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At least 829 records · Page 46Linked to original sources
PubMed1989

Low-temperature unfolding of a mutant of phage T4 lysozyme. 1. Equilibrium studies.

The mutant protein I3C-C97/C54T of phage T4 lysozyme is free of sulfhydryl groups and has a genetically engineered disulfide bridge between positions 3 and 97 (Perry & Wetzel, 1986). This protein has a maximum stability at 12 degrees C in 3 M guanidinium chloride and undergoes reversible high- and low-temperature melting at 28 and -3 degrees C, respectively, in this medium. The free energy of stabilization of the protein has been studied over a range of temperature that includes both melting transitions. The stability curve fits a constant delta Cp model over the entire range, permitting an unusually complete determination of the thermodynamic parameters of the protein and demonstrating that the low-temperature unfolded form of the protein may be interpreted as an extrapolation with constant delta Cp of the high-temperature unfolded form. The free energy of unfolding is a linear function of guanidinium concentration within experimental error which permits a rough estimate of the stability of the protein at low temperatures and of the differential interaction of the unfolded protein with guanidinium chloride. These equilibrium studies provide a basis for the interpretation of the kinetic studies reported in the following paper.

Calorimetry↗
PubMed1999

The thumb domain of the P51-subunit is essential for activation of HIV reverse transcriptase.

The biologically relevant and active form of human immunodeficiency virus reverse transcriptase is a heterodimer produced in a two-step dimerization process. Dimerization involves first the rapid association of the two subunits, followed by a slow conformational change yielding a fully active form. In the present study, we demonstrate that the interaction between the thumb domain of p51 and the RNase-H domain of p66 plays a major role in an essential conformational change required for proper folding of the primer/template and the tRNA-binding site, for maturation and for activation of heterodimeric reverse transcriptase. A synthetic peptide derived from the sequence within the thumb domain of p51, which forms the interface with the RNase-H domains of p66, binds heterodimeric reverse transcriptase with an apparent dissociation constant in the nanomolar range and selectively inhibits activation of heterodimeric reverse transcriptase with an inhibition constant of 1.2 microM. A detailed study of the mechanism of inhibition reveals that this peptide does not require dissociation of heterodimeric RT for efficient inhibition and does not affect subunit association, but interferes with the conformational change required for activation of heterodimeric reverse transcriptase, resulting in a decrease in the affinity of reverse transcriptase for the tRNA and an increase in the stability of the primer/template/reverse transcriptase complex. We have previously proposed that the dimeric nature of reverse transcriptase represents an interesting target for the design of antiviral agents. On the basis of this work, we propose that the conformational changes involved in the activation of reverse transcriptase similarly represent an important target for the design of novel antiviral compounds.

Amino Acid Sequence↗
PubMed2007

Potent knock down of HIV-1 replication by targeting HIV-1 Tat/Rev RNA sequences synergistically with catalytic RNA and DNA.

OBJECTIVE: Ribozymes (Rzs) and DNA-enzymes (Dzs) possess the ability to prevent gene expression by cleaving target RNA in a catalytic and sequence-specific manner. Although Rzs or Dzs have been used earlier for HIV-1 gene suppression, the present study explored the possibility of using catalytic RNA and DNA simultaneously in a synergistic manner with the hope that this novel approach will allow more potent inhibition for a longer duration. METHODS: In order to achieve long-term inhibition of HIV-1 replication, a novel non-GUX hammerhead Rz was designed by standard recombinant DNA technology and cloned it under the powerful CMV promoter containing expression vector. A 10-23 catalytic motif containing Dz that was targeted against the conserved second exon of HIV-1 Tat/Rev region was also assembled. RESULTS: Both Rz and Dz possessed sequence-specific cleavage activities individually and simultaneously cleaved target RNA in a synergistic manner under the same in vitro cleavage conditions. These catalytic molecules inhibited HIV-1 replication in macrophages individually and exhibited potent inhibitory effects when used in combination. CONCLUSIONS: The combination strategy described here can be widely used against any target RNA to achieve more effective gene inhibition that exploits the simultaneous sequence-specific cleavage potentials of catalytic RNA and DNA.

