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Biomedical subjects

Mark Young

Publications and source records attributed to Mark Young.

32 records · Page 2Linked to original sources
PubMed2005

Gene therapy for oral cancer: efficient delivery of a 'suicide gene' to murine oral cancer cells in physiological milieu.

Gene therapy is a new therapeutic modality in which defective genes are replaced with functional ones, or genes are delivered that can specifically kill cancer cells. Efficient gene delivery is an important component of gene therapy approaches. Potential safety problems with viral vectors necessitate the development of efficient nonviral vectors. DNA complexes with synthetic cationic liposomes or polymers constitute a simple means of transferring DNA into target cells. Gene delivery mediated by many nonviral vectors, however, is inhibited by serum components, and this is expected to limit the efficiency of gene delivery in vivo. In this study, the authors examined two novel gene transfection reagents, Metafectene and GeneJammer, for their ability to deliver a reporter gene to SCCVII murine squamous cell carcinoma cells in the presence of high concentrations of mouse serum. After establishing conditions that achieved significant gene delivery, the authors introduced the Herpes Simplex Virus Thymidine kinase (HSV-tk) gene into the cells using the cationic liposome reagent, Metafectene, followed by the administration of ganciclovir. After seven days of incubation, 90 percent and 82 percent cytotoxicity was observed in 0 percent and 60 percent mouse serum, respectively. The authors' observations suggest that Metafectene may be useful for the gene therapy of oral squamous cell carcinoma in a murine model involving the induction of oral tumors by SCCVII cells.

Animals↗
PubMed2004

Photocatalytic synthesis of copper colloids from CuII by the ferrihydrite core of ferritin.

The iron-storage protein ferritin encapsulates a nanoparticle of iron oxide. The size and properties of these nanoparticles can be adjusted by controlled oxidative hydrolysis reactions of Fe(II). This mineralized ferritin protein cage has previously been shown to act as an effective photocatalyst for reduction of Cr(VI). In the present work, we demonstrate that Fe(O)OH-mineralized ferritin catalyzes the photoreduction of Cu(II) to form a stable, air-sensitive, colloidal dispersion of Cu(0). In addition, the particle sizes of the Cu colloids can be controlled by varying the ratio of Cu(II) to ferritin. This illustrates an important principle, namely that the properties of one preformed material can be utilized for the specific synthesis of a second material, thus tailoring the desired physical properties of the final products. This procedure represents a multistep materials synthesis: the formation of a new nanomaterial from a catalytic precursor.

Catalysis↗
PubMed2004

The structure of a thermophilic archaeal virus shows a double-stranded DNA viral capsid type that spans all domains of life.

Of the three domains of life (Eukarya, Bacteria, and Archaea), the least understood is Archaea and its associated viruses. Many Archaea are extremophiles, with species that are capable of growth at some of the highest temperatures and extremes of pH of all known organisms. Phylogenetic rRNA-encoding DNA analysis places many of the hyperthermophilic Archaea (species with an optimum growth > or = 80 degrees C) at the base of the universal tree of life, suggesting that thermophiles were among the first forms of life on earth. Very few viruses have been identified from Archaea as compared to Bacteria and Eukarya. We report here the structure of a hyperthermophilic virus isolated from an archaeal host found in hot springs in Yellowstone National Park. The sequence of the circular double-stranded DNA viral genome shows that it shares little similarity to other known genes in viruses or other organisms. By comparing the tertiary and quaternary structures of the coat protein of this virus with those of a bacterial and an animal virus, we find conformational relationships among all three, suggesting that some viruses may have a common ancestor that precedes the division into three domains of life >3 billion years ago.

Capsid↗
PubMed2004

Thermobaculum terrenum gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia.

A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65-92 degrees C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 degrees C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1(T) (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).

Base Composition↗
PubMed2004

Heterologous expression of the modified coat protein of Cowpea chlorotic mottle bromovirus results in the assembly of protein cages with altered architectures and function.

We have developed methods for producing viral-based protein cages in high yield that are amenable to genetic modification. Expression of the structural protein of Cowpea chlorotic mottle bromovirus (CCMV) using the yeast-based Pichia pastoris heterologous expression system resulted in the assembly of particles that were visibly indistinguishable from virus particles produced in the natural host. We have shown that a collection of non-infectious CCMV coat protein mutants expressed in the P. pastoris system assemble into viral protein cages with altered architectures and function. This provides an alternative to other heterologous expression systems for production of viral structural proteins in which expression has resulted in unassembled cages. Heterologous expression in P. pastoris further enhances the development of viral-based protein cages as biotemplates for nanotechnology and for future studies examining details of icosahedral virus assembly.

Bromovirus↗
PubMed2004

Structure of D-63 from sulfolobus spindle-shaped virus 1: surface properties of the dimeric four-helix bundle suggest an adaptor protein function.

