Expression and purification of a peptide fragment of 660 - 1468 amino acids in Neisseria gonorrhoeae NGO2105 protein as well as preparation and identification of its polyclonal antibody / 中华皮肤科杂志

Objective:To prokaryotically express a peptide fragment of 660 - 1468 amino acids in Neisseria gonorrhoeae NGO2105 protein, and to prepare and identify its polyclonal antibody. Methods:The pCold TF-NGO2105 660-1468 aa recombinant plasmid was transformed into the bacterium Escherichia coli BL21 (DE3) for protein expression. After the inclusion body protein was denatured and renatured, the target protein was purified. Then, BALB/c mice were immunized with the target protein to prepare a polyclonal antiserum; the antibody potency was evaluated by enzyme-linked immunosorbent assay, the specificity of the antibody against NGO2105 protein in Neisseria gonorrhoeae was analyzed by Western blot analysis, the affinity of the antiserum with Neisseria gonorrhoeae was analyzed by flow cytometry, and adhesion inhibition assay was performed to evaluate the inhibitory effect of anti-NGO2105 660-1468 aa antibody on the adhesion of Neisseria gonorrhoeae to human cervical epithelial ME-180 cells. Comparisons between different groups were performed by using t test. Results:The NGO2105 660-1468 aa protein was expressed as the inclusion body, and the soluble target protein was obtained by denaturation, renaturation, and purification. After immunization of mice with the target protein, the antiserum titer was 5.12 × 10 6, and flow cytometry showed that the antibody bound well to the Neisseria gonorrhoeae NGO2105 660-1468 aa. Adhesion inhibition assay showed that the anti-NGO2105 660-1468 aa antibody significantly inhibited the adhesion of Neisseria gonorrhoeae to ME-180 cells, and the inhibitory effect was concentration-dependent to some extent, with the adhesion rates of Neisseria gonorrhoeae treated with 20- and 40-fold dilutions of the anti-NGO2105 660-1468 aa antibody being 52.9% and 79.2% respectively, significantly lower than the adhesion rate in the untreated group (100%, t = 8.40, 5.29, P < 0.001, = 0.006, respectively) . Conclusion:The NGO2105 660-1468 aa protein was successfully expressed and purified, and a highly potent polyclonal antibody was prepared, which had a good affinity with Neisseria gonorrhoeae and an adhesion inhibition ability.

Mechanism of Regulating BAFF Signaling Pathway by Act1 in B Cell Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the mechanism of regulating B cell-activating factor (BAFF) signalling pathway by NF-κB activator 1 (Act1) in B cell lymphoma so as to provide a new thinking for treatment of B cell lymphoma.</p><p><b>METHODS</b>The human B cell lymphoma cell lines including Raji, Daudi and BALL-1 were cultured, when the cells were in logarithmic phase, the RNA was extracted, and the Act1 was amplified by RT-PCR; and pTT5-Act1 expression plasmid was constructed and was transfected into cells; the Act1 was silenced by using Act1 mRNA; the NF-κB signaling pathway protein was detected by Western blot.</p><p><b>RESULTS</b>After silence or overexpression of Act1, the proliferation levels of Raji, Daudi and BALL-1 cells were up- or down-regulated, respectively. Overexpression and silence of Act1 could down-or up-regulate BAFF-R expression level, furthermore could inhibit or activate of NF-κB signalling pahway, respectively.</p><p><b>CONCLUSION</b>Among 3 above-mentioned B cell lymphoma cell lines, Act1 plays negative regulating role, indicating that the Act1 may be a promising therapeutic target for treating B cell lymphoma.</p>

Spd1672 gene knockout significantly attenuates the virulence of Streptococcus pneumoniae / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of spd1672 gene in the infection process of Streptococcus pneumoniae.</p><p><b>METHODS</b>BALB/c mice were intraperitoneally infected by a spd1672 knockout strain and a D39 wild-type strain of S. pneumoniae, and the survival time of mice and blood bacterial counts were recorded. The adhesion and invasion ability of S. pneumoniae strains were assessed in A549 cells. Bactericidal assays were carried out to determine the resistance of spd1672 knockout strains and D39 wild strains, and the serum levels of inflammatory cytokines were detected in the infected mice.</p><p><b>RESULTS</b>The mice infected with spd1672 knockout strains showed a significantly longer median survival time, a higher survival rate, and a lower blood bacterial load than the wild strain-infected mice (P<0.05). Having a similar cell adhesion ability to the wild-type strain (P>0.05), the spd1672 knockout strain showed significantly lower cell invasion ability than the wild-type strain (P<0.05). The spd1672 knockout strain also had a reduced resistance to whole blood cells, and thw mice infected with spd1672 knockout strain exhibit lower levels of serum inflammatory cytokines than those infected with the wild-type strain.</p><p><b>CONCLUSION</b>Spd1672 gene is importantly related to the virulence of S. pneumoniae and plays important roles in modulating bacterial invasion, resistance to whole blood cells and proinflammatory responses.</p>

