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At least 919 records · Page 51Linked to original sources
PubMed2005

A modular and supramolecular approach to bioactive scaffolds for tissue engineering.

Bioactive polymeric scaffolds are a prerequisite for the ultimate formation of functional tissues. Here, we show that supramolecular polymers based on quadruple hydrogen bonding ureido-pyrimidinone (UPy) moieties are eminently suitable for producing such bioactive materials owing to their low-temperature processability, favourable degradation and biocompatible behaviour. Particularly, the reversible nature of the hydrogen bonds allows for a modular approach to gaining control over cellular behaviour and activity both in vitro and in vivo. Bioactive materials are obtained by simply mixing UPy-functionalized polymers with UPy-modified biomolecules. Low-molecular-weight bis-UPy-oligocaprolactones with cell adhesion promoting UPy-Gly-Arg-Gly-Asp-Ser (UPy-GRGDS) and the synergistic UPy-Pro-His-Ser-Arg-Asn (UPy-PHSRN) peptide sequences are synthesized and studied. The in vitro results indicate strong and specific cell binding of fibroblasts to the UPy-functionalized bioactive materials containing both UPy-peptides. An even more striking effect is seen in vivo where the formation of single giant cells at the interface between bioactive material and tissue is triggered.

3T3 Cells↗
PubMed1998

Attenuation of influenza A virus mRNA levels by promoter mutations.

We have engineered influenza A/WSN/33 viruses which have viral RNA (vRNA) segments with altered base pairs in the conserved double-stranded region of their vRNA promoters. The mutations were introduced into the segment coding for the neuraminidase (NA) by using a reverse genetics system. Two of the rescued viruses which share a C-G-->A-U double mutation at positions 11 and 12' at the 3' and 5' ends of the NA-specific vRNA, respectively, showed approximately a 10-fold reduction of NA levels. The mutations did not dramatically affect the NA-specific vRNA levels found in virions or the NA-specific vRNA and cRNA levels in infected cells. In contrast, there was a significant decrease in the steady-state levels of NA-specific mRNAs in infected cells. Transcription studies in vitro with ribonucleoprotein complexes isolated from the two transfectant viruses indicated that transcription initiation of the NA-specific segment was not affected. However, the majority of NA-specific transcripts lacked poly(A) tails, suggesting that mutations in the double-stranded region of the influenza virus vRNA promoter can attenuate polyadenylation of mRNA molecules. This is the first time that a promoter mutation in an engineered influenza virus has shown a differential effect on influenza virus RNA transcription and replication.

Animals↗
PubMed1998

Cre-loxP-mediated DNA flip-flop in mammalian cells leading to alternate expression of retrovirally transduced genes.

While DNA excision by Cre-loxP homologous recombination has been exploited for mammalian genetic engineering, it has not been reported whether DNA inversion is achievable by the same mechanism in mammalian cells. To investigate whether Cre-loxP-mediated DNA inversion takes place in mammalian cells, a novel retroviral vector, NT(FF), was constructed. The vector carries a marker gene cassette consisting of the neo and tk genes linked tail-to-tail to each other and flanked by an inverted repeat of loxP sequences. In NT(FF)-transduced Rat2 cells, the marker gene cassette was inverted reversibly in a Cre-dependent manner, leading to DNA "flip-flop" associated with alternate expression of the neo and tk genes. This study provides the first example of Cre-loxP-mediated DNA inversion in mammalian cells facilitating regulation of retrovirally transduced genes, suggesting the usefulness of the system for genetic engineering.

Animals↗
PubMed2006

Quantification of homogentisate-1,2-dioxygenase expression in a fungus degrading ethylbenzene.

A quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) assay was utilized to quantify the expression of ElHDO in the fungus Exophiala lecanii-corni during the biodegradation of ethylbenzene and other volatile organic pollutants. The assay was applied to measure the impact of pollutant mixtures on ElHDO expression relative to that of a housekeeping gene (18S rRNA). Three compounds were tested in mixtures with ethylbenzene: methyl propyl ketone, phenylacetate and o-xylene. These chemicals repressed, induced, or had no effect on ethylbenzene degradation, respectively. The results demonstrate that the gene target expression value (T(N)) is a useful parameter for evaluating the effect of pollutant mixtures on gene expression. T(N) was found to reflect macroscopic changes in ethylbenzene utilization rates although these two parameters were not related in a linear fashion for all compounds. The assay was log-linear over 5 orders of magnitude of RNA concentration and reproducible between samples (the largest T(N) standard deviation was 20%). The comparative qRT-PCR assay used in this research represents a viable alternative to absolute quantification methods to monitor in situ fungal gene expression in natural and engineered environmental systems.

