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At least 1,117 records · Page 62Linked to original sources
PubMed2000

Sodium sulphate reactivates a protein A minidomain with a short elastin beta-turn.

Elastin polymer sequences derived from muscle exhibit temperature and salt-induced reversible contractions and expansions. A folded and contracted beta-turn helical elastin structure is stabilised by increased intramolecular hydrophobic interactions. We have generated a switchable binding protein by inserting a typical elastin turn sequence, GVPGVG, between the two IgG-binding helices of an engineered globular minidomain from Protein A. This mutant showed increased binding of IgG-molecules compared to the wild-type sequence in the presence of sodium sulphate, as measured by surface plasmon resonance spectroscopy. Analysis by circular dichroism revealed a salt-induced folding of the mutant minidomain to a native type I beta-turn, likely stabilised as a result of the two interacting dehydrated valines across the beta-turn. Since sodium sulphate can be successfully used to regulate the folding/unfolding or binding/dissociation of this minidomain, this suggests an alternative protein purification method.

Amino Acid Sequence↗
PubMed1990

A putative approach for gene therapy against human immunodeficiency virus (HIV).

It is proposed here that a form of intracellular immunity can be devised which would protect cells from virus infection and, in particular, could be used as a treatment for the human immunodeficiency virus (HIV) infected individual. Following in vitro immunization of naive human B lymphocytes with reverse-transcriptase (RT) or HIV transactivator protein (tat), messenger RNA (mRNA) would be isolated from these cells. Using the mRNA molecules as templates, copy DNA (cDNA) molecules encoding the RT or tat-specific immunoglobulins, are prepared and amplified by the polymerase chain reaction. After engineering of the antibody encoding cDNAs to provide appropriate intracellular addressing information, the cDNAs would be used to transfect stem cells of HIV infected individuals in vitro. The presence, in the cytoplasm and nucleus, of antibodies which had been selected to interfere with the reproduction of the virus, would protect these cells from infection. Autologous transplantation of such cells would confer resistance against HIV replication by these stem cells and their progeny in the treated individual. Such a strategy may also be useful against other retroviruses and could provide resistance against retrovirally triggered leukemia.

DNA↗
PubMed2026

Light-activated CRISPR/dCas9 nanomedicine for programmable control of renal fibrosis.

Renal fibrosis is the final common pathway of progressive chronic kidney disease and is maintained by spatially heterogeneous interactions among injured epithelial cells, activated fibroblasts, immune cells, extracellular matrix remodeling, metabolic stress, and persistent profibrotic transcriptional programs. Current therapies slow renal functional decline but do not directly control the regulatory circuits that stabilize maladaptive repair. Photoresponsive renal nanomedicine offers a potential strategy to add external control to anti-fibrotic intervention by combining kidney-directed delivery with light-gated release or activation of molecular payloads. This review examines the emerging interface between photoresponsive nanomaterials and CRISPR/dCas9-based gene regulation for renal fibrosis, with emphasis on upconversion nanoparticles, photoresponsive polymers, ROS- and pH-responsive matrices, optogenetic switches, and renal-compartment-directed carrier design. We argue that the most defensible therapeutic objective is not permanent genome editing or autonomous organ regeneration, but spatially confined, temporally limited, and reversible regulation of validated fibrotic or protective gene programs using CRISPRa, CRISPRi, or dCas9-based epigenome editors. The review therefore evaluates material requirements, optical-dosimetry constraints, payload architecture, renal biodistribution, target-selection logic, safety risks, and preclinical validation criteria. By defining the engineering and biological conditions required for controlled anti-fibrotic regulation, this framework positions photoresponsive renal nanomedicine as a translationally testable route toward localized modulation of fibrotic cell states rather than an overextended claim of kidney regeneration.

Anti-fibrotic gene regulation↗
PubMed1995

Functional significance of a protein conformation change at the cytoplasmic end of helix F during the bacteriorhodopsin photocycle.

