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Viral-based modelling and correction of neurodegenerative diseases by RNA interference.

Experimental recapitulation of recessive human genetic neurodegenerative disease in rodents can be classically addressed through genetic disruption of the related gene. Although very informative, this specific gene targeting is restricted to mice and precludes a species scale-up towards non-human primates. Concomitantly, this requirement to silence a specific gene in a broad range of animal models is important in the design of therapeutic approaches to dominantly inherited neurodegenerative diseases. The emergence of RNA interference (RNAi), a highly specific mechanism of post-translational gene silencing, has opened a plethora of biological application ranging from reverse genetic analysis to therapeutic schemes. Recombinant viral vectors, by promoting a long-lasting delivery of genetic instructions in a broad range of cellular types of different species origins, represent potential platforms mandating silencing of specific gene products through RNAi. This review aims at providing an overview of the different viral systems engineered so far for efficient in vitro and in vivo delivery of RNAi instructions. Additionally, the potential of RNAi for functional analysis and therapy for polyglutamine disorders or amyotrophic lateral sclerosis is discussed.

Animals↗

Revertant seedlings from crown gall tumors retain a portion of the bacterial Ti plasmid DNA sequences.

BT37 is a crown gall teratoma incited on tobacco by Agrobacterium tumefaciens containing pTi-T37, a nopaline-type Ti plasmid. Treatment of this cloned tumor tissue with kinetin at 1 mg/liter results in the formation of relatively normal-appearing shoots. These shoots can be induced to root and set viable seed. In contrast to BT37 tissue, the derived tissues are not phytohormone independent and do not produce nopaline. The reverted plants, like normal tobacco plants, are susceptible to infection by A. tumefaciens. This loss of tumorous traits is accompanied by the loss of most of the Ti plasmid sequences (T-DNA) found in BT37 DNA. Southern blot analysis indicates that the revertant tissues have lost the central portion of the T-DNA, which contains the "common DNA" sequences, a highly conserved region of the Ti plasmid that has been found to be incorporated into all tumors studied. Thus, these sequences appear necessary for oncogenicity and tumor maintenance and their loss is probably directly related to tumor reversal. The reverted plants as well as the plants obtained from seed, however, do retain sequences homologous to the ends of the T-DNA present in the parental teratoma. The persistence of foreign DNA sequences during the process of meiosis and seed formation has important implications for the possibility of the genetic engineering of plants.

Journal Article↗

Insulin-secreting pituitary GH3 cells: a potential beta-cell surrogate for diabetes cell therapy.

In a companion article, we describe the engineering and characterization of pituitary GH3 cell clones stably transfected with a furin-cleavable human insulin cDNA (InsGH3 cells). This article describes the performance of InsGH3 (clones 1 and 7) cell grafts into streptozotocin (STZ)-induced diabetic nude mice. Subcutaneous implantation of 2 x 10(6) InsGH3 cells resulted in the progressive reversal of hyperglycemia and diabetic symptoms, even though the progressive growth of the transplanted cells (clone 7) eventually led to glycemic levels below the normal mouse range. Proinsulin transgene expression was maintained in harvested InsGH3 grafts that, conversely, lose the expression of the prolactin (PRL) gene. Elevated concentrations of circulating mature human insulin were detected in graft recipients, demonstrating that proinsulin processing by InsGH3 cells did occur in vivo. Histologic analysis showed that transplanted InsGH3 grew in forms of encapsulated tumors composed of cells with small cytoplasms weakly stained for the presence of insulin. Conversely, intense insulin immunoreactivity was detected in graft-draining venules. Compared to pancreatic betaTC3 cells, InsGH3 cells showed in vitro a higher rate of replication, an elevate resistance to apoptosis induced by serum deprivation and proinflammatory cytokines, and significantly higher antiapoptotic Bcl-2 protein levels. Moreover, InsGH3 cells were resistant to the streptozotocin toxicity that, in contrast, reduced betaTC3 cell viability to 50-60% of controls. In conclusion, proinsulin gene expression and mature insulin secretion persisted in transplanted InsGH3 cells that reversed hyperglycemia in vivo. InsGH3 cells might represent a potential beta-cell surrogate because they are more resistant than pancreatic beta cells to different apoptotic insults and might therefore be particularly suitable for encapsulation.

