HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.
Cloning, Molecular ; Escherichia coli ; metabolism ; HIV Integrase ; metabolism ; HIV-1 ; physiology ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; Protein Binding ; Virus Replication

China ; Critical Care ; history ; methods ; trends ; History, 20th Century ; History, 21st Century ; Humans

Objective To summarize the clinical characteristics and diagnosis and treatment of hemangioma of small intestine in children.Methods Location,pathology,clinical manifestation,diagnosis and treatment of 3 cases from our hospital and 44 cases reported in our courtry with hemangioma of the small bowel were analyzed from 1994 to 2004.Results These tumors locating in jejunum,ileum,duodenum as well as parts small intestine were 31.9%,42.6%,4.2% and 21.3%,respectively.Solitary and multiple tumors were 29.8% and 70.2% separately.The histopatholical report of 10 cases revealed that capillary,cavernous,mixed type hemangioma and hematolymphangioma were 2,6,1 and 1 cases,respectively.Thirty nine children presented with recurrent black stool.Five patients manifest in the form of intussusception.One child passed a bloody stool so massive as to cause shock.Small-bowel obstruction occurred in 5 cases.The hemoglobin of 38 patients were lower than 90 g/L.The incidence of preoperative diagnosis was only 10.6%.All of patients were performed operation.Conclusions The frequent locations of hemangioma of small intestine are in ileum and jejunum,and multiple tumors are common.The diseases are characterized as recurrent hematochezia with painlessness.Preoperative diagnosis of a small bowel hemangioma can be very difficult.The segments of small bowel with hemangioma resection are the most method of the treatment.

Objective: To analyze the efficiency of ureteroscope and biopsy in the diagnosis of tumor grade, muscle-invasiveness and multifocality in suspected upper tract urinary carcinoma (UTUC) patients in order to find out whether it can be used in the risk stratification of UTUC patients.Methods: A retrospective study of 76 UTUC patients who underwent preoperative ureteroscope and/or biopsy and received radical nephroureterectomy in Peking University Third Hospital during January 2014 to December 2016 was undertaken.Results: In this study, 76 patients were included.There were 31 males (40.8%), and 45 females (59.2%).The median age was 64.5 years (31-88), and 51 patients had the symptom of hematuresis.The tumor was located in renal pelvis in 39 patients, and in ureter in 37 patients.Post-operative pathology confirmed that all the 76 patients included in this study suffered from UTUC, of whom 21 (21.6%) were of low-grade, 51 (67.1%) were of high-grade, 4 (5.3%) were undetermined, and 47 (61.9%) patients were muscle-invasive, and 27 (35.5%) were not, and 2 (2.6%) were undetermined.Among the 50 patients, in whom the grade of the tumor could be diagnosed by biopsy, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value for low-grade tumor was 88.2%, 69.7%, 76.0%, 60.0% and 92.0%, respectively.Among the 27 patients, in whom the muscle-invasiveness could be diagnosed by biopsy, 5 patients were diagnosed with muscle-invasiveness, all confirmed by pathology after surgery and 22 patients were diagnosed with none muscle-invasiveness, turned out to be 50% muscle-invasive and 50% none-muscle invasive after surgery.The accuracy was 59.3%.The accuracy of ureteroscopic biopsy to diagnosis multifocality was 61.0%.On univariate ana-lysis, biopsy grade was associated with postoperative pathology (P=0.001), while gender, age, side, body mass index (BMI), hematuresis, preoperative estimated glomerular filtration rate (eGFR), hydronephrosis, tumor size, location, multifocality and sessile were not associated with postoperative pathology grade.Biopsy grade (P=0.02), preoperative eGFR<90 mL/(min·1.73 m2)(P=0.025) and tumor located in pelvis (P=0.049) were associated with muscle invasiveness.Gender, age, side, BMI, hematuresis, hydronephrosis, tumor size, multifocality and sessile were not significantly associated with muscle invasiveness.Conclusion: Ureteroscope and biopsy can assist risk stratification in upper tract urothelial carcinoma patients.

