For metabolic engineering of cyanobacteria, there is an urgent need to construct a group of efficient heterologous gene expression platforms and to evaluate their expression efficiencies. Here we constructed three integrative vectors, the pKW1188-derived pFQ9F, pFQ9R and pFQ20, for integration of heterologous genes into the genome of the model cyanobacteria strain Synechocystis sp. strain PCC6803. The pFQ16, an RSF1010-derived broad host range shuttle vector, was constructed for conjugative transfer of genes to various cyanobacteria strains. All the four platforms constructed here applied the rbc (encodes Ribulose-1, 5-bisphosphate carboxylase/oxygenase) and the rbc terminator to promote and terminate the gene transcription. Besides, a "Shine-Dalgarno -AUG" fusion translation strategy was used to keep the high protein translation efficiency. Using lacZ as a reporter gene, the expression efficiency of pFQ20 was evaluated and showed a strong beta-galactosidase expression (109 Miller). Furthermore, the platform pFQ20 was used to express the E. coli tesA' gene and showed significant protein bands through the Western Blot test. The expression platforms constructed in this study offer useful molecular tools for metabolic engineering of cyanobacteria in the future.
Genetic Vectors ; genetics ; Industrial Microbiology ; methods ; Metabolic Engineering ; methods ; Palmitoyl-CoA Hydrolase ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Synechocystis ; genetics ; metabolism ; beta-Galactosidase ; biosynthesis ; genetics

OBJECTIVEThe effect of angelica sinensis polysaccharides (ASP) on the production of reactive oxygen specie (ROS), the capability of total anti-oxidant (T-AOC), and the expression of p16 in mRNA level in mice hematopoietic stem cells (HSCs) were observed to explore the underlying mechanism that ASP delay aging of HSCs in vivo.

METHODC57BL/6J mice were randomly divided into normal group, aging group, and the above groups treated with ASP. Mice were uniformly explored in X-ray (3.0 Gy/8 F) to erect model of aging. Normal and aging ASP intervention groups mice were treated with ASP by intragastric administration, while normal and aging groups were treated with equal-volume NS during X-ray irradiation. Mice HSCs were isolated by magnetic cell sorting and cultured in vitro. Senescence-associated beta-galactosidase (SA-beta-Gal) staining was used to detect aging HSCs. Cell cycles analysis and CFU-Mix cultivation were used to evaluate the capability of self-renewing and colony forming in HSCs. The production of ROS in HSCs was evaluated by flow cytometry analysis and immunofluorescence assess, respectively. T-AOC was detected by chemical colorimetric method. The expression of p16 was determined by real-time quantitative PCR (qRT-PCR).

RESULTExogenous X-ray irradiation induced HSCs aging was compared with normal group without irradiation. Biological feature of HSCs in aging group with X-ray irradiation as follows: The percentage of SA-beta-Gal positive cells, the ratio of G1 stages and the production of ROS were significantly increased , the expression of p16 in mRNA level was also upregulated. The capacility of colony forming and T-AOC in HSCs were decreased. ASP could significantly decrease the percentage of SA-beta-Gal positive cells, the ratio of G1 stages and the production of ROS in HSCs, and downregulate the expression of p16 in mRNA level in HSCs contrast to aging group without ASP treatment. In addition, ASP could remarkably increase T-AOC and the capacility of colony forming in HSCs compared with aging group without ASP treatment.

CONCLUSIONX-ray (3.0 Gy/8 F) could induce mice HSCs aging. ASP could delay senescence HSCs aging which maybe partly ascribed to the inhibition of oxidative damage and the downregulation of p16 mRNA expression.

Aging ; drug effects ; radiation effects ; Angelica sinensis ; chemistry ; Animals ; Cell Cycle ; drug effects ; radiation effects ; Cells, Cultured ; Cellular Senescence ; drug effects ; radiation effects ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Female ; Flow Cytometry ; Gene Expression ; drug effects ; radiation effects ; Hematopoietic Stem Cells ; drug effects ; metabolism ; radiation effects ; Male ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; drug effects ; radiation effects ; Polysaccharides ; pharmacology ; Random Allocation ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; X-Rays ; beta-Galactosidase ; metabolism

DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.
Antigen-Antibody Complex ; metabolism ; Antigens, Nuclear ; genetics ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Cellular Senescence ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; DNA-Activated Protein Kinase ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; HeLa Cells ; Homologous Recombination ; genetics ; physiology ; Humans ; Ku Autoantigen ; Nicotinamide Phosphoribosyltransferase ; genetics ; metabolism ; physiology ; Nuclear Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; beta-Galactosidase ; metabolism

OBJECTIVETo observe the effect of extracts from Radix Ginseng, Radix Notoginseng and Rhizoma Chuanxiong (EXT) on delaying vascular smooth muscle cells (VSMCs) aging in aged rats.