Animals↗

The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage.

Commercially available 125I-labeled erythropoietin, obtained by genetic engineering from a human gene, was used to characterize receptors for this hormone on the cell surface of rat erythroid progenitor cells. A low number of high affinity binding sites (487 +/- 32 sites/cell, Kd = 167 +/- 14 pm) were found. Nonerythroid cells and erythrocytes did not exhibit specific binding. The high affinity binding was reversible and displaced by unlabeled erythropoietin, but not by other hormones and growth factors. After incubation at 37 degrees C, nearly 35% of the specifically bound erythropoietin seemed to be internalized, as judged by resistance to acidic buffer treatment. Thus, binding showed characteristics of a hormone-receptor association. 125I-Erythropoietin-labeled cells were treated with the bifunctional reagent dissucinimidyl suberate. Analysis of the cellular extracts by polyacrylamide gel electrophoresis under denaturing and reducing conditions revealed that erythropoietin can be cross-linked to two molecules of 94 and 78 kDa, respectively. Both labeled bands disappeared when the cells were labeled in the presence of an excess of unlabeled erythropoietin. Under nonreducing conditions, a cross-linked band of 230-255 kDa was observed. The relationships between these bands are discussed.

Animals↗

Dynamic and reversible changes of interstitial cell phenotype during remodeling of cardiac valves.

BACKGROUND AND AIM OF THE STUDY: The roles of cardiac valvular interstitial cells (VIC) in extracellular matrix remodeling in fetal development, adaptation and response to injury are largely unknown. METHODS: The phenotype of VIC was studied in health (normal adult human and sheep), development (fetal human and sheep), disease (human mitral valves with myxomatous degeneration), adaptation (clinical pulmonary to aortic valve autografts) and tissue-engineered heart valves matured in vitro and remodeled in vivo. Cell phenotype was assessed using expression of vimentin (V), alpha-smooth muscle actin (SMA, A), matrix metalloproteinase (MMP)-13/collagenase-3 (M), and SMemb (S). RESULTS: VIC in normal adult valves were predominantly quiescent fibroblasts immunoreactive to vimentin (89.7 +/- 2.5%), but not MMP-13 or SMemb, with only 2.5 +/- 0.4% of alpha-SMA-positive cells ('normal/quiescent' phenotype: V+/A-/M-/S-). In contrast, fetal VIC were mostly activated myofibroblasts ('developing/activated' phenotype: V+/A+/M+/S+), with 62.1 +/- 5.0% of cells staining positive for alpha-SMA. VIC in myxomatous valves, short-term autografts and engineered valves in vitro were also activated myofibroblasts with coexpression of vimentin, alpha-SMA (36.2 +/- 3.7%, 19.3 +/- 2.4%, and 60.3 +/- 9% positive cells, respectively), strong MMP-13 activity indicative of collagen remodeling, and SMemb ('remodeling/activated' phenotype: V+/A+/M+/S+). In contrast, VIC in long-term pulmonary autografts and engineered valve explants had a mostly fibroblast-like phenotype, with sparse alpha-SMA expression (6.0 +/- 1% and 5.4 +/- 1.0% positive cells) (V+/A-/M-/S-). CONCLUSION: Most VIC in normal valves were quiescent with a fibroblast-like phenotype. VIC in developing, diseased, adapting and engineered valves adjust to a dynamic environment through VIC activation and secretion of proteolytic enzymes mediating extracellular matrix remodeling ('developing/ remodeling/activated' phenotype), followed by a normalization of phenotype.

Actins↗

Effect of passage number and matrix characteristics on differentiation of endothelial cells cultured for tissue engineering.

