finger and leucine zipper domains and are thus potential regulators of transcription. Expression of MLrgW and MLrgS were robustly upregulated following LPS treatment, and the proteins were localized predominantly in the nuclear membrane and cytoplasm. In stable transfectants over-expressing MLrgW the proportion of cells in G1 phase was significantly reduced, while in cells over-expressing MLrgS the proportion of cells in G2 was significantly increased; both proteins are thus potential regulators of cell cycle progression. Upregulation of MLrgW and MLrgS may be an important component of the LPS inflammatory pathway and of the host response to infection with GNB.

[Soluble expression, purification and bioactivity of recombinant human Era protein].

AIM: To prepare a soluble human Era(hEra) protein and to measure its bioactivity. METHODS: Human era cDNA gene from pUC19 plasmid was subcloned into the expression plasmid pMAL-p2x. pMAL-hEra was transducted to E.coli TB1 and the strain was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). RESULTS: The expressed MBP-fused protein existed in a soluble form. The fused protein made up 23.9% of the total cell lysate. It was purified by amylose affinity chromotography and digested with Factor X. Although the fused segment was dissected, the remained hEra protein was unstable in the solution with the passage of time. The activity assay showed that hEra was a GTPase that could bind GTP and hydrolyze GTP to GDP. CONCLUSION: Human Era protein can be expressed in a soluble form and it has been proved to be a kind of G protein by the experiments in vitro. The study is important to further research into the function of human era gene.

Expression and regulation of the yggG gene of Escherichia coli.

Our previous study indicated that Era, a membrane-associated GTPase essential for the survival of Escherichia coli, binds with the product of the yggG gene. However, the expression, regulation, and function of the yggG gene have not been established. In this study, the transcript of the yggG gene was determined by analysis of the 5'-end of the yggG mRNA using 5'-RACE (5'-rapid amplification of cDNA ends) method. The promoter and transcription regulatory regions of yggG were analyzed through systematic analysis of the transcriptional activity of the fragment containing a 339-bp 5' flanking sequence of yggG mRNA. The results showed that the sequence -39/-1 upstream of the transcriptional start site of the yggG gene contained a core promoter required for the expression of 25-kDa YggG protein, whereas the -106/-40 region was associated with transcriptional upregulation of yggG under heat shock. Immunocytochemistry and subcellular fraction analysis showed that YggG was a membrane-associated protein. Based on these results, we confirmed that the -39/780 region contains the whole set of the promoter and coding sequence of the yggG gene. The expression regulation of the yggG gene under stress conditions and the YggG protein located on cell membrane are consistent with the bioinformatics analysis that YggG, a metallopeptidase, is functional as a heat shock protein that is associated with Era functions.

Up-regulation of yggG promotes the survival of Escherichia coli cells containing Era-1 mutant protein.

Era is a highly conserved GTPase essential for bacterial growth. Using a digoxigenin-labeled Era protein to screen a phage expression library of Escherichia coli genomic DNA, yggG, a gene that encodes a putative zinc metalloprotease was isolated and characterized. The deduced amino acid sequence of YggG showed high degrees of similarity to some reported heat shock proteins. In this study, the direct interaction between Era and YggG was confirmed, and it was found that the yggG gene, encoding a 25 kDa heat shock protein, was up-regulated at the mRNA level in partially defective Era GTPase mutants (era-1) and in E. coli cells overproducing Era-1. The delta yggG strain displayed the same growth rate as wild-type strain under normal growth conditions and after heat shock. Overexpression of Era-1 in the delta yggG strain resulted in a stronger growth-inhibitory effect than that in the wild-type strain, while coexpression of YggG partially restored the bacterial growth rate. The results indicated that YggG expression is significantly increased in response to stress caused by the Era-1 mutant protein in E. coli, thus promoting the growth of E. coli.

[Expression of mouse lipopolysaccharide response protein LRP in E. coli and preparation of rabbit anti-mLRP serum].

AIM: To express mouse lipopolysaccharide response protein (mLRP) and prepare rabbit anti-mLRP serum. METHODS: The predicted mouse lrp cDNA sequence was obtained by splicing homologous ESTs by comparing human lrp cDNA with mouse ESTs. Then the primers were designed. mlrp cDNA from NIH3T3 cells stimulated with lipopolysaccharide (LPS) was amplified by RT-PCR and was cloned into prokaryotic expression vector pTAT to construct recombinant expression vector pTAT-mlrp. The His-TAT-mLRP fusion protein was expressed in E. coli BL21(DE3) and was used to immunize the rabbits to get rabbit anti-mLRP serum. The anti-serum was purified by the acetone precipitation method. The specificity of the rabbit anti-mLRP serum was determined by Western blot. RESULTS: The predicted length of mlrp cDNA was 1905 bp. The encoding region of the cloned mlrp cDNA, 1554 bp, was inserted into pTAT. The His-TAT-mLRP fusion protein was expressed successfully in E. coli. The rabbit anti-mLRP serum was prepared by immunizing the rabbit with mLRP protein. CONCLUSION: The successful expression of mLRP and the preparation of rabbit anti-mLRP serum lays the foundation for further study of the function of mLRP.

[Expression of a candidate human Era binding protein A19 and the preparation of its polyclonal antibody].

AIM: To express a candidate hEra binding protein A19 in Escherichia coli and to prepare anti-A19 antibody. METHODS: A19 gene was amplified by PCR from the plasmid containing A19 gene and was cloned into the expression vector pGEX-4T3 which was then transformed into E.coli. The A19 protein was expressed under IPTG induction. Antiserum was prepared by immunizing rabbits with the expressed A19 protein. The titer and specificity of polyclonal antibody were detected by Western blot. RESULTS: The expressed A19 accounted for about 30.2% of total bacterial protein. The titer of the antiserum was about 1:4 000. Western blot analysis indicated that the antiserum had high specificity. CONCLUSION: A19 fusion protein was highly expressed. The specific anti-A19 antiserum was prepared successfully.

[Expression and purification of two kinds of alternative splicing mouse Era].

AIM: To express and purify two alternative splicing mouse Era proteins and detect whether anti-human Era antibody can be used in the study of mouse Era proteins. METHODS: Two fusion protein expression vectors, pMAL-meraW and pMAL-meraS, were constructed, then the MBP-mEra proteins were expressed in E. coli. The target proteins were purified by amylose affinity chromatography. The specificity of rabbit anti-human Era antibody to the proteins was identified by Western blot. RESULTS: The expressed MBP-mEraW and MBP-mEraS proteins constituted approximately 17% and 19% of the total bacterial proteins. The purity of the fused proteins was 67% and 61% respectively after amylose affinity chromatography. Rabbit anti-human Era antibody had high specificity to these two kinds of splicing mouse Era proteins. CONCLUSION: Two fusion mera genes could be expressed in E. coli by using gene recombination technique. The high specificity of rabbit anti-human Era antibody to the two splicing mouse ERA proteins indicates that this antibody can be used to study the function of these two kinds of splicing mouse Era.