A central portion of nifS, designated nifS', was amplified from Azotobacter vinelandii. The nifS' was cloned into pUC18 to make pUCS. Then pUCS was integrated into Azotobacter vindandii chromosome DNA by homologous recombination. This nifS disruption mutants were generated by single cross-over event and selected by Amp resistence on BBGN medium. The nifS disruption mutant (named SU1) was affirmed by southern blot and PCR amplification. SU1 grows rapidly on BBGN, but very slowly on Burk's N-free medium. This phenomenon showes that SU1 nearly lost its nitrogen fixation ability because of the disruption of nifS. The successful construction of SU1 is helpful for further research on the effect of nifS on the structure and function of nitrogenase component-Ⅰ and Ⅱ.

The aim of this research is to prepare high quality polyclonal antibodies against Ecp, a recently identified leucine-zipper protein. The full length cDNA of ecp was amplified by PCR, cloned into pGEX-4T-1(His)6 and transformed into E. coli DH 5alpha. After induction with IPTG, the GST-Ecp fusion protein from the lysate was bound to glutathione-Sepharose 4B and digested with thrombin. The released Ecp protein was further purified through Ni-NTA affinity chromatography to homogeneity. A rabbit was immunized with the purified Ecp, and the antibody generated against Ecp was purified by affinity chromatography. The results of the Western blot showed that Ecp is present in various development stages of Drosophila melanogaster, from larvae to adult.
Animals ; Antibodies ; immunology ; isolation & purification ; metabolism ; Blotting, Western ; Chromatography, Affinity ; Drosophila Proteins ; genetics ; immunology ; Drosophila melanogaster ; growth & development ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Gene Expression Regulation, Developmental ; genetics ; physiology ; Green Fluorescent Proteins ; genetics ; metabolism ; Peptide Initiation Factors ; genetics ; immunology ; Polymerase Chain Reaction ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo study the nerve electromyogram results by analysing the pathological characters of 4 cases diagnosed as peripheral neuropathy caused by n-hexane and arsenic.

METHODSThe nerve electromyogram examination and pathology data of 4 patients, who had been diagnosed as toxic chemicals peripheral neuropathy, were studied retrospectively.

RESULTSTwo patients in this group were exposed to n-hexane, their nerve electromyogram examinations and biopsy pathology of superficial peroneal nerve indicated the peripheral neuropathy was mainly manifests the lesion of medullary sheath. Another two patients were exposed to arsenic, their nerve electromyogram examinations showed axonal degeneration associated with demyelination, and their biopsy pathology showed the peripheral neuropathy was mainly axonal degeneration.

CONCLUSIONAxonal degeneration and demyelination always coexist in peripheral neuropathy caused by chemicals.

Arsenic Poisoning ; pathology ; physiopathology ; Female ; Hexanes ; poisoning ; Humans ; Male ; Middle Aged ; Peripheral Nervous System Diseases ; chemically induced ; pathology ; physiopathology ; Retrospective Studies ; Young Adult

OBJECTIVETo investigate the testicular blood flow in patients with testicular microlithiasis (TM) and its correlation with the seminal profile in infertile men.

METHODSWe selected 88 infertile men and examined them by testicular color Doppler and routine seminal tests.

RESULTSTesticular microlithiasis was found in 19 (19.3%) of the patients, classic testicular microlithiasis (CTM) in 7 (8.0%), and limited testicular microlithiasis (LTM) in 10 (11.3%). No significant differences were observed in the age of onset, bilateral testicular volume, resistance index (RI) of bilateral testicular arteries, semen amount and the rate of teratospermia. The bilateral testicular peak systolic velocity (PSV), sperm count and sperm motility were significantly lower in the CTM than in the LTM group (P < 0.05), but showed no statistically significant difference between the LTM and the non-calcification group.

CONCLUSIONTM may be one of the causes of poor sperm function in infertile men.