Benzene Derivatives↗
PubMed2005

Selective inhibition of type X collagen expression in human mesenchymal stem cell differentiation on polymer substrates surface-modified by glow discharge plasma.

Recent evidence indicates that a major drawback of current cartilage- and disc-tissue engineering is that human mesenchymal stem cells (MSCs) rapidly express type X collagen-a marker of chondrocyte hypertrophy associated with endochondral ossification. Some studies have attempted to use growth factors to inhibit type X collagen expression, but none to date has addressed the possible effect of the substratum on chondrocyte hypertrophy. Here, we sought to examine the growth and differentiation potential of human MSCs cultured on two polymer types, polypropylene and nylon-6, both of which have been surface-modified by glow discharge plasma treatment in ammonia gas. Cultures were performed for up to 14 days in Dulbecco's modified Eagle medium + 10% fetal bovine serum. Commercial polystyrene culture dishes were used as control. Reverse transcriptase-polymerase chain reaction was used to assess the expression of types I, II, and X collagens and aggrecan using gene-specific primers. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. Types I and X collagens, as well as aggrecan, were found to be constitutively expressed by human MSCs on polystyrene culture dishes. Whereas both untreated and treated nylon-6 partially inhibited type X collagen expression, treated polypropylene almost completely inhibited its expression. These results indicate that plasma-treated polypropylene or nylon-6 may be a suitable surface for inducing MSCs to a disc-like phenotype for tissue engineering of intervertebral discs in which hypertrophy is suppressed.

Aged↗
PubMed2000

Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes.

The search for biocompatible materials that can support the growth and phenotypic expression of osteoblasts and chondrocytes is a major challenge in the application of tissue engineering techniques for the repair of bone and cartilage defects. Chitosan, a copolymer of glucosamine and N-acetylglucosamine, may provide an answer to this search. Chitosan is the deacetylated product of chitin, a ubiquitous biopolymer found in the exoskeleton of insects and marine invertebrates. Little is known about the utility of chitosan in propagating human osteoblasts and chondrocytes. In this study, we test the hypothesis that chitosan promotes the survival and function of osteoblasts and chondrocytes. Chitosan (4%, w/v in 2% HAc) was coated onto plastic coverslips that had been fitted into 24-well plates. Human osteoblasts and articular chondrocytes were seeded on either uncoated or chitosan-coated coverslips at 1 x 10(5)/cells per well. Cultures were incubated at 37 degrees C, 5% CO(2) for a period of 7 days. Cell viability was assessed at that time using a fluorescent molecular probe. The phenotypic expression of osteoblasts and chondrocytes was analyzed by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Osteoblasts and chondrocytes appeared spherical and refractile on chitosan-coated coverslips. In contrast, greater than 90% of cells on plastic coverslips were elongated and spindle shaped after 7 days of culture. Similar to cells propagated on uncoated control wells, greater than 90% of human osteoblasts and chondrocytes propagated on chitosan remained viable. Human osteoblasts propagated on chitosan films continued to express collagen type I whereas chondrocytes expressed collagen type II and aggrecan, as shown by reverse transcriptase-polymerase chain reaction analysis and immunostaining. The present in vitro work demonstrates the biocompatibility of chitosan as a substrate for the growth and continued function of human osteoblasts and chondrocytes. Chitosan may have potential use as a tissue engineering tool for the repair of osseous and chondral defects.

Aggrecans↗
PubMed2005

Ribonucleoprotein particle formation is necessary but not sufficient for LINE-1 retrotransposition.

Long interspersed elements (LINE-1s or L1s) are abundant non-LTR retrotransposons that mobilize through an RNA intermediate by target site primed reverse transcription. The L1-encoded proteins (ORF1p and ORF2p) preferentially associate with their encoding transcript to form a ribonucleoprotein particle (RNP), which is a proposed retrotransposition intermediate. Here, we have used epitope tagging to discriminate the proteins encoded by engineered L1s from those encoded by endogenously expressed L1s. We demonstrate that an L1 containing an epitope tag at the carboxyl terminus of ORF1p remains retrotransposition-competent and that tagged ORF1p and its encoding RNA localize to cytoplasmic RNPs. We also identified two classes of ORF1p mutants, one that severely decreased RNP formation and blocked retrotransposition, and another that allows RNP formation but reduces retrotransposition by 100-fold. Thus, these data indicate that RNP formation is important but not sufficient for L1 retrotransposition and suggest that ORF1p also may function at downstream steps in the L1 retrotransposition pathway.

