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At least 1,711 records · Page 95Linked to original sources
PubMed2004

Finding a new vaccine in the ricin protein fold.

Previous attempts to produce a vaccine for ricin toxin have been hampered by safety concerns arising from residual toxicity and the undesirable aggregation or precipitation caused by exposure of hydrophobic surfaces on the ricin A-chain (RTA) in the absence of its natural B-chain partner. We undertook a structure-based solution to this problem by reversing evolutionary selection on the 'ribosome inactivating protein' fold of RTA to arrive at a non-functional, compacted single-domain scaffold (sequence RTA1-198) for presentation of a specific protective epitope (RTA loop 95-110). An optimized protein based upon our modeling design (RTA1-33/44-198) showed greater resistance to thermal denaturation, less precipitation under physiological conditions and a reduction in toxic activity of at least three orders of magnitude compared with RTA. Most importantly, RTA1-198 or RTA1-33/44-198 protected 100% of vaccinated animals against supra-lethal challenge with aerosolized ricin. We conclude that comparative protein analysis and engineering yielded a superior vaccine by exploiting a component of the toxin that is inherently more stable than is the parent RTA molecule.

Circular Dichroism↗
PubMed2002

Efficient expression and secretion of recombinant hirudin III in E. coli using the L-asparaginase II signal sequence.

One of the hirudin variants HV3 was efficiently expressed in Escherichia coli using the L-asparaginase II signal sequence and the product was secreted into the culture medium. For the secretory manufacture of HV3, the L-asparaginase II signal sequence containing a single NheI restriction site at its 3' end was designed using the degenerate codons and PCR-amplified from E. coli chromosomal DNA. The synthetic HV3 coding sequence was fused to the signal sequence in-frame by its 5' NheI restriction site. The above signal-HV3 fusion gene was inserted into an expression vector pTA, which was derived from pkk223-3 such that its expression was under the control of the tac promotor. The resulting HV3 secretion expression vector pTASH thus constructed was introduced into an E. coli host cell AS1.357 with high L-asparaginase II producing level. After inducing with IPTG, the expression product was efficiently secreted into the culture medium and shake-flask culturing gave a yield of approximately 5 x 10(5)ATU/L (approximately 60mg/L). The secreted HV3 was easily purified from culture supernatant using ultrafiltration, ion-exchange column chromatography, and FPLC reverse-phase chromatography. The purified rHV3 from the culture supernatant had the expected N-terminal amino sequence and strong antithrombin activity, suggesting that the signal sequence was completely removed and the product was processed accurately during the secretion process.

Amino Acid Sequence↗
PubMed2002

BetaCore, a designed water soluble four-stranded antiparallel beta-sheet protein.

BetaCore is a designed approximately 50-residue protein in which two BPTI-derived core modules, CM I and CM II, are connected by a 22-atom cross-link. At low temperature and pH 3, homo- and heteronuclear NMR data report a dominant folded ('f') conformation with well-dispersed chemical shifts, i, i+1 periodicity, numerous long-range NOEs, and slowed amide hydrogen isotope exchange patterns that is a four-stranded antiparallel beta-sheet with nonsymmetrical and specific association of CM I and CM II. BetaCore 'f' conformations undergo reversible, global, moderately cooperative, non-two-state thermal transitions to an equilibrium ensemble of unfolded 'u' conformations. There is a significant energy barrier between 'f' and 'u' conformations. This is the first designed four-stranded antiparallel beta-sheet that folds in water.

Amino Acid Sequence↗
PubMed2006

Evolution of the uniquely adaptable lentiviral envelope in a natural reservoir host.

BACKGROUND: The ability of emerging pathogens to infect new species is likely related to the diversity of pathogen variants present in existing reservoirs and their degree of genomic plasticity, which determines their ability to adapt to new environments. Certain simian immunodeficiency viruses (SIVcpz, SIVsm) have demonstrated tremendous success in infecting new species, including humans, resulting in the HIV-1 and HIV-2 epidemics. Although SIV diversification has been studied on a population level, the essential substrates for cross-species transmission, namely SIV sequence diversity and the types and extent of viral diversification present in individual reservoir animals have not been elucidated. To characterize this intra-host SIV diversity, we performed sequence analyses of clonal viral envelope (env) V1V2 and gag p27 variants present in individual SIVsm-infected sooty mangabeys over time. RESULTS: SIVsm demonstrated extensive intra-animal V1V2 length variation and amino acid diversity (le38%), and continual variation in V1V2 N-linked glycosylation consensus sequence frequency and location. Positive selection was the predominant evolutionary force. Temporal sequence shifts suggested continual selection, likely due to evolving antibody responses. In contrast, gag p27 was predominantly under purifying selection. SIVsm V1V2 sequence diversification is at least as great as that in HIV-1 infected humans, indicating that extensive viral diversification in and of itself does not inevitably lead to AIDS. CONCLUSION: Positive diversifying selection in this natural reservoir host is the engine that has driven the evolution of the uniquely adaptable SIV/HIV envelope protein. These studies emphasize the importance of retroviral diversification within individual host reservoir animals as a critical substrate in facilitating cross-species transmission.

