[Effect of HIF-2α mediated FoxM1 expression on proliferation of pulmonary artery smooth muscle cells in hypoxic rats].

Objective: To investigate the effect of hypoxia-inducible factor 2α (HIF-2α) gene on the expression of Forkhead box M1 (FoxM1) protein in the proliferation of hypoxic rat pulmonary artery smooth muscle cells (PASMC). Methods: HIF-2α overexpression lentiviral vector (LV-HIF-2α) and silencing RNA (siRNA) were constructed and transfected into rat PASMC under normoxia and hypoxia, respectively. The PASMC under normoxia were classified into normoxic control group, normoxia + LV-HIF-2α empty group, normoxia + LV-HIF-2α group; the PASMC under hypoxia were classified into hypoxic control group, hypoxia + siRNA-HIF-2α empty group, hypoxia + siRNA-HIF-2α group. The expression of HIF-2α and its downstream proteins FoxM1, cyclin D1 and Aurora A expressions were detected by Western blot. 5-Ethyny-2'-deoxyuridine (EdU) cell proliferation assay was used to evaluate the effect of overexpression and inhibition of HIF-2α expression on the proliferation of rat PASMC. Results: The expression of HIF-2α in normoxia + LV-HIF-2α group was significantly higher than that in normoxic control group and normoxia+LV-HIF-2α empty group (0.17±0.02 vs 0.09±0.01 and 0.07±0.00), while the expression of HIF-2α in PASMC of hypoxia + siRNA-HIF-2α group was significantly lower than that of hypoxic control group and hypoxia + siRNA-HIF-2α empty group (0.28±0.01 vs 0.35±0.02 and 0.30±0.01) (all P<0.05); the expression of FoxM1 protein, cyclinD1 and cell proliferation-related Aurora A protein in normoxia+LV-HIF-2α group were significantly higher than that in normoxic control group and normoxia+LV-HIF-2α empty group (0.40±0.03 vs 0.24±0.01 and 0.30±0.01, 0.22±0.02 vs 0.09±0.01 and 0.08±0.02, 0.29±0.02 vs 0.04±0.01 and 0.07±0.01, respectively) (all P<0.05); the expressions of FoxM1 protein, cyclinD1 and Aurora A protein in hypoxia + siRNA-HIF-2α group were significantly lower than those in hypoxic control group and hypoxia + siRNA-HIF-2α empty group (0.23±0.01 vs 0.36±0.02 and 0.32±0.01, 0.15±0.01 vs 0.31±0.01 and 0.28±0.03, 0.14±0.02 vs 0.33±0.03 and 0.27±0.02, respectively) (all P<0.05); the positive expression rate of EdU in the normoxic control group was significantly lower than that in the normoxia+LV-HIF-2α group [(30.77±2.43)% vs (55.56±3.01)%], while the hypoxic control group was significantly higher than the hypoxic+siRNA-HIF-2α group [(65.28±3.21)% vs (44.64±2.78)%] (both P<0.05). Conclusion: HIF-2α up-regulates the expression of FoxM1 and promotes the proliferation of pulmonary artery smooth muscle cells in hypoxic rats.

[Effect of Krüppel like zinc finger transcription factor 2 on γ-glutamylcysteine synthetase in bronchial epithelial cells of chronic obstructive pulmonary disease].