The second half of the photocycle of the light-driven proton pump bacteriorhodopsin includes proton transfers between D96 and the retinal Schiff base (the M to N reaction) and between the cytoplasmic surface and D96 (decay of the N intermediate). The inhibitory effects of decreased water activity and increased hydrostatic pressure have suggested that a conformational change resulting in greater hydration of the cytoplasmic region is required for proton transfer from D96 to the Schiff base, and have raised the possibility that the reversal of this process might be required for the subsequent reprotonation of D96 from the cytoplasmic surface. Tilt of the cytoplasmic end of helix F has been suggested by electron diffraction of the M intermediate. Introduction of bulky groups, such as various maleimide labels, to engineered cysteines at the cytoplasmic ends of helices A, B, C, E, and G produce only minor perturbation of the decays of M and N, but major changes in these reactions when the label is linked to helix F. In these samples the reprotonation of the Schiff base is accelerated and the reprotonation of D96 is strongly retarded. Cross-linking with benzophenone introduced at this location, but not at the others, causes the opposite change: the reprotonation of the Schiff base is greatly slowed while the reprotonation of D96 is accelerated. We conclude that, consistent with the structure from diffraction, the proton transfers in the second half of the photocycle are facilitated by motion of the cytoplasmic end of helix F, first away from the center of the protein and then back.

Bacteriorhodopsins↗
PubMedDate not supplied

A cysteine-linkable, short cleavable photoprobe with dual functionality to explore protein-protein interfaces.

We developed a bifunctional photoprobe with dual functionality, that can be specifically tethered to cysteinyl residues of peptides and proteins through a short cleavable disulfide bond. Thus, an aryldiazonium moiety is positioned at approximately 8.5 A from the modified cysteinyl alpha-carbon, leading to one of the shortest cleavable linkages. In a sodium azide-containing buffer, the aryldiazonium moiety is transformed into an aryl azide. Therefore, with one bifunctional photoprobe two types of photogenerated species can be obtained: a hydrophilic and positively charged arylcation or a hydrophobic nitrene. We coupled the aryldiazonium probe, in a site-directed manner, to a nicotinic acetylcholine receptor competitive antagonist, obtained by chemical engineering of an analogue of a snake alpha-neurotoxin. In this molecule, Arg33, which is known to interact with the receptor, was replaced by a cysteine residue, where the photoprobe could be attached. Under inactinic light, this novel photosensitive snake toxin behaved as a reversible ligand on the Torpedo acetylcholine receptor. However, when irradiated at 391 nm, it generated a highly reactive arylcation which labeled mostly the receptor alpha-subunit, confirming the location of the tip of the second toxic loop near this receptor subunit. Finally, we showed that reduction of the disulfide bond, linking the ligand to the photocoupled receptor, allowed introduction of radioactivity on the labeled residue(s), opening the way to further characterization and avoiding the synthesis of a radioactive bifunctional photoprobe.

Animals↗
PubMed2006

Baculovirus transduction of human mesenchymal stem cell-derived progenitor cells: variation of transgene expression with cellular differentiation states.

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs). In this study, we further demonstrated, for the first time, that baculovirus can transduce adipogenic, chondrogenic and osteogenic progenitors originating from MSCs. The transduction efficiency (21-90%), transgene expression level and duration (7-41 days) varied widely with the differentiation lineages and stages of the progenitors, as determined by flow cytometry. The variation stemmed from differential transgene transcription (as revealed by real-time reverse transcription-polymerase chain reaction), rather than from variability in virus entry or cell cycle (as determined by quantitative real-time PCR and flow cytometry). Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic, osteogenic and chondrogenic pathways. The susceptibility to baculovirus transduction was higher for adipogenic and osteogenic progenitors, but was lower for chondrogenic progenitors. In particular, the duration of transgene expression was prolonged in the transduced adipogenic and osteogenic progenitors (as opposed to the MSCs), implicating the possibility of extending transgene expression via a proper transduction strategy design. Taken together, baculovirus may be an attractive alternative to genetically modify adipogenic and osteogenic progenitors in the ex vivo setting for cell therapy or tissue engineering.

Adipocytes↗
PubMed2000

A plant virus vector for systemic expression of foreign genes in cereals.