Blotting, Western↗
PubMed2006

Growth factor effects on passaged TMJ disk cells in monolayer and pellet cultures.

OBJECTIVES: Previously, we demonstrated rapid changes in temporomandibular joint (TMJ) disk gene expression during monolayer expansion. This study's objective was to investigate the ability of pellet culture and growth factors to rescue TMJ disk gene expression changes. DESIGN: Temporomandibular joint disk cells were isolated from mature porcine tissue and passaged up to five times. At each passage, 300 000 cells were placed in a monolayer or pellet culture environment before being exposed to transforming growth factor-beta 3 (TGF-beta3) (5 ng/ml), TGF-beta1 (5 ng/ml), and insulin-like growth factor I (IGF-I) (10 ng/ml). OUTCOME MEASURE: After 24 h, gene expression was analyzed via reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Pelleting was detrimental to TMJ disk gene expression, marked by gene expression decreases in collagen type I (5.5-fold), aggrecan (1.4-fold), decorin (0.73-fold), and biglycan (0.73-fold) relative to monolayer cultures. IGF-I, TGF-beta1, and TGF-beta3 demonstrated limited ability to rescue TMJ disk gene expression in the pellet culture. In monolayer, TGF-beta3 and TGF-beta1 increased decorin and biglycan gene expression relative to passaged controls. Collagen type I expression, the TMJ disk's primary matrix constituent, was highest in TGF-beta3 cultures; however, differences were not statistically significant. CONCLUSION: These results indicate that pellet cultures are a poor choice for TMJ disk tissue engineering, and the effects of TGF-beta1, TGF-beta3, and IGF-I on TMJ disk gene expression are minimal relative to passaging and pelleting effects.

Aggrecans↗
PubMed2005

Crystal engineering of nonporous organic solids for methane sorption.

The low density polymorph of the well-known host p-tert-butylcalix[4]arene absorbs more methane than p-tert-pentylcalix[4]arene at room temperature and 1 atm pressure, but the order of absorption is reversed at 38 atm with p-tert-pentylcalix[4]arene absorbing more.

Journal Article↗
PubMed2005

Evolution of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes in the yeast Saccharomyces cerevisiae.

The shikimate pathway resulting in three aromatic amino acids is initiated in different organisms by two and three 3-deoxy-d-arabino-heptulosonate-7-phosphate synthases, respectively. Aro3p and Aro4p are the yeast enzymes feedback-inhibited by phenylalanine and tyrosine, respectively. A yeast strain deficient in the general control transcriptional regulatory system of amino acid biosynthesis is unable to live in the presence of high amounts of phenylalanine and tyrosine. Here, we show that this yeast strain can be rescued by the expression of aroH from Escherichia coli encoding the tryptophan-regulated AroH as third isoenzyme. Yeast carrying Ec AroH as the only enzyme for the initial step of the shikimate pathway can grow in the absence of tryptophan. Without aromatic amino acids, this yeast strain survives only when the yeast ARO3 promoter instead of the ARO4 promoter drives E. coli aroH. The detailed analysis of Aro3p and Aro4p revealed a triple feedback control by tyrosine/phenylalanine and tryptophan. Dissecting this control allowed engineering of Aro4p S195A as an enzyme, which is inhibited like AroH only by tryptophan. In addition, Aro4p variants were constructed that show an equally strong inhibition by tyrosine and tryptophan (Aro4p P165G Q302R) and in which the regulation by tyrosine and tryptophan was reversed (Aro4p P165G). Our data suggest that yeast possesses only two instead of three isogenes encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthases because both isoenzymes can be fine tuned by tryptophan as additional effector and because transcriptional regulation by the general control system can be induced as backup when aromatic amino acids in the environment are imbalanced.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2000

Paracrine delivery of IL-12 against intracranial 9L gliosarcoma in rats.