Sulfolobus spindle-shaped virus 1 (SSV1) and its fusellovirus homologues can be found in many acidic (pH or=70 degrees C) around the world. SSV1 contains a 15.5-kb double-stranded DNA genome that encodes 34 proteins with greater than 50 amino acids. A site-specific integrase and a DnaA-like protein have been previously identified by sequence homology, and three structural proteins have been isolated from purified virus and identified by N-terminal sequencing (VP1, VP2, and VP3). The functions of the remaining 29 proteins are currently unknown. To assign functions to these proteins, we have initiated biochemical and structural studies on the SSV1 proteome. Here we report the structure of SSV1 D-63. The structure reveals a helix-turn-helix motif that dimerizes to form an antiparallel four-helix bundle. Mapping residues conserved among three fusellovirus isolates onto the structure shows that one face of the rod-shaped molecule is highly conserved. This conserved surface spans the dimer axis and thus exhibits 2-fold symmetry. Two smaller conserved patches, also related by 2-fold symmetry, are found on the opposite face of the molecule. All of these conserved surfaces are devoid of clefts or pockets typically used to bind small molecules, suggesting that D-63 may function as an adaptor protein in macromolecular assembly.

Amino Acid Sequence↗
PubMed2004

Crystal structure of F-93 from Sulfolobus spindle-shaped virus 1, a winged-helix DNA binding protein.

Sulfolobus spindle-shaped viruses (SSVs), or Fuselloviridae, are ubiquitous crenarchaeal viruses found in high-temperature acidic hot springs around the world (pH </=4.0; temperature of >/=70 degrees C). Because they are relatively easy to isolate, they represent the best studied of the crenarchaeal viruses. This is particularly true for the type virus, SSV1, which contains a double-stranded DNA genome of 15.5 kilobases, encoding 34 putative open reading frames. Interestingly, the genome shows little sequence similarity to organisms other than its SSV homologues. Together, sequence similarity and biochemical analyses have suggested functions for only 6 of the 34 open reading frames. Thus, even though SSV1 is the best-studied crenarchaeal virus, functions for most (28) of its open reading frames remain unknown. We have undertaken biochemical and structural studies for the gene product of open reading frame F-93. We find that F-93 exists as a homodimer in solution and that a tight dimer is also present in the 2.7-A crystal structure. Further, the crystal structure reveals a fold that is homologous to the SlyA and MarR subfamilies of winged-helix DNA binding proteins. This strongly suggests that F-93 functions as a transcription factor that recognizes a (pseudo-)palindromic DNA target sequence.

Amino Acid Sequence↗
PubMed2004

Comparative genomic analysis of hyperthermophilic archaeal Fuselloviridae viruses.

The complete genome sequences of two Sulfolobus spindle-shaped viruses (SSVs) from acidic hot springs in Kamchatka (Russia) and Yellowstone National Park (United States) have been determined. These nonlytic temperate viruses were isolated from hyperthermophilic Sulfolobus hosts, and both viruses share the spindle-shaped morphology characteristic of the Fuselloviridae family. These two genomes, in combination with the previously determined SSV1 genome from Japan and the SSV2 genome from Iceland, have allowed us to carry out a phylogenetic comparison of these geographically distributed hyperthermal viruses. Each virus contains a circular double-stranded DNA genome of approximately 15 kbp with approximately 34 open reading frames (ORFs). These Fusellovirus ORFs show little or no similarity to genes in the public databases. In contrast, 18 ORFs are common to all four isolates and may represent the minimal gene set defining this viral group. In general, ORFs on one half of the genome are colinear and highly conserved, while ORFs on the other half are not. One shared ORF among all four genomes is an integrase of the tyrosine recombinase family. All four viral genomes integrate into their host tRNA genes. The specific tRNA gene used for integration varies, and one genome integrates into multiple loci. Several unique ORFs are found in the genome of each isolate.

Archaeal Viruses↗
PubMed2003

Constrained synthesis of cobalt oxide nanomaterials in the 12-subunit protein cage from Listeria innocua.

The protein cage of the 12-subunit ferritin-like protein from Listeria innocua has been utilized as a size and shape constrained reaction environment for the synthesis of two cobalt oxide minerals, Co(3)O(4) and Co(O)OH. Reaction of Co(II) with H(2)O(2) at pH 8.5 under either elevated temperature (65 degrees C) or ambient temperature (23 degrees C) resulted in the formation of cobalt oxide nanoparticles encapsulated within the protein cage. At elevated temperatures, Co(3)O(4) was formed while at lower temperature the oxyhydroxide Co(O)OH was found. Mineral particles, commensurate in size with the internal dimensions of the protein (5 nm), were imaged by transmission electron microscopy and shown to be surrounded by the intact protein cage. The minerals were investigated by electron diffraction and revealed a crystalline Co(3)O(4) phase and an amorphous Co(O)OH phase. Further investigation of these composite materials using size exclusion chromatography, gel electrophoresis, dynamic light scattering, and zeta potential measurements indicated that the mineral was encapsulated within the protein cage giving rise to properties of both the mineral and protein components.

