Objective: To clone the 5＇-flanking region of the human CD34 gene containing transcriptional regulatory sequence (TRS). Methods: According to the registered 5＇-flanking region of CD34 gene, two pairs of primers were designed and net-PCR was used to amplify 661 bp long TRS of CD34 gene. The CD34 TRS fragment was cloned into reported plasmid pEGFP-1. The role of the regulating the specific expression of recombinant plasmid pCD34 EGFP in hematopoietic and non-hematopoietic cells was observed. Results: Restrictive endonuclease identification and DNA sequencing provedthat the CD34 promoter cloned was consistent with the sequence reported to a large extent. It could induce the EGFP gene to express in hematopoietic cell line K562 specifically, while has no effect on hepatocellular carcinoma cell HepG-2. Conclusion: The cloned CD34 gene TRS has the effect of regulating gene expression specifically. The study established the fundament for the construction of specific gene expression vector used in hematopoietic system cells.

Objective To investigate the effects of glycyrrhizin on gene expression of transforming growth factor(TGF)-?signaling pathway of hepatic stellate cell(HSC)in mice by gene microarray tech- nique.Methods The HSCs were isolated from mice and cultured in vitro.Then the mice were divided into control group,TGF-?_1 group(5 ng/ml) or TGF-?_1(5 ng/ml)combined with glycyrrhizin(100?mol/L) group.The cells were collected after 10 hours to extract RNA.A cDNA microarray(GEArray~(TM) Q) targeting TGF-?/BMP signal transduction was used to screen the genes which showed significant changes in expression of TGF-?pathway of HSC by glycyrrhizin.Results The microarray analysis showed that 16 genes(16.7%),such as Smad2,Smad3,Smad7,CoL3A1,CoL1A2,and PAI-1,were upregulated by TGF-?_1 and then down-regulated by glycyrrhizin.Five genes(5.2%)(including BMP7,IGFbp3 and etc.)downregulated by TGF-?_1,were then up-regulated by glycyrrhizin.Finally,2 genes upregulated by TGF-?_1 were then up-regulated predominantly by glycyrrhizin in HSC(T?R2,betaglycan).Changes in some genes,such as Smad2 Smad3,Smad7,were further confirmed to be coincided with cDNA microarray by semi-quantitative RT-PCR.Conclusions The anti-fibrosis mechanisms of glycyrrhizin may be through to interference of TGF-?signaling pathway,decrease the synthesis and increase the degradation of collagen.

Bacillus subtilis ZC-7 was obtained by implantation with N+ ions beam to B.subtilis AS1.398,and compared with the AS1.398 neutral protease,the enzyme activity of ZC-7 neutral protease was about 1 timeshigher in previous research.A neutral protease was purified from the culture of B.Subtilis ZC-7 by the procedures including amoninium sulfate precipitation,ultrafiltration,DEAE-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography.By multi-step purification,the ZC-7 neutral protease was purified to 78.5 folds and its yield was 27.7%,at last,the specific activity of ZC-7 neutral protease was up to 4.1?105U/mg.Analysed by SDS-PAGE,the purified protease has shown a molecular mass of about 42kDa.The Km for casein hydrolysis was 3.67?10-3?g/ml and the Vmax was 12.21?g/min.The optimum pH and temperature forhydrolysis of casein were 7.0 and 55℃,respectively.This protease was stable up to 40℃ within the pH range of 6.5 and 8.0.EDTA,isopropanol and alcohol nearly inhibited its activity while some ions such as Ca2+,Mg2+,Fe3+ can improve its activity.In addition,it could resist 1 mol/L H2O2.

Objective:Aldose reductase,involved in the pathogenesis of diabetic complications,was recombinated with an adeno associated virus vector pSNAV2.0,and it was transfected into human embryonic kidney 293(HEK 293)cells.The gene engineering produced AR would be used as a target protein to screen aldose reductase inhibitors.Restriction endonuclease digestion and ligation procedures were performed to construct the AR expression plasmid vector pSNAV-hAR.Methods:After confirmation the recombinant plasmid by PCR,restriction endonuclease digestion,and DNA sequencing,pSNAV-hAR was transfected into HEK293 cells.Western blot and immunofluorescence analysis were performed to detect the expression of AR and its enzyme activity.Results:The results of a series of analysis including AR activity assay,Western blot and immunofluorescence analysis shown the expressed protein mediated by the adeno associated virus vector transfecting HEK 293 cells,was functional AR.The traditional aldose reductase inhibitors,Sobinil and Zopolrestat,were used to test and verify the constructed cell model.Conclusion:The established AR expression model can be used in mechanismresearch of activation of polyol pathway on diabetic complications and screening potential aldose reductase inhibitors.

