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At least 1,225 records · Page 68Linked to original sources
PubMed2005

Optimization of adenovirus serotype 35 vectors for efficient transduction in human hematopoietic progenitors: comparison of promoter activities.

Adenoviral gene transfer to hematopoietic stem cells (HSCs)/progenitors would provide a new approach to the treatment of hematopoietic diseases and study of the hematopoietic system. We have previously reported that an adenovirus (Ad) vector composed of whole Ad serotype 35 (Ad35), which belongs to subgroup B, shows efficient gene transfer into human bone marrow CD34+ cells. However, Ad35 vector-mediated transduction into human HSCs/progenitors has not yet been fully optimized. In the present study, we have systematically examined promoter activity in the context of Ad35 vectors in human bone marrow CD34+ cells and primitive CD34+ subsets to optimize the transduction efficiency in human hematopoietic stem/progenitor cells. In the first of the transduction experiments, the improved in vitro ligation method was applied to Ad35 vector construction to allow for simple and efficient production of an E1/E3-deleted Ad35 vector. Using this method, we constructed a series of Ad35 vectors encoding the enhanced green fluorescence protein (GFP) under the control of a variety of strong viral and cellular promoters. Of the six types of promoters tested, significantly higher transduction efficiencies were achieved with the human elongation factor 1alpha promoter (EF1alpha promoter), the human cytomegalovirus (CMV) immediate-early 1 gene enhancer/beta-actin promoter with beta-actin intron (CA promoter), and the CMV promoter/enhancer with the largest intron of CMV (intron A) (CMVi promoter) in the human CD34+ cells and the immature subsets (CD34+ CD38(low/-) and CD34+ AC133+ subsets). In particular, the CA promoter was found to allow for the highest transduction efficiencies in both the whole human CD34+ cells and the immature hematopoietic subsets. Furthermore, the CA promoter-mediated GFP-expressing cells differentiated into progenitor cells of all lineages. These results indicate the construction of an optimized Ad35 vector backbone for efficient transduction into HSCs/progenitors.

Actins↗
PubMed2006

AAV serotype-1 mediates early onset of gene expression in mouse hearts and results in better therapeutic effect.

Adeno-associated viral vectors (AAV) are attractive tool for gene therapy for coronary artery disease. However, gene expression in myocardium mediated by AAV serotype 2 (AAV2) does not peak until 4-6 weeks after gene transfer. This delayed gene expression may reduce its therapeutic potential for acute cardiac infarction. To determine whether earlier gene expression and better therapeutic effect could be achieved using a different serotype, CMV promoter driving the EPO gene (AAV-EPO) was packaged into AAV serotypes 1-5 capsids and injected into mouse myocardium. EPO expression was studied by measuring the hematocrits and EPO mRNA. After we found that AAV1 mediates the highest gene expression after 4 days of gene transduction, AAV-LacZ (CMV promoter driving LacZ gene expression) and MLCVEGF (hypoxia-inducible and cardiac-specific VEGF expression) were packaged into AAV1 and 2 capsids. LacZ expression was detected in AAV1-LacZ but not in AAV2-LacZ-injected hearts 1 day after vector injection. Compared to AAV2-MLCVEGF that mediated no significant VEGF expression, AAV1-MLCVEGF mediated 13.7-fold induction of VEGF expression in ischemic hearts 4 days after gene transduction and resulted in more neovasculatures, better cardiac function and less myocardial fibrosis. Thus, AAV1 mediates earlier and higher transgene expression in myocardium and better therapeutic effects.

Animals↗
PubMed1998

High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

Arabidopsis↗
PubMed2004

Xenoestrogens and the induction of proliferative effects in breast cancer cells via direct activation of oestrogen receptor alpha.