Adult ; Blood Flow Velocity ; Calcinosis ; complications ; physiopathology ; Humans ; Infertility, Male ; complications ; physiopathology ; Male ; Middle Aged ; Regional Blood Flow ; Semen ; cytology ; Sperm Count ; Sperm Motility ; Testicular Diseases ; complications ; physiopathology ; Testis ; blood supply ; pathology

To explore the protective effect of Angelica sinensis polysaccharides(ASP) on subacute renal damages induced by D-galactose in mice and its mechanism. Male C57BL/6J mice were randomly divided into 3 groups, with 10 mice in each group. The D-galactose model group was subcutaneously injected with D-galactose (120 mg x kg(-1)), qd x 42; the ASP + D-galactose model group was intraperitoneally injected with ASP since the 8th day of the replication of the D-galactose model, qd x 35; and the normal control group was subcutaneously injected with saline at the same dose and time. On the 2nd day of after the injection, the peripheral blood was collected to measure the content of BUN, Crea, UA, Cys-C; paraffin sections were made to observe the renal histomorphology by HE staining; senescence-associated β-g-alactosidase (SA-β-Gal) stain was used to observe the relative optical density (ROD) in renal tissues; transmission electron microscopy was assayed to observe the renal ultrastructure; the renal tissue homogenate was prepared to measure the content of SOD, GSH-PX, MDA; the content of AGEs and 8-OH-dG were measured by ELISA. According to the result, compared with the D-galactose model group, the ASP + D-galactose model group showed obviously decreases in the content of BUN, Crea, UA, Cysc, AGES, 8-OH-dG, the number of hardening renal corpuscle, renal capsular space and renal tubular lumen, ROD of SA-β-Gal staining positive kidney cells, mesangial cells, basement membrane thickness, podocyte secondary processes fusion and MDA and increases in the number of normal renal corpuscle, ribosome and rough endoplasmic reticulum in podocytes, the activity of SOD and GSH-PX. In Conclusion, A. sinensis polysaccharides can antagonize kidney subacute damages induced by D-galactose in mice. Its protective mechanism may be correlated with the inhibition of the oxidative stress injury.
Angelica sinensis ; chemistry ; Animals ; Deoxyguanosine ; analogs & derivatives ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Galactose ; adverse effects ; Humans ; Kidney ; anatomy & histology ; drug effects ; injuries ; Kidney Diseases ; chemically induced ; drug therapy ; metabolism ; prevention & control ; Male ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; drug effects ; Polysaccharides ; administration & dosage ; Protective Agents ; administration & dosage

Leukemia is a type of malignant tumors of hematopoietic system with the abnormal increased immature leukemia cells showing metastasis and invasion ability. Liver is one of the main targets of the leukemia cells spread to, where they may continue to proliferate and differentiate and cause liver function damage, even liver failure. Our previous studies showed that Angelica polysscharides (APS), the main effective components in Angelica sinensis of Chinese traditional medicine, was able to inhibit the proliferation and induced differentiation of the leukemia cells, however, its effect on the liver during the treatment remains elucidated. In the present study, the human leukemia NOD/SCID mouse model were established by implantation human leukemia K562 cells line, then the leukemia mouse were treated with APS, Ara-c or APS + Ara-c respectively by peritoneal injection for 14 days, to explore the effect and mechanism of the chemicals on the mouse liver. Compared to the human leukemia NOD/SCID mouse model group with the treatments of APS, Ara-c and APS + Ara-c, We found that severe liver damage and pathological changes of the liver were able to alleviate: First, the number of white blood cells in the peripheral blood was significantly lower and with less transplanted K562 leukemia cells; Second, liver function damage was alleviated as liver function tests showed that alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBiL) were significantly reduced, while the albumin (Alb) was notably increased; Third, liver antioxidant ability was improved as the activities of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were significantly increased, and the contents of GSH and malonaldehyde (MDA) were decreased significantly in the liver; Fourth, the inflammation of the liver was relieved as the level of IL-1beta and IL-6, the inflammatory cytokines, were decreased significantly in the liver. Fifth, liver index was increased as the pathological observation showed that leukemia cells with diffused infiltration into the liver lobules were significantly reduced and with a remarkable increase of apoptotic positive cell rate by TUNEL test. Furthermore, the APS + Ara-c combined administration showed an even more significant positive effect. In conclusion, the APS, Ara-c therapy reduced the accumulation of leukemia cells within the liver, reduced the liver function damage and levels of inflammatory factors, improved antioxidant capacity of the liver tissue and thus alleviate the pathological changes of the liver. Moreover, the APS + Ara-c combination therapy may have an additive effect.
Angelica ; chemistry ; Animals ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Cell Line, Tumor ; Cytarabine ; administration & dosage ; Humans ; K562 Cells ; Leukemia ; drug therapy ; Liver ; drug effects ; Male ; Mice ; Mice, SCID ; Polysaccharides ; administration & dosage

OBJECTIVETo explore the feasibility of autologous fascia as a scaffold for the reconstruction of skeletal muscle in vivo.

