By reverse transcription-polymerase chain reaction (RT-PCR), an open reading frame of pathogenesis-related protein 1 (PR1) was isolated from Panax notoginseng and named as PnPR1. Molecular and bioinformatic analyses of PnPR1 revealed that an open reading frame of 501 bp was predicted to encode a 166-amino acid protein with a deduced molecular mass of 18.1 kD. Homology analysis showed that the deduced amino acid sequence of PR1 protein of Panax notoginseng had a high similarity with other higher plants had the same conservative structure domain of cysteine-rich secretory protein (CAP). The recombinant expressed plasmid pET28a(+)-PnPR1 was expressed in Escherichia coli BL21. The expression conditions were optimized by induction at different times, different temperatures, different IPTG concentrations and different giving times. The optimum expression condition was 0.4 mmol.L-1 IPTG at 28 degrees C for 20 h. The successful expression of PnPR1 provides some basis for protein purification and preparation of the monoclonal antibody.
Amino Acid Sequence ; Cloning, Molecular ; Escherichia coli ; metabolism ; Molecular Weight ; Open Reading Frames ; genetics ; Panax notoginseng ; chemistry ; Phylogeny ; Plant Proteins ; genetics ; metabolism ; Plants, Medicinal ; chemistry ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Alignment

To compare the growth characteristics of non-hematopoietic adult stem cells (NASC) derived from rat fetal blood and rat bone marrow in vitro, and to study the differentiation of these stem cells into neuron-like cells in vitro, the fetal blood of pregnant rats and bone marrow of adult rats were sterilely collected; mononuclear cells (MNC) were isolated by using standard Ficoll-hypague techniques and then cultured in DMEM/LG containing 10% fetal bovine serum (FBS). The acquired NASCs were subcultured for passage. The immunophenotype of NASCs was detected by flow cytometry. The expanded NASCs were induced to differentiate into neurons-like cells by beta-mercaptoethanol (beta-ME), dimethylsulfoxide (DMSO), butylated hydroxyanisole (BHA). The specific markers of these neuron-like cells were detected by immunocytochemistry. The results showed that two kinds of subcultured NASCs showed homogeneous spindle-shaped and expressed antigens CD44 and CD54, but did not expressed CD11b and CD45. The both induced cells were similar to neuron in morphology and were positive for nestin and neuron-specific enolase (NSE), but negative for glial fibrillary acidic protein (GFAP). It is concluded that no significant difference of NASCs derived from pregnant rat fetal blood and adult rat bone marrow found in cell morphology and biological characteristics. NASCs of both origins can be induced to differentiate into neuron-like cells, so fetal blood can be regarded as another resource of NASC.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; physiology ; Female ; Fetal Blood ; cytology ; Male ; Multipotent Stem Cells ; cytology ; physiology ; Neurons ; cytology ; Pregnancy ; Rats ; Rats, Wistar ; Stem Cells ; cytology

OBJECTIVETo describe a HPLC method for assessing betaine in Fufang Guilu granule.

METHODThe content of betainephenaxcyl bromide in Fufang Guilu granule was determined by HPLC. The analytical column was a shim-pack CLC-ODS (6.0 mm x 150 mm) filling a 5 microm stationary phase; The mobile phase consisted of acetonitrile-water(35:65) with 0.1 mol x L(-1) NaClO4; The flow-rate was 1 mL x min (-1); The detector was set at 254 nm.

RESULTThe calibration curve was linear over the range of 0.09-0.585 microg (r = 0.9997). The average recovery of the method was 98.4%, RSD 2.5% (n = 5).

CONCLUSIONThe results showed that this method was reliable and accurate, and can be used for quality control of Fufang Guilu granule.

Betaine ; analysis ; Chromatography, High Pressure Liquid ; methods ; Codonopsis ; chemistry ; Drug Combinations ; Drugs, Chinese Herbal ; chemistry ; Lycium ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Reproducibility of Results

OBJECTIVECord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk.

METHODSCB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo.

RESULTSDifferent results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice.

CONCLUSIONEx vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.

Animals ; Antigens, CD34 ; Cell Culture Techniques ; methods ; Cells, Cultured ; Culture Media ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Megakaryocytes ; cytology ; Mice ; Mice, SCID

ence of precancerous pathological changes.

OBJECTIVETo evaluate the application of different variable number tandem repeats (VNTR) locus in genotyping of Mycobacterium tuberculosis (M.tuberculosis) strains isolated from eight provinces in China, and to find the suitable locus-set of VNTR for epidemical strains in China.