Environmental contamination with a variety of industrial products has been associated with developmental and reproductive abnormalities in wildlife species. Increasing evidence has suggested that bisphenol A (BPA) and 4-nonylphenol (NPH), two major endocrine-disrupting chemicals, might be responsible for adverse effects on humans as a consequence of ubiquitous use together with potential oestrogen-like activity. To provide insight into the oestrogen-like nature of BPA and NPH, their ability to activate a reporter gene construct via an oestrogen response element in the hormone-dependent breast cancer cell lines MCF7 and T47D was ascertained. Both compounds transactivated the endogenous oestrogen receptor (ER) alpha in a direct fashion since the anti-oestrogen 4-hydroxytamoxifen abolished the response. In addition, using steroid-receptor-negative HeLa cells engineered to express ERalpha and ERbeta and the hormone-binding domains of both ERalpha and ERbeta, BPA and NPH confirmed the direct transcriptional activity. Interestingly these properties were supported in MCF7 cells by the ability to autoregulate ERalpha expression as well as to induce its nuclear compartmentalization. We therefore evaluated by reverse transcriptase polymerase chain reaction the expression of oestrogen-controlled genes such as cathepsin D and TFF1 (formerly pS2), which were increased by both chemicals tested. The agonistic effects exhibited in all assays performed prompted the evaluation of a more complex biological response such as the proliferation of MCF7 and T47D cells. The same concentration of xenoestrogens eliciting substantial transcriptional activity significantly stimulated the proliferation of both breast cancer cell lines, although with a reduced effectiveness with respect to the natural hormone 17beta-oestradiol. The results indicate that the biological action of environmental oestrogen such as BPA and NPH should be taken into account for the potential impact on human disease-like hormone-dependent breast cancer. However, further studies are needed to clarify their bioavailability and metabolism as well as whether compound mixtures could produce noticeable effects by synergistic activity.

Benzhydryl Compounds↗
PubMed1989

Peristaltic transport of a particle-fluid suspension.

Peristaltic pumping by a sinusoidal traveling wave in the walls of a two-dimensional channel filled with a viscous incompressible fluid in which are distributed identical rigid spherical particles, is investigated theoretically. A perturbation solution is obtained which satisfies the momentum equations for the case in which amplitude ratio (wave amplitude/channel half width) is small. The results show that the fluid phase mean axial velocity decreases with increase in the particle concentration. The phenomenon of reflux (the mean flow reversal) is discussed. A reversal of velocity in the neighborhood of the centerline occurs when the pressure gradient is greater than that of the critical reflux condition. It is found that the critical reflux pressure is lower for the particle-fluid suspension than for the particle-free fluid. It is further observed that the mean flow reversal is strongly dependent on the particle concentration and the presence of particles in the fluid favors the reversal flow. A motivation of the present analysis has been the hope that such a theory of two-phase flow process is very useful in understanding the role of peristaltic muscular contraction in transporting bio-fluid behaving like a particle-fluid mixture. Also the theory is important to the engineering applications of pumping solid-fluid mixtures by peristalsis.

Gastrointestinal Motility↗
PubMed1970

Behavioral engineering: control of posture by informational feedback.

The effects of informational feedback on a socially undesirable behavior were studied. The feedback was a mild vibrotactile stimulus and the response was slouching. When subjects slouched, a behavioral engineering apparatus provided vibrotactile stimulation to the shoulder. All subjects slouched less when stimulation was provided. A procedural control revealed that slouching will decrease because of the informational aspect of the stimulus consequence and not because of its aversive properties. When the subjects were instructed to slouch, the effects of feedback were reversed: feedback increased, rather than decreased, slouching. These results indicate that the effect of feedback for a response depends on the subject's motivation to perform that response. It is suggested that informational feedback could be used more widely as a therapeutic procedure to modify human behaviors, but only those behaviors that a patient is strongly motivated to change.

Journal Article↗
PubMed2006

[A potential use of collagen-hyaluronan-chondroitin sulfate tri-copolymer scaffold for cartilage tissue engineering].

OBJECTIVE: To evaluate collagen(Col)-hyaluronan (HA)-chondroitin sulfate (CS) tri-copolymer as a new biomimetic biodegradable polymer scaffold for application of the articular cartilage tissue engineering. METHODS: The Col-HA-CS tri-copolymer was prepared by freezing and lyophilization and was cross-linked by 1-ethyl-3-(3-dimethy inaminoproyl) carbodiimide (EDC). The morphological characteristics of the matrices were evaluated by the SME and HE stainings. The rabbit chondrocytes were isolated and seeded in the tri-copolymer scaffold. Morphology, proliferation and differentiation of glycosaminoglycan (GAG), and phenotypic expression of the rabbit articular chondrocytes cultured within the tri-copolymer scaffold were indicated by the histological examination, SEM, biochemical analysis, and reverse transcriptase PCR for collagen type II (Col II). RESULTS: The chondrocytes proliferated and differentiated well, and they preserved the phenotypic expression of Col II in the Col-HA-CS scaffold. After the 21-day cell culture within the Col-HA-CS scaffolds, the cartilage-specific morphology and the structural characteristics such as lacunae appeared, and DNA and GAG contents increased with the time. In addition, DNA and GAG contents were significantly higher in the Col-HA-CS matrix than in the collagen matrix alone (P < 0.05). CONCLUSION: These results show that the Col-HA-CS tri-copolymer matrices can provide an appropriate environment for the generation of cartilage-like tissues and have a potential application in the cartilage tissue engineering scaffold field.

