Four small GTPase genes which may be relative to sclerotial development were firstly cloned from medicinal fungus Polyporus umbellatus using rapid amplification of cDNA end PCR (RACE) method. The results showed that full-length cDNA of PuRhoA was 698 bp contained 585 bp ORF, which was predicted to encode a 194 amino acid protein with a molecular weight of 21.75 kD with an isoelectric point (pI) of 6.44; the full length cDNA of PuRhoA2 was 837 bp in length and encoded a 194 amino acid protein with a molecular weight of 21.75 kD and an isoelectric point (pI) of 6.33; the full length cDNA of Puypt1 was 896 bp in length and encoded a 204-aa protein with a molecular weight of 22.556 kD and an isoelectric point (pI) of 5.75; the full length cDNA of PuRas was 803 bp in length and encoded a 212-aa protein with a molecular weight of 23.821 kD and an isoelectric point (pI) of 5.2. There are fani acyl transferase enzyme catalytic site and myrcene-transferase enzyme catalytic site in PuRhoA1 while the PuRhoA2 only possess myrcene-transferase enzyme catalytic site. Puypt1 contains the Rab1-Ypt1 conserved domain of small GTPase family and PuRas contains the fani acyl transferase enzyme catalytic site. According to the phylogenetic analysis all these four small GTPase clustered with basidiomycete group. Quantitative real-time PCR analysis revealed that Puypt1, PuRas and PuRhoA1 transcripts were significantly higher in the beginning of sclerotial formation than that in the mycelia, whereas the transcripts levels of PuRhoA2 gene were particularly lower in sclerotia than that in mycelia, suggesting that these four genes might be involved in P umbellatus selerotial development.

10.3969/j.issn.1000-8179.2013.12.005

Cytochrome P450 (CYP450) is a key element in the Ganoderma triterpenoid biosynthetic pathway. The catalytic reaction process for CYP450 requires NADPH / NADH for electron transfer. After searching the genome dataset of Ganoderma lucidum, the unique sequence encoding CYP450 and NADPH were discovered, separately. The open reading frames of GLCYP450 and GLNADPH were cloned separately using RT-PCR strategy from G lucidum. The appropriate restriction enzyme cutting sites were introduced at the 5' and 3' ends of gene sequence. The genes of GLCYP450 and GLNADPH were recombined into the yeast expression vector pESC-URA, leading to the formation of the yeast expression plasmid pESC-GLNADPH-GLCYP450. This study provides a foundation for researching Ganoderma triterpene biosynthesis using the approach of synthetic biology.

OBJECTIVE To evaluate theefficacy of the pedicled nasoseptal flap for anterior skull base reconstruction after endoscopic resection of sinonasal malignancies involving the skull base.METHODS From September 2008 to May 2016, 31 patients with sinonasal malignancies involving the skull base were treated via transnasal endoscopic surgery and then two type mucoperiosteal flap of contralateral nasal septum were used to repair the anterior skull base defect according to the actual situation, one is a flap supplied by the posterior nasal septal artery and the other is supplied by the anterior and posterior ethmoidal arteries.RESULTS Successful anterior skull base reconstruction was obtained in all 31 cases. Complications included 3 cases of intracranial infection without hemorrhage or hematoma. In addition, cerebrospinal fluid leakage occurred in one case because of tumor recurrence, and leakage was healed by vertebral draining for one week. Another one had occurred as a result of removing the support form nasal cavity.The follow-up lasted from 3 to 66 months, there were no necrosis of the flap or meningoencephalocele occurred and mucoperiosteal flap healed up well.CONCLUSION The vascularized nasoseptal flap is a reliable and preferred repairing material for anterior skull base reconstruction.

Objective To analyze the clinicopathologic characteristics of atypical polypoid adenomyoma (APA) of endometrium,and investigate the special characteristics of cancerous transformation from APA.Methods Fourteen cases of APA were collected in General Hospital of People' s Liberation Army from January 2007 to March 2013.The clinical data,morphologic features,immunohistochemistry and the related literature were reviewed.Results The median age of the 14 patients was 38 years (ranged from 23 to 72 years),only 1 patient was postmenopausal.The most common symptom was irregular vaginal bleeding (4/14),and 4 patients were identified during routine physical examination for infertility.Among 14 cases,4 cases were diagnosed as well differentiated endometrioid adenocarcinoma originating from APA,and their median age was 35 years (ranged from 28 to 41 years); color Doppler flow imaging (CDFI) of ultrasound showed rich blood flow signal.The tumors with cancerous components were obviously larger than the usual APA (mean diameter:4.7 versus 1.8 cm).Histologically,irregular and branched glands were embedded in fibromuscular stroma and the glandular epithelium were atypical hyperplasia in varying degrees.While carcinoma developed in the APA,the sieve,solid and papillary structures were noticeable,and necrosis were common.Conclusions APA is a rare lesion of the uterus.Although the clinical behavior is benign in most cases,there may be possible for some cases developing carcinomas.If the APA mass is more than 4 cm in diameter,and microscopically demonstrates prominent sieve,solid,papillary structures and necrosis,the diagnosis of carcinoma developed from APA can be made.Thorough analysis should be done before the most proper therapeutic regimen is drawn up.

