The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k (cat)/K (m)) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.
Aeropyrum ; chemistry ; enzymology ; Archaeal Proteins ; genetics ; metabolism ; Binding Sites ; Biocatalysis ; Caprylates ; metabolism ; Cloning, Molecular ; Dimethyl Sulfoxide ; chemistry ; Escherichia coli ; Hot Temperature ; Industrial Microbiology ; methods ; Kinetics ; Laurates ; metabolism ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; methods ; Peptide Hydrolases ; genetics ; metabolism ; Protein Binding ; Protein Conformation ; Recombinant Proteins ; genetics ; metabolism ; Solvents ; chemistry ; Substrate Specificity

Objective Construction and expression of recombinant plasmid fusing encoding prME protein derived from Japanese encephalitis virus （JEV） and GM-CSF of BALB/c mouse. Methods GM-CSF gene was ampli/ied by nested-RT-PCR from BALB/c spleen cells. JEV prME protein gene was eluted by the digestion with restrict/on endonucleases BamH I/EcoR I from pJME piasmid. Genetic fusion of prME protein and GM-CSF are subcloned info pcDNA3.1 （＋） eukaryotic vector, and named as pJME/GM-CSF.The recombinant was confirmed by restriction enzymes digestion and DNA sequencing, and then transfected into China hamster ovary （CHO） cells by Lipofectamine2000. pJME/ GM-CSF expression in CHO cells was examined by Western blot. Results We observed 2001 bp and 2472 bp DNA fragments when pJME/GM-CSF was digested with BamHI/EcoRI and BamHI/NotI respectively as expected. The estimated molecular weight of the fusion protein was 85kD. Conclusion The recombinant pJME/GM-CSF was constructed and transfected into CHO cells successfully with pJME/GMCSF stably expressed.

Objective To study the adjuvant effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) coding gene on cellular immunity induced by Japanese encephalitis (JE) virus DNA vaccine. Methods GM-CSF coding gene was amplified by nested-reverse transcriptase-polymerase chain reaction (RT-PCR) technique from BALB/c murine spleen cells. Recombinant plasmids pJME/GM-CSF and pGM-CSF were constructed by JE virus (JEV) prM-E protein with GM-CSF coding gene or GM-CSF coding gene only, respectively. The plasmids were transfected into China hamster ovary (CHO) cells by Lipofectamine 2000. The coding protein expressions and distributions were detected by immunofluorescence. The BALB/c mice were vaccinated with indicated immunogens with or without GM-CSF gene. The changes of T lymphocyte subsets in the spleen and levels of intracellular cytokines, such as interferon (IFN)-γ and interleukin (IL)-4 of splenic cells from mice immunized with different immunogens were evaluated by flow cytometry. The cytotoxicity T lymphocyte (CTL) activity was assessed by lactate dehydrogenase (LDH). The data were compared by one-factor analysis of variance and least significant difference. Results The constructed recombinant pGM-CSF and pJME/GM-CSF were confirmed by restrict enzyme digestion and DNA sequencing. The expressions of the above proteins were mainly in the cytoplasm and minor on cell membrane. The percentage of CD4+ T lymphocytes in pJME/GM-CSF vaccinated group was (33.90±0.79)%, which was significantly higher than that of in other groups (t values were 9. 818, 6. 804, 6.594, 10.061, 9.380, and 17.675, all P<0.05). The percentages of CD4+T lymphocytes in pJME +pGM-CSF (0) and pJME+pGM-CSF (-3) vaccinated groups were (29.83±0.61)% and (29.70±0.51)%, respectively, which were both higher than that in pJME+pGM-CSF (+3) vaccinated group of (27.69+0.50)% (t=3.466, t=3.255, both P<0.05). The percentages of CD8+ T cells in pJME/GM-CSF and pJME+pGM-CSF vaccinated groups were both higher than that in empty vector (pcDNA 3.1+) group and JE inactivated vaccine vaccinated group (t values were 3.811, 2.627, 10.537, and 3.811, all P<0.05). The CTL activity in pJME/GM-CSF vaccinated group was (51.48±0.10)%, which was higher than those in other groups (t values were 22.868, 13.823, 5.377, 32.287, 34.632, and 53.795, all P<0.05). The IFN-γ/IL-4 ratios in pJME/GM-CSF, pJME+pGM-CSF (0) and pJME + pGM-CSF (-3) vaccinated groups were (19.13±1.36), (12.32±0.82) and (7.05±0.43), respectively, which were higher than those in other groups (P<0.05). Conclusion GM-CSF coding gene could enhance the cellular immune response induced by Japanese encephalitis DNA vaccine.