Inserts bearing the coding sequences of NPT II and beta-glucuronidase (GUS) were placed between the nuclear inclusion b (NIb) and coat protein (CP) domains of the wheat streak mosaic virus (WSMV) polyprotein ORF. The WSMV NIb-CP junction containing the nuclear inclusion a (NIa) protease cleavage site was duplicated, permitting excision of foreign protein domains from the viral polyprotein. Wheat, barley, oat and maize seedlings supported systemic infection of WSMV bearing NPT II. The NPT II insert was stable for at least 18-30 days post-inoculation and had little effect on WSMV CP accumulation. Histochemical assays indicated the presence of functional GUS protein in systemically infected wheat and barley plants inoculated with WSMV bearing GUS. The GUS constructs had greatly reduced virulence on both oat and maize. RT-PCR indicated that the GUS insert was subject to deletion, particularly when expressed as a GUS-NIb protein fusion. Both reporter genes were expressed in wheat roots at levels comparable to those observed in leaves. These results clearly demonstrate the utility of WSMV as a transient gene expression vector for grass species, including two important grain crops, wheat and maize. The results further indicate that both host species and the nature of inserted sequences affect the stability and expression of foreign genes delivered by engineered virus genomes.

Carrier Proteins↗
PubMed2001

The degree of attenuation of tick-borne encephalitis virus depends on the cumulative effects of point mutations.

An infectious clone (pGGVs) of the tick-borne encephalitis complex virus Vasilchenko (Vs) was constructed previously. Virus recovered from pGGVs produced slightly smaller plaques than the Vs parental virus. Sequence analysis demonstrated five nucleotide differences between the original Vs virus and pGGVs; four of these mutations resulted in amino acid substitutions, while the fifth mutation was located in the 3' untranslated region (3'UTR). Two mutations were located in conserved regions and three mutations were located in variable regions of the virus genome. Reverse substitutions from the conserved regions of the genome, R(496)-->H in the envelope (E) gene and C(10884)-->T in the 3'UTR, were introduced both separately and together into the infectious clone and their biological effect on virus phenotype was evaluated. The engineered viruses with R(496) in the E protein produced plaques of smaller size than viruses with H(496) at this position. This mutation also affected the growth and neuroinvasiveness of the virus. In contrast, the consequence of a T(10884)-->C substitution within the 3'UTR was noticeable only in cytotoxicity and neuroinvasiveness tests. However, all virus mutants engineered by modification of the infectious clone, including one with two wild-type mutations, H(496) and T(10884), showed reduced neuroinvasiveness in comparison with the Vs parental virus. Therefore, although the H(496)-->R and T(10884)-->C substitutions clearly reduce virus virulence, the other mutations within the variable regions of the capsid (I(45)-->F) and the NS5 (T(2688)-->A and M(3385)-->I) genes also contribute to the process of attenuation. In terms of developing flavivirus vaccines, the impact of accumulating apparently minor mutations should be assessed in detail.

3' Untranslated Regions↗
PubMed1995

T-cell recognition of antigen. A process controlled by transient intermolecular interactions.

As recently as ten years ago, the nature of the T-cell receptor for antigen was a mystery, as was the precise role of histocompatibility molecules in antigen-presentation to T cells. Although T-cell receptors have now been cloned and crystal structures of MHC/peptide molecules exist, our understanding of the parameters that characterize this interaction and other interactions relevant to T-cell immunity are still unclear. The engineering of soluble forms of proteins that mediate T-cell recognition of antigen has allowed the first measurements of these parameters. Interestingly, many of these interactions are of a transient nature, with very rapid off-rates. These data suggest a model whereby highly reversible intermolecular interactions mediate the cell-cell association. The association of adhesion molecules is probably the first step in the stabilization of a conjugate, because they are more numerous than any antigen-specific interaction, followed later by TCR-MHC engagements. Diffusion within each lipid bilayer should allow the congregation of MHC/TCR interactions at the cell-cell interface, with peptide-specific TCR interactions outcompeting irrelevant interactions. Rapid off-rates for both the antigen-specific and nonspecific interactions may be necessary to maintain reversibility, yet allow a rapid approach to equilibrium and consequent signaling when a specific antigen is present or disengagement when it is not.

