In this assay, the reaction kinetics, optimum temperature, pH and various influential factors of ATP microbial rapid detection reagent by bioluminescence were studied. The results showed that it's enough for detection system to have 40 ~ 50?g/mL D-Luciferin. The light production decreased fastest in the first minute of reaction, then began to decay slowly. The optimal reaction temperature was 24℃~25℃and the optimal pH was pH 7.2 -7.4 in the reaction system. In addition, when stored at 4℃for 45h, the dissolved reagent solution could keep its 86% activity. When preserved at 25℃, the enzyme activity decreased less for 1h, and degraded gradually as time went by and only left 53. 5% of its activity after 6. 5h. While stored at 33℃, the enzyme activity decreased quickly with the time and only left 59. 1% after 1. 5h. The result indicated that storage temperature was a very important influential factor to the activity of reagent Meanwhile, different chemical substance such as acid, alkali, salt and surfactants inhibited the ATP bioluminescent reaction. When the concentration of NaCl reached 1. 5g/L, it could inhibit 52. 5% light production. Triton X-100, acid, and alkali also had some effects on the reaction, while CTAB, SDS and TCA would inhibit the bioluminescent reaction seriously.

Human tissue kallikrein-binding protein (Kallistatin, KAL), a secretory protein that participates in the regulation of multiple signaling pathways by binding to the extracellular receptor, however, at present has not been reported about the intracellular activity, and whether it has the similar biological activity with extracellular activity. Here we constructed no signal peptide KAL (NSK) into the adeno-associated virus vector to explore the intracellular activity of KAL. Both the endothelial cell and lung cancer cells could express KAL, but not secreted after rAAV2-NSK transfection. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) were inhibited, but the apoptosis rate was not affected. The proliferation rates, mobility and tubule formation of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited to different extents. This cellular study not only confirmed the intracellular activity, but also suggested it may serve as a kind of "balance factor" in multi-targeted controlling, which may provide a new train of thoughts to explain the regulatory contradiction in PI3K-Akt signaling pathways by KAL.
Apoptosis ; Cell Proliferation ; Dependovirus ; Genetic Vectors ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; Serpins ; metabolism ; Signal Transduction ; Transfection

OBJECTIVE: To study the morphological characteristics of femurs of adult human and 11 kinds of adult animals from cattle, horses, pigs, goats, sheep, dogs, cats, rabbits, geese, ducks, chickens, and to establish an effective species identification method among various species. METHODS: The 4 cm mid-diaphyseal segment of the femur from adult human (older than 20 years old) at autopsy was obtained. Addi-tionally, the 4 cm ones from 11 kinds of adult animals were obtained. After decalcification, all femurs were made into slices, and then were observed by optical microscope. The 25 indexes were selected and analyzed by step discriminant analysis according to differences between human and mammal, human and poultry, and human and 11 kinds of animals. RESULTS: The histological structure of bone mineral density of middle part of femur had obvious characteristics among the species. And the morphology and number of osteon showed the trend of obvious biological evolution. There were 11 indexes with significant differences between human and 11 kinds of animals to establish some mathematical models to discriminate all species. The correct discrimination rate was 96.3% between human and mammal. The correct discrimination rate was up to 100% between human and poultry, and was 89.4% among human, mammal and poultry. CONCLUSION The mathematical models have good correct discrimination rate among human and the other animals, which could be applied in the practical species identification cases.
Adult ; Animals ; Autopsy ; Bone Density ; Cadaver ; Cats ; Cattle ; Chickens ; Discriminant Analysis ; Dogs ; Femur/ultrastructure* ; Forensic Anthropology ; Haversian System/ultrastructure* ; Horses ; Humans ; Sheep ; Species Specificity ; Swine

OBJECTIVETo explore the influence of exogenous putrescine and cadaverine on pro-inflammatory factors in the peripheral blood of rabbits.

METHODSForty ordinary adult New Zealand rabbits were divided into saline, necrotic tissue homogenate (NTH), putrescine, and cadaverine groups according to the random number table, with 10 rabbits in each group. Saline, NTH, 10 g/L putrescine, and 10 g/L cadaverine were respectively peritoneally injected into rabbits of corresponding group in the amount of 1 mL/kg. The blood sample in the volume of 2 mL was collected from the central artery of rabbit ears before injection and at 2, 6, 12, 24, 30, 36, 48, 60 hours post injection (PIH). Contents of TNF-α, IL-1, and IL-6 in the serum were determined with enzyme-linked immunosorbent assay. Data were processed with repeated measurement data analysis of variance and Spearman correlation analysis, and cubic model curve was applied in curve fitting for the contents of inflammatory factors.