METHODSTwenty-eight healthy New Zealand rabbits were employed in the study. The anterior tibial muscle in both legs were divided to create a gap of 10 mm in each muscle. One leg was used in the experiment (E, n = 28), while the contralateral as self-control (C). The legs in C group were further divided into 3 groups (C1, C2 and C3). While defects in the midportion of anterior tibial muscle in the hind legs were created in all rabbits. In E group, each defect was filled with a tubule made of autologous fascia lata, and the fascial tubule was filled with tiny muscular granules (< 1 mm x 1 mm x 1 mm). In C1 group (n = 10), the defect was also filled with fascial tubule but with no muscle filling. The defect in C2 group (n = 10) was only filled with muscle granules without fascial tubule. The defect in C3 group (n = 8) received no treatment. The survival rate of the transplantation was grossly observed, and the tissue samples were harvested for histological and ultra-structural examination and immunohistochemical identification of desmin at 2, 3, 4, 6 and 9 post-operation weeks. The expression level of alpha-actin DNA in the tissue samples from the midportion of grafted fascia was assessed by RT-PCR (reverse transcription polymerase chain reaction) in E and C1 groups.

RESULTS(1) Survival rate of the transplantation: In E group, it was 93.33% with near normal tissue contour in the grafting area. The muscle defects were not completely repaired in C1, C2 and C3 groups. (2) Under light and electronic microscopy, marked proliferation of muscular cells surrounding fibrous tissue could be discerned at 2 and 3 post-operation weeks in E group, while only necrotic tissue and fibrosis were observed in C1 and C2 groups, and no definite tissue could be discernible in C3 group. (3) Immunohistochemical staining revealed that over 85% of the cells were positive for desmin in E group, while only less than 25% in C1 group. (4) The expression level of alpha-actin DNA was significantly higher in E group than that in C2 group (P < 0.05).

CONCLUSIONThese results suggested that autologous fascia as a scaffold is beneficial for skeletal muscle reconstruction in vivo.

Animals ; Disease Models, Animal ; Fascia ; transplantation ; Muscle, Skeletal ; surgery ; Rabbits ; Soft Tissue Injuries ; surgery ; Tissue Culture Techniques ; Tissue Scaffolds ; Transplantation, Autologous

OBJECTIVETo observe the incidence, causes and deviation angle of axial offset in patients with fracture ununited treated by Ilizarov bone transport technology.

METHODSFrom January 2007 to December 2012, 10 patients with fracture ununited were treated by Ilizarov bone transport including 8 males and 2 females with an average age of (30.3 ± 10.6) years old ranging from 18 to 49 years old. The segment of bone defect involved upper tibial in 2 cases, medial tibia in 2 cases, lower tibial in 5 cases, upper femoral in 1 case. For Paley type of bone defect, 6 cases were type B1, 4 cases were B3. The incidence and deviation angle of axial offset after Ilizarov bone transport technology were observed and evaluated on bone result by Paley assessment.

RESULTSAll patients were followed up from 19 to 32 months with an average of (22.0 ± 5.6) months. Three cases were natural healed at fracture ends, the other 7 cases were healed after bone graft. The time of external fixator was 16 to 28 months. At the last follow-up, there were 3 cases occurred coronal angulation of angle 5° to 11° with an average of (8.7 ± 3.2). Sagittal angulation was in 4 cases, angle 6° to 9° with an average of (8.5 ± 2.1)°. There were 4 cases occurred axial offset. In the last follow-up, according to Paley evaluation criteria, osseous results were excellent in 7 cases, good in 3 cases; functional results were excellent in 6 cases, good in 4 cases.

CONCLUSIONAxial deviation after the Ilizarov bone transport treatment is relatively common, which will result in delayed healing of bone and poor limb alignment. In order to improve the bone healing, corresponding measurements should be taken to avoid or reduce the incidence of axial deviation during and after the operation.