Amino Acid Sequence↗
PubMed2000

Low-friction nanoscale linear bearing realized from multiwall carbon nanotubes

We demonstrate the controlled and reversible telescopic extension of multiwall carbon nanotubes, thus realizing ultralow-friction nanoscale linear bearings and constant-force nanosprings. Measurements performed in situ on individual custom-engineered nanotubes inside a high-resolution transmission electron microscope demonstrated the anticipated van der Waals energy-based retraction force and enabled us to place quantitative limits on the static and dynamic interwall frictional forces between nested nanotubes. Repeated extension and retraction of telescoping nanotube segments revealed no wear or fatigue on the atomic scale. Hence, these nanotubes may constitute near perfect, wear-free surfaces.

Journal Article↗
PubMed1993

Diesel asthma. Reactive airways disease following overexposure to locomotive exhaust.

While some of the gaseous and particulate components of diesel exhaust can cause pulmonary irritation and bronchial hyperreactivity, diesel exhaust exposure has not been shown to cause asthma. Three railroad workers developed asthma following excessive exposure to locomotive emissions while riding immediately behind the lead engines of caboose-less trains. Asthma diagnosis was based on symptoms, pulmonary function tests, and measurement of airways hyperreactivity to methacholine or exercise. One individual's peak expiratory flow rates fell in a work-related pattern when riding immediately behind the lead diesel engine. None had a previous history of asthma or other respiratory disease and none were current smokers. All three developed persistent asthma. In two cases, physiologic abnormalities suggesting reversible restriction were observed. This is the first report implicating diesel exhaust as a cause of reactive airways disease.

Adult↗
PubMed1998

Functional reverse transcriptases encoded by A-type mouse LINE-1: defining the minimal domain by deletion analysis.

Long interspersed elements, or LINEs, are retrotransposons that move via an RNA intermediate. In mice, one polymorphic variant of L1 has amplified relatively recently, giving rise to the A-type subfamily in species belonging to the genus and subgenus Mus. Retrotransposition of LINE-1 (L1) requires the function of the L1-encoded reverse transcriptase that is produced from open reading frame 2 (ORF2). Here, we employ a convenient yeast genetic assay to determine the reverse transcriptase activity of the ORF2 obtained from three A-type L1 elements: one, a cDNA from the RNA in ribonucleoprotein particles; another with a purported inactivating mutation; and the third, a hypothetical ancestral construct. Because there are no examples of A-type elements that have transposed recently to inactivate a gene, this assay is the first step towards demonstrating the functional capability of mouse A-type LINE-1 elements. One of the three elements was believed to have been inactivated during evolution by the substitution of leucine for a highly conserved phenylalanine or tryptophan residue among known reverse transcriptases. This mutation did not inactivate the L1 reverse transcriptase in the yeast assay; thus, all three of the elements tested encoded reverse transcriptase activity. We further examined the minimal reverse transcriptase domain within ORF2 by creating a series of deletions. The results demonstrate that removal of the L1 endonuclease domain from the N-terminal region of ORF2 does not affect reverse transcriptase activity as determined by this assay, and that approximately half of the ORF2 coding sequence from mouse A-type L1 elements is required for functional reverse transcriptase.

Animals↗
PubMed2006

Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament.

This study investigated four different connective tissue cell types to determine which cell type should be the source for seeding a tissue-engineered anterior cruciate ligament (ACL) replacement. Cells derived from the ACL, medial collateral ligament (MCL), achilles tendon (AT), and patellar tendon (PT) of New Zealand White rabbits were isolated and cultured. Each cell type was cultured in vitro after seeding on three-dimensional (3-D) braided polymer scaffolds and on tissue culture polystyrene that served as a control. Samples were evaluated and compared for their morphology, proliferation, and gene expression of fibronectin, type I and type III collagen. Scanning electron microscopy (SEM) photomicrographs verified cell attachment of all four types of connective tissue fibroblasts to the scaffolds. Preliminary results comparing proliferation indicate that cells obtained from the PT and AT have the fastest proliferation. Whereas gene expression of the phenotypic markers measured using real-time reverse transcription polymerase chain reaction (RT-PCR) indicates ACL cells have the highest gene expression for the matrix markers. This leads to the question of which cell type should be the cell source for tissue-engineering of ligament, the highly proliferating cells or the differentiated matrix producing cells. This study would suggest that ACL differentiated matrix producing cells are the most suitable cells for further study and development of a tissue-engineered ligament.