RESULTS(1) The serum contents of TNF-α, IL-1, and IL-6 were increased in NTH, putrescine, and cadaverine groups in different degrees at most post injection time points. There was no significant change in the concentrations of the three pro-inflammatory factors in saline group, and they were significantly lower than those of the other three groups at most post injection time points (with F values from 3.49 to 13.58, P values all below 0.05). The serum contents of TNF-α, IL-1, and IL-6 in putrescine group began to increase at PIH 2, 6, and 6, which was similar to the trend of NTH group, but the changes were delayed compared with those of cadaverine group(all at PIH 2). The peak values of TNF-α, IL-1, and IL-6 in putrescine group were respectively (339 ± 36), (518 ± 44), and (265.9 ± 33.5) pg/mL, which were significantly lower than those of cadaverine group [ (476 ± 86), (539 ± 22), and (309.4 ± 27.1) pg/mL], with F values respectively 5.11, 1.90, and 5.56, P values all below 0.05. (2) The period of time in which contents of TNF-α, IL-1, and IL-6 began to increase (PIH 3-4) and the peaking time of the three pro-inflammatory cytokines (PIH 18-30) in putrescine group appeared later than those of cadaverine group (PIH 2 and 12-30). The duration of peaking time of the three pro-inflammatory cytokines in putrescine group was shorter than that of cadaverine group (PIH 18-30 vs. PIH 12-30). The increasing period and the duration of peaking time of TNF-α, IL-1, and IL-6 in putrescine group were close to those of NTH group (PIH 3-5 and 18-30). The correlation coefficient test analysis showed that the trends of changes in contents of three pro-inflammatory cytokines in putrescine group were significantly correlated with those of NTH group (r(TNF-α) = 0.933, P < 0.01; r(IL-1) = 0.967, P < 0.01; r(IL-6) = 0.950, P < 0.01). The obvious correlation between cadaverine group and NTH group was only found in the contents of IL-1 and IL-6 (r(IL-1) = 0.913, P < 0.01; r(IL-6) = 0.883, P < 0.05).

CONCLUSIONSBoth exogenous putrescine and cadaverine can cause inflammatory reaction in rabbits. The trend of the inflammatory reaction induced by putrescine was similar with that by NTH, suggesting that putrescine may play a leading role in the inflammatory reaction induced by necrotic tissue decomposition.

Animals ; Cadaverine ; adverse effects ; Inflammation ; blood ; Interleukin-1 ; blood ; Interleukin-6 ; blood ; Necrosis ; blood ; Putrescine ; adverse effects ; Rabbits ; Tumor Necrosis Factor-alpha ; blood

This study was aimed to develop and establish an efficient method for high through-put automatically extracting genomic DNA from EDTA-anticoagulated whole blood samples, and to utilize this method in routine rSSO HLA genotyping by luminex flow array assay, the genomic DNA was extracted automatically from 400 microl blood samples by using TECAN DNA workstation and 96-well plate with 2 ml volume per well. The yield and purity of each DNA sample was tested by UV-spectrophotometer, the integrity of these DNA samples were run electrophoresis on the agarose gel. Each DNA sample was subjected to PCR amplification and hybridization using One lambda rSSO HLA-A, -B and -DRB1 commercial kit, the fluorescent intensity for positive bead and negative bead hybridized with HLA-A, -B and -DRB1 PCR products were calculated and analyzed. The results showed that the mean yield and purity (A260/A280) of genomic DNA extracted from 400 microl whole blood samples were 3.217+/-0.715 microg and 1.710+/-0.103 respectively. The molecular weight was more than 15 kb in size and the fluorescent intensity for positive bead hybridized with HLA-A, -B and -DRB1 PCR products of each sample was >600 RFU, however, the fluorescent intensity for negative bead for each sample was <50 RFU. It is concluded that the highly qualified genomic DNA can be extracted automatically from blood samples of marrow-donors by using TECAN DNA workstation, and the extracted DNA samples are suitable for high through-put HLA genotyping by luminex flow array assay and other downstream transplant immunological and molecular biological experiments.
Biological Specimen Banks ; Bone Marrow ; DNA ; isolation & purification ; DNA Primers ; Genotype ; HLA Antigens ; genetics ; High-Throughput Screening Assays ; methods ; Humans ; Living Donors ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; methods