Studies on expression, purification, crystal growth and optimization of putative transcription factor LytR from Streptococcus pneumoniae / 生物医学工程学杂志

The aim of the present study was to obtain the crystal of transcription factor LytR of streptococcus pneumoniae for X-ray crystal structure and function analysis. The LytR gene of D39 strains from Streptococcus pneumoniae (S. pn) was cloned into the prokaryotic expression vector pET32a(+), then overexpression was obtained in the E. coli BL21 (DE3) through transformation of the recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with His-tag highly expressed by the induction of 0.5 mmol/L IPTG and was purified by a three step procedure, the purity of the purified LytR recombinant protein was over 90%. Preliminary screening of crystallization conditions was performed using the hanging-drop vapour-diffusing method with Hampton Crystal screen and PEG screen kits. The protein crystals X-ray diffraction data were collected from a single crystal and more stick crystals whose X-ray diffraction reached 4.0 A were obtained. These works laid the foundation for further research on the 3D structure of putative transcription factor LytR and its biological aspects.

Cytogenetic and clinical analysis of 340 Chinese patients with primary amenorrhea / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To analyze the relationship between karyotypes and clinic features of patients with primary amenorrhea.</p><p><b>METHODS</b>G banding was done for 340 patients with primary amenorrhea to facilitate individual chromosome identification, and if specific staining for certain portions of the chromosome was necessary, C banding was used. The clinical data were recorded by physical examination and ultrasound scanning.</p><p><b>RESULTS</b>Karyotype analysis of the 340 patients revealed that 180 (52.94%) patients had normal female karyotypes and 160 (47.06%) patients had abnormal karyotypes. The abnormal karyotypes included abnormal X chromosome (150 patients), mosaic X-Y chromosome (4 patients), abnormal autosome (5 patients), and X-autosome translocation (1 patient). The main clinical manifestations in patients with primary amenorrhea were primordial or absent uterus (95.9%), invisible secondary sex features (68.8%), little or absent ovary (62.6%), and short stature (30.0%). The incidence of short stature in patients with X chromosome aberration (46%, 69/150) was significangly higher that in patients with 46, XX (9.44%, 17/180) as well as 46, XY (6.67%, 3/45; Chi square = 146.25, P=0.000). All primary amenorrhea patients with deletion or break-point at Xp1 1.1-11.4 were short statures.</p><p><b>CONCLUSIONS</b>One of the main reasons of primary amenorrhea is choromosome abnormality, especially heterosome abnormality. It implies the need to routinely screen chromosomal anomalies for such patients. There might be relationship between Xp1 1.1-11.4 integrity and height improvement.</p>

Membrane testosterone receptors in cultured vascular smooth muscle cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To determine the presence of membrane testosterone receptors in cultured vascular smooth muscle cells (VSMC), and investigate their relationship with classical intracellular androgen receptors (iAR).</p><p><b>METHODS</b>VSMCs were cultured from the thoracic aorta of male Sprague-Dawley rats by the explant method. Subconfluent VSMCs were incubated with serum-free medium for 24 h to obtain quiescent non-dividing cells, and then treated with the indicated agents. The aliquots of VSMCs were labeled with testosterone-BSA-FITC (T-BSA-FITC) and analyzed by flow cytometry. Classical iARs in intact- and permeabilized-cells were detected with anti-iAR antibodies and FITC-labeled secondary antibodies by immunofluorescence, followed by flow cytometry analysis.</p><p><b>RESULTS</b>Incubation of VSMCs with T-BSA-FITC obviously increased their relative fluorescence intensity at 10 sec as compared with the untreated controls (P < 0.01), and so did it at 10 min in comparison with the treatment with BSA-FITC alone or together with free testosterone (P < 0.01). Pretreatment with iAR antagonist flutamide exhibited no significant influence on the relative fluorescence intensity of VSMCs (P = 0.318). Traditional iARs were not detectable on the surface of intact VSMCs, although permeabilized cells contained iARs.</p><p><b>CONCLUSION</b>VSMCs contain testosterone receptors in the plasma membrane, and these membrane receptors are not identical to classical iARs.</p>