Achilles Tendon↗
PubMedDate not supplied

Fabrication and endothelialization of collagen-blended biodegradable polymer nanofibers: potential vascular graft for blood vessel tissue engineering.

Electrospun collagen-blended poly(L-lactic acid)-co-poly(epsilon-caprolactone) [P(LLA-CL), 70:30] nanofiber may have great potential application in tissue engineering because it mimicks the extracellular matrix (ECM) both morphologically and chemically. Blended nanofibers with various weight ratios of polymer to collagen were fabricated by electrospinning. The appearance of the blended nanofibers was investigated by scanning electron microscopy and transmission electron microscopy. The nanofibers exhibited a smooth surface and a narrow diameter distribution, with 60% of the nanofibers having diameters between 100 and 200 nm. Attenuated total reflectance-Fourier transform infrared spectra and X-ray photoelectron spectroscopy verified the existence of collagen molecules on the surface of nanofibers. Human coronary artery endothelial cells (HCAECs) were seeded onto the blended nanofibers for viability, morphogenesis, attachment, and phenotypic studies. Five characteristic endothelial cell (EC) markers, including four types of cell adhesion molecule and one EC-preferential gene (von Willebrand factor), were studied by reverse transcription-polymerase chain reaction. Results showed that the collagen-blended polymer nanofibers could enhance the viability, spreading, and attachment of HCAECs and, moreover, preserve the EC phenotype. The blending electrospinning technique shows potential in refining the composition of polymer nanofibers by adding various ingredients (e.g., growth factors) according to cell types to fabricate tissue-engineering scaffold, particularly blood vessel-engineering scaffold.

Biocompatible Materials↗
PubMed2003

Human keratocytes cultured on amniotic membrane stroma preserve morphology and express keratocan.

PURPOSE: To develop a new method of expanding human corneal keratocytes in serum while maintaining their characteristic morphology and keratocan expression. METHODS: Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/mL of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. On confluence, cells on AM were continuously subcultured for six passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were reseeded on AM. Cellular morphology and cell-cell networks were assessed by phase-contrast microscopy and a cell viability assay, respectively. Expression of keratocan was determined by RT-PCR and Western blot analysis. RESULTS: Trephined stroma yielded 91,600 +/- 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM, even in 10% FBS but fibroblastic on plastic. Such a difference in morphology correlated with expression of keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM, even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passages 3 and 11 did not reverse their morphology or synthesize keratocan when reseeded on plastic in 1% FBS or on AM. CONCLUSIONS: Human keratocytes maintain their characteristic morphology and keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.

Amnion↗
PubMedDate not supplied

Gene expression is altered in perfused arterial segments exposed to cyclic flexure ex vivo.

Certain regions of coronary and other arteries undergo cyclic flexure due to attachment to the heart or crossing of joints. Such motion gives rise to fluctuations in transmural stress and luminal shear stress. It is well known that cyclic variation of these biomechanical forces influences many aspects of vascular cell biology including gene expression. The purpose of this work was to investigate the hypothesis that cyclic flexure of arterial segments influences their gene expression. Bilateral porcine femoral arteries were obtained fresh from the abattoir. One vessel was mounted in an ex vivo perfusion system and subjected to an intraluminal pressure of 60 mmHg and flow of 50 ml/min to serve as a control. The other vessel was mounted in a second perfusion system with similar hemodynamic conditions, but also subjected to controlled cyclic bending consistent with that found in coronary arteries in vivo. Reverse transcriptase-polymerase chain reaction analysis demonstrated that E-selectin and matrix metalloproteinase-1 (MMP-1) were consistently and significantly downregulated in the specimens subjected to 4 h of cyclic bending as compared to the control (n = 8, p < 0.05). Our results show that cyclic flexure of arterial segments in vitro may influence their gene expression. Further investigation should follow this novel observation and focus on other known mediators to more carefully elucidate the consequence of cyclic flexure on arterial pathobiology.

Actins↗
PubMed2001

De novo design of fibrils made of short alpha-helical coiled coil peptides.