Animals↗
PubMed2005

The potential role of mesenchymal stem cell therapy for intervertebral disc degeneration: a critical overview.

Low-back pain is the most common health problem for men and women between 20 and 50 years of age, resulting in 13 million doctor visits in the US annually, with significant costs to society in terms of lost time from work and direct and indirect medical expenses. Although the exact origin of most cases of low-back pain remains unknown, it is understood that degenerative damage to the intervertebral disc (IVD) plays a central role in the pathogenic mechanism leading to this disorder. Current treatment modalities for disc-related back pain (selective nerve root blocks, surgical discectomy and fusion) are costly procedures aimed only at alleviating symptoms. Consequently, there is growing interest in the development of novel technologies to repair or regenerate the degenerated IVD. Recently, mesenchymal stem cells (MSCs) have been found to possess the capacity to differentiate into nucleus pulposus-like cells capable of synthesizing a physiological, proteoglycan-rich extracellular matrix characteristic of healthy IVDs. In this article, the authors review the use of MSCs for repopulation of the degenerating IVD. Although important obstacles to the survival and proliferation of stem cells within the degenerating disc need to be overcome, the potential for MSC therapy to slow or reverse the degenerative process remains substantial.

Animals↗
PubMed2001

Analysis of estrogenic hormones in municipal wastewater effluent and surface water using enzyme-linked immunosorbent assay and gas chromatography/tandem mass spectrometry.

Although the estrogenic hormones 17 beta-estradiol and 17 alpha-ethinyl estradiol can be quantified in polluted waters by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/tandem mass spectrometry (GC/MS/MS), the compounds often are present at concentrations below detection limits. Enzyme-linked immunosorbent assays (ELISAs) provide a sensitive and robust means of quantifying estrogenic hormones in wastewater effluents and surface waters. Results from ELISA analysis of estrogenic hormones in secondary wastewater effluent indicate concentrations comparable to those that cause vitellogenesis in fish. Confirmatory analyses by GC/MS/MS are consistent with ELISA results. Effluent filtration, using sand filtration or microfiltration, removes approx. 70% of the hormones from secondary effluent, while advanced treatment, using reverse osmosis, removes more than 95% of hormones. The detection limits for estrogenic hormones are approx. 0.1 ng/L in wastewater effluent and 0.05 ng/L in surface water. The ELISA technique provides a relatively simple and practical method of assessing the fate of estrogenic hormones in engineered and natural systems.

Chromatography, High Pressure Liquid↗
PubMed2001

Circularly permuted green fluorescent proteins engineered to sense Ca2+.

To visualize Ca(2+)-dependent protein-protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini. The cpGFP was fused to calmodulin and its target peptide, M13. The chimeric protein, which we have named "pericam," was fluorescent and its spectral properties changed reversibly with the amount of Ca(2+), probably because of the interaction between calmodulin and M13 leading to an alteration of the environment surrounding the chromophore. Three types of pericam were obtained by mutating several amino acids adjacent to the chromophore. Of these, "flash-pericam" became brighter with Ca(2+), whereas "inverse-pericam" dimmed. On the other hand, "ratiometric-pericam" had an excitation wavelength changing in a Ca(2+)-dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca(2+) dynamics, such as Ca(2+) oscillations in the cytosol and the nucleus. Ca(2+) imaging using high-speed confocal line-scanning microscopy and a flash-pericam allowed to detect the free propagation of Ca(2+) ions across the nuclear envelope. Then, free Ca(2+) concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca(2+) transients caused rapid changes in the concentration of mitochondrial Ca(2+). Finally, a "split-pericam" was made by deleting the linker in the flash-pericam. The Ca(2+)-dependent interaction between calmodulin and M13 in HeLa cells was monitored by the association of the two halves of GFP, neither of which was fluorescent by itself.

Calcium↗
PubMed1999

TGF-beta-independent induction of immunogenicity by decorin gene transfer in human malignant glioma cells.