Objective To characterize the audiological features in the infants with otitis media with effusion(OME)and to investigate the utility of variety of objective andiometry methods in diagnosis and intervention on OME.Methods Fifry six infants(40 males and 16 females)were investigated,who were referred to our clinic at the General Hospital of Chinese People's Liberation Army by the other hospitals from December 2004 to June 2007 when the infants were diagnosed or highly suspected of OME.The ages at the initial diagnosis ranged from 42 days to three years,with an average of five months.The infants,after receiving the conventional otolaryngological exams,were subjected to the tests of auditory brainstem response (ABR),otoacoustic emission(OAE),tympanometry(226 Hz and 1000 Hz)and behaviors audiometry.Results Among 56 affected infants.87 ears were diagnosed with OME,of which 31 infants were affected bilateral and 25 with monaural.For the 49 infants who received hearing screening at birth.36 infants were referred at the initial screening.For the 52 infants who received repeated screening.all subjects were referred.Six infants without receiving hearing screening came to clinic when their parents observed their kids'hearing impairment.Among the 52 cases(104 ears)who received tympanometry test,20 subjects (28 ears)showed B or C type tympanometry curve.Thirty-nine cases(78 ears)were given tympanometry test at 1000 Hz,of which 38 cases(55 ears)showed abnormal hearing.Among 56 infants(112 ears)with ABR test,49 subjects(74 ears)exhibited prolonged ABR type Ⅰ curve.All 56 infants(112 ears)received OAE test,of which 55 subjects(81 ears)were referred Four infants(8 ears)accepted the behavior test and all of them showed A-B Gap.Conclusions The combined tympanometry test at both 226 Hz and 1000 Hz,ABR latency or threshold test,infant's behavior test and OAE,used jointly,enable characterizing better OME in infants.thus helping early diagnosis of this hearing disorder.

To investigate JAK2V617F mutation and its clinical significance in patients with idiopathic myelofibrosis (IMF), genomic DNA was extracted from peripheral blood cell samples of 12 IMF cases. Allele-specific PCR (AS-PCR) was performed to identify JAK2V617F mutation, and the results were confirmed by sequence analysis. A retrospective study was performed to explore the correlation between JAK2V617F mutation and the clinical, hematologic features. The results showed that in follow-up for 2 to 15 months, the occurrence of the positive point mutation in 12 patients with IMF was 50%, and the half of these positive patients had thrombosis. Patients with JAK2V617F point mutation had a higher counts of platelets and megakaryocytes in bone marrow than those in patients without JAK2V617F point mutation. Out of other 6 IMF patients without JAK2V617F point mutation only 1 patient had thrombosis, and lower counts of platelets in peripheral blood and megakaryocytes in bone marrow. It is concluded that majority of IMF patients with positive JAK2V617F point mutation have typical clinical and hematologic features, higher incidence of thrombosis, and higher counts of platelets in peripheral blood and megakaryocytes in bone marrow.
Adult ; Aged ; Base Sequence ; Bone Marrow ; pathology ; Female ; Follow-Up Studies ; Humans ; Janus Kinase 2 ; genetics ; Male ; Megakaryocytes ; pathology ; Middle Aged ; Molecular Sequence Data ; Platelet Count ; Point Mutation ; Primary Myelofibrosis ; genetics ; Retrospective Studies

Humans ; Leukemia, Myeloid, Acute ; Mutation ; Prognosis ; Remission Induction ; fms-Like Tyrosine Kinase 3

BACKGROUNDCa(2+) in the central nervous system plays important roles in brain physiology, including neuronal survival and regeneration in rats with injured facial motoneurons. The present research was to study the modulations of intracellular free Ca(2+) concentrations by cholinergic receptors in rat facial nucleus, and the mechanisms of the modulations.

METHODSThe fluorescence intensity of facial nucleus in Fluo-3 AM loaded acute brainstem slices was detected by applying intracellular free Ca(2+) measurement technique via confocal laser scanning microscope. The changes of fluorescence intensity of facial nucleus indicate the average changes of intracellular free Ca(2+) levels of the neurons.