Cells can sense the physical and chemical properties of artificial materials used as scaffolds for tissue engineering and regulate their behavior. Therefore, biomimetic and biospecific molecules are coated on materials to regulate function of cells on the tissue-engineered product. These bioactive molecules can be attached in a defined spectrum, concentration and spatial distribution in order to control adhesion, growth, viability, differentiation, and function of the cells. When autologous cells are used for tissue engineering, initially limited cells obtained may often need an amplification of cell number by passage in tissue culture before they are seeded on a biomaterial or scaffold. We have conducted this study to understand how the characteristics of bioactive molecule coating might affect proliferation, apoptosis and differentiation when endothelial cell (EC) is serially passaged. Proliferation was assessed by proliferating cell nuclear antigen (PCNA) staining along with counting of cells harvested from confluent monolayer. Apoptosis was assessed by Annexin V staining and differentiation by semi quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for von Willebrand factor (vWF) expression and quantification of its release using enzyme linked immunosorbant assay (ELISA), and thrombogenicity by comparing platelet adhesion to EC monolayer Dacron grafts (DG) with specific protein coating. The results indicate that ECs easily lose its proliferation potential when they are cultured repeatedly on gelatin, turn apoptotic and over express the prothrombotic protein- vWF. Whereas, when it is grown on a matrix composed of fibrin, fibronectin, gelatin and vascular EC growth factor (VEGF), the cells retained their ability to proliferate, remained viable and were relatively less thrombogenic, even when passage number progressed. It is concluded that if ECs are grown on the composite matrix that mimics natural vessel scaffold, the cell number can be amplified without affecting its normal physiological function and may be used to generate effective tissue-engineered cardiovascular constructs.

Cell Adhesion↗

Encapsulation of chondrocytes in photopolymerizable styrenated gelatin for cartilage tissue engineering.

We have developed a photopolymerizable styrenated gelatin that can cross-link through polymerization induced by irradiation with visible light. The purpose of this study was to investigate the feasibility of using photopolymerizable styrenated gelatin as a cell carrier in chondrocyte transplantation. As visible light activates camphorquinone added as a photoinitiator, free radicals induce the polymerization of the gelatin macromer; the styrenated gelatin then becomes cross-linked. Rabbit articular chondrocytes were cultured in styrenated gelatin hydrogels and also in collagen gels as a control. After being cultured in the gels, the cells were collected from both gels and counted. Reverse transcriptase-polymerase chain reaction, histological examination, and quantification of the synthesized glycosaminoglycan were performed. On average, 26% of the embedded cells were collected from the gelatin hydrogel immediately after the crosslinking reaction. The surviving chondrocytes expressed the mRNA of type II collagen and aggrecan core protein and produced a cartilaginous matrix throughout the gelatin after 3 weeks. A slightly insufficient accumulation of the matrix was found in the internal region of the gelatin hydrogels, suggesting that less permeability for nutrients due to the high concentration and closely packed structure resulted in less cell viability. Although some limitations became evident, these results indicate that it may be possible to use photopolymerizable styrenated gelatin as a cell carrier in chondrocyte transplantation.

Animals↗

Microbial conversion of glycerol to 1,3-propanediol: physiological comparison of a natural producer, Clostridium butyricum VPI 3266, and an engineered strain, Clostridium acetobutylicum DG1(pSPD5).

Clostridium acetobutylicum is not able to grow on glycerol as the sole carbon source since it cannot reoxidize the excess of NADH generated by glycerol catabolism. Nevertheless, when the pSPD5 plasmid, carrying the NADH-consuming 1,3-propanediol pathway from C. butyricum VPI 3266, was introduced into C. acetobutylicum DG1, growth on glycerol was achieved, and 1,3-propanediol was produced. In order to compare the physiological behavior of the recombinant C. acetobutylicum DG1(pSPD5) strain with that of the natural 1,3-propanediol producer C. butyricum VPI 3266, both strains were grown in chemostat cultures with glycerol as the sole carbon source. The same "global behavior" was observed for both strains: 1,3-propanediol was the main fermentation product, and the qH2 flux was very low. However, when looking at key intracellular enzyme levels, significant differences were observed. Firstly, the pathway for glycerol oxidation was different: C. butyricum uses a glycerol dehydrogenase and a dihydroxyacetone kinase, while C. acetobutylicum uses a glycerol kinase and a glycerol-3-phosphate dehydrogenase. Secondly, the electron flow is differentially regulated: (i) in C. butyricum VPI 3266, the in vitro hydrogenase activity is 10-fold lower than that in C. acetobutylicum DG1(pSPD5), and (ii) while the ferredoxin-NAD+ reductase activity is high and the NADH-ferredoxin reductase activity is low in C. acetobutylicum DG1(pSPD5), the reverse is observed for C. butyricum VPI 3266. Thirdly, lactate dehydrogenase activity is only detected in the C. acetobutylicum DG1(pSPD5) culture, explaining why this microorganism produces lactate.