Objective To study the effect of lgG Fc gene on JEV DNA vaccine immunity. Methods Gene encoding IgG Fc was amplified by nested-RT-PCR technique from BALB/c murine spleen cells. JEV prME protein gene was obtained with restriction endonuclease BamH Ⅰ/EcoR Ⅰ from the eukaryotic recombinant named after pJME, which was constructed by us before. Recombinant, named after pJME/IgG Fc, with above two genes encoding JEV prME protein and BALB/c murine IgG Fc was constructed, and was tested by restriction enzymes analysis and DNA sequencing, then was transfected into China hamster ovary (CHO) cells by Lipo-fectAMINE 2000. Distribution and expression of the fusion proteins encoded by JEV prME protein and BALB/c murine IgG Fc genes in transfected CHO cells were detected by immunofluorescence and Western blot. The BALB/c micc were vaccinated with pJME/IgG Fc via intramuscular injection. Then the cytotoxic T lymphocyt (CTL) activity were assessed by lactic dehydrogenase (LDH) and the neutralizing antibody titer were assessed by 80% plaque reduction neutralization test. Results Molecular weights (2001 bp, 2730 bp) of the two in- serts released from pJME/IgG Fc with two group of restriction analysis associated with BamH 1/EcoR I and BamH Ⅰ/Not Ⅰ were correlated to the expected theoretic results respectively. It was estimated that molecular weight (Mr) of the fusion protein was 101 x 103. The expression of the above fusion protein was mainly distribu- ted in endochylema of transfected CHO cells,and not much in membrane of transfected CHO cells. CHO cells transfected with pJME/IgG Fc could express the fusion protein at the 32th cell passage. After immunization, the CTL activity and the neutralizing antibody titer in the pJMF/IgG Fc vaccinated group increased significantly compared with other vaccinated groups(P <0.05). Conclusion The recombinant pJME/IgG Fc was construc- ted and transfected into CHO cells successfully, and CHO cellular lines expressed fusion protein encoded by JEV prME protein and BALB/c murine lgG Fc genes stably were obtained. IgG Fc gene could reinforce the cellular immunity and humoral immunity of JEV DNA vaccine.

BACKGROUND: Human amniotic membrane (HAM) contains various ingredents such as collagen, glycoprotein,proteoglycan, integrin and laminated body, and so on, and expresses many kinds of growth factors and mRNA-associated proteins. And these ingredents can supply abundant nutriments for cellular proliferation and differentiation, and benefit cells to grow and propagate. Whether or not HAM can load porcine bone marrow-derived mesenchymal stem cells (BMSCs) to well grow on it deserves to be further investigated.OBJECTIVE: To set up a method of tissue engineering of human amniotic membrane loading porcine BMSCs and observe the morphological characteristics of growth and proliferation of BMSCs seeded on HAM.DESIGN: Randomized controlled observation.SETTING: State Key Laboratory of Trauma, Burn and Combined Injury, General Institute of Combined Injuries, Academy of Preventive Medicine, Third Military Medical University of Chinese PLA.MATERIALS: This experiment was carried out in the State Key Laboratory of Trauma, Burn and Combined Injury,General Institute of Combined Injuries, Academy of Preventive Medicine, Third Military Medical University of Chinese PLA between January and November 2003. Three Guizhou minipigs of either gender, aged 2 to 3 months, weighing from 6 to 8 kg, were provided by the Experimental Animal Center, Third Military Medical University of Chinese PLA. Main reagent:ISCOVE'S modified DULBECCO'S medium (IMDM) culture medium (Hyclone, USA); high-quality fetal bovine serum PAA (Germany); haematoxylin (China); Eosin B (Sigma, USA) and OCT embedding medium (USA). Main instruments: BX51 stereoscopic fluorescence microscope (Olympus, JaPan); IX70 inverted fluorescence microscope (Olympus, Japan);cryostat (2700-Frigcut, Germany); myeloid puncture needle (Jiangsu); superclean bench (Sujing Bloc Antai Company);CO2 constant-temperature incubator (QUEUE, USA).METHODS: HAM was prepared as previously described. The BMSCs of Guizhou minipigs isolated and cultured according to method described previously were primarily cultured and passaged, then they were inoculated to the stromal surface of HAM at different densities (0.84×105 cells/cm2,1.54×105 cells/cm2,2.75×105 cells/cm2); The growth and proliferation of BMSCs of different densities were observed under an inverted microscope and scanning electron microscope; BMSCs of the second or the third passages were inoculated on HAM held with tissue-holding device at a density of 1.54×105 cells/cm2, and they were cultured for 18 days at most. The HAM was daily rolled, sliced and stained by HE for observing the growth of BMSCs loaded on HAM under the light, scanning and transmission electron microscopes.MAIN OUTCOME MEASURES: The growth of BMSCs on HAM was examined at different densities and different time points.RESULTS: ① Comparison of growths of BMSCs promoted by different densities of HAM: BMSCs,which were planted on HAM at the density of 0.84×105 cells/cm2 were irregular and scattered under an invert microscope. Distances between BMSCs were biggish. BMSCs seeded on HAM at the density of 1.54×105 cells/cm2 were regular in arrangement and moderate in density, with clear cell outline and good cell activity before 24 hours, and seeded at the density of 2.75×105 cells/cm2 were congested with many nonattached cells and the longer the growing time of the cells was, the more the cellular debris were observed. BMSCs,which were planted on HAM at the density of 0.84×105 cells/cm2 under the scanning electron microscope, scatted on HAM presented in shapes of irregular, long, thin and flat polygon. Their membrane protuberances presented in shapes of thick and thin, and the distances between cells were biggish. BMSCs,which were planted on HAM at the density of 1.54×105 cells/cm2 have similar appearance of their bodies and membrane protuberances, and the membrane protuberances were more compared with the BMSCs planted at the density of 0.84×105 cells/cm2. Their membrane protuberances intercrossed each other, and the margin of some BMSCs overlapped each other. BMSCs planted at the density of 2.75×105 cells/cm2, arraved on HAM crowdedly and overlappedly with many debris. Their membrane protuberances were not obviously. The margin of some BMSCs was overlapped.② Comparisonof growths of BMSCs promoted by HAM at different time points: Under the inverted microscope, the BMSCs adhered quickly to HAM after being incubated for about 30 minutes. All of BMSCs adhered to HAM within 24 hours, and formed monolayer on it within 48 hours, and grew densely on HAM after being cultured for 4 to18 days. Under the light and electron microscopes, HE results revealed that BMSCs adhered tightly and grew on HAM in different arrays, such as emitting, whirlpool or parallel,and their nuclei located in middle, dense in staining, were big and clear. The shapes of BMSCs were comparatively consistent on HAM. HAM loaded with BMSCs grew 4 days, and BMSCs covered HAM completely. The densities of BMSCs on HAM were suitable, and their bodies were large, and presented irregular, long,thin and flat polygon under the scanning electron microscope. The margin of some BMSCs overlapped each other. The protuberances of cellular membrane of BMSCs were abundant in the shapes of thick and thin. Some protuberances intercrossed each other in the shape of net. BMSCs adhered tightly to HAM through these protuberances. HAM loading BMSCs grow 4 days; most of BMSCs grew on HAM in double layers with the shapes of cambiform under the transmission electron microscope, Their nucleoli were clear. The protuberances of cellular membrane of BMSCs, which situated at two sides of nuclei and overlapped each other, were long. Most of chromatins of BMSCs were autosome.Abundant organell such as rough endoplasmic reticulum (RER),mitochondria could be observed in BMSCs.CONCLUSION:HAM is able to promote the proliferation of BMSCs significantly. BMSCs may be cultured on HAM ex vivo.HAM is a good carrier of BMSCs.