METHODSAll 140 M.tuberculosis strains were randomly selected from 2800 M.tuberculosis strains isolated from eight provinces in China, 27 VNTR loci were used for typing all isolates. Discriminatory power (Hunter-Gaston Index, HGI) of every locus and different locus-set were analyzed by BioNumerics software. Meanwhile, Spoligotyping was used to identify Beijing family and non-Beijing family. Then the HGI of different locus-sets in two families was also evaluated.

RESULTSAll 140 isolates were clustered into Beijing kindred (112 strains, 80%) and non-Beijing kindred (28 strains, 20%) by Spoligotyping. The discriminatory power of Spoligotyping in 140 isolates was 0.4589. Every locus showed different polymorphism and HGI were from 0 to 0.809. The number of VNTR loci with HGI higher than 0.5 in all strains, Beijing family and non-Beijing family was 8, 7 and 14 respectively. 27 loci were combined into four groups which included 8, 12, 15 and 24 VNTR loci respectively. Four locus-sets showed different polymorphism, HGI of eight-locus, 12-locus, 15-locus, and 24-locus set in 140 strains was 0.9991, 0.9882, 0.9980 and 0.9986, and their discriminatory power were calculated in Beijing kindred (HGI: 0.9987, 0.9318, 0.9969 and 0.9975) and non-Beijing kindred (HGI: 1, 0.9894, 1 and 1).

CONCLUSIONDifferent VNTR locus and locus-set showed different discriminatory power in the selected M.tuberculosis strains isolated from China. Eight-locus set can be used in molecular epidemiological study of M.tuberculosis in China after standardization.

Bacterial Typing Techniques ; DNA, Bacterial ; genetics ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Tandem Repeat Sequences

OBJECTIVETo compare the efficacy, tolerance and safety between oral sodium phosphate(NaP) and polyethylene glycol(PEG) on bowel preparation.

METHODSOne hundred and fifteen inpatients were randomly divided into NaP group and PEG group. The questionnaire was designed for scoring by patients and doctors regarding to tolerance, taste, side effects and cleaning degree etc.

RESULTSCompared with PEG group, NaP presented better tolerance, lower side effects and higher rate of adequate cleaning quality(P<0.05). NaP could cause electrolytic alterations, such as hyperphosphatemia, hypernatremia, hypocalcemia and hypopotassemia, but these changes were transient and without clinical significance.

CONCLUSIONSodium phosphate is safe and effective for bowel preparation, and is better than polyethylene glycol in tolerance.

Administration, Oral ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Phosphates ; therapeutic use ; Polyethylene Glycols ; therapeutic use ; Preoperative Care ; methods ; Prospective Studies ; Young Adult

OBJECTIVETo study the changes of the intramembrane protein particles of erythrocyte from Duchenne muscular dystrophy (DMD) patients and the gene carriers and to explore the pathogenesis of DMD and the diagnostic value of erythrocyte freeze-fracture technology.

METHODSThe fixed erythrocyte mass was treated to form replica membrane by means of the freeze-fracture technology. Then the replica membrane was observed and a picture was taken under electron microscope. The protein particles of extracellular face(EF) and protoplasmic face(PF) per square were counted. The statistical comparative analysis was performed.

RESULTSThe protein particle counts of EF face and PF face of erythrocyte membrane from DMD patients and DMD carriers decreased obviously in comparison with the normal control group (P<0.001).

CONCLUSIONThe erythrocyte freeze-fracture electron microscopic technology may serve as a method for accessory examination of diagnosing DMD patients and a method for detecting DMD carriers. This investigation material supplies reliable evidence for the theory of the systemic membrane defect of DMD.

Erythrocyte Membrane ; metabolism ; ultrastructure ; Female ; Heterozygote ; Humans ; Male ; Membrane Proteins ; metabolism ; Microscopy, Electron ; Muscular Dystrophy, Duchenne ; diagnosis ; genetics ; metabolism