Clostridium acetobutylicum↗

Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments.

Bone marrow stromal cells (BMSCs) play a central role in the repair and regeneration of mesenchymal tissues. For tissue engineering of ligaments and tendons, both stimulation of cell proliferation and differentiation with increased expression of essential extracellular matrix proteins and cytoskeletal elements are desirable. This study analyzes the effect of low-dose (3 ng/mL) fibroblast growth factor 2 (FGF-2) and high-dose FGF-2 (30 ng/mL) on proliferation (bromodeoxyuridine content, spectrophotometry), differentiation (transcription of collagen I, collagen III, fibronectin, elastin, alpha-smooth muscle actin, and vimentin, reverse transcription-polymerase chain reaction, and cell density and apoptosis (annexin V, fluorescence-activated cell sorting) of human BMSCs, and compares the results with those of a control group without FGF-2. Low-dose FGF-2 triggered a biphasic BMSC response: on day 7, cell proliferation reached its maximum and was significantly higher compared with the other groups. On days 14 or 28, collagen I, collagen III, fibronectin, and alpha- smooth muscle actin mRNA expression was significantly enhanced in the presence of low-dose FGF-2. In contrast, high-dose FGF-2 did not stimulate differentiation or proliferation. Vimentin mRNA was expressed only in cultures with low-dose and high-dose FGF-2 after 14 and 28 days. Cell density was significantly higher in cultures with low-dose FGF-2 compared with the group with high-dose FGF-2 on days 7, 14, and 28. The apoptosis rate remained stable, at a rather high level, in all groups. Microscopic investigation of the cell cultures with low-dose FGF-2 showed more homogeneous, dense, fibroblast-like, spindle-shaped cells with long cell processes compared with cultures with high-dose, or no FGF-2. Low-dose FGF-2 may be useful for tissue engineering of ligaments and tendons by increasing BMSC proliferation and stimulating mRNA expression of specific extracellular matrix proteins and cytoskeletal elements.

Bone Marrow Cells↗

Novel food-grade plasmid vector based on melibiose fermentation for the genetic engineering of Lactococcus lactis.

The alpha-galactosidase gene (aga) and a gene coding for a putative transcriptional regulator from the LacI/GalR family (galR) of Lactococcus raffinolactis ATCC 43920 were cloned and sequenced. When transferred into Lactococcus lactis and Pediococcus acidilactici strains, aga modified the sugar fermentation profile of the strains from melibiose negative (Mel(-)) to melibiose positive (Mel(+)). Analysis of galA mutants of L. lactis subsp. cremoris MG1363 indicated that the putative galactose permease GalA is also needed to obtain the Mel(+) phenotype. Consequently, GalA may also transport melibiose into this strain. We demonstrated that when aga was associated with the theta-type replicon of a natural L. lactis plasmid, it constituted the selectable marker of a cloning vector named pRAF800. Transcriptional analysis by reverse transcriptase PCR suggests that this vector is also suitable for gene expression. The alpha-galactosidase activity conferred by pRAF800 was monitored in an industrial strain grown in the presence of various carbon sources. The results indicated that the enzymatic activity was induced by galactose and melibiose, but not by glucose or lactose. The gene encoding the phage defense mechanism, AbiQ, was cloned into pRAF800, and the resulting clone (pRAF803) was transferred into an industrial L. lactis strain that became highly phage resistant. The measurements of various growth parameters indicated that cells were not affected by the presence of pRAF803. Moreover, the plasmid was highly stable in this strain even under starter production conditions. The L. raffinolactis aga gene represents the basis of a novel and convenient food-grade molecular tool for the genetic engineering of lactic acid bacteria.