Objective To introduce an optimized practical method of making and detecting pipettes for microinjection.Methods Transfer pipette was made from hard glass capillary. We softened the hard glass capillary by rotating it in a spirit-lamp flame,then moved out from the flame and quickly pulled it into two transfer pipettes.After broken by a grinding wheel,the tip of the pipette was fire-polished by quickly touching the flame to make a fine opening.A hard glass capillary (1.0 mm,ouside diametre)was pulled into two holding pipettes by pipette Puller.The pipette shoulder was broken at desired position with a grinding wheel,then the fine pipette tip opening was heated by a microforge and shrinked into a diameter -15 μm.Injection pipette could be made directly from a capillary with filament by Puller.The solution loaded injection pipette and holding pipette were assembled into the micromanipulator and could be checked before use.We transfered both pipettes into the zygotes media drop,touched the holding pipette with the tip of the injection pipette to make a "suitable"opening.Then we switched injection pipette to the mineral oil and applied injection pressure through the injector to check whether the solution could come out of the tip smoothly and at a proper speed.It could be further verified by pronucleus microinjection of zygotes.Results The results showed that the method introduced in this paper could produce suitable pipettes for zygote microinjection.In particular,the method of detecting the opening of the injection pipette was helpful for achieving high efficiency of zygote microinjection.Conclusion The method introduced here to make and detect pipettes for microinjection is very helpful for establishing a standard microinjection manipulation procedure and improving the efficiency of zygote microinjection.

OBJECTIVETo study the clinical and epidemiologic characteristics and the serum types of enterovirus of hand, foot and mouth disease (HFMD) in children.

METHODSThe RT-nPCR method was established with universal primers within 5' untranslated region of enterovirus and VP1 region of Coxsackievirus A16 (CAV16) and enterovirus 71 (EV 71). Enteroviruses were detected with RT-nPCR in 237 children with HFMD. Clinical and epidemiologic characteristics and serum types of enterovirus of the patients with HFMD were studied.

RESULTSThe patients'age ranged from 7 months to 11 years (mean 4.2 +/- 0.5 years). The majority (94.5%) were less than 6 years old. HFMD was mostly seen in spring and winter (67.9%). Oral mucosal pox or ulcer as well as hand and foot rashes were observed in all 237 patients. Fever occurred in 141 patients (59.5%). Of the 237 patients, 133 (56.1%) were RT-nPCR positive. Of the 133 cases, 38 were positive for EV71, 64 were positive for CAV16, and 31 were negative for both EV71 and CAV16. The patients infected by different types of enteroviruses had similar clinical characteristics. Gene colon and sequence analysis for 12 strains of enteroviruses PCR positive products presented as EV71 (n=5), CAV16 (n=5), ECHO13 (n=1), and CAV5 (n=1).

CONCLUSIONSHFMD tends to occur in younger children less than 6 years old. The majority are affected in spring and winter. EV71 and CAV16 are common pathogens of HFMD. There is no relationship between clinical characteristics and serum types of enteroviruses in HFMD patients.

Child ; Child, Preschool ; Enterovirus ; classification ; Female ; Hand, Foot and Mouth Disease ; virology ; Humans ; Infant ; Male ; Reverse Transcriptase Polymerase Chain Reaction ; Seasons ; Serotyping

OBJECTIVETo elucidate the roles of Toll-like receptor 3 (TLR3) on dendritic cells (DCs) in HBV infection.

METHODSPeripheral blood mononuclear cells (PBMCs) were isolated from 48 healthy volunteers (HV) and 50 chronically HBV-infected patients (CH). DCs were induced and proliferated in a culture medium with rhGM-CSF and rhIL-4. We stimulated DCs with poly I:C and then TLR3, HLA-DR, and CD86, and CD1a expressions were examined by flow cytometry at 0 h, 12 h, 24 h and 48 h. The mRNA expressions of TLR3 were quantified by real-time PCR.

