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At least 541 records · Page 30Linked to original sources
PubMedDate not supplied

Deletions in the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase are observed in HIV-1 isolated from subjects during long-term antiretroviral therapy.

OBJECTIVES: To examine the effect of in-frame deletions in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on plasma viremia and phenotypic resistance to antiretroviral drugs. STUDY DESIGN/METHODS: Plasma HIV-1 RNA was isolated from 168 antiretroviral therapy-experienced subjects for quantification of plasma viremia, RT sequence analysis, and phenotypic resistance assays. RESULTS: Four patients were found to harbor HIV-1 strains possessing in-frame, 3-nucleotide deletions at RT codons 67, 69, and 70. In these subjects, phenotypic resistance and high plasma viremia were observed only in a background of multiple resistance mutations. A recombinant virus engineered with an in-frame deletion of RT codon 67 did not have increased resistance to nucleoside reverse transcriptase inhibitors (NRTIs). CONCLUSIONS: Selection for deletions within the beta3-beta4 hairpin loop of the HIV-1 RT is an uncommon event most likely to occur in subjects with long-term antiretroviral experience. The codon 67 deletion does not appear to cause increased phenotypic resistance or increased viremia in the absence of concomitant RT mutations.

Adult↗
PubMed2005

A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes.

Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.

Animals↗
PubMed2002

Efficient transfection method for primary cells.

Transfection of primary cells and stem cells is a problem in the laboratory routine and further in tissue engineering and gene therapy. Most methods working effectively for cell lines in culture fail to transfect primary cells. Here we describe the use of the Nucleofector technology developed by amaxa biosystems. We were able to transfect primary human melanocytes, human coronary smooth muscle cells, human chondrocytes, and human mesenchymal stem cells with high efficiencies (28.9-45.3%). All primary cell types failed to be transfected satisfactorily by methods based on liposome-mediated transfection in our hands. The viability of the transfected cells varied between 11.2% and 75% in comparison to untreated cells. Only 200,000 cells per transfection sample were needed. In summary, this method presents an effective and fast mean for transfection of primary and stem cells demonstrated by four cell types which are only transfected with low efficiency by other methods.

Cell Survival↗
PubMed2005

Irreversible photoinhibition of photosystem II is caused by exposure of Synechocystis cells to strong light for a prolonged period.

Irreversible photoinhibition of photosystem II (PSII) occurred when Synechocystis sp. PCC 6803 cells were exposed to very strong light for a prolonged period. When wild-type cells were illuminated at 20 degrees C for 2 h with light at an intensity of 2,500 micromol photons m(-2) s(-1), the oxygen-evolving activity of PSII was almost entirely and irreversibly lost, whereas the photochemical reaction center in PSII was inactivated only reversibly. The extent of irreversible photoinhibition was enhanced at lower temperatures and by the genetically engineered rigidification of membrane lipids. Western and Northern blotting demonstrated that, after cells had undergone irreversible photoinhibition, the precursor to D1 protein in PSII was synthesized but not processed properly. These observations may suggest that exposure of Synechocystis cells to strong light results in the irreversible photoinhibition of the oxygen-evolving activity of PSII via impairment of the processing of pre-D1 and that this effect of strong light is enhanced by the rigidification of membrane lipids.

Cell Membrane↗
PubMed2004

The voltage-gated Na+ channel NaVBP has a role in motility, chemotaxis, and pH homeostasis of an alkaliphilic Bacillus.

The prokaryotic voltage-gated Na(+) channel, NaChBac, is one of a growing channel superfamily of unknown function. Here we show that Na(V)BP, the NaChBac homologue encoded by ncbA in alkaliphilic Bacillus pseudofirmus OF4, is a voltage-gated Na(+) channel potentiated by alkaline pH. Na(V)BP has roles in motility, chemotaxis, and pH homeostasis at high pH. Reduced motility of bacteria lacking functional Na(V)BP was reversed by restoration of the native channel but not by a mutant Na(V)BP engineered to be Ca(2+)-selective. Motile ncbA mutant cells and wild-type cells treated with a channel inhibitor exhibited behavior opposite to the wild type in response to chemoeffectors. Mutants lacking functional Na(V)BP were also defective in pH homeostasis in response to a sudden alkaline shift in external pH under conditions in which cytoplasmic [Na(+)] is limiting for this crucial process. The defect was exacerbated by mutation of motPS, the motility channel genes. We hypothesize that activation of Na(V)BP at high pH supports diverse physiological processes by a combination of direct and indirect effects on the Na(+) cycle and the chemotaxis system.