Animals↗
PubMed2006

Adult rat liver cells transdifferentiated with lentiviral IPF1 vectors reverse diabetes in mice: an ex vivo gene therapy approach.

AIMS/HYPOTHESIS: We examined a clinical model of ex vivo transdifferentiation of primary adult hepatocytes to insulin-secreting cells for the treatment of type 1 diabetes. MATERIALS AND METHODS: Isolated rat hepatocytes were transduced in primary culture with a human lentivirus containing pancreatic duodenal homeobox 1 (PDX1, now known as insulin promoter factor 1, homeodomain transcription factor [IPF1]). Insulin expression and secretion of the newly engineered cells were assessed in vitro by RT-PCR, in situ hybridisation, immunostaining and radioimmunoassay. PDX1-transduced hepatocytes were further studied in vivo by injecting them under the renal capsule of diabetic SCID mice. RESULTS: Isolated rat hepatocytes were efficiently transduced with the lentiviral vector, as assessed by green fluorescent reporter gene expression. The transduced cells exhibited insulin at both mRNA (RT-PCR, in situ hybridisation) and protein levels (immunostaining and radioimmunoassay). Moreover, insulin secretion by the engineered cells was dependent on glucose and sulfonylurea. Other beta cell genes, including those encoding solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a2), glucokinase (Gck), ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (Abcc8), the potassium inwardly-rectifying channel, subfamily J, member 11 (Kcnj11) and proprotein convertase subtilisin/kexin type 1 (Pcsk1) were also expressed. The PDX1-transduced hepatocytes expressed several pancreatic transcription factors related to early pancreatic endocrine development (endogenous Pdx1, neurogenic differentiation factor 1 [Neurod1], and NK6 transcription factor related, locus 1 [Nkx6-1]) as well as the late-stage pancreatic transcription factors (paired box gene 4 [Pax4], paired box gene 6 [Pax6], and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A [Mafa]). Transplantation of 3 x 10(6) transdifferentiated liver cells under the renal capsule of seven streptozotocin-induced diabetic SCID mice resulted in significant reduction of non-fasting blood glucose levels from 30.7 +/- 1.3 to 8.7 +/- 3.7 mmol/l (mean +/- SEM, p = 0.01), in 6 to 8 weeks. Removal of the graft resulted in severe hyperglycaemia. CONCLUSIONS/INTERPRETATION: Ex vivo lentiviral-mediated PDX1 expression in isolated adult liver cells represents a potential model for type 1 diabetes mellitus therapy.

Animals↗
PubMed2002

Mutants of Arabidopsis reveal many roles for membrane lipids.

Polyunsaturated acyl lipids constitute approximately 50% of the hydrophobic membrane barriers that delineate the compartments of cells. The composition of these lipids is critically important for many membrane functions and, thus, for proper growth and development of all living organisms. In the model plant Arabidopsis, the isolation of mutants with altered lipid compositions has facilitated biochemical and molecular approaches to understanding lipid metabolism and membrane biogenesis. Just as importantly, the availability of a series of plant lines with specific changes in membrane lipids have provided a new resource to study the structural and adaptive roles of lipids. Now, the sequencing of the Arabidopsis genome, and the development of reverse-genetics approaches provide the tools needed to make additional discoveries about the relationships between lipid structure and membrane function in plant cells.

Arabidopsis↗
PubMed2004

From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza.

Over the past eight years, cases of human infection with highly pathogenic avian influenza viruses have raised international concern that we could be on the brink of a global influenza pandemic. Many of these human infections have proved fatal and if the viruses had been able to transmit efficiently from person to person, the effects would have been devastating. How can we arm ourselves against this pandemic threat when these viruses are too dangerous to use in conventional vaccine production? Recent technological developments (reverse genetics) have allowed us to manipulate the influenza virus genome so that we can construct safe, high-yielding vaccine strains. However, the transition of reverse-genetic technologies from the research laboratory to the manufacturing environment has presented new challenges for vaccine manufacturers as well as veterinary and public health authorities.

Disease Outbreaks↗
PubMed2004

Germ-line engineering and late-onset diseases: the ethics of self-evolution.