Objective To assess the feasibility of anticoagulation therapy after mechanical valve replacement in grass-root health institutions.Methods One hundred and sixty one patients with mechanical valve replacement received anticoagulation therapy with warfarin,including 79 cases receiving the therapy in grass-root health institutions (test group) and 82 cases in the tertiary hospitals (control group).The patients were followed up for 12 months after operation;the rate of anticoagulation efficacy,the anticoagulationrelated complications,and the anticoagulation-related cost were documented and compared between two groups.Results The international normalized ratio (INR) tests were performed for 1 021 times in test group and 717 times were up to anticoagulation standard (70.2 ％,717/1 021),while INR tests in control group were performed for 965 times and 688 times were up to standard (71.3％,688/965);there were no significantly differences in efficacy rate between two groups (P ＞ 0.05).There were no significant differences in rate of bleeding events and thrombosis between two groups [16.5％ (13/79) vs.12.2％ (10/82),6.3％(5/79) vs.4.9％(4/82),respectively,x2 =0.596,P=0.44,x2 =0.161,P=0.69].The anticoagulation-related cost per month and per patient in test group was significantly lower than those in control group [(63.1 ±.12.8) vs.(176.6 ± 16.4) yuan,t =48.716,P ＜0.05].Conclusion Compared with the tertiary hospital,the anticoagulation therapy in grass-root institutions can accomplish the similar clinical outcomes and significantly reduce the medical cost in patients with mechanical valve replacement.

Objective To investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on voltage-gated calcium channels on the cell membrane of pheochromocytoma cell 12 (PC 12 cells) induced by ischemic/hypoxia and its mechanisms.Methods PC12 cells at logarithmic phase were collected,which were challenged with hypoxia for 6 hours and reoxygenation for 12 hours to stimulate the nerve cells suffered ischemia/reperfusion pathological process in vitro.PC12 cells were divided into non-infection group,infected by lentivirus containing green fluorescent protein (GFP) without HSP70 gene lentivirus control group (GFP lentivirus control group),and infected by lentivirus containing HSP70 and GFP gene recombined lentiviral infection group (HSP70 recombined lentiviral infection group).Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to determine mRNA and protein expressions of L-type voltage-gated Ca2+ channel subunits cav1.2 and cav1.3,receptor gated channel subunits NR1 and NR2,and Na+/Ca2+ exchange (NCX) in PC12 cells.Results After being challenged with hypoxia/reoxygenation,the mRNA and protein expressions of cav1.2,NR1 and NR2 in the PC12 cells were significantly lower in HSP70 recombined lentiviral infection group than those of GFP lentivirus control group and non-infection group [cav1.2 mRNA (2-△△Ct):3.13 ± 0.46 vs.5.12 ± 0.52,5.13 ± 0.66;NR1 mRNA (2-△△△Ct):1.61 ± 0.44 vs.3.23 ±0.82,3.31 ±0.78;NR2 mRNA (2-△△Ct):2.09±0.41 vs.3.91 ±0.64,3.88±0.62;cav1.2 protein (gray value):2.82±0.39 vs.3.98±0.23,3.96±0.24;NR1 protein (gray value):1.84±0.35 vs.2.79±0.21,2.86±0.23;NR2 protein (gray value):0.87±0.24 vs.1.57±0.31,1.33±0.44;all P ＜ 0.01].But there were no statistical differences in the mRNA and protein expressions of cav1.2,NR1 and NR2 between GFP lentivirus control group and non-infection group (all P ＞ 0.01).There were no statistical differences in the mRNA and protein expressions of cav1.3 and NCX among non-infection group,GFP lentivirus control group and HSP70 recombined lentiviral infection group [cav1.3 mRNA (2-△△Ct):4.82 ± 0.32,4.72 ± 0.36,4.82 ± 0.29;NCX mRNA (2-△△Ct):3.49 ± 0.78,3.47 ± 0.71,3.56 ± 0.65;cav 1.3 protein (gray value):2.63±0.40,2.64±0.39,2.68±0.39;NCX protein (gray value):3.27±0.48,3.34±0.48,3.31 ±0.42;all P ＞ 0.01].Conclusion Exogenous HSP70 affects the expression of L-type voltage-gated Ca2+ channel subunit cav1.2 and receptor gated channel subunits NR1 and NR2,which may protect PC12 cells from the injury caused by ischemic/hypoxia.