OBJECT: Interleukin-12 (IL- 12) has potential for the treatment of tumors because it can stimulate an antitumor immune response and possesses antiangiogenic properties. In the study reported here, the authors investigated the therapeutic role of locally delivered IL-12 in a malignant brain tumor model. METHODS: After genetically engineering 9L gliosarcoma cells to express IL-12 (9L-IL12 cells), the authors used these cells as a source of locally delivered cytokine. First, they investigated the behavior of these cells, which were implanted with the aid of stereotactic guidance into the rat brain, by using serial magnetic resonance imaging and histopathological examination. Second, they assessed the antitumor efficacy of proliferating, as well as nonproliferating (irradiated), 9L-IL12 cells by implanting these cells in animals challenged by wild-type 9L gliosarcoma (9Lwt) cells. The IL-12 expression in brain regions injected with 9L-IL12 was confirmed by reverse transcription-polymerase chain reaction. Last, the authors explored whether animals treated with 9L-IL12 cells developed an antitumor immunological memory by rechallenging the survivors with a second injection of 9Lwt cells. The authors demonstrated that local delivery of IL-12 into the rat brain by genetically engineered cells significantly prolongs survival time in animals challenged intracranially with a malignant glioma. CONCLUSIONS: These findings support continued efforts to refine local delivery systems of IL-12 in an attempt to bring this therapy to clinical trials.

Animals↗
PubMed1996

Native and non-native structure in a protein-folding intermediate: spectroscopic studies of partially reduced IGF-I and an engineered alanine model.

The structure of a metastable folding intermediate of human insulin-like growth factor I (IGF-I) and an engineered model are investigated by circular dichroism and two-dimensional 1H NMR spectroscopy. The intermediate, which contains two of three native disulfide bonds, was trapped by acid quenching and isolated by reverse-phase HPLC. The reduced cysteine residues were mapped to residues 47 and 52 (corresponding to A6-A11 in insulin). In the native state this disulfide bridge anchors an adjoining amphipathic alpha-helix (helix 2; residues 42 to 49) against the hydrophobic core. Comparison of CD and 1H-NMR spectra demonstrates that the acid-quenched intermediate is partially folded and contains elements of native secondary and tertiary structure. Spectra are similar to those of an equilibrium model in which the reduced cysteine residues are replaced by alanine. Complete 1H-NMR sequential assignment of the alanine model has been obtained and demonstrates that removal of the disulfide bond is associated with local unfolding of the adjoining alpha-helix. Native secondary structure (including helices 1 and 3) is otherwise retained and defines a folded subdomain. Long-range nuclear Overhauser effects (NOE) within this subdomain are similar to those of native IGF-I; no non-native NOE is observed. Our results support the hypothesis that folding of the insulin motif is directed by a subset of native structural elements and that these elements form at an early step in the pathway. Formation of helix 2, despite its prominence in the native state, is likely to represent a late step. Hydrophobic collapse of this segment appears to precede helix formation.

Alanine↗
PubMed2004

Crystal structure of a human VH: requirements for maintaining a monomeric fragment.

The variable domain of dromedary immunoglobulins comprises only the heavy chain and is missing the light-chain variable domain. This single domain is sufficient for antigen recognition and binding-half that required by other mammals. Human antibody-VHs have previously been camelized to be soluble stable fragments that retain antigen binding. Such engineered VHH are of interest in drug development, since they are nonimmunogenic, and in other biotechnology applications. We present the structure of a camelized human antibody fragment (cVH), which is a competitive and reversible inhibitor of the NS3 serine protease of the hepatitis C virus (HCV). In solution, this cVH undergoes a concentration-dependent monomer-dimer equilibrium. The structure confirms the minimum mutational requirements of the VL-binding face. The fragment also suggests a means by which the observed dimerization occurs, highlighting the importance of the composition of the CDR3 in maintaining a truly camelized VH.

Amino Acid Sequence↗
PubMed2000

Membrane processing of fruit juices and beverages: a review.

Membrane technology for the processing of fruit juices and beverages has been applied mainly for clarification using ultrafiltration and microfiltration, and for concentration using reverse osmosis. The effects of product preparation, membrane selection, and operating parameters are important factors influencing filtration rate and product quality. Technological advances related to the development of new membranes, improvement in process engineering, and better understanding of fruit beverage constituents have expanded the range of membrane separation processes. Developments in novel membrane processes, including electrodialysis and pervaporation, increased the array of applications in combination with other technologies for alternate uses in fruit juices and beverages.