Platelet-derived growth factors (PDGFs) are a member of family of glycoprotein dimers (Mr 27000～35000) that exert potent mitogenic and chemotactic activities toward cells of mesenchymal origin. The PDGFs possess a myriad of critical roles in embryonic development,cellular differentiation and response to tissue damage. It is one of the growth factors,which appear early during the process of wound healing. Especially,PDGFs can effectively promote the healing of some chronic refractory wounds,such as diabetes mellitus ulcer,chronic venous ulcer,bedsore,radioactivity ulcer,etc..

BACKGROUND: As a kind of semitransparent membrane, human amniotic membrance contains many kinds of nutrients, which is a good biological material loaded with keratinocytes.OBJECTIVE: To construct epidermal substitute of the skin from human amniotic membrane loaded with porcine keratinocytes and examine the morphological characteristics of the growth and proliferation of keratinocytes seeded on human amniotic membrane.DESIGN: Single sample study and repetitive measured observation based on the cells.SETTING: Institute of Combined Injuries of Chinese PLA, Academy of Preventive Medicine, Third Military Medical University of Chinese PLA.MATERIALS: The experiment was completed in the State Key Laboratory of Trauma, Burn and Combined Injury and Institute of Combined Injuries of Chinese PLA, Academy of Preventive Medicine, Third Military Medical University of Chinese PLA from January to November 2001. Porcine keratinocytes was collected from Guizhou minipigs aged 3 weeks.METHODS: The primarily cultured keratinocytes of Guizhou minipigs were subcuhured, expanded and bred on the stroma surface of human amniotic membrance at the density of 1.63 × 105/cm2. The growth and proliferation of keratinocytes were observed under inverted microscope every day. From the 3rd day and the 15th day after being cultured, the growth of keratinocytes on human amniotic membrane was examined under light microscope and electron microscope.MAIN OUTCOME MEASURES: The growth of keratinocytes on human amniotic membrane was examined RESULTS: Keratinocytes evidently adhered to the stroma surface of human amniotic membrane about 30 minutes after being cultured, which was observed under inverted microscope. Most keratinocytes grew and adhered to the stroma surface of human amniotic membrane within 24 hours. Monolayer of keratinocytes formed and completely covered human amniotic membrane within 3 days. It was observed under the light microscope that the monolayer of keratinocytes adhered to human amniotic membrane and arrayed tightly. The keratinocytes presented in the shape of polygon, and plasmalemmas of keratinocytes formed many pseudopods under the observation with scanning electron microscope. Keratinocytes adhered to human amniotic membrane well and with many keratinofilaments in them under the observation with transmission electron microscope. Keratinocytes arrayed on human amniotic membrane densely with many cellular debris and some keratinocytes formed cavitations in them due to aging after growth for 15 days under the observation with inverted microscope.CONCLUSION: Human amitotic membrane is a good carrier of keratinocytes cultured on it in vitro, and is able to promote the proliferation of keratinocytes significantly. However, when keratinocytes were loaded on the human amniotic membrane for 15 days, some keratinocytes formed cavitations in them due to aging.

