Objective With the sole object of glycyrrhizic acid products,the methods were investigated for Glycyrrhiza uralensis genomic DNA transformation into Hansenula anomala by nitrogen and argon ion implantation.Methods The genomic DNA from G.uralensis was randomly transferred into H.anomala by nitrogen and argon ion bombardment.The recombined yeasts were cultured by the slant and liquid cultivation,in which the contents of glycyrrhizic acid and glycyrrhetic acid were determined by acetic anhydried-H2SO4 qualitative test and RP-HPLC determination.Results Five recombined yeast strains that produced glycyrrhizic acid and glycyrrhetic acid were obtained.After cultured in liquid medium for 96 h,the highest content of glycyrrhizic acid in the cultivation liquid was 114.49 mg/L and that of 18?-glycyrrhetic acid and 18?-glycyrrhetic acid were respectively 0.56 and 0.81 mg/L by RP-HPLC.A kind of unknown red component was found in the cultivation liquid of one recombined strain by TLC.Conclusion The recombined yeast strains of producing glycyrrhizic acid could be obtained G.uralensis genomic DNA transformation into yeast mediated by the ion implantation.

BackgroundHuman retinal gliocytes play an important role in proliferative diseases,which are the basis of in vitro studies.Researchers have cultured human retinal gliocytes in the past.In our study,we found that the cells we cultured presented a unique shape different from those by other researchers.ObjectiveThis study was to design to produce a new culture and purification method for retinal gliocyte in vitro.Methods Retinal tissue was isolated from human eyeballs and digested using the two-step digestion method (2％ pancreatin and 0.133％collagenase Ⅵ) to harvest the retinal glio cytes.The cells were collected and cultured in endothelial cell-targeted nutrient culture containing 10％ fetal calf serum and supplemented with β-endothelial cell growth factor (ECGF) and sodium heparin,and the culture dishes were coated with fibronectin(FN) to promote the attachment of retinal gliocyte.During the culturing process,the gliocytes were identified by the observation of morphological characteristic and regular histological examination.The identification of the cells also was performed by immunochemistry targeting glial fibrillary acidic protein (GFAP),Vimentin,neuron specific enolase ( NSE ),S-100,CD34,and Ⅷ factor.Results Retinal gliocytes were isolated successfully from the human retina by the two-step digestion method.Primary cultured cells attached after 72 hours and achieved confluency between day 9 and 10 that were aligned petaliform in shape.Regular histological examination after H&E staining showed blue cell nuclei and light red cytoplasm.The target cells presented with strong responses for GFAP and Vimentin and no response for NSE,S-100,CD34 and Ⅷ factor.ConclusionsLarge amount of purified human retinal gliocytes can be obtained by two-step digestion and cultured in endothelial cells-targeted culture medium supplemented with β-ECGF and sodium heparin in plates coated with FN.The cultured cells expressed markers for retinal gliocytes.However,specific features of these cells remain to be further elucidated.