RESULTSTLR3 expression on DCs before the poly I:C stimulation and afterwards on the 12 h, 24 h, and 48 h were 69.2%+/-20.4%, 76.0%+/-18.6%, 78.2%+/-19.5% and 85.5%+/-6.9% respectively in the CH group, and 70.8%+/-11.2%, 67.5%+/-20.9%, 86.3%+/-14.7%, 68.6%+/-16.9% in the HV group. The expressions of TLR3 were up-regulated significantly at 24 h and 48 h after stimulation with poly I:C in the HV group, and in the CH group they were not significantly increased at 24 h but obviously increased at 48 h. The mRNA expressions of TLR3 increased significantly at 12 h in the HV groups, and at 48 h in CH group. The rate of CD86 expressions increased after poly I:C stimulation, and the increased rates were 12.6%+/-9.8%, 23.8%+/-20.0%, 20.7%+/-14.3% in the CH group, and 31.0%+/-25.0%, 43.4%+/-24.7%, 44.6%+/-25.5% in the HV group at 12 h, 24 h and 48 h after poly I:C stimulation. There was a marked increase of the expression level of CD86 in the HV group. In contrast, the level was only slightly increased in the CH group (31.0% vs 12.6%). The differences between the two groups were significant at 24 h and 48 h. No significant differences were detected in HLA-DR and CD1a between the two groups.

CONCLUSIONSThe increase of expression level of TLR3 is slower in the CH group than that in the HV group. A marked increase of the expression level of CD86 is observed in the HV group. Our results suggest that abnormal expression of TLR3 and CD86 may relate to the persistence of HBV infection.

Adult ; B7-2 Antigen ; metabolism ; Dendritic Cells ; immunology ; metabolism ; Female ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; Humans ; Male ; Middle Aged ; Toll-Like Receptor 3 ; metabolism ; Young Adult

The genetic diversity of 279 indivdiuals from 10 populations in Shandong Province was investigated using inter-simple sequence repeat (ISSR) markers. As a result, 116 bands were amplified by 10 informative and reliable primers, of which 101 were polymorphic loci. A relatively high level of genetic diversity was revealed: PPL = 87.07, He = 0.2697, H0 = 0.3999 (at the species level); PPL = 64.58, He = 0.2004, H0 = 0.3010 (at the population level). A higher level of genetic differentiation was detected among populations with Nei's G(ST) analysis and the analysis of molecular variance (AMOVA; G(ST) = 0.2414, F(ST) = 0.2224). Habitat fragmentation and gene flow may result in genetic differentiation. UPGMA cluster analysis indicated that the four populations from Linshu, Junan, Tancheng and Feixian grouped together, whereas Laiyang populations clustered in an isolated clade. The results showed that a mixed mating system was possibly the main factor influencing the genetic structure of this species. These results, combined with other information about Castanea mollissima, may provide a valuable basis for proposing conservation strategies.
China ; Fagaceae ; genetics ; Genetic Markers ; genetics ; Genetic Variation ; Genetics, Population ; Microsatellite Repeats

OBJECTIVETo compare therapeutic effects between moxibustion and infrared therapy for the treatment of knee osteoarthritis.

METHODSFrom January 2007 to June 2008 period, 65 patients with knee osteoarthritis were divided into treatment and control groups randomly uniform random number table generated from SAS statistical software. Among 35 patients in the treatment group, 17 patients were male and 18 patients were female, ranging in age from 45 to 75 years, with an average of (61.2+/-6.4) years; the course of disease ranged from 9 to 43 months, with a mean of (23.6+/-13.8) months; the preoperative Lysholm score ranged from 19 to 28 scores, averaged (24.3+/-3.3) scores. In the control group, there were 30 patients, including 13 males and 17 females, ranging in age from 47 to 79 years, with an average of (62.5+/-9.3) years; the course of disease ranged from 8 to 45 months, with a mean of (24.6+/-16.6) months; the preoperative Lysholm score ranged from 20 to 29 scores, averaged (25.9+/-3.0) scores. The patients in the treatment group were treated with moxibustion, and the patients in control group were treated with infrared therapy. All the patients were followed up for 4 weeks. The Lysholm scores were compared between the two groups.

RESULTSAccording to Lysholm score for clinical efficacy, treatment group got (87.5+/-5.6) scores and the control group were (85.9+/-3.5) scores, the Lysholm score of the treatment group was higher than that of the control group (P<0.05). Among pain score, joint flexion and extension score, joint stability score, and up and down stairs score, the pain and joint stability scores of patients in the treatment group were higher than those of control group (P<0.05).

CONCLUSIONCompared with infrared therapy, moxibustion treatment for knee osteoarthritis can get better joint function, which is effect to alleviate the patient's pain, improve joint stability, improve the efficacy, and is valued to be promoted.

Aged ; Female ; Humans ; Male ; Middle Aged ; Moxibustion ; methods ; Multicenter Studies as Topic ; Osteoarthritis, Knee ; therapy ; Treatment Outcome