OBJECTIVETo study the effects of dosages of total body irradiation on the healing process of cutaneous wounds and to observe the changes of wound area at different periods after injury.

METHODSThe entire body irradiation from a (60)Co gamma-ray source was performed on Wistar rats. The single dosage varied from 1 to 8 Gy. Within 1 h after irradiation, two whole thickness circular cutaneous wounds corresponding to 2.5% of total body surface area (Phi = 22 mm) were produced on the back of the animals (combined injury groups). Same wounds were produced on rats with no irradiation (single wound group). Wound healing was observed at different points after injury.

RESULTSAfter total body irradiation with the dose of 1, 2, 3, 4, 5, 6, 7 or 8 Gy, the wound healing was obviously retarded as the dosages increased. The wound area remained was larger in the large dosage groups than in the small dosage groups. Seven days after injury, there was 33.5% wound surface left unhealed in the single wound group, whereas in the combined injury groups, 35.4%, 38.1%, 41.6%, 48.8%, 53.9%, 63.7%, 69.2% and 73.9% of the wound surfaces remained unhealed, respectively. Statistical analysis showed marked correlations between the various times after total body irradiation and various dosages to the percentage of unhealed wound surface. Nine dose-effect relation formulae were deduced according to the statistical results.

CONCLUSIONSIn soft tissue trauma combined with radiation injury, the delay of wound healing is related to the dose of radiation inflicted. It is also related to the time between injury and time of observation.

Animals ; Dose-Response Relationship, Radiation ; Female ; Male ; Rats ; Rats, Wistar ; Time Factors ; Whole-Body Irradiation ; Wound Healing ; radiation effects

Objective To clone platelet-derived growth factor A chain (PDGF-A) gene and insert PDGF-A gene into. Enhanced green fluorescent protein (EGFP) vector and then transformed into dermis-drived mesenchymal stem cells (DMSCs). Methods cDNA clones encoding human PDGF-A gene were isolated from a human hepatoma cell line mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The PCR amplified fragment of PDGF-A gene was cloned into pMD18-T vector. The eukaryotic expression vector pEGFP-N1/PDGF-A was constructed by subcolone PDGF-A gene into pEGFP-N1 vector. PDGF-A gene was transfected into DMSCs with the help of Fugene 6 transfection reagent. Results Full cDNA sequence encoding human PDGF-A gene had been cloned, which sequence was consistent with the reported sequence in GenBank by sequence assaying. Conclusion cDNA sequence encoding human PDGF-A gene was successfully cloned into pEGFP-N1. The transient expression of PDGF-A gene in DMSCs has been realized.

Objective To further determine their possible synergistic effect on accelerating wound healing, adenovirus vector containing recombinant human hPDGF-A and hBD2 genes was constructed and the expression of exogenous genes in transformed mesenchymal stem cells derived from rat bone marrow was observed. Methods By putting IRES in the middle of hPDGF-A and hBD2, these two genes were expected to be expressed individually. The shuttle vector was named as pAdTrack-hPDGF-A-IRES2-hBD2, which homologously recombinated with Adeasy-1 in BJ5183 cells and formed the mammalian expression vector pAdeasy-hPDGF-A-IRES2-hBD2. Furthermore, the recombinant vector was packaged in 293 cells into infectious recombinant adenovirus, which were used to infect BMSCs. The expression of hPDGF-A and hBD2 in BMSCs was detected by RT-PCR. Results We successfully constructed recombinant adenovirus vector that simultaneously expressed hPDGF-A and hBD2. The expressions of hPDGF-A and hBD2 were confirmed by RT-PCR on transformed BMSCs. Conclusion The established BMSCs that overexpressed hPDGF-A and hBD2 provide a new strategy of combining cell therapy and gene therapy to promote wound healing, especially the chronic one.