To ascertain whether connective tissue growth factor (CTGF) participates in the remodeling of pulmonary artery at the early-stage of bleomycin (BLM)-induced pulmonary fibrosis, mean pulmonary arterial pressure, the expression of type I and type III collagens, and the expression and location of CTGF in pulmonary artery and arteriole were investigated in the present study. Sprague-Dawley rats received instillation of BLM [5 mg/kg body weight, in 0.5 mL of normal saline (NS)] or instillation of the same amount of NS as control. Mean pulmonary arterial pressure was detected via a catheter in the pulmonary artery. Type I and type III collagens were examined with Sirius red staining under polarized light. CTGF expression was investigated by using immunohistochemistry, and was represented as average optical density and percentage of positive area of CTGF. The mean pulmonary arterial pressure was higher in rats on day 14 after BLM instillation [(19.5+/-2.9) mmHg] than that in the control rats [(14.8+/-1.2) mmHg] (P<0.05). The type I and type III collagens were increased both in pulmonary artery and arteriole of rats on day 14 after BLM instillation, compared with those in the control rats (P<0.05, P<0.01, respectively). The ratio of type I/III collagens in pulmonary artery was also higher in BLM-treated rats than that in the control rats (P<0.05). The values of average optical density of positive CTGF staining were increased both in pulmonary artery (0.37+/-0.02) and arteriole (0.40+/-0.03) of rats on day 14 after BLM instillation, compared with those in the control rats (artery, 0.34+/-0.01; arteriole, 0.29+/-0.01) (both P<0.05). The percentages of positive area of CTGF were higher in pulmonary artery (8.40+/-1.13) and arteriole (12.4+/-2.0) of rats on day 14 after BLM instillation than those in the control rats (artery: 1.42+/-0.63; arteriole: 1.16+/-0.34), respectively (both P<0.05). The increased positive CTGF staining areas were mainly located in the endothelium and smooth muscle layer. It is therefore concluded that CTGF expression increases in the endothelium and smooth muscle layer of pulmonary artery and arterioles during high pulmonary arterial pressure and remodeling of pulmonary artery at the early-stage of BLM-induced pulmonary fibrosis, and that the increased CTGF might be one of the mechanisms of maintenance and development of pulmonary hypertension.
Animals ; Bleomycin ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Connective Tissue Growth Factor ; metabolism ; Hypertension, Pulmonary ; Pulmonary Artery ; metabolism ; Pulmonary Fibrosis ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the prognosis and the factors affecting the prognosis in children with acute respiratory distress syndrome (ARDS).

METHODSSeventy-eight children with ARDS were enrolled. The states of their survival within 30 days were followed-up.

RESULTSOf the 78 children with ARDS, 51 cases demised, 27 cases survived, with a 30-days survival rate of 35%. The average survival time was 14.4 days (median: 8 days). The peak of death appeared within 3 days after ARDS. There were significant differences in aspects of age, primary disease, percentage of neonatal hyaline membrane disease, pediatric critical illness score (PCIS), duration of mechanical ventilation, oxygenation index (PaO(2)/FiO(2)), white blood cell count and number of involved organs between the died and survived children (P<0.05 or 0.01). The Cox multiple factors analysis showed that the age (HR 3.924~3.938), primary disease (HR=1.817) and PCIS (HR=0.469) were the risk factors of death.

CONCLUSIONSThe peak of death usually appears within 3 days after ARDS. Age, primary disease and PCIS are the independent factors of prognosis in children with ARDS.

Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Multivariate Analysis ; Prognosis ; Respiratory Distress Syndrome, Adult ; mortality ; Survival Rate

Streptoverticillum caespitosus ATCC27422 is a producing strain of mitomycin A for cancer therapy. Taking the advantage of the conserved sequences of genes flanking the oriC of high G + C Gram-positive bacteria, a 1.3 kb DNA fragment containing oriC and its flanking region was cloned by PCR. Nuleotide sequence comparisons revealed that the cloned fragment is more than 80% identical to the same region of S. coelicolor. There are 22 DnaA-boxes in the oriC region, and the conserved sequence of DnaA-box is TTGTCCACA. The plasmid containing the oriC of S. caespitosus was constructed (pMJ9), and it was able to transform the protoplast of Streptomyces lividans ZX7 at the frequency of 3.2 x 10(2) transformants/micrograms plasmid DNA. The colony and mycelia's morphology of the transformants are normal. The constructed plasmid can exist stable in the host as a low copy extra-chromosome replicon. The high rate of the homology and the cross genus replication initiation activity suggests close relationship between Streptomyces and Streptoverticillum in the evolution. While the maximum likelihood phylogenetic tree based upon the oriC of S. caespitosus and several Streptomyces spp. revealed that S. caespitosus differed extensively from the Streptomyces spp. This result supports the separation of Streptoverticillum from Streptomyces.
Actinomycetales ; genetics ; Base Sequence ; Blotting, Southern ; Cloning, Molecular ; Conserved Sequence ; Molecular Sequence Data ; Plasmids ; Replication Origin ; genetics ; Streptomyces ; genetics ; Transformation, Bacterial

BACKGROUNDThrombin is a multifunctional serine protease that plays a crucial role in hemostasis following tissue injury. In addition to its procoagulation effect, thrombin is also a potent mesenchymal cell mitogen, therefore it plays important roles in the local proliferation of mesenchymal cells in the tissue repair process. Reactive oxygen species (ROS) can induce some human cells to proliferate at lower rates while at higher concentrations they promote cells to undergo apoptosis or necrosis. Accumulative evidence suggests that thrombin can induce some cells to produce ROS. Based on these observations, we provide a hypothesis that thrombin can stimulate human lung fibroblasts to produce ROS, which play an important role in human lung fibroblast proliferation.