While some claim that germ-line engineering is a definite possibility, the law in Israel and in most countries states that it should be avoided. This paper suggests that using GLE in order to 'self-evolve' (when it becomes safe) is not only inevitable but also morally justified. This paper argues that, The great achievements of healthcare during the last century, enabling longer life, have made almost everyone prey to late-onset diseases. The conundrum of healthcare allocation is worsening, partly due to late-onset dysfunctional genes that have escaped the barriers of natural selection, Trying to free future generations from late-onset diseases (such as Alzheimer's for instance) may be considered as 'eugenics' but, if pursued freely and justly, is a noble goal. We will be affecting future generations whether or not we use GLE. By definition, GLE might be reversible; it follows therefore that GLE may not necessarily represent the dramatic change inserted in the germ line forever--as is usually suggested. Reproductive freedom and justice are paramount in this scenario. These values are not necessarily incompatible if the right policies are in place.

Age of Onset↗
PubMed2004

[Age-related changes in growth and metabolism function of human costal chondrocytes cultured in vitro].

OBJECTIVE: To investigate the expressive changes of collagen I, II and aggrecan during the aging of the human costal chondrocytes cultured in vitro, in order to select the best chondrocytes for cartilage engineering. METHODS: The human costal chondrocytes from the different passages (P1-P5) were harvested. The morphological changes and cell proliferation rate were observed, and the quantity of glycosaminoglycans (GAGs) was examined in each passage with alcian blue precipitation. The expression of collagen I, II and aggrecan was measured with immunohistochemistry and RT-PCR method. RESULTS: From the third passage, the human costal chondrocytes were transformed into fibroblast-like cell morphology; and the GAGs content in each passage was decreased and became significantly low in the third passage. The expression of the collagen I, II was consistent in the protein level with that in the mRNA level. Before the second passage, the expression of collagen II was high while the collagen I was low. After the second passage, the expression of collagen II decreased while the collagen I increased. The expression of aggrecan was high before the third passage, and then became low in the fourth passage. CONCLUSION: With the examinations of the cell proliferating rate and cell functions in human chondrocytes cultured in vitro, the chondrocytes of the second passage seem suitable for human cartilage engineering.

Aggrecans↗
PubMedDate not supplied

Assessing laser-tissue damage with bioluminescent imaging.

Effective medical laser procedures are achieved by selecting laser parameters that minimize undesirable tissue damage. Traditionally, human subjects, animal models, and monolayer cell cultures have been used to study wound healing, tissue damage, and cellular effects of laser radiation. Each of these models has significant limitations, and consequently, a novel skin model is needed. To this end, a highly reproducible human skin model that enables noninvasive and longitudinal studies of gene expression was sought. In this study, we present an organotypic raft model (engineered skin) used in combination with bioluminescent imaging (BLI) techniques. The efficacy of the raft model was validated and characterized by investigating the role of heat shock protein 70 (hsp70) as a sensitive marker of thermal damage. The raft model consists of human cells incorporated into an extracellular matrix. The raft cultures were transfected with an adenovirus containing a murine hsp70 promoter driving transcription of luciferase. The model enables quantitative analysis of spatiotemporal expression of proteins using BLI. Thermal stress was induced on the raft cultures by means of a constant temperature water bath or with a carbon dioxide (CO2) laser (lambda=10.6 microm, 0.679 to 2.262 Wcm2, cw, unfocused Gaussian beam, omegaL=4.5 mm, 1 min exposure). The bioluminescence was monitored noninvasively with an IVIS 100 Bioluminescent Imaging System. BLI indicated that peak hsp70 expression occurs 4 to 12 h after exposure to thermal stress. A minimum irradiance of 0.679 Wcm2 activated the hsp70 response, and a higher irradiance of 2.262 Wcm2 was associated with a severe reduction in hsp70 response due to tissue ablation. Reverse transcription polymerase chain reaction demonstrated that hsp70 mRNA levels increased with prolonged heating exposures. Enzyme-linked immunosorbent protein assays confirmed that luciferase was an accurate surrogate for hsp70 intracellular protein levels. Hematoxylin and eosin stains verified the presence of the thermally denatured tissue regions. Immunohistochemical analyses confirmed that maximal hsp70 expression occurred at a depth of 150 microm. Bioluminescent microscopy was employed to corroborate these findings. These results indicate that quantitative BLI in engineered tissue equivalents provides a powerful model that enables sequential gene expression studies. Such a model can be used as a high throughput screening platform for laser-tissue interaction studies.

Animals↗
PubMed2006

Engineering of primary carbohydrate metabolism for increased production of actinorhodin in Streptomyces coelicolor.