METHODSVSMCs were obtained by the modified tissue explants technique and were shown to be positive for smooth muscle α-actin (SM-α-actin) by immunohistochemistry staining. VSMCs obtained from the young rats were served as the young control group; VSMCs obtained from the old rats were treated with no drug (the old group), with low dose extracts (20 mg/L, the EXT low-concentration group) and high dose extracts (40 mg/L, the EXT high concentration group), and with Probucal (10(-6) mol/L, the Probucal group) as a positive control. All groups were cultured for 24 h in the medium with 10% serum for 24 h followed by another 24 h in the serum-free medium. At the end of the 48-h culture, the following analyses were performed including determination of senescence-associated β-galactosidase (SAβ-Gal) activity, flow cytometry analysis of cell cycle, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analyses of p16, Cyclin D1, cyclin-dependent kinase 4 (CDK4) and retinoblastoma (Rb) mRNA expression, and Western blotting analyses of p16, cyclin D1, CDK4 and phosphoretinoblastoma (pRb) protein expressions.

RESULTS(1) In comparison to the younger rats, VSMCs from aged rats had significantly more SA&beta;-Gal positive cells (P<0.01) and more cells in S phase (P<0.05). VSMCs from the all treated groups showed a significant decrease in both SA&beta;-Gal positive cells (P<0.05) and S phase (P<0.05) compared to the old rats. (2) Compared with the young group, VSMCs in the old group had a significant decrease in p16 and Rb mRNA expression and a significant increase in Cyclin D1 and CDK4 mRNA expression. Compared with the old group, VSMCs in the treated groups had a significant increase in p16 and Rb mRNA expression and a significant decrease in Cyclin D1 and CDK4 mRNA expression (P<0.05). (3) Compared with the young group, VSMCs in the old group had a significant decrease in p16 protein expression and a significant increase in Cyclin D1, CDK4 and pRb protein expressions (P<0.05). Compared with the old group, VSMCs in the treated groups had a significant increase in p16 protein expression and a significant decrease in cyclinD1, CDK4 and pRb protein expressions (P<0.05).

CONCLUSIONSVSMCs obtained from old rats showed typical signs of cellular senescence and vascular aging. EXT had an effect on delaying senescence of VSMCs in vitro by altering the p16-cyclinD/CDK-Rb pathway.

Aging ; drug effects ; Animals ; Aorta ; cytology ; Cell Cycle ; drug effects ; Cellular Senescence ; drug effects ; Cyclin D1 ; genetics ; metabolism ; Cyclin-Dependent Kinase 4 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Flow Cytometry ; Gene Expression Regulation ; drug effects ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Panax ; Plant Extracts ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Retinoblastoma Protein ; genetics ; metabolism ; beta-Galactosidase ; metabolism

OBJECTIVETo report the results of clinical characteristics, enzyme activity determination and mutation analysis of GLB1 gene in a Chinese patient with mucopolysaccharidosis (MPS) type IVB (Morquio B disease).

METHODA 14-year-old Chinese boy with MPS type IVB was firstly diagnosed by blood leucocytes galactosamine-6-sulfate sulfatase (GALNS) and &beta;-galactosidase (GLB1) determination, who was characterized by short stature, multiplex skeletal abnormalities, difficulty in walking. PCR-sequencing analysis was applied to detect the mutations in GLB1 of the patient.

RESULTThe patient was characterized by dwarfism, pectus carinatum, kyphosis, normal intelligence, and no neurologic damage of spasms, linguistic capacity and so on. The patient had normal GALNS enzyme activity and very low GLB1 enzyme activity [5.03 nmol/(h·mg) vs. normal value 118 - 413 nmol/(h·mg) ] in leukocytes. A compound heterozygous missense mutations c.442C > T(p.R148C)/c.1454A > G(p.Y485C) in GLB1 gene were detected in this patient. The mutation p.Y485C is a novel variant. With the method of gene analysis of new variant, the mutation p.Y485C was considered to be a pathogenic mutation.

CONCLUSIONThe MPS IVB patient showed severe multiple skeletal deformities, normal intelligence, no neurologic damage and very low GLB1 enzyme activity, who carries compound heterozygous mutations p.R148C/p.Y485C. The mutation p.Y485C in GLB1 gene may be a novel pathologic mutation of MPS type IVB.