Ectopic expression of the proteoglycan, decorin, abrogates the growth of experimental C6 gliomas in the rat. Since gliomas release large amounts of transforming growth factor-beta (TGF-beta) and since decorin is a TGF-beta antagonist, decorin gene transfer-mediated abrogation of glioma growth in vivo may involve enhanced immunogenicity of the tumor cells. Here, we report that human glioma cells stimulate alloreactive immune responses when engineered to express decorin whereas parental glioma cells are non-immunogenic in vitro. The alloreactive immune response is mediated by CD8+ and CD4+ T cells as well as by NK cells. The immunosuppression exerted by parental or mock-transfected glioma cells is mediated by soluble factors and can in part be mimicked by exogenous TGF-beta. However, neutralizing anti-TGF-beta antibodies do not reverse glioma-mediated immunosuppression, suggesting that decorin abrogates glioma-induced immune cell inhibition by interfering with the activity of other, so far unidentified glioma-secreted mediators. We conclude that enhanced immunogenicity may mediate the antineoplastic effects of decorin gene therapy for malignant glioma but that factors other than TGF-beta may be responsible for glioma-induced immunosuppression.

Adenoviridae↗
PubMed2005

Nanografting de novo proteins onto gold surfaces.

The immobilization of novel proteins onto addressable locations on a flat surface has potential applications in a range of biotechnologies. Here we describe the nanopatterning of a de novo protein onto a gold surface. Patterning was achieved using a technique called nanografting, in which the tip of an atomic force microscope is used to disrupt a preexisting monolayer of alkanethiol molecules on a gold surface, thereby facilitating exchange with alternative thiol-linked molecules from the surrounding solution. The protein used for these studies was chosen from a designed combinatorial library of de novo sequences expressed in E. coli and was engineered to have a glycine-glycine-cysteine tag at its C-terminus, thereby enabling attachment to the gold surface through a single cysteine thiol. The average height of the grafted protein patterns was found to be somewhat higher than expected from the known NMR structure of the protein. Compression of the nanografted patches by an external force (below 10 nN) was reversible but showed some hysteresis. Interestingly, both the energy required to deform the immobilized protein patterns and the energy defined by the hysteresis loop were found to be of the same order as the energy required to unfold the monomeric protein in solution. These studies demonstrate the possibility of preparing nanometer scale protein arrays, lowering significantly the volume requirements of the protein samples necessary to fabricate protein-based biosensor arrays and thereby providing a base for increasing their sensitivity.

Adsorption↗
PubMed2003

Contribution of surface salt bridges to protein stability: guidelines for protein engineering.

The small globular protein, ubiquitin, contains a pair of oppositely charged residues, K11 and E34, that according to the three-dimensional structure are located on the surface of this protein with a spatial orientation characteristic of a salt bridge. We investigated the strength of this salt bridge and its contribution to the global stability of the ubiquitin molecule. Using the "double mutant cycle" analysis, the strength of the pairwise interactions between K11 and E34 was estimated to be favorable by 3.6kJ/mol. Further, the salt bridge of the reverse orientation, i.e. E11/K34, can be formed and is found to have a strength (3.8kJ/mol) similar to that of the K11/E34 pair. However, the global stability of the K11/E34 variant of ubiquitin is 2.2kJ/mol higher than that of the E11/K34 variant. The difference in the contribution of the opposing salt bridge orientations to the overall stability of the ubiquitin molecule is attributed to the difference in the charge-charge interactions between residues forming the salt bridge and the rest of the ionizable groups in this protein. On the basis of these results, we concluded that surface salt bridges are stabilizing, but their contribution to the overall protein stability is strongly context-dependent, with charge-charge interactions being the largest determinant. Analysis of 16 salt bridges from six different proteins, for which detailed experimental data on energetics have been reported, support the conclusions made from the analysis of the salt bridge in ubiquitin. Implications of these findings for engineering proteins with enhanced thermostability are discussed.

Circular Dichroism↗
PubMed2006

Chondrocyte phenotype in engineered fibrous matrix is regulated by fiber size.