Animals↗
PubMed2005

Expression of a major house dust mite allergen gene from Dermatophagoides farinae in Lotus japonicus accession Miyakojima MG-20.

Transformation of a model legume Lotus japonicus accession Miyakojima MG-20 was examined using Agrobacterium tumefaciens with a binary expression vector. Using the improved transformation method, we introduced a major allergen gene from a house dust mite, Der f 1, into MG-20. Analyses by Southern hybridization, reverse transcription (RT)-PCR, and Western blotting showed that the Der f 1 gene was integrated into the genome of L. japonicus, expressing the gene product in the T1 lines. Our results imply future application of oral allergen-specific immunotherapy using legume plants.

Antigens, Dermatophagoides↗
PubMed2005

Expression of MMPs and TIMPs in primary epithelial cell cultures of the upper aerodigestive tract seeded on the surface of a novel polymeric biomaterial.

INTRODUCTION: Using standard cell biological and biochemical experimental approaches we were able to test the ability of a particular polymer construct to support the adhesion, proliferation, and the cellular acitivity of pharyngeal cells. The delicate balance between Matrix Metalloproteinases (MMPs) and their endogenous inhibitors (Tissue Inhibitor of MMPs, TIMPs) have a decisive function in the remodeling of the extracellular matrix during cellular ingrowth. Novel polymeric biomaterials may be useful to develop new therapeutic options in head and neck surgery. METHODS: Primary cell cultures of the pharynx of Sprague-Dawley rats were seeded on the surface of a thermoplastic multi-block copolymer and on a polystyrene surface as control. Conditioned media of the primary cells was analyzed for MMPs and TIMPs. The MMP and TIMP expression was analysed by zymography and a radiometric enzyme assay. RESULTS: No statistically significant differences in the levels of MMP-1, MMP-2, MMP-9 and TIMPs were detected between cells grown on the novel polymer surface versus control. CONCLUSION: An appropriate understanding of the molecular machinery that regulates gene expression and cellular growth in tissue engineered constructs is the requirement for an optimal adaptation of biodegradable biomaterials to develop new therapeutic options in otolaryngology and head and neck surgery.

Animals↗
PubMed1986

Viruses as environmental mutagenic factors.

Injections into Drosophila melanogaster males of 9 DNA- and RNA-containing viruses non-infectious for this insect, proved that all of them are highly mutagenic, inducing numerous gene mutations or microdeletions but no gross chromosome rearrangements. Relatively few specific loci are affected, they vary per virus, and the mutation rate for such a locus is extraordinarily high. These peculiarities of the mutagenic action of viruses closely resemble those of the mutagenic action of exogenous non-viral DNA earlier studied by the author and his co-workers. It was shown that the mutagenic element of a virus is its nucleic acid; viral proteins completely lack mutagenic properties. Virus-induced gene mutations are probably due to insertions of fragments of viral DNA (or cDNA) into the host chromosomes; at least some of these mutations are capable of transpositions and reversions. On the other hand, chromosome aberrations which arise in virus-infected cells are evidently produced secondarily by pathological disturbances of metabolic processes in the infected cells. Our results and literature data lead to the conclusion that a significant share of hereditary defects in man might be caused by viruses, even by those apathogenic for humans, particularly by attenuated live viral vaccines. It is, therefore, important to work on the creation of purely proteinaceous viral vaccines free from nucleic acids and thus genetically safe. The hazards of viral mutagenesis should also be kept in mind when preparing recombinant DNA molecules used in genetic engineering.

Animals↗
PubMed1995

Retrovirus-mediated expression of an artificial beta-endorphin precursor in primary fibroblasts.