Animals↗
PubMedDate not supplied

B7.1/NHS76: a new costimulator fusion protein for the immunotherapy of solid tumors.

Tumor evasion from immune surveillance is due to the anergic status of tumor-infiltrating lymphocytes, especially T cells. Inappropriate or absent expression of costimulatory molecules such as B7.1 and B7.2 lead to anergy and apoptosis of tumor-infiltrating T cells. To reverse this situation, a tumor-targeted fusion protein, human B7.1/NHS76, was generated by molecular engineering, which retains both the costimulatory activity of B7.1 and the tumor-targeting ability of NHS76 antibody. NHS76 is a human tumor necrosis therapy monoclonal antibody derived from phage display, and is capable of binding intracellular antigens, which are accessible and abundant in necrotic regions of tumors. As human B7.1 can interact functionally with murine B7.1 counter-receptors, the immunotherapeutic potential of this fusion protein was tested in 3 mouse tumor models (Colon 26, RENCA, and MAD109), and animal studies showed a 35% to 55% reduction in tumor volume. To modulate the immune inhibitory microenvironment in tumors, naturally occurring CD4+ CD25+ Treg cells were depleted by cytotoxic CD4 or CD25 antibodies. Combination therapy with anti-Treg and B7.1/NHS76 produced complete regression of established tumors and was associated with increased effector T-cell infiltration in tumors. Rechallenge experiments performed 3 months after mice attained complete remission by combination therapy showed that immunologic memory was established by these treatments. These studies indicate that the targeting of B7.1 to necrotic areas of tumors, where both the release of tumor antigens and infiltrating lymphocytes are prevalent, may be a new approach for the immunotherapy of solid tumors. Our results also suggest that the manifestation of immune-inhibitory factors such as the presence of Treg cells at the tumor site and associated draining lymph nodes may be a major cause for immune system failure to eradicate solid tumors.

Animals↗
PubMed2002

Sister chromatid exchange analysis and chromosoma aberration studies in interventional cardiology laboratory workers: one war follow up study.

Invasive cardiology laboratory workers are occupationally exposed to chronic ionizing radiation. It is known that ionizing radiation has a damaging effect on chromosomes. In present study. we investigated the frequency of sister chromatid exchange (SCE) and chromosomal aberrations in 11 invasive cardiology laboratory workers and 11 healthy controls. After a vacation period, we took blood samples for chromosome analysis in months 0, 4, 8 and 12 (last two month period was the nonradiation time). The SCE frequencies did not change significantly after exposure to ionizing radiation in any worker. Our study has revealed that non-specific structural chromosome aberrations such as gaps, isogaps, acentric chromosomes, chromatids and chromosome breakage could be in the 4th and 8th months after ionizing radiation exposure in the metaphase plaques. All abnormal chromosomal effects had disappeared by the end of the two month non-exposure period in each worker. In conclusion, the results suggest that SCE frequencies are not significantly affected in invasive cardiology laboratory workers who are exposed occupationally to ionizing radiation, although some degree of reversible chromosomal aberrations did appear.

Adult↗
PubMed2000

Human amniotic epithelial cells are promising transgene carriers for allogeneic cell transplantation into liver.