The objectives of the current studies were to determine the roles of key enzymes in central carbon metabolism in the context of increased production of antibiotics in Streptomyces coelicolor. Genes for glucose-6-phosphate dehydrogenase and phosphoglucomutase (Pgm) were deleted and those for the acetyl coenzyme A carboxylase (ACCase) were overexpressed. Under the conditions tested, glucose-6-phosphate dehydrogenase encoded by zwf2 plays a more important role than that encoded by zwf1 in determining the carbon flux to actinorhodin (Act), while the function of Pgm encoded by SCO7443 is not clearly understood. The pgm-deleted mutant unexpectedly produced abundant glycogen but was impaired in Act production, the exact reverse of what had been anticipated. Overexpression of the ACCase resulted in more rapid utilization of glucose and sharply increased the efficiency of its conversion to Act. From the current experiments, it is concluded that carbon storage metabolism plays a significant role in precursor supply for Act production and that manipulation of central carbohydrate metabolism can lead to an increased production of Act in S. coelicolor.

Acetyl-CoA Carboxylase↗
PubMed1998

Resveratrol arrests the cell division cycle at S/G2 phase transition.

Resveratrol (3,5,4'-trihydroxystilbene) is a naturally occurring phytoalexin, found in grapes and wine, which has been reported to exert a variety of important pharmacological effects. We have investigated the activity of resveratrol on proliferation and differentiation of the promyelocitic cell line HL-60. A concentration as low as 30 microM causes a complete arrest of proliferation and a rapid induction of differentiation towards a myelo-monocytic phenotype. Analyses by flow cytometry showed the absence of the G2/M peak and the accumulation of cells in G1 and S phases. Moreover, at the concentrations employed, a very low amount of apoptotic cells was evidenced. A detailed biochemical analysis demonstrated that the G1 phase of the cell division cycle engine was completely unmodified by resveratrol addition, thus indicating that the G1 --> S transition occurs normally. Conversely, after only 24 h treatment, a significant increase of cyclins A and E could be observed along with the accumulation of cdc2 in the inactive phosphorylated form. These data demonstrate that resveratrol causes a complete and reversible cell cycle arrest at the S phase checkpoint.

Anticarcinogenic Agents↗
PubMed2002

A fusion protein system for the recombinant production of short disulfide-containing peptides.

A recombinant fusion protein system for the production, oxidation, and purification of short peptides containing a single disulfide bond is described. The peptides are initially expressed in Escherichia coli as a fusion to an engineered mutant of the N-terminal SH2 domain of the intracellular phosphatase, SHP-2. This small protein domain confers several important properties which facilitate the production of disulfide-containing peptides: (i) it is expressed at high levels in E. coli; (ii) it can be purified via a hexahistidine tag and reverse-phase HPLC; (iii) it contains no endogenous cysteine residues, allowing the formation of an intrapeptide disulfide bond while still attached to the fusion partner; (iv) it is highly soluble in native buffers, facilitating the production of very hydrophobic peptides and the direct use of fusion products in biochemical assays; (v) it contains a unique methionine residue at the junction of the peptide and fusion partner to facilitate peptide cleavage by treatment with cyanogen bromide (CNBr). This method is useful for producing peptides, which are otherwise difficult to prepare through traditional chemical synthesis approaches, and this has been demonstrated by preparing a number of hydrophobic disulfide-containing peptides derived from phage-display libraries.

Amino Acid Sequence↗
PubMed2007

Mass spectrometric identification of serum peptides employing derivatized poly(glycidyl methacrylate/divinyl benzene) particles and mu-HPLC.

Biomarkers play a key role in preclinical screening and diagnosis of a disease. Various support materials are utilized for this task, in combination with MALDI-TOF-MS. The way to effectively bind serum contents and their profiling is well-elaborated by the material-enhanced laser desorption ionization (MELDI) approach. In this particular work, focus is placed on the development of a strategy to identify low molecular weight serum peptides. Poly(GMA/DVB) is derivatized in a way to achieve an affinity termed as immobilized metal ion affinity chromatography (IMAC). Iminodiacetic acid (IDA) is used as a chelating ligand, whereas copper (Cu2+) acts as a metal ion for complexing peptides and proteins out of blood serum. Polymer binds the serum compounds over a broad mass range, which includes low mass peptides and high mass albumin (66 kDa). Bound contents are eluted from material by an acetonitrile/trifluoroacetic acid mixture, which proves the reversible nature of metal and amino acid linkage. Polystyrene/divinyl benzene (PS/DVB) monolithic capillary column is used for fractionation through RP-HPLC, prior to the target spotting. The tandem TOF fragment ion mass spectra of each fraction is acquired and used to search against the Swiss-Prot database, using the Mascot search engine for the identification of peptides.

Amino Acid Sequence↗