Peptides are of potential interest in the field of gene therapy but require modification by genetic engineering to facilitate their secretion. Amino terminal addition of a signal peptide is not always sufficient to achieve this goal, as found in this study for beta-endorphin. To overcome this problem, addition of the pre-pro-sequence of mouse nerve growth factor to beta-endorphin was tested. Retrovirus-mediated expression of a hybrid construct of the pre-pro-sequence of nerve growth factor and human beta-endorphin in primary fibroblasts resulted in the secretion of beta-endorphin immunoreactivity at a rate of 620 pg/h/10(6) cells. Analysis of the secreted beta-endorphin immunoreactivity with reverse-phase HPLC, immunoassays using three different antibodies, and an assay for the specific displacement of [3H][D-Ala2,N-MePhe4,Gly-ol5]enkephalin from mu-opioid receptors suggests that the pre-pro-sequence is cleaved off from the pre-pro-sequence/beta-endorphin construct prior to secretion, resulting in bona fide beta-endorphin. Transplantation of beta-endorphin-secreting cells into brain or spinal cord may provide a gene therapy approach for the treatment of chronic, opioid-sensitive pain states.

3T3 Cells↗
PubMed1997

Mutants of human beta2-microglobulin map an immunodominant epitope within the three-stranded beta-pleated sheet.

Genetically engineered structural variants of human beta2-microglobulin (beta2m) were produced by sequence exchange with mouse beta2m for the purpose of examining species-specific antigenic determinant expression. For aggregate mapping, mouse and human beta2m, which differ by 30% in their primary sequence of 99 amino acids, were prepared as chimeric (human X mouse) molecules and expressed in the FO-1 beta2m-null human melanoma cell line. A chimera containing residues 1-69 from human beta2m (and residues 70-99 from mouse beta2m) induced expression of the epitopes defined by the anti-beta2m monoclonal antibodies (mAb) BBM.1, NAMB-1, and L368; the reverse chimera did not, although HLA class I heavy chain was evident on the cell surface as determined with the TP25.99 mAb. For fine dissection of the epitopes defined by these mAbs, site-directed mutants of beta2m were prepared by replacement of individual amino acids in human beta2m with the dimorphic residue from mouse beta2m. Substitutions were made at each divergent residue between positions 1 and 66 and, as controls for COOH-terminal modification, a series of residues between positions 75 and 94. Replacement of amino acids 38, 44, and 45, but not 16 other dimorphic residues in the linear stretch from residue 1 to residue 66, resulted in the loss of, or gross reduction in, binding by mAbs BBM.1 and NAMB-1. A reduction in binding was also observed for mAb L368. These data provide strong evidence that the antigenic epitopes defined by these mAb map to a region including S3 and its adjacent intra-beta-strand turn of the three-stranded beta-pleated sheet of beta2m. The mapping of these epitopes is consistent with their accessibility in the assembled major histocompatibility complex class I molecule and indicates that the region from amino acid 38 to 45 is an important structural feature in the "foreignness" of human and mouse beta2m.

Animals↗
PubMed2004

Toward construction of a self-sustained clock-like expression system based on the mammalian circadian clock.

Despite recent advances in circadian biology, detailed understanding of how a biological pacemaker system is assembled, maintained, and regulated continues to be a significant challenge. We have assembled and characterized a first-generation, regulatable, self-sustained clock-like expression system based on key components of the mammalian circadian clock. The molecular setup of the clock-like oscillator was reduced to the core set of positive and negative elements common to all known circadian pacemakers. Sophisticated tetracycline-responsive multi-cistronic expression integrated with forefront lentiviral transduction tools enabled autoregulated reporter transgene expression in a human cell line. We characterized transgene expression kinetics of an artificial oscillator and showed that its expression profiles could be modulated by a serum shock and administration of regulating tetracycline antibiotics. Design of a generic mammalian clock-like expression system will offer novel opportunities to study circadian biology and may provide a unique tool for rhythmic expression of desired transgenes fostering advances in biopharmaceutical manufacturing, gene therapy, and tissue engineering.

ARNTL Transcription Factors↗
PubMed2006

Dexamethasone-functionalized gels induce osteogenic differentiation of encapsulated hMSCs.