Adolescent ; Adult ; Female ; Fracture Healing ; Fractures, Ununited ; surgery ; Humans ; Ilizarov Technique ; adverse effects ; Male ; Middle Aged

OBJECTIVETo explore the biological mechanisms underlying Angelica sindsis polysaccharide (ASP) -induced aging of human-derived leukemia stem cells (LSCs) in vitro.

METHODAcute myelogenous leukemia stem cells were isolated by magnetic activated cell sorting (MACS). The ability of LSC proliferation treated by various concentration of ASP(20-80 mg · L(-1)) in vitro for 48 hours were tested using cell counting Kit-8 ( CCK8) , colony forming were evaluated by methylcellulose CFU assay. The ultra structure changes of AML CD34+ CD38- cells were analyzed by transmission electron microscopy. The aging cells were detected with senescence-β-galactosidase Kit staining. Expression of aging-related p53, p21, p16, Rb mRNA and P16, Rb, CDK4 and Cyclin E protein were detected by quantitative reverse transcription polymerase chain reaction( qRT-PCR) and Western blotting, respectively.

RESULTThe purity of the CD34 + CD38 - cells is (91.15 ± 2.41)% after sorted and showed good morphology. The proliferation of LSC was exhibited significantly concentration-dependent inhibited after exposure to various concentration of ASP. Treated by 40 mg · L(-1) ASP for 48 hours, the percentage of positive cells stained by SA-β-Gal was dramatically increased (P < 0.01) and the colony-formed ability has been weakened (P < 0.01). The observation of ultrastructure showed that cell heterochromatin condensation and fragmentation, mitochondrial swelling, lysosomes increased in number. Aging-related p53, p21, p16, Rb and P16, Rb were up-regulated, protein regulatory cell-cycle CDK4 and Cyclin E were down-regulated. ASP may induce the senescence of LSCs effectively in vitro, P16-Rb cell signaling pathway play a significant role in this process.

CONCLUSIONASP can induce human leukemia stem cell senescence in vitro, the mechanism involved may be related to ASP regulation P16-Rb signaling pathways.

Angelica sinensis ; chemistry ; Cell Cycle ; drug effects ; Cell Cycle Proteins ; genetics ; metabolism ; Cells, Cultured ; Cellular Senescence ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; Leukemia ; drug therapy ; genetics ; metabolism ; physiopathology ; Neoplastic Stem Cells ; cytology ; drug effects ; Polysaccharides ; pharmacology ; Signal Transduction ; drug effects

OBJECTIVETo investigate the role of p38 mitogen-activated protein kinase (MAPK) signal transduction pathway in the Kupffer cells production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in severely burns rats.

METHODSMale health adult Sprague-Dawley rats were randomized into four groups: sham burn rats given vehicle, sham burn rats given the p38 MAP kinase inhibitor SB203580, rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24 h after burn or sham burn and Kupffer cells (KCs) were isolated. After 18 h incubation, KCs next were stimulated with 50 ng/ml of LPS for 18 h. After stimulation, supernatants were removed for analysis of TNF-alpha and IL-1beta levels by ELISA. The TNF-alpha and IL-1beta mRNA expressions (by quantitative real-time RT-PCR) and the activities of p38 MAPK and JNK (by Western blot analysis) in KCs were examined.

RESULTSEighteen hours after 50 ng/ml LPS stimulation, KCs from burn rats released significantly higher levels of TNF-alpha and IL-1beta than did shams. The mRNA levels of TNF-alpha and IL-1beta in KCs increased significantly postburn. Western blot analysis suggested that expression of phosphorylated p38 MAPK and JNK were increased in KCs harvested from burn group after stimulation with LPS compared with those from sham group. In vivo administration of SB203580 markedly suppressed both the release of TNF-alpha and IL-1beta and the mRNA expressions of TNF-alpha and IL-1beta in KCs from both sham and burn rats. p38 MAPK activity in KCs was abolished by administration with SB203580, whereas JNK was not.

CONCLUSIONSp38 MAPK signal transduction pathway mediates KCs production of proinflammatory cytokines TNF-alpha and IL-1beta in severely burned rats.

Animals ; Blotting, Western ; Burns ; enzymology ; physiopathology ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Interleukin-1 ; genetics ; metabolism ; Kupffer Cells ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tumor Necrosis Factor-alpha ; genetics ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism ; physiology