2,5-DKG reductase I was purified from cell-free extracts of a recombinant,ER97 by a procedure involving ammonium sulfate precipitation and successive column chromatography on DEAE-Sepharose CL-6B and Phenyl Sepharose CL-4B with 5 fold purification,27 % recovery and 3418 U/mg specific activity.The molecular weight of the enzyme estimated by SDS-PAGE was 34kD.The isoelectric point was estimated to be 6.0 by PAG-IEF.The optimum pH was 7.0 and the optimum temperature was about 40℃.The enzyme can catalyze the stereospecific NADPH-dependent reduction of 25-DKG to 2-KLG.The michaelis-menten constant(Km) for 2,5-DKG and NADPH were 0.29 mmol/L and 14.7 mmol/L respectively.The enzyme is specific for NADPH and 2,5-DKG,1 mmol/L Cu~(2+) or Zn~(2+) could highly inhibited the enzyme activity.EDTA and ?-Mercaptoethanol have no effect on the enzyme activity.

Because of the neonatal immune system is immature,particularly in preterm and low birth weight infants,the neonate is prone to some infections.Neonatal sepsis usually has the slight performance and atypical clinical symptoms,which raise the great interest of searching for some better biomarkers.With the development of testing technology,the research of serum inflammation marker has made remarkable progress at home and abroad in recent years,however,the research of umbilical blood inflammation marker is still in the early stage of exploration.Umbilical blood detection have the advantages of early,noninvasive,safe and convenient compare to the traditional serum detection.This paper mainly discusses the application of umbilical blood detection to early onset neonatal sepsis.

Objective: To analyze the constituents and metabolites of Radix Sa poshnikoviae (RS) in rat plasma and urine by rapid-resolution liquid chromatography-time of flight mass spectrometry (RRLC-TOF/MS), so as to explore the active ingredients and metabolites of RS in vivo. Methods: The separation was performed on a Angilent Zorbax Extend-C14 (5 μm, 250 mm x 4.6 mm id) column, with a methanol-water mobile phase system used for gradient elution. Time-of-flight mass spectrometer (TOF/MS) was applied for qualitative analysis under positive ion mode. Based on the accurate molecular weight of TOF/MS detection and the compound list of RS established previously, the constituents and metabolites of RS in different matrix in vivo were identified. Results: Six constituents of RS were identified in the plasma, sucrose, prim-O-glucosylcimifugin, cimifugin, nodakenetin, 5-O-methylvisamminol, and 3′-O-i-butyrylhammaudol. Eight constituents were identified in the urine, prim-O-glucosylcimifugin, divaricatacid, cimifugin, 4′-O-glucosyl-5-O- methylvisamminol, (3S)-2,2-dimethyl-3, 5-dihydroxy-8-hydroxymethyl-3, 4-dihydro-2H, 6H-benzo-[1, 2-b: 5, 4-b′] dipyran-6-one, 5-O-methylvisamminol, see-O-β-D-glucosylhammaudol, and wogonin. Two metabolites were identified in the urine, glucuronide of cimifujin and an isomer of it. Conclusion: The present method is reliable and effective for identifying compounds of RS in vivo, and it can provide a reference and evidence for the further pharmacodynamics experiments.

Humans ; Tuberculosis, Hepatic

Diagnostic Errors ; Dyspnea ; diagnosis ; Female ; Humans ; Infant ; Male

BACKGROUND: Clusterin has many biological functions, especially the great protection effects on histiocytes in pathological conditions. It will be helpful to discuss the expression of clusterin in the spinal cord injury tissue to further identify the mechanism of secondary spinal cord injury and to provide possible treatments.OBJECTIVE: To observe the expression of clusterin in acute spinal cord injury tissue.DESIGN: Randomized controlled animal study.SETTING: Department of Orthopaedics, First Affiliated Hospital, China Medical University.MATERIALS: The experiment was performed in Experimental Animal Department, China Medical University from January 2003 to January 2006.A total of 65 adult healthy SD rats of 250-300 g provided by Experimental Animal Department, China Medical University were selected. METHODS: All the rat models by modified Allen assay for acute spinal cord injury were randomized into injury group, sham operation group and normal control group with 30, 30 and 5 rats in each group, respectively.The rat injury models in dorsal spinal cord were prepared by modified Allen assay: the target injury site was T10 segment; the sham operation group was given T10 total laminectomy. The normal control group received no operation. Rats in the injury group and sham operation group were killed at hour 12, days 1, 3, 7, 14 and 21 after spinal injury and operation with 5 rats at each time point, respectively. Frozen sections of the injury part were made at different time point after injury. Degeneration and necrosis of spinal cord injury tissues were observed with hematoxylin-eosin (HE) staining. Deposition and disposition of positive reactants of clusterin were observed with clusterin immunohistochemical staining. Average gray value of positive reactants of clusterin was detected with semi-quantitative image analysis (went with the intensity of clusterin immunoreaction in inverse ratio).MAIN OUTCOME MEASURES: ①Degeneration and necrosis as well as positive expression of clusterin of spinal cord injury tissues of rats in each group. ②Average gray value of clusterin positive reactants in spinal cord injury tissues of rats in each group.RESULTS: A total of 65 rats were involved in the result analysis, without dropout. ①In the injury group, positive expression of clusterin began to increase at day 1 after injury, reached peak at about day 7, declined gradually at day 14 and tended to be stable at day 21 after injury. This dynamic course of change was along with the prolongation of time. The expression of clusterin appeared in the sham operation group and normal control group at each time point, and the expression was stable. There was no dynamic change with the prolongation of time. ②In the injury group average gray value of positive reactants of clusterin was significantly lower at days 1, 3,7, 14 and 21 as compared with that in the sham operation group and normal control group (t=6.33-15.57, P ＜ 0.01 ). There was no significant difference in average gray value of positive reactants of clusterin at each time point in the sham operation group as compared with the normal control group (P ＞ 0.05).CONCLUSION: The expression of clusterin increases greatly in acute spinal cord injury tissues, and has dynamic change, which is the same with spinal cord injury.