Synthetic hydrogels represent highly controlled environments for three-dimensional culture of human mesenchymal stem cells (hMSCs). Encapsulated hMSCs are presented with a "blank" environment, and this environment can be closely controlled in order to elicit an osteogenic response. In vitro, dexamethasone is an efficient and reliable factor that leads to the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The aim of this work was to develop a dexamethasone-releasing poly(ethylene glycol) (PEG)-based hydrogel scaffold to deliver dexamethasone to encapsulated cells in a sustained manner. To accomplish this goal, dexamethasone was covalently linked to a photoreactive mono-acrylated PEG molecule through a degradable lactide bond, and this molecule was covalently incorporated into the PEG hydrogel during photopolymerization. Over time, hydrolysis of the ester bonds resulted in dexamethasone release from the gel. The biological activity of the released dexamethasone was verified in monolayer cell culture and in three-dimensional culture (i.e., in the gel) by the ability of hMSCs to express osteogenic genes, including alkaline phosphatase, osteopontin, and core binding factor alpha 1, as measured using real-time reverse transcription polymerase chain reaction (RT-PCR). These studies indicate that encapsulated hMSCs are capable of osteogenic differentiation in response to released dexamethasone.

Alkaline Phosphatase↗
PubMed1999

Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant.

The event that initiates the extrinsic pathway of blood coagulation is the association of coagulation factor VIIa (VIIa) with its cell-bound receptor, tissue factor (TF), exposed to blood circulation following tissue injury and/or vascular damage. The natural inhibitor of the TF.VIIa complex is the first Kunitz domain of tissue factor pathway inhibitor (TFPI-K1). The structure of TF. VIIa reversibly inhibited with a potent (Ki=0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been determined at 2.1 A resolution. When bound to TF, the four domain VIIa molecule assumes an extended conformation with its light chain wrapping around the framework of the two domain TF cofactor. The 5L15 inhibitor associates with the active site of VIIa similar to trypsin-bound BPTI, but makes several unique interactions near the perimeter of the site that are not observed in the latter. Most of the interactions are polar and involve mutated positions of 5L15. Of the eight rationally engineered mutations distinguishing 5L15 from BPTI, seven are involved in productive interactions stabilizing the enzyme-inhibitor association with four contributing contacts unique to the VIIa.5L15 complex. Two additional unique interactions are due to distinguishing residues in the VIIa sequence: a salt bridge between Arg20 of 5L15 and Asp60 of an insertion loop of VIIa, and a hydrogen bond between Tyr34O of the inhibitor and Lys192NZ of the enzyme. These interactions were used further to model binding of TFPI-K1 to VIIa and TFPI-K2 to factor Xa, the principal activation product of TF.VIIa. The structure of the ternary protein complex identifies the determinants important for binding within and near the active site of VIIa, and provides cogent information for addressing the manner in which substrates of VIIa are bound and hydrolyzed in blood coagulation. It should also provide guidance in structure-aided drug design for the discovery of potent and selective small molecule VIIa inhibitors.

Amino Acid Chloromethyl Ketones↗
PubMed1994

A molecular genetic approach to the study of Venezuelan equine encephalitis virus pathogenesis.

Viral pathogenesis can be described as a series of steps, analogous to a biochemical pathway, whose endpoint is disease of the infected host. Distinct viral functions may be critical at each required step. Our genetic approach is to use Venezuelan equine encephalitis virus (VEE) mutants blocked at different steps to delineate the process of pathogenesis. A full-length cDNA clone of a virulent strain of VEE was used as a template for in vitro mutagenesis to produce attenuated single-site mutants. The spread of molecularly cloned parent or mutant viruses in the mouse was monitored by infectivity, immunocytochemistry, in situ hybridization and histopathology. Virulent VEE spread through the lymphatic system, produced viremia and replicated in several visceral organs. As virus was being cleared from these sites, it began to appear in the brain, frequently beginning in the olfactory tracts. A single-site mutant in the E2 glycoprotein appeared to block pathogenesis at a very early step, and required a reversion mutation to spread beyond the site of inoculation. The feasibility of combining attenuating mutations to produce a stable VEE vaccine strain has been demonstrated using three E2 mutations.

Amino Acid Sequence↗
PubMed2006

In vivo gene transfer into the honeybee using a nucleopolyhedrovirus vector.

The honeybee Apis mellifera L. is a social insect and one of the most industrially important insects. We examined whether a baculovirus-mediated retrotransposon is applicable to in vivo transfer of exogenous genes to the honeybees. Honeybee larvae and pupae were injected with two types of recombinant Autographa californica nucleopolyhedrovirus (AcNPV) vectors, one that includes the enhanced green fluorescent protein gene (egfp) as a reporter to be inserted into the honeybee genome, and another that includes the reverse transcriptase gene responsible for the insertion. Fluorescence was observed in most of the viral-injected larvae and pupae. Reverse transcription-polymerase chain reaction and immunoblotting confirmed egfp mRNA and eGFP expression in these honeybees, although egfp insertion into the honeybee genome was not confirmed. These results indicate that AcNPV vectors can be used for the transfer and transient expression of an exogenous gene in the larval and pupal honeybees.