Objective: To research the regulation effects of Krüppel like zinc finger transcription factor 2 (KLF2) on γ-glutamylcysteine synthetase (γ-GCS) in airway epithelial cells of chronic obstructive pulmonary disease (COPD). Methods: (1) Human specimen experiment: lung tissue of pulmonary lobectomy patients with lung cancer with or without COPD was collected from Department of Thoracic Surgery of Hunan Cancer Hospital from December 2008 to December 2009. The patients were divided into COPD group and control group without COPD. The levels of KLF2, γ-GCS mRNA and protein expression in lung tissues were measured by immunohistochemistry and in situ hybridization (ISH). Then, the correlation between KLF2 and γ-GCS mRNA and protein expression levels were analyzed, as well as the correlation between KLF2 or γ-GCS protein and smoking index, percentage of forced expiratory volume in one second to predicted value (FEV1%), percentage of forced expiratory volume in one second to forced vital capacity (FVC/FEV1). (2) Animal experiment: the primary bronchial epithelial cells of rats were extracted by enzyme digestion. After 6 hours of incubation with 10% tobacco smoke extract (TSE), cellular glutathione (GSH) was measured by enzyme linked immunosorbent assay (ELISA) method. The cells were transfected by specific inhibitor of KLF2 through the liposom, which inhibited the protein expression of KLF2. Then, the cells were divided into KB group (blank control group without any treatment), KB+ TSE group (treated with TSE), NC group (control group transfected with miRNA), NC+ TSE group (treated with miRNA and TSE), 92a group (transfected with KLF2 inhibitor), 92a+ TSE group (treated with KLF2 inhibitor transfection and TSE) based in the treatment. After that, the changes of KLF2 and γ-GCS mRNA and protein expression in the cells of each group were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot method. Results: (1) Human specimen experiment: The expressions of KLF2 mRNA, protein and γ-GCS mRNA, protein in the lung tissue of COPD patients were strong positive and higher than those in control group (0.32±0.04 vs 0.19±0.03, 0.35±0.05 vs 0.22±0.03; 0.28±0.03 vs 0.16±0.03, 0.31±0.05 vs 0.21±0.03; all P<0.01). Linear correlation analysis showed that KLF2 mRNA and protein were positively correlated with γ-GCS mRNA and protein (r=0.705, 0.722; both P<0.01). The KLF2 and γ-GCS protein were positively correlated with smoking index, FEV1% and FEV1/FVC (r=0.552, 0.728, 0.670, and r=0.631, 0.727, 0.657; all P<0.01). (2) Animal experiment: The level of GSH in KB+ TSE group was significantly higher than that in KB group[(28.05±2.04) vs (7.27±0.33) nmol/mg, P<0.01]. The KLF2 mRNA, protein and γ-GCS mRNA, protein in KB+ TSE group (1.715±0.026, 1.842±0.028 and 2.117±0.067, 1.879±0.065) were higher than those in KB group (1.130±0.017, 1.177±0.033 and 1.378±0.053, 1.177±0.042; all P<0.05), and those in 92a group (0.472±0.028, 0.634±0.025 and 0.582±0.025, 0.554±0.021) were significantly lower than those in KB group, NC group (1.047±0.056, 1.092±0.045 and 1.303±0.037, 1.252±0.037), and those in TSE+ 92a group (0.262±0.017, 0.288±0.017 and 0.337±0.022, 0.321±0.022) were significantly lower than those in KB+ TSE group, 92a group and NC+ TSE group (1.576±0.036, 1.646±0.066 and 1.948±0.093, 1.843±0.078) (all P<0.05). Conclusion: KLF2 exerts antioxidative effect by regulating the expression of γ-GCS in the bronchial epithelial cells of chronic obstructive pulmonary disease.

[Root canal anatomy of maxillary second premolars at various ages observed by cone-beam CT].

Objective: To observe the morphological changes of root canals in maxillary second premolars at various ages by using cone-beam CT (CBCT) in order to provide imaging and theoretical reference for clinical treatments. Methods: The digital CBCT data of the maxillary second premolars in 440 cases from the patients in Department of Stomatology, The First Affiliated Hospital, Jinan University during March 2011 and December 2017 were collected. The CBCT images were divided into 4 groups according to the patients' ages: groups ≤20, 21-40, 41-60 and>60 years old, respectively. Changes of morphologies of root canals with aging including such parameters as types of the root canal, incidence of double root canals in single rooted teeth, distance between both root canal orifices of double rooted canals, and morphological change of the cross section of roots. Chi-square test and liner trend test were adopted in statistical analysis in the present study. Results: Most maxillary second premolars had only one root [95.2% (419/440)]. Type Ⅰ of the root canals was the most common type [57.3% (252/440)], and the following prevalent groups were type Ⅱ[16.8% (74/440)], type Ⅳ [10.2% (45/440)] and type Ⅲ [8.9% (39/440)]. The distribution of type Ⅰ~Ⅳ of the root canals were significantly different amongst various aged groups (P<0.05). Along with aging, the percentages of type Ⅰ decreased while type Ⅱ increased. However, there were no remarkable changes of type Ⅲ and type Ⅳ observed. The incidence of double canal in single rooted teeth gradually increased with aging especially in 20-year-old and above groups, e.g. 13.1% (13/99) in group of ≤20 years old and 45.0% (86/191) in group of 21-40 years old. However, there was no significant increase observed after the age of 40. The distance between two root canal orifices of double rooted canals became shorter with aging except in groups of 40-year-old and above. The morphologies of the cross sections of root canals in most groups were flat shaped [57.8% (1 121/1 938)] and oval shape [31.3% (607/1 938)]. Along with aging, the percentage of circular shape gradually increased while flat and oval shapes decreased. Conclusions: The morphology of root canal could be clearly showed by the CBCT images. Most maxillary second premolars had only one root and one apical foramen. Along with aging, the morphology of the root canals became more and more complicated.