BACKGROUND: The alpha-helical coiled coil structures formed by 25-50 residues long peptides are recognized as one of Nature's favorite ways of creating an oligomerization motif. Known de novo designed and natural coiled coils use the lateral dimension for oligomerization but not the axial one. Previous attempts to design alpha-helical peptides with a potential for axial growth led to fibrous aggregates which have an unexpectedly big and irregular thickness. These facts encouraged us to design a coiled coil peptide which self-assembles into soluble oligomers with a fixed lateral dimension and whose alpha-helices associate in a staggered manner and trigger axial growth of the coiled coil. Designing the coiled coil with a large number of subunits, we also pursue the practical goal of obtaining a valuable scaffold for the construction of multivalent fusion proteins. RESULTS: The designed 34-residue peptide self-assembles into long fibrils at slightly acid pH and into spherical aggregates at neutral pH. The fibrillogenesis is completely reversible upon pH change. The fibrils were characterized using circular dichroism spectroscopy, sedimentation diffusion, electron microscopy, differential scanning calorimetry and X-ray fiber diffraction. The peptide was deliberately engineered to adopt the structure of a five-stranded coiled coil rope with adjacent alpha-helices, staggered along the fibril axis. As shown experimentally, the most likely structure matches the predicted five-stranded arrangement. CONCLUSIONS: The fact that the peptide assembles in an expected fibril arrangement demonstrates the credibility of our conception of design. The discovery of a short peptide with fibril-forming ability and stimulus-sensitive behavior opens new opportunities for a number of applications.

Amino Acid Motifs↗
PubMed1991

Novel guanosine requirement for catalysis by the hairpin ribozyme.

THERE is much interest in the development of 'designer ribozymes' to target destruction of RNAs in vitro and in vivo. Engineering of ribozymes with novel specificities requires detailed knowledge of the ribozyme-substrate interaction, and a rigorous evaluation of sequence specificity. The hairpin ribozyme catalyses an efficient and reversible site-specific cleavage reaction. We have used mutagenesis and in vitro selection strategies to show that RNA cleavage and ligation has an absolute requirement for guanosine immediately 3' to the cleavage-ligation site. This G is not required for efficient substrate binding, rather, its 2-amino group is an essential component of the active site required for catalysis.

Base Sequence↗
PubMed2000

Emerging cell and molecular strategies for the study and treatment of painful peripheral neuropathies.

Pharmacologic treatment for the symptoms of painful neuropathy has been problematic, because there has been limited understanding of the underlying etiologies and systemic levels that an effective dose can have on multiple side effects. The use of molecular methods, such as gene deletion from knockout mice and cellular minipumps for delivery of biologic antinociceptive molecules, has led to a better understanding of the underlying mechanisms of the induction of intractable neuropathic pain. The initiation of an excitatory cascade after injury or disease leads to the induction of various second messenger systems, loss or down-regulation of the endogenous inhibitory spinal GABA system and central sensitization, causing such pain. The development and use of cellular minipumps, immortalized cell lines bioengineered to secrete various antinociceptive molecules for the reversal of neuropathic pain, makes cellular therapy a strategy for clinical use in the next few years. The development of molecular "disimmortalization" technologies will make the use of such engineered cell lines safe for human use. Direct somatic gene transfer for neuropathic pain will eventually overcome the problems associated with transplantation of non-autologous and xenogenic cells. These virus-mediated methods, although at the early stages of evolution and use, offer large-scale production of biologic agents that can be conveniently and confidently used for the long-term relief of chronic neuropathic pain in a clinical setting, without systemic effects or surgical interventions.

Animals↗
PubMed2000

Phase I clinical trial with IL-2-transfected xenogeneic cells administered in subcutaneous metastatic tumours: clinical and immunological findings.

Various studies have emphasized an immunodepression state observed at the tumour site. To reverse this defect and based upon animal studies, we initiated a phase I clinical trial of gene therapy in which various doses of xenogeneic monkey fibroblasts (Vero cells) genetically engineered to produce human IL-2 were administered intratumorally in 8 patients with metastatic solid tumours. No severe adverse effect was observed in the 8 patients analysed during this clinical trial even in the highest dose (5 yen 107 cells) group. This absence of toxicity seems to be associated with rapid elimination of Vero-IL-2 cells from the organism. Indeed, exogenous IL-2 mRNA could no longer be detected in the peripheral whole blood 48 hours after Vero-IL-2 cell administration. In addition, we did not find any expression of exogenous IL-2 mRNA in post-therapeutic lesions removed 29 days after the start of therapy. A major finding of this trial concerns the two histological responses of two treated subcutaneous nodules not associated with an apparent clinical response. The relationship between local treatment and tumour regression was supported by replacement of tumour cells by inflammatory cells in regressing lesions and marked induction of T and natural killer cell derived cytokines (IL-2, IL-4, IFNg ...) in post-therapeutic lesions analysed 28 days after the start of Vero-IL-2 administration. Gene therapy using xenogeneic cells as vehicle may therefore present certain advantages over other vectors, such as its complete absence of toxicity. Furthermore, the in vivo biological effect of immunostimulatory genes, i.e IL-2-, may be potentiated by the xenogeneic rejection reaction.