Animals↗
PubMed2004

OptiRNAi, an RNAi design tool.

RNA interference (RNAi), a recently developed reverse genetics tool, has many advantages compared to traditional gene knockout methods. Appropriate selection of double stranded RNAs identical to a specific region(s) of the target gene is critical for the successful implementation of this technology. Recently, Elbashir et al. [Methods 26 (2002) 199] has established empirical criteria for siRNA sequence selection that significantly improved the success rate for RNAi attempts. We have developed OptiRNAi, a computational tool, which uses the Elbashir et al. criteria to predict appropriate target sequences for siRNA production. Specificity of these siRNAs for the target of interest can then be assessed by the investigator using the embedded Blast search engine optimized for RNAi design. Thus, OptiRNAi is an efficient and user friendly tool for RNAi design based on criteria that are more stringent than other available tools.

Animals↗
PubMed1999

Primary structure of the antigen-binding domains of a human oligodendrocyte-reactive IgM monoclonal antibody derived from a patient with multiple sclerosis.

Several murine IgM monoclonal antibodies (mAbs) promoting remyelination in mice were shown to be germline gene-encoded natural autoantibodies that react with oligodendrocytes and intracellular antigens. Here, we show that human oligodendrocyte-reactive IgM mAb DS1F8 derived from a patient with multiple sclerosis targets microtubule-like structures similar to the murine mAbs. Sequencing of the cDNAs of the variable regions revealed that the antigen-binding domains are also encoded by germline genes. These similarities of mAb DS1F8 to the murine mAbs promoting remyelination suggest that this human mAb is a natural autoantibody. This may imply that the engineering of human autoantibodies for therapy of demyelinating diseases is feasible.

Amino Acid Sequence↗
PubMed1998

Partially unfolded proteins efficiently penetrate cell membranes--implications for oral drug delivery.

We have previously reported on the biological activity of members of a library of low molecular weight compounds (carriers) that enable the oral delivery of proteins (Milstein, Proceedings of the 1995 Miami Bio/Technology Winter Symposium on Protein Engineering and Structural Biology, IRL Press at Oxford University Press, 1995, p. 13; Leone-Bay et al., J. Med. Chem. 38 (1995) 4263-4269; Leone-Bay et al., J. Med. Chem. 39 (1996) 2571-2578; [1-3]). When rats or primates are orally administered a solution of carrier and either recombinant human alpha-interferon (rhIFN), insulin or recombinant human growth hormone (rhGH) significant serum concentrations of the proteins are detectable. The transport activity of these compounds is positively correlated with their structural effects on the protein molecules. Direct measurement of the interaction of these carrier molecules with the proteins indicates that they reversibly destabilize the native state of the molecule favoring a partially unfolded conformation. Apparently these intermediate protein conformations are transport competent and are able to be absorbed through the intestinal tissue and into the bloodstream. Since the measured binding of the carriers to the partially unfolded proteins is relatively weak (Kb = 100 M(-1)) and the systemic activity of the proteins appears to be unaffected, the changes in the structure of the proteins are manifestly reversible.

Administration, Oral↗
PubMed2003

A forward look at therapy for pediatric movement disorders.

The mainstay of current therapy for pediatric movement disorders is oral symptomatic medication, unless a reversible etiology can be found. However, this approach is apt to pale in comparison with innovative strategies on the clinical forefront. Classical pharmacotherapy is restricted by the blood-brain barrier, which prevents access to the brain of potentially therapeutic molecules. Recent developments in molecular biotechnology include antibody-mediated drug release, feedback-responsive delivery systems, carrier-mediated transport, microspheres composed of polymers and liposomes, permeabilizers, and selective delivery to localized sites and vectors. Neuroprotective strategies for delivering neurotrophic factors and antiapoptotic and antioxidant molecules in neurodegenerative disorders are currently under study in clinical trials. Stem cell transplantation has great potential for tissue engineering and also as a carrier for gene therapy, although its use raises complex societal issues. These approaches, together with a plethora of transgenic knockout animal models of neurodegenerative disorders, offer real promise for a previously untreatable group of movement disorders.

Animals↗