Adolescent ; Amino Acid Sequence ; Asian Continental Ancestry Group ; genetics ; Chondroitinsulfatases ; genetics ; metabolism ; DNA Mutational Analysis ; Humans ; Joints ; pathology ; Male ; Molecular Sequence Data ; Mucopolysaccharidosis IV ; enzymology ; genetics ; pathology ; Mutation, Missense ; Pedigree ; Polymerase Chain Reaction ; Radiography ; Spine ; diagnostic imaging ; pathology ; beta-Galactosidase ; genetics ; metabolism

Biliverdin reductase A (BLVRA), an enzyme that converts biliverdin to bilirubin, has recently emerged as a key regulator of the cellular redox cycle. However, the role of BLVRA in the aging process remains unclear. To study the role of BLVRA in the aging process, we compared the stress responses of young and senescent human diploid fibroblasts (HDFs) to the reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). H2O2 markedly induced BLVRA activity in young HDFs, but not in senescent HDFs. Additionally, depletion of BLVRA reduced the H2O2-dependent induction of heme oxygenase-1 (HO-1) in young HDFs, but not in senescent cells, suggesting an aging-dependent differential modulation of responses to oxidative stress. The role of BLVRA in the regulation of cellular senescence was confirmed when lentiviral RNAitransfected stable primary HDFs with reduced BLVRA expression showed upregulation of the CDK inhibitor family members p16, p53, and p21, followed by cell cycle arrest in G0-G1 phase with high expression of senescence-associated beta-galactosidase. Taken together, these data support the notion that BLVRA contributes significantly to modulation of the aging process by adjusting the cellular oxidative status.
Age Factors ; Blotting, Western ; *Cell Aging ; Cell Cycle ; Cells, Cultured ; Enzyme Induction ; Fibroblasts/physiology ; G1 Phase ; Heme Oxygenase-1/metabolism ; Humans ; Hydrogen Peroxide/pharmacology ; *Oxidative Stress ; Oxidoreductases Acting on CH-CH Group Donors/*metabolism ; Protein Kinase Inhibitors/metabolism ; RNA, Small Interfering ; Reactive Oxygen Species/metabolism ; beta-Galactosidase/genetics/metabolism

BACKGROUNDIt has been proven that ultrasonic destruction of microbubbles can enhance gene transfection efficiency into the noncardiac cells, but there are few reports about cardiac myocytes. Moreover, the exact mechanisms are not yet clear; whether the characteristic of microbubbles can affect the gene transfection efficiency or not is still controversial. This study was designed to investigate whether the ultrasound destruction of gene-loaded microbubbles could enhance the plasmids carried reporter gene transfection in primary cultured myocardial cell, and evaluate the effects of microbubbles characteristics on the transgene expression in cardiac myocytes.

METHODSThe &beta;-galactosidase plasmids attached to the two types of microbubbles, air-contained sonicated dextrose albumin (ASDA) and perfluoropropane-exposed sonicated dextrose albumin (PESDA) were prepared. The gene transfection into cardiac myocytes was performed in vitro by naked plasmids, ultrasound exposure, ultrasonic destruction of gene-loaded microbubbles and calcium phosphate precipitation, and then the gene expression and cell viability were analyzed.

RESULTSThe ultrasonic destruction of gene-loaded microbubbles enhanced gene expression in cardiac myocytes compared with naked plasmid transfection ((51.95 ± 2.41) U/g or (29.28 ± 3.65) U/g vs. (0.84 ± 0.21) U/g, P < 0.01), and ultrasonic destruction PESDA resulted in more significant gene expression than ASDA ((51.95 ± 2.41) U/g vs. (29.28 ± 3.65) U/g, P < 0.05). Ultrasonic destruction of microbubbles during calcium phosphate precipitation gene transfection enhanced &beta;-galactosidase activity nearly 8-fold compared with calcium phosphate precipitation gene transfection alone ((111.35 ± 11.21) U/g protein vs. (14.13 ± 2.58) U/g protein, P < 0.01). Even 6 hours after calcium phosphate precipitation gene transfection, ultrasound-mediated microbubbles destruction resulted in more intense gene expression ((35.63 ± 7.65) U/g vs. (14.13 ± 2.58) U/g, P < 0.05).

CONCLUSIONSUltrasonic destruction of microbubbles might be a promising method for the delivery of non-viral DNA into cardiac myocytes, and the gene tranfection is related to the characteristics of microbubbles.

Albumins ; Animals ; Cell Survival ; genetics ; physiology ; Cells, Cultured ; Microbubbles ; Myocytes, Cardiac ; cytology ; metabolism ; Rats ; Rats, Wistar ; Transfection ; methods ; Ultrasonics ; methods ; beta-Galactosidase ; genetics ; metabolism

