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Biomedical subjects

M G Hill

Publications and source records attributed to M G Hill.

At least 19 recordsLinked to original sources
PubMed2003

Biological control of the larger grain borer Prostephanus truncatus (Coleoptera: Bostrichidae) in Kenya using a predatory beetle Teretrius nigrescens(Coleoptera: Histeridae).

The establishment of the predatory histerid beetle, Teretrius nigrescens Lewis, following its field release in south eastern Kenya in 1992, and its impact on populations of the larger grain borer, Prostephanus truncatus (Horn), in woodland habitats is evaluated. Pheromone trap catch data show a single strong peak of P. truncatus from November to January, coincident with the short rains. Comparisons of P. truncatus pheromone trap catches before and after predator establishment, and in areas with and without T. nigrescens, show a sustained decline of over 80% in P. truncatus abundance over a 5-year period, following the establishment T. nigrescens. Concurrent laboratory studies of the impact of T. nigrescens on populations of P. truncatus raised on a wood substrate showed a 77% reduction in the growth of the pest population. These results are compared with published findings of the impact of T. nigrescens as a biological control agent of P. truncatus in west Africa.

Animals↗
PubMed2001

Synthesis and spectroelectrochemistry of Ir(bpy)(phen)(phi)(3+), a tris(heteroleptic) metallointercalator.

A tris(heteroleptic) phenanthrenequinone diimine (phi) complex of Ir(III), Ir(bpy)(phen)(phi)(3+), was synthesized through the stepwise introduction of three different bidentate ligands, and the Lambda- and Delta-enantiomers were resolved and characterized by CD spectroscopy. Like other phi complexes, this tris(heteroleptic) iridium complex binds avidly to DNA by intercalation. Electrochemical studies show that Ir(bpy)(phen)(phi)(3+) undergoes a reversible one-electron reduction at E(0) = -0.025 V in 0.1 M TBAH/DMF (versus Ag/AgCl), and spectroelectrochemical studies indicate that this reduction is centered on the phi ligand. The EPR spectrum of electrochemically generated Ir(bpy)(phen)(phi)(2+) is consistent with a phi-based radical. The electrochemistry of Ir(bpy)(phen)(phi)(3+) was also probed at a DNA-modified electrode, where a DNA binding affinity of K = 1.1 x 10(6) M(-1) was measured. In contrast to Ir(bpy)(phen)(phi)(3+) free in solution, the complex bound to DNA undergoes a concerted two-electron reduction, to form a diradical species. On the basis of UV-visible and EPR spectroscopies, it is found that disproportionation of electrochemically generated Ir(bpy)(phen)(phi)(2+) occurs upon DNA binding. These results underscore the rich redox chemistry associated with metallointercalators bound to DNA.

DNA↗
PubMed2001

Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary.

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet(7)]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the alpha-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet(7)]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by approximately 45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.

Amino Acid Sequence↗
PubMed2001

Electrochemistry at DNA-modified surfaces: new probes for charge transport through the double helix.

Electrochemistry at DNA-modified surfaces provides an alternative approach to photochemistry or radiation biology for studying charge migration through the double helix. Using short duplexes self-assembled onto gold, electrochemical reduction of redox-active reporter molecules has been observed through DNA films more than 50 A thick, with heterogeneous rate constants as great as approximately 100 s(-1). Though apparently insensitive to base content and sequence, even small distortions in the electronic structure of the pi-stack (caused, for example, by single-base mismatches and other DNA lesions) attenuate the rate of electron transport. Understanding the role of conformational dynamics within the double helix, as well as the cooperative effects of self-assembling individual duplexes into ordered superlattices remain important challenges for theory and experiment.

Base Pair Mismatch↗
PubMed2000

Mutation detection by electrocatalysis at DNA-modified electrodes.