As human amniotic epithelial tissue is formed on about the eighth day after fertilization, human amniotic epithelial cells (hAEC) may have multipotency to differentiate into various organs, such as brain, heart, or liver. In this study, we showed evidence of the synthesis and excretion of albumin by hAEC, by immunostaining and enzyme-linked immunoassay. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses revealed the expression of albumin mRNA and protein, respectively. In addition, hAEC also demonstrated immunoreactivity to genetic markers of liver lineage, such as human serum albumin and alpha-fetoprotein. Transplanted hAEC to Scid mouse liver showed positive immunoreactivity to albumin and alpha-fetoprotein. Genetically modified cells containing the beta-galactosidase (LacZ) gene (AxCALacZ) were integrated in liver parenchyma. Human polymorphic gene analysis in Scid mouse liver after the implantation of hAEC showed that these Scid mouse livers obviously contained this human-specific gene until day 7 after the cell transplantation. As hAEC do not cause any acute rejection by allotransplantation, we conclude that hAEC may be useful as a transgene carrier to treat patients with inherited liver diseases.

Albumins↗
PubMed2005

Targeted protein degradation.

The ubiquitin-proteasome pathway plays a major role in cellular protein destruction and regulates fundamental cellular processes such as the cell cycle, cell signaling, and development. By altering the substrate recognition of ubiquitin-protein ligases, their robust proteolytic activity can be re-directed to recruit and accelerate the degradation of other cellular targets. Two approaches have been applied for targeted proteolysis: one entails designing a chimeric substrate receptor for recruitment of the target protein, the other involves the construction of peptide-small-molecule hybrids that bridge the interaction between the intended target and the substrate receptor of the known ubiquitin-protein ligases. The engineered ubiquitin-proteolytic apparatus operates at the post-translational level, and thus provides a new tool of reverse genetics to dissect complicated protein functions at a higher resolution than knockout or knockdown approaches functioning at the level of DNA or RNA. It also sheds light on novel therapeutic strategies for the amelioration of human disease.

F-Box Proteins↗
PubMed2005

An in-vitro study on regeneration of human nucleus pulposus by using gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer scaffold.

Tissue engineering approaches for treating degenerative intervertebral discs aim to promote tissue regeneration then retard or even reverse the degenerative process. A gelatin/chondroitin-6-sulfate/hyaluronan tri-copolymer was developed to serve as a bioactive scaffold that could help human nucleus pulposus (NP) cells to preserve their cell viability/proliferation and promote matrix synthesis. Each scaffold was seeded with 1 x 10(6) monolayer-expanded human NP cells and then cultured in vitro. Over a 4-week cultivation period, cell-scaffold hybrids demonstrated active cell viability/proliferation and a progressive increase in net production of glycosaminoglycans. In comparison to monolayer cells, scaffold-cultured cells showed significantly higher mRNA expression in collagen II, aggrecan, Sox9, TGFbeta1, and TIMP1. Expression of mRNA was significantly suppressed in collagen I, collagen X, IL1, and Fas-associating death domain protein. Histological studies showed newly synthesized glycosaminoglycans deposits and collagen II in scaffolds. These results indicate that the tri-copolymer scaffold could be considered as a promising bioactive scaffold for regenerating human NP.

Adjuvants, Immunologic↗
PubMed2005

Structure and catalytic properties of an engineered heterodimer of enolase composed of one active and one inactive subunit.

Enolase is a dimeric enzyme that catalyzes the interconversion of 2-phospho-D-glycerate and phosphoenolpyruvate. This reversible dehydration is effected by general acid-base catalysis that involves, principally, Lys345 and Glu211 (numbering system of enolase 1 from yeast). The crystal structure of the inactive E211Q enolase shows that the protein is properly folded. However, K345 variants have, thus far, failed to crystallize. This problem was solved by crystallization of an engineered heterodimer of enolase. The heterodimer was composed of an inactive subunit that has a K345A mutation and an active subunit that has N80D and N126D surface mutations to facilitate ion-exchange chromatographic separation of the three dimeric species. The structure of this heterodimeric variant, in complex with substrate/product, was obtained at 1.85 A resolution. The structure was compared to a new structure of wild-type enolase obtained from crystals belonging to the same space group. Asymmetric dimers having one subunit exhibiting two of the three active site loops in an open conformation and the other in a conformation having all three loops closed appear in both structures. The K345A subunit of the heterodimer is in the loop-closed conformation; its Calpha carbon atoms closely match those of the corresponding subunit of wild-type enolase (root-mean-squared deviation of 0.23 A). The kcat and kcat/Km values of the heterodimer are approximately half those of the N80D/N126D homodimer, which suggests that the subunits in solution are kinetically independent. A comparison of enolase structures obtained from crystals belonging to different space groups suggests that asymmetric dimers can be a consequence of the asymmetric positioning of the subunits within the crystal lattice.