PURPOSE: This study was designed to investigate whether transduction of lentiviral vectors (LV) carrying human coagulation factor VIII (hFVIII) cDNA into skeletal muscle could increase circulating hFVIII concentrations. MATERIALS AND METHODS: A LV containing bacterial LacZ gene as a control or human FVIII gene was intramuscularly administered into the thigh muscle of 5 weeks old Sparague-Dawley rats. The plasma human FVIII concentration and neutralizing anti-FVIII antibodies were measured for up to 12 weeks in these experimental animals. RESULTS: The plasma human FVIII levels in the rats injected with LV carrying FVIII cDNA peaked at post-injection 1st week (5.19 +/- 0.14 ng/mL vs. 0.21 +/- 0.05 ng/mL in control rats , p < 0.05). Elevated hFVIII concentrations were maintained for 4 weeks (2.52 +/- 0.83 ng/mL vs. 0.17 +/- 0.08 ng/mL in control rats, p < 0.05) after a single intramuscular injection. In the Bethesda assay, neutralizing antibodies for FVIII protein were detected only in FVIII-LV injected rats by the 10th week, but not in control rats. CONCLUSION: This study suggested that a single administration of an advanced generation LV carrying the human FVIII cDNA resulted in elevation of FVIII level in immune competent rats, and that this gene transfer approach to the skeletal muscle could be an effective tool in treatment of hemophilia A.
Animals ; Antibodies/blood/immunology ; Factor VIII/genetics/immunology/*metabolism ; Gene Therapy ; Genetic Vectors/genetics ; Hela Cells ; Hemophilia A/therapy ; Humans ; Lentivirus/genetics ; Male ; Muscle, Skeletal/*metabolism ; Rats ; Rats, Sprague-Dawley ; Transduction, Genetic ; beta-Galactosidase

OBJECTIVETo evaluate the efficacy of gene delivery to the right lateral lobe of the rat liver via a branch of the bile duct using naked DNA.

METHODSWe evaluated regional gene delivery to the right lateral lobe of the rat liver via a branch of the bile duct, using naked DNA, including pGL3, pCMV beta and Cy3 labeled CMV beta.

RESULTSGene expression was observed in right lateral lobe of both the damaged and the normal rats liver. The gene delivery efficiency was similar in two groups (P > 0.05). Gene expression was found in the right lateral lobe of damaged and normal livers. Fluorescence was observed in the region of the portal triads, and occasionally, in the lobule.

CONCLUSIONRetrograde infusion of naked DNA via the bile duct is an effective way to deliver genes to in both damaged and normal rat liver.

Animals ; Bile Ducts ; metabolism ; DNA ; genetics ; Disease Models, Animal ; Female ; Galactose ; Gene Expression ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Therapy ; methods ; Genetic Vectors ; Liver ; metabolism ; pathology ; Liver Diseases ; genetics ; pathology ; therapy ; Luciferases ; genetics ; metabolism ; Plasmids ; genetics ; Rats ; Rats, Inbred Lew ; beta-Galactosidase ; genetics ; metabolism

OBJECTIVETo investigate the mechanism involved in aging process of immortalized human keratinocyte (HaCaT) and primary human epidermis keratinocyte of adults (HEKa) irradiated by ultraviolet B(UVB).

METHODSHEKa and HaCaT were repeatedly exposed to UVB at a subcytotoxic level. SA-beta-Gal staining was performed to evaluate the senescence state; flow cytometry was applied to detect the changes of apoptosis, necrosis and cell cycle. Intracellular levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by ELISA method. Western blot was performed to detect the expression pattern of redox protein p66Shc and RT-PCR was performed to determine the mRNA level of human telomerase reverse transcriptase (hTERT).

RESULTStrong positive SA-beta-Gal staining was observed in both HEKa cell and HaCaT cells after UVB irradiation. Apoptosis rate increased from (1.81 +/-0.25)% to (4.43 +/-0.28)% and necrosis rate increased from (0.05 +/-0.01)% to (0.10 +/-0.03)% in HaCaT cell, but no marked arrest of cell cycle was observed during UVB irradiation. As a contrast, apoptosis rate of in HEKa cells significantly increased from (0.65 +/-0.05)% to (59.53 +/-2.35)%, and the necrosis rate in HEKa cells also reached (3.89 +/-0.24)%(P<0.05). Growth arrest in G0/G1 phase was also found in HEKa cells. In both cell lines, intracellular level of SOD decreased and MDA increased remarkably after UVB exposure, and an increased expression of p66Shc protein was also observed. High level of hTERT mRNA was detected in HaCaT cells and UVB exposure had little effect on its expression.

CONCLUSIONThe stress-induced premature senescence (SIPS) in HaCaT and HEKa cell lines by UVB irradiation might be closely associated with increased intracellular levels of oxidative stress, not related to the telomerase expression.

Apoptosis ; Cell Line ; Cells, Immobilized ; radiation effects ; Cellular Senescence ; physiology ; radiation effects ; Humans ; Keratinocytes ; cytology ; radiation effects ; Malondialdehyde ; metabolism ; Skin ; cytology ; Superoxide Dismutase ; metabolism ; Telomerase ; genetics ; metabolism ; radiation effects ; Ultraviolet Rays ; beta-Galactosidase ; pharmacology