Detection of mutations and damaged DNA bases is important for the early diagnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in fully hybridized duplexes, based on charge transport through DNA films. Gold electrodes modified with preassembled DNA duplexes are used to monitor the electrocatalytic signal of methylene blue, a redox-active DNA intercalator, coupled to [Fe(CN)6]3-. The presence of mismatched or damaged DNA bases substantially diminishes the electrocatalytic signal. Because this assay is not a measure of differential hybridization, all single-base mismatches, including thermodynamically stable GT and GA mismatches, can be detected without stringent hybridization conditions. Furthermore, many common DNA lesions and "hot spot" mutations in the human p53 genome can be distinguished from perfect duplexes. Finally, we have demonstrated the application of this technology in a chip-based format. This system provides a sensitive method for probing the integrity of DNA sequences and a completely new approach to single-base mismatch detection.

Base Pair Mismatch↗
PubMed2000

Integrity of thermus thermophilus cytochrome c552 synthesized by Escherichia coli cells expressing the host-specific cytochrome c maturation genes, ccmABCDEFGH: biochemical, spectral, and structural characterization of the recombinant protein.

We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene. Key features are (1) construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2) coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC552, and native cytochrome c552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba3, a Thermus heme-copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1H-NMR spectroscopies. The 1.7 A resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629-644). This approach may be generally useful for expression of alien cytochrome c genes in E. coli.

Amino Acid Sequence↗
PubMed1999

Single-base mismatch detection based on charge transduction through DNA.

High-throughput DNA sensors capable of detecting single-base mismatches are required for the routine screening of genetic mutations and disease. A new strategy for the electrochemical detection of single-base mismatches in DNA has been developed based upon charge transport through DNA films. Double-helical DNA films on gold surfaces have been prepared and used to detect DNA mismatches electrochemically. The signals obtained from redox-active intercalators bound to DNA-modified gold surfaces display a marked sensitivity to the presence of base mismatches within the immobilized duplexes. Differential mismatch detection was accomplished irrespective of DNA sequence composition and mismatch identity. Single-base changes in sequences hybridized at the electrode surface are also detected accurately. Coupling the redox reactions of intercalated species to electrocatalytic processes in solution considerably increases the sensitivity of this assay. Reporting on the electronic structure of DNA, as opposed to the hybridization energetics of single-stranded oligonucleotides, electrochemical sensors based on charge transport may offer fundamental advantages in both scope and sensitivity.

Base Pair Mismatch↗
PubMed1999

Support plasmids and support proteins required for recovery of recombinant respiratory syncytial virus.

Respiratory syncytial virus (RSV) can be recovered from plasmids that separately encode antigenomic RNA and the N, P, L, and M2-1 proteins of the nucleocapsid. However, in a recent study the inclusion of a separate M2-1 expression plasmid was found to be unnecessary (H. Jin, D. Clarke, H. Zhou, X. Cheng, K. Coelingh, M. Bryant, and S. Li, Virology 1998, 251, 206-214). This suggested that the M2-1 protein, which is a transcription antitermination factor, is not required to reconstitute the minimum unit of infectivity, namely a nucleocapsid fully functional for viral transcription and RNA replication. Here we show that the antigenomic plasmid is remarkably efficient as a substitute for an M2-1 expression plasmid in supporting processive transcription by an RSV minigenome. Thus, the simple expedient of omitting an expression plasmid is invalid for evaluating recovery requirements. The issue of the requirement of M2-1 for the recovery of infectious RSV is discussed.

Cells, Cultured↗
PubMed1999

Selenomethionine-substituted Thermus thermophilus cytochrome ba3: characterization of the CuA site by Se and Cu K-EXAFS.