Objective To observe the changes of image quality after adjusting the space location and the scan parameters of MRS.Methods 65 patients were scanned with magnetism resonance spectrum head examination.The location,size and other scan parameters of the region interested were changed intentionally.The quality of the images was preliminarily evaluated and the relationship between the parameters and the quality of MRS were probed.Results In the 65 cases,60 were with better shape of wave lines and metabolite peak positions,5 with aberrant lines and shifted peaks.Among the recorded data: the range of half high width value is 5~23,water suppression level 92~99.With the 60-case group,all those half high width values were within the given range of the system,on the contrast,within the 5-case group half high width values were 7,9,16 and 23,and water suppression levels were 95,96,97.Conclusion High-quality wave spectrum depends on high uniformity of magnetic field.The value of half high width is an important factor that reflects the uniformity of magnetic field.

The aim of this study was to observe and compare the endogenous circadian rhythm and photoresponse of Clock gene transcription in the suprachiasmatic nucleus (SCN) and pineal gland (PG) of rats. With free access to food and water in special darkrooms, Sprague-Dawley rats were housed under the light regime of constant darkness (DD) for 8 weeks (n=36) or 12 hour-light: 12 hour-dark cycle (LD) for 4 weeks (n=36), respectively. Then, their SCN and PG were dissected out every 4 h in a circadian day, 6 rats at each time (n=6). All animal treatments and sampling during the dark phases were conducted under red dim light (<0.1 lux). The total RNA was extracted from each sample and the semi-quantitative RT-PCR was used to determine the temporal mRNA changes of Clock gene in the SCN and PG at different circadian times (CT) or zeitgeber times (ZT). The grayness ratio of Clock/H3.3 bands was served as the relative estimation of Clock gene expression. The experimental data were analyzed by the Cosine method and the Clock Lab software to fit original results measured at 6 time points and to simulate a circadian rhythmic curve which was then examined for statistical difference by the amplitude F test. The main results are as follows: (1) The mRNA levels of Clock gene in the SCN under DD regime displayed the circadian oscillation (P<0.05). The endogenous rhythmic profiles of Clock gene transcription in the PG were similar to those in the SCN (P>0.05) throughout the day with the peak at the subjective night (CT15 in the SCN or CT18 in the PG) and the trough during the subjective day (CT3 in the SCN or CT6 in the PG). (2) Clock gene transcription in the SCN under LD cycle also showed the circadian oscillation (P<0.05), and the rhythmic profile was anti-phasic to that under DD condition (P<0.05). The amplitude and the mRNA level at the peak of Clock gene transcription in the SCN under LD were significantly increased compared with that under DD (P<0.05), while the value of corresponding rhythmic parameters in the PG under LD were remarkably decreased (P<0.05). (3) Under LD cycle, the circadian profiles of Clock gene transcription induced by light in the PG were quite different from those in the SCN (P<0.05). Their Clock transcription rhythms were anti-phasic, i.e., showing peaks at the light phase ZT10 in the SCN or at the dark time ZT17 in the PG and troughs during the dark time ZT22 in the SCN or during the light phase ZT5 in the PG. The findings of the present study indicate a synchronous endogenous nature of the Clock gene circadian transcriptions in the SCN and PG, and different roles of light regime in modulating the circadian transcriptions of Clock gene in these two central nuclei.
Animals ; CLOCK Proteins ; genetics ; Circadian Rhythm ; physiology ; Male ; Photoreceptor Cells, Vertebrate ; physiology ; Pineal Gland ; physiology ; Rats ; Rats, Sprague-Dawley ; Suprachiasmatic Nucleus ; physiology ; Transcription, Genetic

OBJECTIVETo evaluate the value of magnetic resonance imaging (MRI) in displaying the parotid gland segments of the facial nerve.

METHODSSixteen volunteers (9 males and 7 females) and 132 surgically confirmed patients with parotid tumors locating in the deep or shallow lobe of the parotid gland (including 89 with benign and 43 with malignant tumors) underwent MRI using T1WI and T2WI. The transverse images were obtained with the plane tilted 35 degrees to the foot, and the coronal images were acquired using conventional scanning.

RESULTSOn transverse T1WI, the parotid gland segments of the facial nerve displayed low signal with arc-shaped curve in the cross-section, showing a symmetrical dot-like low signal in the coronal plane. The facial nerve in 63% of the patients with parotid tumors in the cross-section could be displayed, but in the coronal plane the proportion reached 83%. MRI could accurately reveal the position of the parotid tumors in the deep or shallow lobe of the parotid gland.