Testosterone at physiological level inhibits PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the acute effects of testosterone at the physiological level on PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells (VSMCs).</p><p><b>METHODS</b>VSMCs from the thoracic aorta of male Sprague-Dawley rats were cultured using the explant method. The subconfluent VSMCs were incubated with serum-free medium for 24 hours to obtain quiescent non-dividing cells and then treated with the indicated agents. For the measurement of [Ca2+]i, the VSMCs were loaded with fura-2. Changes of [Ca2+]i were determined ratiometrically with a Nikon TE-2000E system.</p><p><b>RESULTS</b>The resting level of [Ca2+]i was around 100 nmol/L in the VSMCs. Exposing cells to perfusate containing 10 micromol/L PGF2alpha triggered an immediate and transient peak in [Ca2+]i, which gradually decreased afterwards. Interference at the peak with the physiological concentration (40 nmol/L) of testosterone significantly decreased the peak-to-baseline time of [Ca2+]i, compared with ethanol vehicle (104.9 +/- 27.0 s vs 153.5 +/- 40.4 s, P < 0.01). Pretreatment with testosterone at 40 nmol/L for 2 minutes also reduced the peak-to-baseline time of [Ca2+]i significantly in comparison with the ethanol control (120.6 +/- 32.0 s vs 151.4 +/- 27.4 s, P < 0.01), but it had no significant effect on the peak level of PGF2alpha-induced intracellular Ca2+ (390.0 +/- 126.0 nmol/L vs 403.4 +/- 160.7 nmol/L, P > 0.05).</p><p><b>CONCLUSION</b>Testosterone at physiological concentration inhibits PGF2alpha-induced Ca2+ fluxes, probably via receptor-operated calcium channels by a non-genomic mechanism in VSMCs, which may be involved in the vasodilatory effect of testosterone.</p>

Testosterone therapy improves cardiac function of male rats with right heart failure / 中华男科学杂志

<p><b>OBJECTIVE</b>Clinical studies have shown decreased levels of sexual hormones, particularly testosterone deficiency, in men with chronic heart failure (CHF). The authors aimed to investigate the effect of testosterone on cardiac function and the possible mechanism of androgen protecting the heart in male rats.</p><p><b>METHODS</b>Forty-three male SD rats were randomly divided into 3 groups: right heart failure (RHF, n = 15), physiologic testosterone treatment (TT, n = 15) and control (n = 13). The RHF group was given intraperitoneal injection of monocrotaline at 60 mg/kg to make RHF models; the TT group was injected with testosterone at 5 mg/kg 3 days after monocrotaline administration; and the control group received equal volume of saline. The CD34+ cells in the peripheral blood of each rat were counted by flow cytometry. The levels of serum testosterone and tumor necrosis factor alpha (TNF-alpha) were measured by chemiluminescence immunoassay and enzyme linked immunosorbent assay, respectively. The hearts, lungs and livers of all the surviving rats were excised at 6 weeks for pathological and immunohistochemical examinations.</p><p><b>RESULTS</b>The level of serum testosterone was gradually decreased, while that of TNF-alpha obviously increased in the RHF group. After testosterone treatment, the TT group showed a remarkable improvement of cardiac performance and a significant decrease in the level of serum TNF-alpha as compared with the RHF group. Statistically significant differences were observed neither in the CD34+ cell count in the peripheral blood nor in the CD34+ expression of the myocardial cells between the TT and RHF groups.</p><p><b>CONCLUSION</b>Physiological supplementation of testosterone can improve the cardiac function of RHF male rats, probably through its inhibition of TNF-alpha rather than by autologous mobilization of bone marrow stem cells.</p>

Correlative study of carotid transient ischemic attacks and intracranial or extracranial angiostenosis / 中南大学学报(医学版)

OBJECTIVE@#To investigate the relationship between the clinical features of carotid transient ischemic attacks (TIA) and the intracranial or extracranial angiostenosis.@*METHODS@#Location and degree of stenosis of involved arteries were examined by the digital subtraction angiography in 52 patients with carotid TIA.@*RESULTS@#Intracranial or extracranial vascular lesions of different degrees were revealed in 45 patients (86.5%), and 29 out of 45 (64.4%) had more than one site. Severe stenosis and occlusion occurred more frequently in TIA patients with short duration (less than 1 hour) and multiple attacks (more than twice).@*CONCLUSION@#Most patients with TIA of carotid systems have stenosis in intracranial or extracranial arteries. TIA with short duration and multiple attacks always accompany with severe stenosis or occlusion in intracranial or extracranial arteries. Digital subtraction angiography helps to identify the vascular etiology of TIA and provides the instruction of therapeutic regimen.

Significance of NF-?B in immunopathogenesis of Graves disease / 中国病理生理杂志

AIM:To investigate the activity of NF-?B in peripheral blood mononuclear cells (PBMCs) from patients with Graves disease (GD) and the significance in immunopathogenesis of GD.METHODS:Peripheral blood was collected from 22 untreated GD,20 treated GD with tapazole more than 1 year,and 25 healthy volunteers. PBMCs were isolated from the blood by histopaque-1077 density-gradient centrifugation. The activity of NF-?B in PBMCs was analyzed using gel electrophoretic mobility shift assay (EMSA). The contents of IL-1?,IL-6 and TNF-? were tested by radioimmunoassay.RESULTS:The activity of NF-?B in PBMCs of untreated GD group was increased remarkably,compared with that in the treated group and control (P