We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168 of the Thermus thermophilus cytochrome ba3 subunit II [Keightley et al. (1995) J. Biol. Chem. 270, 20345-20358]. The resulting ba3-CuAt10 protein separated into two fractions (A and B) during cation exchange chromatography which were demonstrated to differ only by N-terminal acetylation in fraction A. When the gene was expressed in an Escherichia coli strain that is auxotrophic for methionine and grown in the presence of selenomethionine (Se(Met)), the single methionine of the CuAt10 protein was quantitatively replaced with Se(Met). Native (S(Met)) and Se(Met)-substituted proteins were characterized by electrospray mass, optical absorption, and EPR spectroscopies and by electrochemical analysis; they were found to have substantially identical properties. The Se(Met)-containing protein was further characterized by Se and Cu K-EXAFS which revealed Cu-Se bond lengths of 2.55 A in the mixed-valence form and 2.52 A in the fully reduced form of CuA. Further analysis of the Se- and Cu-EXAFS spectra yielded the Se-S(thiolate) distances and thereby information on the Se-Cu-Cu and Se-Cu-S(thiolate) angles. An expanded EXAFS structural model is presented.

Amino Acid Sequence↗
PubMed1999

Replacement of the F and G proteins of respiratory syncytial virus (RSV) subgroup A with those of subgroup B generates chimeric live attenuated RSV subgroup B vaccine candidates.

Human respiratory syncytial virus (RSV) exists as two antigenic subgroups, A and B, both of which should be represented in a vaccine. The F and G glycoproteins are the major neutralization and protective antigens, and the G protein in particular is highly divergent between the subgroups. The existing system for reverse genetics is based on the A2 strain of RSV subgroup A, and most efforts to develop a live attenuated RSV vaccine have focused on strain A2 or other subgroup A viruses. In the present study, the development of a live attenuated subgroup B component was expedited by the replacement of the F and G glycoproteins of recombinant A2 virus with their counterparts from the RSV subgroup B strain B1. This gene replacement was initially done for wild-type (wt) recombinant A2 virus to create a wt AB chimeric virus and then for a series of A2 derivatives which contain various combinations of A2-derived attenuating mutations located in genes other than F and G. The wt AB virus replicated in cell culture with an efficiency which was comparable to that of the wt A2 and B1 parents. AB viruses containing temperature-sensitive mutations in the A2 background exhibited levels of temperature sensitivity in vitro which were similar to those of A2 viruses bearing the same mutations. In chimpanzees, the replication of the wt AB chimera was intermediate between that of the A2 and B1 wt viruses and was accompanied by moderate rhinorrhea, as previously seen in this species. An AB chimeric virus, rABcp248/404/1030, which was constructed to contain a mixture of attenuating mutations derived from two different biologically attenuated A2 viruses, was highly attenuated in both the upper and lower respiratory tracts of chimpanzees. This attenuated AB chimeric virus was immunogenic and conferred a high level of resistance on chimpanzees to challenge with wt AB virus. The rABcp248/404/1030 chimeric virus is a promising vaccine candidate for RSV subgroup B and will be evaluated next in humans. Furthermore, these results suggest that additional attenuating mutations derived from strain A2 can be inserted into the A2 background of the recombinant chimeric AB virus as necessary to modify the attenuation phenotype in a reasonably predictable manner to achieve an optimal balance between attenuation and immunogenicity in a virus bearing the subgroup B antigenic determinants.

Animals↗
PubMed1998

High-potential states of blue and purple copper proteins.

Electrochemical measurements show that there are high-potential states of two copper proteins, Pseudomonas aeruginosa azurin and Thermus thermophilus CuA domain; these perturbed states are formed in guanidine hydrochloride (GuHCl) solution in which the proteins are still blue (azurin) and purple (CuA). In each case, the high-potential state forms reversibly. Absorption (azurin, CuA), visible circular dichroism (azurin, CuA), resonance-Raman (CuA), and EPR (CuA) spectra indicate that the structure of the oxidized copper site of each high-potential form is very similar to that of the native protein. It is proposed that GuHCl perturbs one or more H-bonds in the blue or purple copper active site, thereby allowing Cu(I) to adopt a more favorable coordination structure than that in the rigid cavity of the native protein.

Azurin↗
PubMed1998

Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: plant-specific mononuclear blue copper proteins.