Objective To summarize the diagnosis and treatment in neonatal cow’s milk protein allergy (CMPA) with sepsis like initial symptom. Methods CMPA patients with the sepsis like initial symptom (n=10) were selected in our hospital from July 2009 to December 2013. History data, clinical manifestation, laboratory results and the treatment outcome of them were retrospectively analyzed. Results Among these 10 cases, 6 have family history of allergy. Main clinical mani-festations include skin, gastrointestinal symptoms and 1 case of anaphylactic shock. IgE mediated 6 cases with acidophilic cells count of (1.40±0.17)×109/L (5%); The rest 4 cases were not mediated by IgE, with acidophilic cells count of (0.71± 0.08)×109/L (0.02-0.03). Blood cultures were all negative;Blood leukocyte count is (24.5±3.3)×109/L;Rod nucleus granulo-cyte/neutrophils count is (0.161±0.035) ×109/L;The platelet count is (655±39)×109/L;Blood interleukin (IL)-6 is 0.31-0.93μg/L;C reactive protein (CRP) is 85-144 mg/L. All 10 cases were with extensively hydrolyzed formular or amino acid formu-lar feeding. Then their clinical symptoms improved or disappeared significantly and the inflammatory indexes returned to nor-mal. Conclusion It is necessary to make the differential diagnosis between sepsis and neonatal CMPA,which is accompa-nied by increased platelet and acidophil. The most effective treatment of neonatal CMPA is hypoallergenic formular replace-ment therapy.

OBJECTIVETo investigate effects of the variation of immune function in high humidity environment in different time, and lay a foundation for further study of the related mechanism.

METHODThirty SD rats were divided into 3 groups (n = 10): 20 day group, 40 day group in 90% relative humidity chamber and control group in normal relative humidity. Peripheral blood and spleens were collected to detect the levels of T lymphocyte subsets by Flow Cytometery.

RESULTSIn peripheral blood of the 20 day group rats, the CD3+ %, CD4+ %, CD8+ % and CD4+/CD8+ were 52.91 +/- 6.27, 37.80 +/- 4.11, 14.85 +/- 3.73 and 2.72 +/- 0.82 separately. Expect CD3+ %, they all had significant differences (P < 0.05). In addition, the data of the 40 day group rats showed no diversity in statistics. In spleen, CD8+ % of the 20 day group rats was 6.23 +/- 2.87 with significant differences (P < 0.05) and IgG, IgA and IgM did not change a lot in blood serum of the high humidity groups except C3 of the 20 days group (P < 0.05).

CONCLUSIONIn high humidity environment, the immune function of the rats increased in the initial stage. As time went on, the immune function gradually went to normal level through the self adjustment.

Four small GTPase genes which may be relative to sclerotial development were firstly cloned from medicinal fungus Polyporus umbellatus using rapid amplification of cDNA end PCR (RACE) method. The results showed that full-length cDNA of PuRhoA was 698 bp contained 585 bp ORF, which was predicted to encode a 194 amino acid protein with a molecular weight of 21.75 kD with an isoelectric point (pI) of 6.44; the full length cDNA of PuRhoA2 was 837 bp in length and encoded a 194 amino acid protein with a molecular weight of 21.75 kD and an isoelectric point (pI) of 6.33; the full length cDNA of Puypt1 was 896 bp in length and encoded a 204-aa protein with a molecular weight of 22.556 kD and an isoelectric point (pI) of 5.75; the full length cDNA of PuRas was 803 bp in length and encoded a 212-aa protein with a molecular weight of 23.821 kD and an isoelectric point (pI) of 5.2. There are fani acyl transferase enzyme catalytic site and myrcene-transferase enzyme catalytic site in PuRhoA1 while the PuRhoA2 only possess myrcene-transferase enzyme catalytic site. Puypt1 contains the Rab1-Ypt1 conserved domain of small GTPase family and PuRas contains the fani acyl transferase enzyme catalytic site. According to the phylogenetic analysis all these four small GTPase clustered with basidiomycete group. Quantitative real-time PCR analysis revealed that Puypt1, PuRas and PuRhoA1 transcripts were significantly higher in the beginning of sclerotial formation than that in the mycelia, whereas the transcripts levels of PuRhoA2 gene were particularly lower in sclerotia than that in mycelia, suggesting that these four genes might be involved in P umbellatus selerotial development.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; Fungal Proteins ; genetics ; GTP Phosphohydrolases ; genetics ; Genes, Fungal ; Mycelium ; Phylogeny ; Polyporus ; enzymology ; genetics ; Real-Time Polymerase Chain Reaction