[Aging changes of the root canal morphology in maxillary first premolars observed by cone-beam computerized tomography].

OBJECTIVE: To observe the morphological changes of root canals with aging in maxillary first premolars by using cone-beam computerized tomography(CBCT)in order to facilitate endodontic management of root canals in various aged patients. METHODS: The digital CBCT data of the maxillary first premolars in 405 cases from the patients in Oral Medical Center of The First Affiliated Hospital, Jinan University from March 2011 to June 2015 were collected. The CBCT images were divided into 6 groups according to the patients' ages: groups 11-20, 21-30, 31-40, 41-50, 51-60 and >60 years-olds, respectively. Changes of morphologies of root canals with aging including such parameters as types of the root canal, incidence of double root canals in single rooted teeth, distance between both root canal orifices of double rooted canals, and morphological change of the cross section of roots. Chi-square test and liner trend test were adopted in statistical analysis in the present study. RESULTS: The distribution of various types of the root canals were significantly different amongst various aged groups(P<0.05). Type Ⅳ is the most common type(210/405, 51.8%), and the following groups were typeⅡ(65/405, 16.0%), typeⅠ(55/405, 13.6%)and type Ⅲ(27/405, 6.7%). Along with aging, the percentages of type Ⅰ and type Ⅲ decreased while type Ⅱ increased. However, there were no remarkable changes of type Ⅳ observed. The incidence of double rooted canals in single rooted teeth gradually increased with aging especially in 20-years-old and above groups, e.g. 51.7%(31/60)in group 11-20 years-olds and 83.0%(44/53)in group 21-30 years-olds. However, there was no significant increase observed after the age of 40. The distance between both root canal orifices of double rooted canals became shorter with aging except in groups of 40-years-olds and above. The morphologies of the cross sections in most aged groups were flat shaped(1 020/2 105, 48.5%)and oval shape(594/2 105, 28.2%). Along with aging, the percentage of circular shape gradually increased while flat and oval shapes decreased. CONCLUSIONS: The morphology of root canal could be clearly showed by the CBCT images. The change of morphologies of the root canals in maxillary first premolars was significantly related to aging. Along with aging, the morphology of the root canal became more and more complicated.

Determination of intrinsic association constant of mouse monoclonal IgM antibody to human hepatitis B surface antigen.

The ultracentrifugation technique utilizing Airfuge was employed to determine the intrinsic association constant of mouse monoclonal IgM antibodies to human hepatitis B surface antigen (HBsAg). The IgM antibody was reduced to Fab fragments by tryptic digestion in the presence of mercaptoethanol, followed by two passages over a Sephacryl S-200 column. The centrifugation conditions were optimized to sediment Fab-HBsAg complexes, leaving the unbound Fab in the supernatant. The analyses of the binding data by Scatchard and Sips methods yielded good agreement, and the intrinsic association constants ranged from 0.13 to 5.3 X 10(7) (l/mol) for three IgM antibodies. The heterogeneity indices deduced from the Sips analysis were 1.0. The present protocol would allow determination of the intrinsic association constant for the binding of a multivalent antibody to a comparatively large antigen bearing multiple combining sites.

Regulation of tyrosine and phenylalanine biosynthesis in Salmonella.

Several types of 4-fluorophenylalanine resistant mutants were isolated. In one type of mutant DAHP synthetase (tyr) and prephenate dehydrogenase were coordinately derepressed. The mutation was linked to aroF and tyrA and was cis- dominant by merodiploid analysis, thus confirming that it is an operator constitutive mutation (tyrOc). A second type of mutation showed highly elevated levels of tyrosine pathway enzymes which were not repressed by L-tyrosine. It was unlinked to tyrA and aroF, and was trans-recessive in merodiploids. These properties were attributed to a mutation in a regulator gene, tyrR (linked to pyr F), that resulted in altered or non-functional aporepressor. Hence tyrO, tyrA, and aroF constitute an operon regulated by tyrR. In a third type of mutation chorismate mutase P-prephenate dehydratase was highly elevated. It was not linked to pheA, was located in the 95--100 min region of the Salmonella chromosome, and was recessive to the wild type gene in merodiploids. A mutation was, therefore, indicated in a regulatory gene, pheR, which specified an aporepressor for regulating pheA. DAHP synthetase (phe), specified by aroG, was not regulated by pheR, but was derepressed in one of the tyrR mutants, suggesting that as in Escherichia coli tyrR may regulate DAHP synthetase(phe) and DAHP synthetase (tyr) with the same aporepressor. A novel mutation in chorismate mutase is described.