Beverages↗
PubMed2000

Membrane processing of fruit juices and beverages: a review.

Membrane technology for the processing of fruit juices and beverages has been applied mainly for clarification using ultrafiltration and microfiltration, and for concentration using reverse osmosis. The effects of product preparation, membrane selection, and operating parameters are important factors influencing filtration rate and product quality. Technological advances related to the development of new membranes, improvement in process engineering, and better understanding of fruit beverage constituents have expanded the range of membrane separation processes. Developments in novel membrane processes, including electrodialysis and pervaporation, increased the array of applications in combination with other technologies for alternate uses in fruit juices and beverages.

Beverages↗
PubMed1999

A direct binding assay for thyrotropin receptor autoantibodies.

There is, at present, no assay in clinical use for the direct assay of autoantibody binding to the thyrotropin receptor (TSHR). We now describe a direct thyrotropin receptor autoantibody binding assay (DTAb) using a secreted form of the TSHR ectodomain (TSHR-289) without the need for antigen purification. The assay compensates for the low TSHR autoantibody concentration in serum by capturing a relatively large amount of patient immunoglobulin G (IgG) on high-capacity beads, a reversal of standard methods that typically first immobilize antigen. TSHR-289 captured by Graves' IgG was detected in a colorimetric reaction using a biotinylated murine monoclonal antibody to the poly-histidine tail engineered into the antigen. By this approach, sera from 11 normal individuals provided a mean optical density (OD) value of 0.20 +/- 0.08 SD (range 0.06-0.33). Of 38 sera from unselected patients with a history of Graves' disease (untreated and treated), 29 (76%) generated OD values > 0.37 (2 SD above the mean for the normal sera), the highest being OD 1.38. Surprisingly, 3 of 13 (23%) sera from TPO autoantibody-positive patients with Hashimoto's thyroiditis also provided values > 2 SD above the normal sera. The extent of direct autoantibody binding to the TSHR correlated closely with the thyrotropin binding inhibition (TBI) values (r = 0.881; p < 0.001). One serum was clearly positive in only the direct binding assay and another in only the TBI assay. The data obtained with the direct binding assay correlated less well with the thyroid-stimulating antibody (TSAb) assay (r = 0.582; p < 0.001). In summary, we describe a new direct DTAb assay that correlates more closely with the TBI than with the TSI assays. Future studies in a large series of clinically defined patients will be needed to evaluate the clinical utility of the DTAb assay.

Autoantibodies↗
PubMed2006

Cx26 inhibits breast MDA-MB-435 cell tumorigenic properties by a gap junctional intercellular communication-independent mechanism.

It has been well established that the restoration of connexin expression in tumor cells often leads to a partial reversion of tumor cell phenotypes and increased growth control. In this study, a less-aggressive variant of MDA-MB-435 cells obtained from the MDA-MB-435 cell line was engineered to express gap junctional intercellular communication (GJIC)-competent Cx26, a GJIC-incompetent cell surface transported GFP-Cx26 chimera, or a Golgi apparatus-localized, disease-linked Cx26 mutant (D66H). Collectively, these cell lines were designed to establish whether Cx26 regulates tumor properties, such as migration, invasion and growth, by (i) a GJIC-dependent pathway; (ii) a mechanism requiring Cx26 transport to the cell surface; or (iii) a mechanism where Cx26 expression alone was sufficient. The expression of Cx26 and green fluorescent protein (GFP)-Cx26 decreased cell proliferation while all three Cx26 variants inhibited anchorage-independent cell growth. All three Cx26 variants also altered the distribution of filamentous actin and significantly reduced cell migration, while only the D66H mutant failed to inhibit cell invasion through matrigel. Furthermore, expression of all the Cx26 variants reduced the levels of total beta1 integrin, and decreased the activity of matrix metalloproteinase-9 (MMP-9) while increasing tissue inhibitors of MMP-1 (TIMP-1) activity. Interestingly, the expression of Cx43 regulated the same gene products without significantly affecting the tumorigenic properties of the MDA-MB-435 cells. Together, these results suggest that Cx26 expression, independent of the necessity for gap junctional intercellular communication, partially reverted MDA-MB-435 cell properties associated with tumorigenesis, and regulated the expression of genes important in cell migration and invasion.

Animals↗