Adenocarcinoma↗
PubMed1996

Comparative analysis of five highly conserved target sites within the HIV-1 RNA for their susceptibility to hammerhead ribozyme-mediated cleavage in vitro and in vivo.

Moloney murine leukemia virus (MMLV)-derived pUCMoTiN-based retroviral vectors were engineered to allow constitutive and Tat (trans-activator of transcription)-inducible expression of five hammerhead ribozymes targeted against highly conserved sequences within the group antigen (Gag), protease (Pro), reverse transcriptase (RT), tat, and envelope (Env) coding regions of human immunodeficiency virus type-1 (HIV-1) RNA. Amphotropic retroviral vector particles were used to infect a human CD4+ lymphocyte-derived MT4 cell line. The pool of stable MT4 transductants expressing these ribozymes were each tested for their susceptibility to HIV-1 infection. RzTat conferred no protection to MT4 cells. RZGag and RzRT completely inhibited virus multiplication for 6 days. RzPro and RzEnv conferred the best protection, as they completely inhibited virus production for 12 and 15 days, respectively. No correlation was found between the degree of HIV-1 resistance conferred and the ability of these ribozymes to cleave their target RNA in vitro. From RzPro-expressing HIV-1-infected cells following virus escape, RzPro and target RNA sequences were amplified and checked for cleavage in vitro. The ribozyme expressed in these cells was shown to cleave the corresponding target RNA. Thus, a mutation in the ribozyme or target RNA does not seem to be the mechanism underlying virus escape.

Base Sequence↗
PubMed2003

Diversity among microbial cyclic lipopeptides: iturins and surfactins. Activity-structure relationships to design new bioactive agents.

A prominent group of bioactive lipopeptides produced by Bacillus species is constituted by iturins, surfactins and lichenysins. Interest in such substances results in their exceptional surfactant power, and their valuable antifungal, antibacterial, antitumoral and anti-Mycoplasma properties. As is typical for peptidic secondary-metabolites synthesized by the polyenzymic pathway, they are produced as mixtures of components varying in the peptidic and/or in the lipidic structure. In the context of structure-activity relationships, it is possible to take advantage of the adaptability of the biosynthesis system by systematically adding selected amino acids in the culture medium of the producing bacterium. When an amino acid is used as the sole nitrogen source, it is inserted directly into selected positions of the peptide sequence, thus amplifying the original structural microheterogeneity via a production of variants. This method revealed very efficient for increasing the amounts of preexisting variants and for building new variants of surfactins and lichenysins but totally inefficient with iturins. In this group, the peptidic diversity strictly depends on the selected strain. So far the screening remained the only method to discover new iturins. Another interesting peculiarity is the common occurrence in a single strain of two lipopeptides with different core structures such as surfactins and iturins. Taken together, these features led to an extensive metabolite pattern. Besides, engineered variants and chemical derivatives enlarged the array of available molecules. Despite the high degree of chemical similarity, the separation of variants and/or homologues was successfully achieved by reversed-phase HPLC leading to well-separated compounds ideally suited to investigation of structure-activity relationships. Improved physical techniques such as 2D-NMR and mass spectrometry allowed to describe efficiently and rapidly the composition of cyclic lipopeptides even in mixtures containing several variants. From NMR, the 3D structure and dynamics gave crucial data for fine structure-activity relationships as well as for understanding of the properties at the membrane and/or at the air/water interface. Here the role of residues was identified in the context of hydrophobic and electrostatic interactions that play a leader role. Such a comprehensive approach, based on both structural and biosynthesis knowledge, opened the way to rational design for enhanced properties and its validity was confirmed with 10 fold higher surfactant efficacy.

Anti-Bacterial Agents↗