The cDNAs encoding plantacyanin from spinach were isolated and characterized. In addition, four new cDNA sequences from Arabidopsis ESTs were identified that encode polypeptides resembling phytocyanins, plant-specific proteins constituting a distinct family of mononuclear blue copper proteins. One of them encodes plantacyanin from Arabidopsis, while three others, designated as uclacyanin 1, 2, and 3, encode protein precursors that are closely related to precursors of stellacyanins and a blue copper protein from pea pods. Comparative analyses with known phytocyanins allow further classification of these proteins into three distinct subfamilies designated as uclacyanins, stellacyanins, and plantacyanins. This specification is based on (1) their spectroscopic properties, (2) their glycosylation state, (3) the domain organization of their precursors, and (4) their copper-binding amino acids. The recombinant copper binding domain of Arabidopsis uclacyanin 1 was expressed, purified, and shown to bind a copper atom in a fashion known as "blue" or type 1. The mutant of cucumber stellacyanin in which the glutamine axial ligand was substituted by a methionine (Q99M) was purified and shown to possess spectroscopic properties similar to uclacyanin 1 rather than to plantacyanins. Its redox potential was determined by cyclic voltammetry to be +420 mV, a value that is significantly higher than that determined for the wild-type protein (+260 mV). The available structural data suggest that stellacyanins (and possibly other phytocyanins) might not be diffusible electron-transfer proteins participating in long-range electron-transfer processes. Conceivably, they are involved in redox reactions occurring during primary defense responses in plants and/or in lignin formation.

Amino Acid Sequence↗
PubMed1996

Transcription elongation factor of respiratory syncytial virus, a nonsegmented negative-strand RNA virus.

RNA synthesis by the paramyxovirus respiratory syncytial virus, a ubiquitous human pathogen, was found to be more complex than previously appreciated for the nonsegmented negative-strand RNA viruses. Intracellular RNA replication of a plasmid-encoded "minigenome" analog of viral genomic RNA was directed by coexpression of the N, P, and L proteins. But, under these conditions, the greater part of mRNA synthesis terminated prematurely. This difference in processivity between the replicase and the transcriptase was unanticipated because the two enzymes ostensively shared the same protein subunits and template. Coexpression of the M2 gene at a low level of input plasmid resulted in the efficient production of full-length mRNA and, in the case of a dicistronic minigenome, sequential transcription. At a higher level, coexpression of the M2 gene inhibited transcription and RNA replication. The M2 mRNA contains two overlapping translational open reading frames (ORFs), which were segregated for further analysis. Expression of the upstream ORF1, which encoded the previously described 22-kDa M2 protein, was associated with transcription elongation. A model involving this protein in the balance between transcription and replication is proposed. ORF2, which lacks an assigned protein, was associated with inhibition of RNA synthesis. We propose that this activity renders nucleocapsids synthetically quiescent prior to incorporation into virions.

DNA, Complementary↗
PubMed1996

The treatment of minor burns in rural Alabama emergency departments.

OBJECTIVE: The purposes of this descriptive survey were to determine the treatments for minor burns in rural Alabama emergency departments, to assess how closely those treatments correspond with recommendations for burn therapy noted in the literature, and to identify specific deficits in the burn research literature. DESIGN: A descriptive survey consisting of open-ended questions was used to gather data on current practices for treating outpatient burn wounds. METHODS: A researcher-developed questionnaire organized into seven categories of burn care was pilot-tested in an urban burn center; the questionnaire was then mailed to 96 rural Alabama hospitals, addressed to the ED nurse manager. The seven categories--initial cooling, analgesics, cleansing agents, topical antimicrobial therapy, wound dressings, blister debridement, and referrals--were identified from both major and minor burn care literature, because little research has been published about minor burns. Simple frequencies were used to analyze results from the 20% (n = 21) of the sample that responded. RESULTS: Seven categories of burn care are common to all respondent facilities, with wide variation in the details. Most practices proceed logically from the treatments recommended for major burn care, with a few notable and potentially dangerous exceptions, but there is no definitive research available to guide the clinician in the care of outpatient burns. Research questions were generated that can provide direction for measurement of outcomes in the care of minor burns treated on an outpatient basis.