METHODSROS were detected in fibroblasts at 30 minutes and 60 minutes following thrombin (20 U/ml) exposure using flow cytometry. The ratio of reduced glutathione/oxidized glutathione (GSH/GSSG) was assayed in lung fibroblasts using a commercial kit following treatment with thrombin at different concentrations. NADPH oxidase and the extracellular regulated kinase1/2 (ERK1/2) signaling pathway were detected by Western blotting after thrombin stimulation to lung fibroblasts.

RESULTSThrombin, at 20 U/ml, stimulated human lung fibroblasts (HLF) to generate ROS in a time dependent manner. The ratio of GSH/GSSG in fibroblasts treated with thrombin showed a significant decrease. NADPH oxidase was activated and the ERK1/2 signal pathway was involved in the proliferation process of fibroblasts treated with thrombin.

CONCLUSIONThe activation of NADPH oxidase by thrombin leads to the production of ROS, which promotes fibroblasts proliferation via activation of the ERK1/2 signaling pathway.

Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; analysis ; physiology ; Fibroblasts ; drug effects ; physiology ; Flow Cytometry ; Glutathione ; metabolism ; Humans ; Lung ; cytology ; NADPH Oxidases ; analysis ; physiology ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology ; Thrombin ; pharmacology

OBJECTIVETo study the clinical characteristics of urticaria in children versus adults, and to provide reference for the etiological analysis, disease evaluation, and treatment of urticaria in children.

METHODSThe clinical data of 2 411 patients with urticaria who visited the Department of Dermatology at Xiangya Hospital of Central South University from January 2013 to May 2017 were collected to study their socio-demographic characteristics. The clinical characteristics of urticaria were compared between the 68 children and 672 adults of the 740 patients with complete follow-up data.

RESULTSAmong the 411 pediatric patients, 314 (76.4%) had acute urticaria; among the 2 000 adult patients, 896 (44.8%) had chronic spontaneous urticaria. The causes of acute urticaria in children included infection (41%, 16/39). The accompanying symptoms of acute urticaria in children mainly included abdominal pain and diarrhea (44%, 17/39), while those in adults mainly included chest distress and shortness of breath (32%, 11/34). Compared with the adult patients, the pediatric patients had significantly lower chronic urticaria activity scores before and after treatment (P<0.05), a significantly higher rate of response to second-generation antihistamines (82.1% vs 62.2%; P<0.05), and a significantly higher proportion of individuals with a personal and family history of urticaria (P<0.05).

CONCLUSIONSAcute urticaria is more commonly seen than chronic urticaria in children with urticaria, and the main accompanying symptoms are abdominal pain and diarrhea, which are different from adults with urticaria. Chronic urticaria has a better treatment outcome in children than in adults. The most frequently seen cause of acute urticaria is infection in children. Atopic children may be susceptible to urticaria.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Female ; Humans ; Male ; Middle Aged ; Urticaria ; drug therapy ; etiology ; Young Adult

OBJECTIVETo compare the clinical, pathological, laboratory test and follow-up data between familial and sporadic patients with distal myopathy with rimmed vacuoles (DMRV) and discuss the characteristics of this disorder in Chinese population.

METHODSThe clinical and pathological features, laboratory data and follow-up results of 33 sporadic and 4 familial cases of pathologically confirmed DMRV were summarized and compared retrospectively.