RESULTSAcetylcholine was effective at increasing the fluorescence intensity of facial nucleus. Muscarine chloride induced a marked increase of fluorescence intensity in a concentration dependent fashion. The enhancement of fluorescence intensity by muscarine chloride was significantly reduced by thapsigargin (depletor of intracellular Ca(2+) store; P < 0.01), rather than Ca(2+) free artifical cerebrospinal fluid or EGTA (free Ca(2+) chelator; P > 0.05). And the increase of fluorescence intensity was also significantly inhibited by pirenzepine (M(1) subtype selective antagonist; P < 0.01) and 4-DAMP (M(3) subtype selective antagonist; P < 0.01). In addition, fluorescence intensity was markedly increased by nicotine. The enhancement of fluorescence intensity by nicotine was significantly reduced by EGTA, nifedipine (L-type voltage-gated Ca(2+) channel blocker), dihydro-beta-erythroidine (alpha4beta2 subtype selective antagonist), and in Ca(2+) free artificial cerebrospinal fluid (P < 0.01), but not in the presence of mibefradil (M-type voltage-gated Ca(2+) channel blocker) or thapsigargin (P > 0.05).

CONCLUSIONSThe data provide the evidence that muscarinic receptors may induce the increase of intracellular free Ca(2+) levels through the Ca(2+) release of intracellular Ca(2+) stores, in a manner related to M(1) and M(3) subtypes of muscarinic receptors in rat facial nucleus. Nicotine may increase intracellular free Ca(2+) concentrations via the influx of extracellular Ca(2+)+ mainly across L-type voltage-gated Ca(2+) channels, in a manner related to the alpha4beta2 subtype of nicotinic receptors.

Acetylcholine ; pharmacology ; Aniline Compounds ; administration & dosage ; Animals ; Brain Stem ; cytology ; drug effects ; metabolism ; Calcium ; metabolism ; Diamines ; pharmacology ; Facial Nerve ; cytology ; Female ; Fluorescent Dyes ; administration & dosage ; In Vitro Techniques ; Male ; Microscopy, Confocal ; Motor Neurons ; drug effects ; metabolism ; Muscarinic Agonists ; pharmacology ; Nicotine ; pharmacology ; Nicotinic Agonists ; pharmacology ; Piperidines ; pharmacology ; Pirenzepine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Cholinergic ; metabolism ; Receptors, Muscarinic ; metabolism ; Receptors, Nicotinic ; metabolism ; Tropicamide ; pharmacology ; Xanthenes ; administration & dosage

OBJECTIVETo explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on the vitality and toxicity of E. coli ATCC 25922, and to analyze the potential mechanism.

METHODS(1) In vitro experiments. Standard strains of E. coli ATCC 25922 were divided into groups A (without addition), B, C, D, and E according to the random number table, and then the latter 4 groups were respectively cultured with 1.2 mmol/L CORM-2, 1.6 mmol/L CORM-2, 1.2 mmol/L inactive CORM-2 (iCORM-2), 1.6 mmol/L iCORM-2, with six samples in each group. After being cultured for 0, 3, 5, 8, 10, 12, 16, 20, 24, 27, 30, 48 hours, proliferative vitality of E. coli was examined (denoted as absorption value under 600 nm wavelength), and bacteria number was counted. Other standard strains of E. coli ATCC 25922 were divided into groups F (without addition) and G (cultured with 0.8 mmol/L CORM-2), the expressions of genes fliA, dnaK, marA, and waaQ related to E. coli were detected by quantitative real-time (qRT)-PCR. (2) In vivo experiments. Other standard strains of E. coli ATCC 25922 were grouped as A', B', C', D', and E' and treated with the same method as that in groups A, B, C, D, and E, and 0.5 mL bacterial liquid of each group were collected when the absorption value of bacterial liquid in group A' was equal to 0.4 (under 600 nm wavelength). Seventy-two C57BL/6 mice were divided into groups, namely blank control (without treatment), H, I, J, K, and L according to the random number table, with 12 mice in each group. The mice in the latter 5 groups were intraperitoneally injected with 0.5 mL bacterial suspension of groups A', B', C', D', and E' respectively. After injection, general condition of mice in groups H, I, J, K, and L was observed. The serum levels of TNF-α and IL-6 were determined at post injection hour (PIH) 6, 12. The liver and lung samples were harvested for determination of myeloperoxidase (MPO) activity at PIH 12. The same process was carried out in blank control group. Data were processed with repeated measure analysis of variance (ANOVA), factorial design ANOVA, one-way ANOVA, and t test.