Alabama↗
PubMed1996

Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus.

Preceding and following each gene of respiratory syncytial virus (RSV) are two conserved sequences, the gene-start (GS) and gene-end (GE) motifs, respectively, which are thought to be transcription signals. The functions and boundaries of these signals and the process of sequential transcription were analyzed with cDNA-encoded RNA analogs (minigenomes) of nonsegmented negative-sense RSV genomic RNA. Two minigenomes were used. The monocistronic RSV-CAT minigenome consists of the chloramphenicol acetyltransferase (CAT) translational open reading frame (ORF) bordered by the GS and GE motifs and flanked by the 3' leader and 5' trailer extragenic regions of genomic RNA. The dicistronic RSV-CAT-LUC minigenome is a derivative of RSV-CAT into which the ORF for luciferase (LUC), bordered by GS and GE motifs, was inserted downstream of the CAT gene with an intergenic region positioned between the two genes. Each minigenome was synthesized in vitro and transfected into RSV-infected cells, where it was replicated and transcribed to yield the predicted polyadenylated subgenomic mRNA(s). The only RSV sequences required for efficient transcription and RNA replication were the 44-nucleotide 3' leader region, the last 40 nucleotides of the 5' trailer region, and the 9- to 10-nucleotide GS and 12- to 13-nucleotide GE motifs. The GS and GE motifs functioned as self-contained, transportable transcription signals which could be attached to foreign sequences to direct their transcription into subgenomic mRNAs. Removal of the GS motif greatly reduced transcription of its gene, and the requirement for this element was particularly strict for the gene in the downstream position. Ablation of the promoter-proximal GS signal was not associated with increased antigenome synthesis. Consistent with its proposed role in termination and polyadenylation, removal of the CAT GE signal in RSV-CAT resulted in the synthesis of a nonpolyadenylated CAT mRNA, and in RSV-CAT-LUC the same mutation resulted in readthrough transcription to yield a dicistronic CAT-LUC mRNA. The latter result showed that a downstream GS signal is not recognized for reinitiation by the polymerase if it is already engaged in mRNA synthesis; instead, it is recognized only if the polymerase first terminates transcription at an upstream termination signal. This result also showed that ongoing transcription did not open the downstream LUC gene for internal polymerase entry. Removal of both the GS and GE signals of the upstream CAT gene in RSV-CAT-LUC silenced expression of both genes, confirming that independent polymerase entry at an internal gene is insignificant. Remarkably, whereas both genes were silent when the CAT GS and GE signals were both absent, restoration of the CAT GE signal alone restored a significant level (approximately 10 to 12% of the wild-type level) of synthesis of both subgenomic mRNAs. This analysis identified a component of sequential transcription that was independent of the promoter-proximal GS signal and appeared to involve readthrough from the leader region.

Animals↗
PubMed1995

Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development.

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.

Base Sequence↗
PubMed1995

Electron transfer in ruthenium-modified proteins.

Photochemical techniques have been used to measure the kinetics of intramolecular electron transfer in Ru(bpy)2(im)(His)2(+)-modified (bpy = 2,2'-bipyridine; im = imidazole) cytochrome c and azurin. A driving-force study with the His33 derivatives of cytochrome c indicates that the reorganization energy (lambda) for Fe2+-->Ru3+ ET reactions is 0.8 eV. Reductions of the ferriheme by either an excited complex, *Ru2+, or a reduced complex, Ru+, are anomalously fast and may involve formation of an electronically excited ferroheme. The distance dependence of Fe2+-->Ru3+ and Cu+-->Ru3+ electron transfer in 12 different Ru-modified cytochromes and azurins has been analyzed using a tunneling-pathway model. The ET rates in 10 of the 12 systems exhibit an exponential dependence on metal-metal separation (decay constant of 1.06 A-1) that is consistent with prediction of the pathway model.

Azurin↗