Bacterial Proteins↗
PubMed2003

2-D array formation of genetically engineered viral cages on au surfaces and imaging by atomic force microscopy.

The preparation and subsequent imaging of a two-dimensional array of a genetically and chemically modified cowpea chlorotic mottle virus (CCMV) is described. The genetic mutation provides symmetrically dispersed exposed thiol groups on the outer surface of the virus capsid. These functional groups can be used to covalently bind the capsid to smooth Au substrate. AFM imaging suggests that the genetic mutation by itself does not promote array formation but, rather, aggregation through disulfide linkages. However, breaking the symmetry of the capsid using a solid-phase approach and chemically passivating the exposed thiol groups with iodoacetic acid results in a capsid with exposed thiols only on one side of the particle. These symmetry-broken capsids were able to form self-assembled monolayers (SAM) on a Au surface.

Alanine↗
PubMed2003

Metal binding to cowpea chlorotic mottle virus using terbium(III) fluorescence.

Metals are thought to play a role in the structure of many viruses. The crystal structure of the T=3 icosahedral cowpea chlorotic mottle virus (CCMV) suggests the presence of 180 unique metal-binding sites in the assembled protein cage. Each of these sites is thought to involve the coordination of the metal by five amino acids contributed from two adjacent coat protein subunits. We have used fluorescence resonance energy transfer (FRET), from tryptophan residues proximal to the putative metal-binding sites, to probe Tb(III) binding to the virus. Binding of Tb(III) was investigated on the wild-type virus and a mutant where the RNA binding ability of the virus was removed. Tb(III) binding was observed both in the wild-type virus ( K(d)=19 microM) and the mutant ( K(d)=17 microM), as monitored by the increase in Tb(III) fluorescence (545 nm) and concomitant decrease in tryptophan fluorescence (342 nm). Competitive binding experiments showed Ca(II) to have about 100-fold less affinity for the binding sites ( K(d)=1.97 mM). This is the first direct evidence of metal binding to the putative metal-binding sites, originally suggested from the crystal structure of CCMV.

Binding Sites↗
PubMed2003

Cost-saving approach to patients on long-term anticoagulation who need endoscopy: a decision analysis.

OBJECTIVES: The management strategies used when patients requiring long-term anticoagulation need endoscopic procedures vary considerably. Two commonly used approaches are a "heparin window" strategy in the inpatient setting and, more recently, a "switch to low molecular weight heparin (LMWH)" strategy for elective procedures. The aim of this study was to determine whether an initial diagnostic endoscopy (visualization only) is a cost-effective strategy in these patients. METHODS: Decision analysis was performed for two scenarios using probability estimates from our retrospective study. Scenario 1: Patients with any (urgent and elective) indication for endoscopy while on anticoagulation. A decision tree was made outlining two strategies: 1) a diagnostic endoscopy on full anticoagulation followed by therapeutic endoscopy if needed using standard practice; and 2) standard approach. Scenario 2: Patients requiring elective endoscopy. Here, the decision tree outlined three strategies: 1) initial diagnostic endoscopy on full anticoagulation followed by a therapeutic endoscopy if needed using a "heparin window"; 2) initial diagnostic endoscopy followed by therapeutic endoscopy if needed using "switch to LMWH" strategy; and 3) "direct switch to LMWH strategy." RESULTS: Initial diagnostic endoscopy is the preferred strategy when patients requiring anticoagulation need endoscopy. In scenario 1 (all patients), the diagnostic endoscopy approach will reduce need for hospital stay and save $85,006 per 100 patients when a therapeutic impact is not predictable before endoscopy. Similarly, in scenario 2, an initial diagnostic endoscopy followed by switch to LMWH strategy is the most cost saving. CONCLUSIONS: In anticoagulated patients, an initial diagnostic endoscopy approach on anticoagulation is the most cost-saving strategy, when a direct therapeutic impact is not predictable.

Anticoagulants↗
PubMed2003

The functionality and cost advantages of high-performance polymers.

Acetals remain extremely important for medical devices, particularly in gears, springs and other mechanisms, although going forward with progressively lower emission targets is likely to require a combination of low-emission grades and tighter processing controls. Nylon and PBT materials have a continued importance in achieving the combination of mechanical performance, biocompatibility (a range of grades are available that have been tested successfully against USP 23 Class VI) and sterilisation performance (dependent on grade and type of sterilisation). Materials such as liquid crystal polymer are progressively more important for their barrier properties, high temperature performance and all-round sterilisation performance. Polycarbonates and cyclic olefin copolymers continue to find new applications, often where clarity is important; transparent Nylons and other olefinic materials are also valuable in this area. With the continuing advances in raw materials and polymer processes, careful choices can produce some worthy advances in device technology, although utilising the technologies effectively still depends on working forwards from the user/patient need and desired functionality. Whether considering developing a new device using plastics, or reconsidering further development of an existing device, engineering polymers can provide the key to something better.

Costs and Cost Analysis↗