Base Sequence↗

Effects of aminoguanidine against renal ischaemia-reperfusion injury in rats.

Aminoguanidine is an inhibitor of nitric oxide synthase (NOS), with high selectivity for the inducible isoform (iNOS). In addition to being an inhibitor of NOS, aminoguanidine also exhibits antioxidant activity. Recent studies suggest that aminoguanidine reduces ischaemia-reperfusion (I/R)-induced damage. However, the role of aminoguanidine, in renal injury associated with I/R remains unknown. This study was designed to investigate the effects of aminoguanidine on renal I/R injury. There were three groups of eight rats each. I/R was induced by occlusion of the left renal vessels for 60 min, followed by 24 h reperfusion in rats. Malondialdehyde (MDA) levels, a stable metabolite of the free radical-mediated lipid peroxidation cascade, were found to be significantly higher in the I/R group (30.3 +/- 0.1 nmol g(-1) tissue) than in the control group (10 +/- 0.05 nmol g(-1)). Aminoguanidine (100 mg kg(-1)) administration to rats significantly reduced the MDA values. We also demonstrated that I/R leads to structural change but aminoguanidine did not reverse this change. Aminoguanidine, according to the biochemical finding is protective but histopathological findings did not reveal protection against I/R injury in kidney. The effects of aminoguanidine on I/R-induced damage remain a subject for future investigations.

Animals↗

Full-speed mammalian genetics: in vivo target validation in the drug discovery process.

The completion of the Human Genome Project has signaled the beginning of the post-genome era, with a corresponding shift in focus from the sequencing and identification of genes to the exploration of gene function. A rate-limiting step in deriving value from this gene sequence information is determining the potential pharmaceutical applications of genes and their encoded proteins. This validation step is crucial for focusing efforts and resources on only the most promising targets. Strategies using reverse mouse genetics provide excellent methods for validating potential targets and therapeutic proteins in vivo in a mammalian model system.

Animals↗

Hydrogenation and hydrocarbonation and etching of single-walled carbon nanotubes.

We present a systematic experimental investigation of the reactions between hydrogen plasma and single-walled carbon nanotubes (SWNTs) at various temperatures. Microscopy, infrared (IR) and Raman spectroscopy, and electrical transport measurements are carried out to investigate the properties of SWNTs after hydrogenation. Structural deformations, drastically reduced electrical conductance, and an increased semiconducting nature of SWNTs upon sidewall hydrogenation are observed. These changes are reversible upon thermal annealing at 500 degrees C via dehydrogenation. Harsh plasma or high temperature reactions lead to etching of nanotubes likely via hydrocarbonation. Smaller SWNTs are markedly less stable against hydrocarbonation than larger tubes. The results are fundamental and may have implications to basic and practical applications including hydrogen storage, sensing, band gap engineering for novel electronics, and new methods of manipulation, functionalization, and etching of nanotubes.

Journal Article↗

Gene silencing by the tRNA maturase tRNase ZL under the direction of small-guide RNA.

We have been developing a unique system for the downregulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3'-processing endoribonuclease (tRNase Z(L)) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase Z(L) in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase Z(L) method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase Z(L) directed by sgRNA not owing to a simple antisense effect. We also show that tRNase Z(L) together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3beta by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.

Animals↗

Electron and energy transfer modulation with photochromic switches.

This tutorial review illustrates how work on the reversible interconversion between the colorless and colored forms of photochromic compounds can be exploited to modulate electron and energy transfer processes. Indeed, a photochrome can be designed to accept electrons or energy from a complementary donor in one of its two states only. Alternatively, the photoinduced transformations associated with a photochromic switch can be engineered to control the relative orientation and distance of donor-acceptor pairs. If either the donor or the acceptor is fluorescent, the photoregulated transfer of energy or electrons results in the modulation of the emission intensity. Thus, these fascinating molecular and supramolecular systems can advance the basic understanding of electron and energy transfer processes, while leading to viable operating principles to control light with light.