Objective To establish a histological classification system for osteophytes by Masson staining and immunohistochemical staining for collagen type II. Methods Osteophytes were collected from knee joints in os?teoarthritis patients after joint replacement surgery. To develop an osteophyte classification ,serial tissue sections were identified using histological(hematoxylin and eosin,Masson ' s trichrome,toluidine blue)and immunohisto?chemical staining(collagen typeⅡ). Joint function was assessed by WOMAC score and radiological findings was measured by Kellgren?Lawrence grading. The consistency between the osteophyte classification and WOMAC/Kell?gren?Lawrence grading were evaluated. Results 31 patients and 101 osteophyte samples were included in this study. Based on the histological and immunohistochemical evaluation,osteophytes can be divided into type A (4.9%),type B(4.0%),type C(26.7%)and type D(64.4%). This histological classification system had better consistency with joint function score(χ2 = 0.108,P = 0.947)and Kellgren?Lawrence grading(χ2 = 3.205,P =0.201). Conclusions This trial establishes a histological classification system for osteoarthritis osteophytes ,types A and B indicating early osteophyte and types C and D indicating advanced osteophyte. The system is helpful in clini?cal and basic research on osteoarthritis.

Objective To explore the immunogenicity of a novel bionic scaffold of nucleus pulposus tissue engineering. Methods The biocompatibility of a subcutaneously implanted scaffold of nucleus pulposus tissue engineering was studied in SD rats by analyzing tissue reactions up to 3 months using histological and ultrastructural methods. The expression of IFN-?, IL-2, IL-4 and IL-10 mRNA was measured by RT-PCR, and the levels of serum antibodies to porcine type Ⅱ collagen were measured by ELISA. Results There was less inflammatory reaction in rats induced by subcutaneously implanted scaffold. By degrees, a granulation tissue had developed within the implant, which had disappeared by 3 months. The expression of IFN-?, IL-2 mRNA by RT-PCR was of no changes. But the expression of IL-4 and IL-10 mRNA increased, which meant the implant induced Th cell into Th2 cell and the induction inhibited the inflammatory reaction. No antibodies to porcine type Ⅱ collagen were found in the sera of implanted rats. Conclusion There was less immunogenicity in rats induced by implanted scaffold of nucleus pulposus tissue engineering.

Brain injury is a kind of wound by violence on head, which is a mechanical distortion of skull, meninx, cerebral vascular and brain tissue due to outside force acting on head. Apolipoproteins E (ApoE ) is a major kind of apolipoprotein s, participating in the metabolismof lipid and regulating bal-ance of cholesterol. Some recent investigations showthat gene polymorphismof ApoE is associated with various kinds of diseases. Also its immunoreactivity is changed regularly with brain injury. In addition, ApoE has remarkable effect in neurological normal growth and reparative process after brain injury. This article reviews the biological characteristics and mechanismof ApoE in the repair of brain injury and application prospect in forensic medicine, which may be able to provide newideas for estimation of the brain injury time and related experimental research.

Guinea pig as a commonly used laboratory animal is widely used in various fields of biomedical research.The stability of genetic quality directly affects its development and application .Genetic testing is designed to confirm the genetic characteristics of each strain , to verify whether there are genetic mutations and other genetic contamination, to ensure that the test object meets the requirements of this strain .Along with the emerge of biochemical and molecular marker technology , a more convenient and reliable means is provided for research of genetic homozygosity , genetic type detection and genetic quality monitoring of guinea pigs .In this paper, the application and research progress of biochemical, cytological and molecular markers in studies of guinea pig diversity will be summarized , and provide some help for genetic testing guinea pig.