A biomaterial scaffold acting as a functional substitute for the native extracellular matrix provides space for cell accommodation. In this study, we seeded chondrocytes, isolated from 4- to 6-month-old calves, in 2 types of poly(L-lactide) scaffolds, composed of micro- and nanofibers, and compared the effects on cellular activities. Scanning electron microscopy revealed a well-spread morphology for chondrocytes grown on microfibers. In contrast, chondrocytes on the nanofibers were found to have a rounded morphology and displayed a disorganized actin cytoskeletal structure compared to the organized cytoskeleton seen in well-spread chondrocytes culture on the microfibrous scaffold. Both scaffolds supported chondrocyte proliferation, with a higher rate seen in cultures in nanofibrous scaffold. Quantitative reverse transcription-polymerase chain reaction analysis showed that both cultures supported expression of collagen types I and II and aggrecan. Biochemical analysis showed a higher level of sulfated glycosaminoglycan in the nanofiber culture, confirmed by more intense alcian blue histologic staining. The nanofiber cultures also showed higher immunostaining for collagen types II and IX, aggrecan, and cartilage proteoglycan link protein. Based on these results, we conclude that chondrocytes respond differently to fibrous scaffolds of varying diameters, and that the scaffolds made of nanofibrous biomaterial promote efficient cell-based cartilage tissue engineering.

Aggrecans↗
PubMed2005

Atomic engineering of mixed ferrite and core-shell nanoparticles.

Nanoparticulate ferrites such as manganese zinc ferrite and nickel zinc ferrite hold great promise for advanced applications in power electronics. The use of these materials in current applications requires fine control over the nanoparticle size as well as size distribution to maximize their packing density. While there are several techniques for the synthesis of ferrite nanoparticles, reverse micelle techniques provide the greatest flexibility and control over size, crystallinity, and magnetic properties. Recipes for the synthesis of manganese zinc ferrite, nickel zinc ferrite, and an enhanced ferrite are presented along with analysis of the crystalline and magnetic properties. Comparisons are made on the quality of nanoparticles produced using different surfactant systems. The importance of various reaction conditions is explored with a discussion on the corresponding effects on the magnetic properties, particle morphology, stoichiometry, crystallinity, and phase purity.

Crystallization↗
PubMed1992

Determination of the excited-state lifetimes of the tryptophan residues in barnase, via multifrequency phase fluorometry of tryptophan mutants.

A multifrequency phase fluorometric study is described for wild-type barnase and engineered mutant proteins in which tryptophan residues have been replaced by less fluorescent residues which do not interfere with the determination of the tryptophan emission spectra and lifetimes. The lifetimes of the three tryptophans in the wild-type protein have been resolved. Trp-35 has a single fluorescence lifetime, which varies in the different proteins between 4.3 and 4.8 ns and is pH-independent between pH 5.8 and 8.9. Trp-71 and Trp-94 behave as an energy-transfer couple with both forward and reverse energy transfer. The couple shows two fluorescence lifetimes: 2.42 (+/-0.2) and 0.74 (+/-0.1) ns at pH 8.9, and 0.89 (+/-0.05) and 0.65 (+/-0.05) ns at pH 5.8. In the mutant Trp-94----Phe the lifetime of Trp-71 is 4.73 (+/-0.008) ns at high pH and 4.70 (+/-0.004) ns at low pH. In the mutant Trp-71----Tyr, the lifetime of Trp-94 is 1.57 (+/-0.01) ns at high pH and 0.82 (+/-0.025) ns at low pH. From these lifetimes, one-way energy-transfer efficiencies can be calculated according to Porter [Porter, G.B. (1972) Theor. Chim. Acta 24, 265-270]. At pH 8.9, a 71% efficiency was found for forward transfer (from Trp-71 to Trp-94) and 36% for reverse transfer. At pH 5.8 the transfer efficiency was 86% for forward and 4% for reverse transfer (all +/-2%). These transfer efficiencies correspond fairly well with the ones calculated according to the theory of Förster [Förster, T. (1948) Ann. Phys. (Leipzig) 2, 55-75].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