RESULTSThe patients age, onset age, or disease duration showed no significant difference between sporadic and familial cases; the onset pattern and affected muscle groups were also similar, but the sporadic cases showed more frequent dysmorphic features than the familial cases. The patients showed mild to moderate elevation of the muscle enzymes by one to three folds, and the familial patients had more significant elevation than the sporadic ones. No correlation was found between the disease duration and the level of muscle enzymes. The pathological findings were similar between the cases, and Gomori staining showed rimmed vacuoles and inclusion bodies without inflammatory cell infiltration. Follow-up results of 29 cases showed no significant difference between the two groups. The disease was slowly progressive and severely affected the quality of life of the patients, but did not produce obvious effect on the life expectancy.

CONCLUSIONThe clinical, pathological and laboratory data of Chinese DMRV patients are basically similar to those of Japanese cases. Sporadic cases tend to show more dysmorphic features than the familial ones, and occasional sporadic cases have early disease onset in early childhood.

Adult ; Asian Continental Ancestry Group ; Distal Myopathies ; classification ; genetics ; pathology ; Female ; Humans ; Inclusion Bodies ; pathology ; Male ; Pedigree ; Retrospective Studies ; Vacuoles ; pathology ; Young Adult

Objective: To explore deep vein thrombosis (DVT) occurrence in patients after right heart catheterization and the effect of anticoagulant therapy. Methods: A total of 171 consecutive patients with electrophysiological study (EPS) and/or radiofrequency catheter ablation (RFCA) in our hospital from 2015-01 to 2016-05 were enrolled. All patients had supra-ventricular tachycardia and completed a venous surgery, they were randomly divided into 2 groups: Anticoagulation group,n=87 and Non-anticoagulation group,n=84. Lower extremity vascular Doppler ultrasonography was performed at (24-48) h post-operation to compare the incidence of DVT between 2 groups. Results: There were 13/171 patients were excluded for not completing post-operative lower extremity vascular Doppler ultrasonography including 9 patients in Anticoagulation group and 4 in Non-anticoagulation group. 158 patients finished post-operative examination and follow-up study. Anticoagulation group had 7/78 (8.97%) patients suffered from DVT, Non-anticoagulation group had 41/80 (51.3%) patients suffered from DVT,P<0.001. Conclusion: The incidence of DVT was higher after right heart catheterization without anticoagulation; heparin treatment may reduce DVT occurrence in relevant patients.

Objective To investigate the relationship between serum progesterone level at the day with human chorionic gonadotrophin (hCG) administration and pregnant outcome from in in-vitro fertilization-embryo transfer(IVF-ET). Methods From Mar. 2002 to Apr. 2007, 786 cycles with serum progesterone measurement on the day of hCG administration for final oocyte maturation in IVF were analyzed retrospectively in Reproductive Medicine Center in First Affiliated Hospital of Nanjing Medical University.All stimulations were down-regulated with gronadotrophin release hormone agonist (GnRH-a) in both long protocols and short protocols before gonadotrophin stimulation. When the thresholds of serum progesterone were set at 5.5, 6.0,6.5,7.0,7.5,8.0,8.5 and 9.0 nmol/L, respectively. If the level of progesterone was less than the thresholds, those patients were in lower progesterone group, on the contrary, more than the threshold value, those patients were in higher progesterone group. The laboratory results and the clinical outcomes between all patients at lower and higher progesterone group at different thresholds value were analyzed. Results The rate of normal fertilization, quality embryos, successful implantation, chemical pregnancy, clinical pregnancy and live birth did not exhibit remarkable difference between patients with higher and lower serum progesterone level at multiple thresholds on the day of hCG administration in the 786 cycles (P >0.05). However, when the thresholds of serum progesterone were at 8.5 and 9.0 nmol/L, early abortion rates of 27.3% (3/11) and 3/7 in higher progesterone group were significantly higher than 8.8% (26/297) and 8.6% (26/301) in lower progesterone group (P<0.05). And the total abortion rates of 3/7 in higher progesterone group were significantly higher than 11.0% (34/301) in lower progesterone group when the thresholds of serum progesterone were 9.0 nmol/L (P<0.05). Conclusions This study did not prove the correlationship between progesterone level at the clay with hCG administration and the probability of clinical pregnancy or live birth. However, early abortion rates or the total abortion rates were associated with higher progesterone level when the thresholds of serum progesterone were at 8.5 nmol/L or 9.0 nmoL/L.