RESULTS(1) In vitro experiments. Compared with those of groups A and D, the proliferative vitality and bacteria number of E. coli in group B were all decreased (with F values respectively 1170.80, 217.52, P values all below 0.01). Compared with those of groups A and E, the proliferative vitality and bacteria number of E. coli in group C were also obviously decreased (with F values respectively 7948.34, 14 432.85, P values all below 0.01). Compared with those in group F, the expression of fliA was downregulated, while the expressions of dnaK, marA, and waaQ were upregulated in group G (with t values 30.28, -165.54, -168.88, -187.28, P values all below 0.01). (2) In vivo experiments. Symptoms including listlessness and tachypnea were observed in mice in groups H, K, and L, and they were ameliorated or not obvious in groups I and J. At PIH 6, the serum levels of TNF-α and IL-6 in groups H and K were respectively (647.3 ± 3.8) pg/mL, (3.44 ± 0.22) ng/mL and (639.3 ± 0.8) pg/mL, (2.47 ± 0.32) ng/mL, which were obviously higher than those in group I [(124.6 ± 10.7) pg/mL, (1.03 ± 0.16) ng/mL, with t values from 15.22 to 84.03, P values all below 0.01]. The serum levels of TNF-α and IL-6 in group J at PIH 6, 12 were also obviously decreased as compared with those in groups H and L (with t values from 19.27 to 245.34, P values all below 0.01). MPO activity of liver and lung tissues were significantly attenuated in group I at PIH 12 as compared with those in groups H and K, and it was also attenuated in group J when compared with those in groups H and L (with t values respectively from 17.36 to 18.92 and 2.35 to 3.61, P values all below 0.01).

CONCLUSIONSCORM-2 can obviously inhibit the vitality and toxicity of E. coli, which might be attributable to regulation of expressions of genes fliA, dnaK, marA, and waaQ of E. coli.

Animals ; Carbon Monoxide ; metabolism ; DNA-Binding Proteins ; metabolism ; Escherichia coli ; drug effects ; metabolism ; physiology ; Escherichia coli Proteins ; metabolism ; Glycosyltransferases ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Interleukin-6 ; blood ; Liver ; enzymology ; Lung ; enzymology ; Mice ; Mice, Inbred C57BL ; Organometallic Compounds ; pharmacology ; Peroxidase ; metabolism ; Sigma Factor ; metabolism ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo observe the effects of Tangweian granule on 5-HT(2A)R in rat model with diabetic gastroparesis (DGP).

METHODThe rats with diabetic gastroparesis induced by injecting alloxan and giving 200% Radix Rehmanniae preparata were divided into four groups randomly: Tangweian high dosage group, Tangweian low dosage group, motilium control group and the model control group, 10 rats each group. Each group was irrigated with drugs during establishing the model. Additionally, we chose 10 rats by way of normal control group. Further more, Tangweian high dosage group were irrigated stomach with gliclazide 20 mg x kg(-1) and Tangweian granule 31.75 g x kg(-1); Tangweian low dosage group were irrigated stomach with gliclazide 20 mg x kg(-1) and Tangweian granule 15.88 g x kg(-1); motilium control group were irrigated stomach with gliclazide 20 mg x kg(-1) and motilium 3.75 mg x kg(-1) and the model control group were irrigated stomach with distilled water. Then the effects of Tangweian granule on 5-HT(2A)R were observed.

RESULTThe curative group had better effects than the control group in lowering the blood sugar and the level of 5-HT(2A)R content (P < 0.01). And there was significant difference between the curative group and control group (P < 0.05).

CONCLUSIONIt is verified that Tangweian granule has obvious effects on lowering the blood sugar and improving the level of 5-HT(2A)R.

Animals ; Blood Glucose ; drug effects ; Diabetes Mellitus, Experimental ; complications ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Gastroparesis ; drug therapy ; metabolism ; Gliclazide ; pharmacology ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Receptor, Serotonin, 5-HT2A ; metabolism