Protein kinase C (PKC) is a critical molecule in cellular signal transduction in mammals. It is involved in many biological processes in embryonic development, including nuclear remodeling, cell cycle adjustment and cellular polarity regulation. The present study aimed to observe the location of PKCα, an important isozyme of PKC, in fertilized, parthenogenetic and tetraploid preimplantation embryos, and compare the expression of PKCα during embryonic compaction in Kunming mice. The location of PKCα was detected by immunochemistry and laser confocal microscopy. Western blot was performed to quantify PKCα expression during embryonic compaction in the three kinds of embryos. In the experiment, fertilized embryos were flushed from oviduct or uterus at 45, 52, 69, 76 and 93 h after injection of human chorionic gonadotrophin (hCG); parthenogenetic embryos were collected by SrCl2 activation of oocytes for 6 h; and tetraploid embryos were produced by electrofusion of 2-cell embryos. Embryos were fixed at different developmental stages for immunofluorescent staining. 8-cell/4-cell embryos and morula were lysed for Western blot. The results showed that PKCα had similar location pattern in different embryos. It was distributed mainly in the nuclear aggregating around chromatin at different developmental stages. However, PKCα expressed strongly in the interphase than in mitotic blastomere. Before embryonic compaction, PKCα was localized at the blastomere boundary. At late blastocyst stage of fertilized embryos, PKCα was localized only in the polar trophoblast, but not in other trophoblast. At late stage of pathenogenetic blastocyst, there was no clear PKCα signal in the polar trophoblast. Tetraploid embryos had larger blastomere than other embryos and compacted after 4-cell stage, but not after 8-cell stage. Meanwhile, there was PKCα signal at the blastomere boundary at 4-cell stage. Our results showed that the expression of PKCα lasted through all the preimplantation stage. Although there were different expression levels among different stages, the expression increased around embryonic compaction. Quantification of expression of PKCα by Western blot demonstrated that the expression increased after compaction, indicating that the compaction was possibly dependent on the relocation of PKCα. Moreover, it was shown that the second relocation of PKCα occurred during the blastocyst formation. PKCα had different expression patterns in the three kinds of preimplantation embryos. However, the effects of PKCα on embryonic development started in early stage. There must be a necessary connection between PKCα relocation and cell adhesion starting at embryonic compaction.
Animals ; Embryonic Development ; Female ; Mice ; Parthenogenesis ; Pregnancy ; Protein Kinase C-alpha ; metabolism ; Tetraploidy ; Trophoblasts ; enzymology

OBJECTIVETo verify houseflies Musca spp. as the intermediate host of Thelazia callipaeda and reveal epidemiological situation of thelaziasis in Hubei province.

METHODSDogs eyes infected with T. callipaeda, 400 houseflies Musca and 259 fruitflies Amiota okadai in the city of Laohekou city (previously named as Guanghua county) of Hubei province had been investigated since September 2000. The newborn larvae of T. callipaeda from Laohekou suburbs were fed to houseflies Musca and A. okadai. Larvae used for the study were isolated from female T. callipaeda in laboratory and the susceptibility to houseflies Musca and A. okadai was observed.

RESULTSTwenty-one dogs from Laohekou, the original epidemic areas of thelaziasis were examined and 7 positive dogs in 21 (33.3%) and 11 T. callipaeda (9 females and 2 males) were identified. From 1975 to 2000, no thelaziasis cases were found through retrospective surveys. These 200 houseflies Musca and 135 A. okadai were dissected for examination but showed all negative with the infection. However, newborn larvae of T. callipaeda were used to experimentally infect 112 houseflies Musca and 84 A. okadai and all infected flies were examined on the 20th day after inoculation. As a consequence, houseflies Musca failed to be infected but 9 in 84 (10.7%) A. okadai were positive. 26 infective larvae of T. callipaeda were obtained and 21 of them were inoculated into right eye of one rabbit. The female worm began to produce newborn larvae in 37 days after infection and 3 adult T. callipaeda (two females and one male) were obtained.

CONCLUSIONSFruitflies A. okadai from Hubei province were susceptible to T. callipaeda, which was similar to the result of experimental studies in Anhui province. This survey further confirmed that A. okadai was the intermediate host of T. callipaeda but not houseflies Musca. Infective resources (adult dogs, for instance) had been under controlled thus human thelaziasis had been eradicated in this rural area.

Animals ; Conjunctivitis ; parasitology ; Disease Reservoirs ; Dog Diseases ; parasitology ; Dogs ; Drosophila ; parasitology ; Eye Infections, Parasitic ; epidemiology ; transmission ; veterinary ; Female ; Host-Parasite Interactions ; Houseflies ; parasitology ; Humans ; Insect Vectors ; parasitology ; Longitudinal Studies ; Male ; Spirurida Infections ; epidemiology ; transmission ; veterinary ; Thelazioidea ; isolation & purification ; physiology