Catalysis↗
PubMed2007

Fibrin gel improved the spatial uniformity and phenotype of human chondrocytes seeded on collagen scaffolds.

A scaffold made of equine collagen type I based material has been assessed for its use in the preparation of tissue-engineered cartilage implants with human articular chondrocytes. Improvements of cell-seeding efficiency and specific gene expression were studied by combining solid scaffold with fibrin glue or human blood plasma. Following 3 weeks of static culture, mRNA expression levels of collagen type I, collagen type II, aggrecan and versican were analyzed by real-time quantitative PCR and compared to those in native cartilage and monolayer cell cultures. Constructs prepared with fibrin glue or plasma showed higher cell seeding efficiencies than those prepared without gel. Chondrocytes seeded directly onto a collagen scaffold appeared fibroblastic in shape while those encapsulated in fibrin gel were spherical. The presence of fibrin glue positively influences on mRNA levels of collagen type II and aggrecan, while blood plasma enhanced only the level of collagen type II expression. Levels of collagen type I and versican decreased in presence of fibrin glue. In orthopaedics, the combination of solid collagen fleece with fibrin gel for implant preparation is seen to be preferred over solid material or even cells in a suspension, since fibrin gel improves seeding capacity of the scaffold, supports equal distribution of cells and stimulates higher chondrogenic phenotype expression.

Adult↗
PubMed1993

Natural tooth intrusion and reversal in implant-assisted prosthesis: evidence of and a hypothesis for the occurrence.

Based on clinical observation, a hypothesis of the mechanism of intrusion of natural teeth in an implant-assisted prosthesis is suggested. Engineering principles are presented that establish an energy absorption model as it relates to the implant-assisted prosthesis. In addition, in the course of patient treatment it has been discovered that the intrusion of natural teeth can be reversed. Patient histories that demonstrate intrusion reversal are reviewed. The possible mechanisms for the intrusion/reversal phenomenon are presented and preventative recommendations are given.

Aged↗
PubMed2004

Dental pulp cells provide neurotrophic support for dopaminergic neurons and differentiate into neurons in vitro; implications for tissue engineering and repair in the nervous system.

Glial cell line-derived neurotrophic factor (GDNF) mRNA is highly expressed by dental pulp cells (DPCs) prior to the initiation of dental pulp innervation. We show that radioactively labelled exogenous GDNF is retrogradely transported from neonatal teeth and vibrissae to the trigeminal neurons, indicating that GDNF acts as a classical neurotrophic factor in the trigeminal system. We also show that DPCs from both rats and humans produce nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and GDNF mRNAs in vitro, promote the survival and phenotypic characteristics of embryonic dopaminergic (DA) neurons and protect DA neurons against the neurotoxin 6-hydroxy-dopamine (6-OHDA) in vitro. By using inhibitory antibodies to NGF, BDNF and GDNF, we show that the promotion of DA neuron survival relates to the production and release of neurotrophic proteins by DPCs in vitro. We suggest that in vivo production of neurotrophic factors by DPCs play roles in tooth innervation. However, continued production of neurotrophic factors by the DPCs might have wider implications. We propose that the dental pulp is a viable source of easily attainable cells with possible potential for development of autologous cell transplantation therapies. We also show that a population of neural crest-derived dental pulp cells acquire clear neuronal morphology and protein expression profile in vitro, indicating the presence of a cell population in the dental pulp with neuronal differentiation capacity that might provide additional benefits when grafted into the CNS.

Adolescent↗
PubMed2006

Characterization of a novel polymeric scaffold for potential application in tendon/ligament tissue engineering.