Benzene Derivatives↗

Complete inhibition and partial Re-activation of single F1-ATPase molecules by tentoxin: new properties of the re-activated enzyme.

During hydrolysis of ATP, the gamma subunit of the rotary motor protein F(1)-ATPase rotates within a ring of alpha(3)beta(3) subunits. Tentoxin is a phyto-pathogenic cyclic tetrapeptide, which influences F(1)-ATPase activity of sensitive species. At low concentrations, tentoxin inhibits ATP hydrolysis of ensembles of F(1) molecules in solution. At higher concentrations, however, ATP hydrolysis recovers. Here we have examined how tentoxin acts on individual molecules of engineered F(1)-ATPase from the thermophilic Bacillus PS3 (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119). We found that inhibition by tentoxin caused a virtually complete stop of rotation, which was partially relieved at higher tentoxin concentrations. Re-activation, however, was not simply a reversal of inhibition; while the torque appears unaffected as compared with the situation without tentoxin, F(1) under re-activating conditions was less susceptible to inhibitory ADP binding but displayed a large number of short pauses, indicating infringed energy conversion.

ATP Synthetase Complexes↗

Improved production by domain inversion of single-chain Fv antibody fragment against high molecular weight proteoglycan for the radioimmunotargeting of melanoma.

Melanoma is among the few cancers with rising incidence. Currently there is no effective treatment for metastatic disease, but improved detection of melanoma has the potential to benefit the management of patients with early disease. Radioimmunodection by imaging with single-chain Fv (scFv) antibody fragments is one such emerging diagnostic method. However, the amount of scFv that can be produced at a scale suitable for use in patients is limiting. We have previously shown that the bacterial expression of a scFv derived from a monoclonal antibody (MAb) specific for melanoma-associated proteoglycan can be increased by light chain shuffling. In this report we show that a further increase in expression yield can be obtained by reversing the usual V(H)-V(L) orientation of scFvs to V(L)-V(H). Such seemingly minor changes have previously been reported to have unexpected effects on the in vitro and in vivo binding properties of recombinant antibodies. Our results show that reversal of the V domain orientation of the scFv improves expression by 150% without an adverse effect on melanoma binding in vitro and tumor targeting in vivo. Therefore, our results show that alteration of V domain orientation can improve the production yield of clinically useful antibody fragments. When used in combination with other antibody engineering approaches for increased antibody production changing the domain orientation is a simple strategy to achieve significant improvements in the production of scFvs for tumor radioimmunodetection for patient studies.

Antibodies, Neoplasm↗

ThermoEnergy Ammonia Recovery Process for municipal and agricultural wastes.

The Ammonia Recovery Process (ARP) is an award-winning, low-cost, environmentally responsible method of recovering nitrogen, in the form of ammonia, from various dilute waste streams and converting it into concentrated ammonium sulfate. The ThermoEnergy Biogas System utilizes the new chemisorption-based ARP to recover ammonia from anaerobically digested wastes. The process provides for optimal biogas production and significantly reduced nitrogen levels in the treated water discharge. Process flows for the ammonia recovery and ThermoEnergy biogas processes are presented and discussed. A comparison with other techniques such as biological nitrogen removal is made. The ARP technology uses reversible chemisorption and double salt crystal precipitation to recover and concentrate the ammonia. The ARP technology was successfully proven in a recent large-scale field demonstration at New York City's Oakwood Beach Wastewater Treatment Plant, located on Staten Island. This project was a joint effort with Foster Wheeler Environmental Corporation, the Civil Engineering Research Foundation, and New York City Department of Environmental Protection. Independent validated plant data show that ARP consistently recovers up to 99.9% of the ammonia from the city's centrate waste stream (derived from dewatering of sewage sludge), as ammonium sulfate. ARP technology can reduce the nitrogen (ammonia) discharged daily into local bodies of water by municipalities, concentrated animal farming operations, and industry. Recent advances to ARP enhance its performance and economic competitiveness in comparison to stripping or ammonia destruction technologies.

Agriculture↗