CONCLUSIONMRI can show the major portion of the parotid gland segments of the facial nerve and has important value in locating the parotid tumors and displaying the relationship between the tumor and facial nerve.

Adolescent ; Adult ; Aged ; Facial Nerve ; pathology ; Female ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Parotid Gland ; innervation ; pathology ; Parotid Neoplasms ; pathology ; Young Adult

OBJECTIVETo investigate the effects of serum containing Gumibao (Chinese character: see text) on the proliferation and differentiation of osteoblast induced by dexamethasone.

METHODSOsteoblasts were extracted from skulls in newly born (within 24 hours) SD rats, and digested with collagenase. The first passage of cells were used for experiments. Cells were cultured in the medium containing different concentrations of dexamethasone (0, 10(-8), 10(-7), 10(-6), 10(-5) ,10(-4) mol/L). Alkaline phosphatase staining were carried out after 1 week and numbers of mineralized nodes with alizarin red staining were observed after 3 weeks. Accordingly, following the treatment of 10(-5) mol/L dexamethasone for 1 week, cells were cultured in the medium with serum containing Gumibao (Chinese character: see text). One week after Cumibao (Chinese character: see text) treatment, cells were stained with Alkaline phosphatase and collagen I and PCNA were examined by Western-blot. However, the observation of numbers of mineralized nodes with alizarin red stain required one more week.

RESULTSHigh concentration of dexamethasone could inhibit the expression of PCNA, collagen I, alkaline phosphatase and reduce the number of mineralized nodes of osteoblast, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.

CONCLUSIONHigh concentration of dexamethasone could inhibit the proliferation and differentiation of osteoblastic cells, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.

Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; analysis ; Dexamethasone ; pharmacology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Female ; Osteoblasts ; cytology ; drug effects ; physiology ; Proliferating Cell Nuclear Antigen ; analysis ; Rats ; Rats, Sprague-Dawley

The selective side-chain cleavage of phytosterol to 4-androstene-3,17-dione(4-AD)and 1,4-androstadiene-3,17-dione(ADD)by Mycobacterium sp.was described.Because of the similarity in chemical structure between 4-AD and ADD,it is difficult to separate them from the fermentation broth.So far,it has been verified that the ADD can be produced by dehydrogenation of 4-AD.In this reaction,3-Ketosteriod-1-Dehydrogenase(ksdD)plays an important role.The gene knocking out method was used to solve the problem.Partial sequence of ksdD was obtained by PCR which was 631bp in length.Then,a targeting vector pUC19-MK was constructed,which was electroporate into the original strain Mycobacterium neoauru.The method of homologous recombination was used to knock out ksdD gene located in the chromosome of Mycobacterium neoauru.In this way,ksdD would lose its enzyme activity.In the result,5 transformants were screened.The experiments of steroid transformation by the transformants were carried out.The productivity of 4-AD reached 17.52% after 144h,which is 192% higher than the original strain.Meanwhile,the productivity of ADD reached 6.12%,which is 89.9% lower than the original strain.

Objective To explore the clinical characteristics of cardiac involvement in children with mitochondriopathies.Methods The clinical data of 23 children with mitochondriopathies were reviewed.The changes of electrocardiography,echocardiography and heart enzymes were analyzed.Results In 15 cases of mitochondrial encephalomyopathy,lactic acidosis,and stroke-like episode(MELAS syndrome),electrocardiography was performed on 9 cases,6 of them showed abnormal electrocardiographic findings,including right bundle branch block,ST-T change,Wolff-Parkinson-White syndrome,et al.On echocardiographic examination in 9 MELAS syndrome ca-ses,only 1 case showed hypertrophy cardiomyopathy.Six cases had increased plasma creatine kinaseMB(CK-MB) mass and only one of 12 MELAS syndrome cases had increased cardiac troponin I(cTnI) level.In 8 cases of subacute necrotizing encephalomyopathy(Leigh syndrome),electrocardiography was performed on 5 cases,4 of them showed abnormal electrocardiographic findings,including sinus tachycardia,ST-T change and low voltage.Two cases showed normal electrocardiography.Three out of 6 cases with Leigh syndrome showed increased plasma CK-MB mass.The molecular genetic examinations were performed in 13 cases of MELAS syndrome and 6 cases of Leigh syndrome.The mitochondrial DNA nt 3243 A→G mutation was found in white blood cells of 9 MELAS syndrome cases,the mutation rate being 37%-60%.The mitochondrial DNA nt 8993 T→C mutation was found in white blood cells of 2 Leigh syndrome cases.Conclusion In children with mitochondriopathies,myocardiac involvement is comparatively common,and even cardiomyopathy can occur.