Unlike braided fabrics, knitted scaffolds have been proven to favor deposition of collagenous connective tissue matrix, which is crucial for tendon/ligament reconstruction. But cell seeding of such scaffolds often requires a gel system, which is unstable in a dynamic situation, especially in the knee joint. This study developed a novel, biodegradable nano-microfibrous polymer scaffold by electrospinning PLGA nanofibers onto a knitted PLGA scaffold in order to provide a large biomimetic surface for cell attachment. Porcine bone marrow stromal cells were seeded onto either the novel scaffolds by pipetting a cell suspension (Group I) or the knitted PLGA scaffolds by immobilizing in fibrin gel (Group II). Cell attachment at 36 hours, cell proliferation and extracellular matrix synthesis at 1 week, and mechanical properties over 2 weeks were investigated. Cell attachment was comparable and cell proliferation was faster in Group I. Moreover, cellular function was more actively exhibited in Group I, as evident by the higher expression of collagen I, decorin, and biglycan genes. Thus, this novel scaffold, facilitating cell seeding and promoting cell proliferation, function, and differentiation, could be applied with promise in tissue engineering of tendon/ligament.

Animals↗
PubMed2006

Osteogenic potentiation of human adipose-derived stem cells in a 3-dimensional matrix.

Adipose-derived stem cells (ADSCs) hold promise for use in tissue engineering. Despite growing enthusiasm for use of ADSCs, there is limited research that has examined their behavior in different in vitro and in vivo systems. The purpose of our study was to evaluate the effect of the extracellular matrix structure and composition on osteogenic differentiation by comparing the osteogenic marker expression of ADSCs grown under 2-dimensional or 3-dimensional cell culture conditions. Group 1 (2-D) included ADSCs raised under conventional cell culture conditions (cells in a 2-D monolayer configuration) (n = 24), and group 2 (3-dimensional) included ADSCs seeded in a collagen gel (cells within a 3-dimensional, biologically active environment) (n = 24). Comparison of ADSC behavior between the 2 groups was analyzed during a 14-day time frame. Osteogenic marker expression (CBFA-1, alkaline phosphatase, osteonectin, osteopontin, Collagen I, and JNK2) was quantified by real-time PCR, and histologic analysis was performed. Histologically, group 1 (2-D) showed cell spreading and deposition of a calcified extracellular matrix. Group 2 (3-dimensional) assumed a disorganized state in the collagen gel, with extension of pseudopodia throughout the matrix. Expression of CBFA-1 was up-regulated immediately in both groups. However, cells in group 2 (3-dimensional) had a more rapid and greater overall expression compared with cells in group 1 (2-D) (250-fold greater at 4 days). At day 14, cells in group 2 (3-dimensional) showed greater expression of all other osteogenic markers than cells in group 1 (2-D) (2.3-fold greater expression of alkaline phosphatase [P < 0.05], 8.4-fold greater expression of osteonectin [P < 0.05], 6.4-fold greater expression of osteopontin [P < 0.05], 2.9-fold greater expression of collagen I [P < 0.05], and 2.5-fold greater expression of JNK2 [P < 0.05]). Our data showed there was a progressive stimulatory effect on ADSCs with regard to osteogenesis when cultured in a 3-dimensional gel compared with a 2-D monolayer.

Adipose Tissue↗
PubMed2005

Evaluation of polymer scaffolds to be used in a composite injectable system for intervertebral disc tissue engineering.

Adult porcine nucleus pulposus cells were seeded onto gelatin, demineralized bone matrix (DBM), and polylactide scaffolds and cultured in vitro. Cellular behavior in response to the scaffolds was analyzed using biochemical assays, histology, and real-time quantitative reverse transcriptase-polymerase chain reaction. Scanning electron microscopy showed pronounced differences in surface texture of the scaffolds. Nucleus pulposus cells attached and assumed an elongated fibroblast-like morphology on the gelatin and DBM scaffolds. The cells cultured on the gelatin and DBM were metabolically active and expressed types I and II collagen and aggrecan. Detached cellular aggregates with a rounded morphology were noted in the culture tubes containing the polylactide scaffolds. Both surface chemistry and texture likely had a role in causing differences in cellular behavior in response to scaffold material. Promising results were observed using the gelatin and demineralized bone scaffolds, but the behavior of cells cultured on these scaffolds will need to be characterized further. This initial research will be used to direct future work involved in developing this composite injectable tissue engineering system.

Animals↗