Biomedical subjects
Publications and source records attributed to A Greenwood.
We report a simple and efficient technique for the covalent immobilisation of a tris(2,2'-bipyridyl)ruthenium(II) derivative suitable for both chemiluminescence and electrochemiluminescence detection.
Three hundred and two oral fluid samples collected in January 2001 from male young offenders aged 18 to 21 years at HMP Hindley were screened for measles and rubella specific IgG, using 'in-house' amplified ELISA assays. MMR (measles/mumps/rubella) vaccine, offered at the same time, was accepted by 68.1% of the prisoners (92.7% of those agreeing to oral fluid antibody testing). Antibody prevalences were 92.1% for rubella and 80.8% for measles. This may, however, underestimate true prevalence as the sensitivity of the oral fluid assays used may be relatively poor in adults. Susceptibility levels are theoretically high enough to result in outbreaks should either infection enter this closed institutional environment. Since a measles outbreak would carry significant morbidity and rubella would represent a risk to associated non-immune women of childbearing age, it is suggested that prison health services should offer MMR to young offenders on entry.
Satellite tobacco mosaic virus (STMV) can undergo at least two physical transitions that significantly alter its mechanical and structural characteristics. At high pH the 17-nm STMV particles expand radially by about 5 A to yield particles having diameters of about 18 nm. This pH-induced transition is further promoted by aging of the virions and degradation of the RNA, so that swollen particles ultimately appear even at neutral pH. While the native 17-nm particles crystallize as orthorhombic or monoclinic crystals which diffract to high resolution (1.8 A), the enlarged 18-nm particles crystallize in a cubic form which diffracts to no better than 5 A. In the transition, not only do the capsid protein subunits move radially outward, but the helical RNA segments with which they interact do as well. This is noteworthy because it demonstrates that the RNA and the protein shell are capable of coordinated movement, and that neither structure is rigidly defined or independent of the other. Using atomic force microscopy, it can be shown that STMV particles, upon drying, lose their mechanical rigidity and undergo deformation. Virions initially 17 nm in diameter shrink to more uniform final sizes than do 18 nm, initially swollen particles. This transition appears to be irreversible, as the particles do not reassume their former size nor structural rigidity upon rehydration. Evidence is also presented that preparations of native virus and their crystals are naturally somewhat heterogeneous and contain a variety of particles of anomalous size.
Satellite tobacco mosaic virus (STMV) was probed using a variety of proteases. Consequences of the degradation were analyzed using gel electrophoresis, quasi-elastic light scattering (QELS), and atomic force microscopy (AFM). Proteolysis rates of 30 minutes for complete degradation of the protein capsid, up to many hours, were investigated. With each protease, degradation of virions 17 nm in diameter was shown by QELS to result in particles of 10 nm diameter, which is that of the RNA core observed in the virion by x-ray diffraction analysis. This was verified by direct visualization with atomic force microscopy. Using QELS, it was further shown that freshly prepared RNA cores remain as individual, stable, 10-nm condensed particles for 12 to 24 h. Clusters of particles then formed, followed by very large aggregates of 500 to 1000 nm diameter. AFM showed that the aggregates were composed of groups of the condensed RNA cores and were not due to unfolding of the nucleic acid. No unfolding of the core particles into extended conformation was seen by AFM until the samples were heated well beyond 90 degrees C. Mass spectrometry of RNA core particles revealed the presence of a major polypeptide whose amino acid sequence corresponded to residues 2 through 25 of the coat protein. Amino acids 13 through 25 were previously observed to be in direct contact with the RNA and are presumably protected from protease digestion. Low resolution difference Fourier analyses indicated the courses of the remainders of the amino terminal strands (amino acids 2-12) in intact virions. Any individual strand appears to have several choices of path, which accounts for the observed disorder at high resolution. These positively charged strands, serving as virtual polyamines, engage the helical segments of RNA. The intimate association of amino acid residues 2 through 25 with RNA likely contributes to the stability of the condensed conformation of the nucleic acid cores.
Desmodium yellow mottle virus is a 28 nm diameter, T=3 icosahedral plant virus of the tymovirus group. Its structure has been solved to a resolution of 2.7 A using X-ray diffraction analysis based on molecular replacement and phase extension methods. The final R value was 0.151 (R(free)=0.159) for 134,454 independent reflections. The folding of the polypeptide backbone is nearly identical with that of turnip yellow mosaic virus, as is the arrangement of subunits in the virus capsid. However, a major difference in the disposition of the amino-terminal ends of the subunits was observed. In turnip yellow mosaic virus, those from the B and C subunits comprising the hexameric capsomeres formed an annulus about the interior of the capsomere, while the corresponding N termini of the pentameric capsomere A subunits were not visible at all in electron density maps. In Desmodium yellow mottle tymovirus, amino termini from the A and B subunits combine to form the annuli, thereby resulting in a much strengthened association between the two types of capsomeres and an, apparently, more stable capsid. The first 13 residues of the C subunit were invisible in electron density maps. Two ordered fragments of single-stranded RNA, seven and two nucleotides in length, were observed. The ordered water structure of the virus particle was delineated and required 95 solvent molecules per protein subunit.
There is evidence that season of birth may predict adult mortality from infectious diseases in rural Gambia. Using data collected over a five-year period from the rural, eastern region of the Gambia, we examined whether the season of birth influences mortality in childhood. 26 894 births and 3776 deaths among children under the age of five years were recorded in this region during the period 1989-1993. The estimated 1-4 year population was 95 355. In children aged 1-4 years, the mortality rate per 1000 per year was 16.1 (95% CI 14.9, 17.2) for those born in the 'harvest' season (January to June), which was not significantly different from the rate of 17.9 (95% CI 16.7, 19.0) recorded for those born in the 'hungry' season (July to December) (age-stratified Mantel-Haenszel mortality ratio 0. 91, 95% CI 0.83, 1.01; p = 0.08). Nearly all deaths of 1-4 year olds were attributed to infectious diseases, with malaria accounting for over 40%. None of the cause-specific child mortality rates differed significantly according to the season of birth. These data suggest that beyond infancy, when it is easier to separate the effect of season on cause of death from that of the season of birth, there is no marked difference in the rate of death between Gambian children born in the harvest season and those born in the hungry season.
The 1990s have witnessed a growth in specialist family planning provision for adolescents including advisory clinics for young people. However there has been no parallel development of teenager-friendly genitourinary medicine (GUM) services despite the prevalence of sexually transmitted disease (STDs) in the adolescent age group. This article profiles a young person's clinic in Morecambe, UK which operates a combined family planning/GUM clinic one night a week in a multi-agency, 'shop front', youth project. Attendances are high, particularly amongst young teenagers and males. The clinic enjoys a 66 per cent follow up rate and 11 per cent of clients had crossed over from one service to the other on succeeding visits. Seventeen cases of chlamydia had been diagnosed, nine to females under 18. The benefits of a 'one stop' clinic and the youth project location are discussed.
The molecular structure of satellite tobacco mosaic virus (STMV) has been refined to 1.8 A resolution using X-ray diffraction data collected from crystals grown in microgravity. The final R value was 0.179 and Rfree was 0.184 for 219,086 independent reflections. The final model of the asymmetric unit contained amino acid residues 13 to 159 of a coat protein monomer, 21 nucleotides, a sulfate ion, and 168 water molecules. The nucleotides were visualized as 30 helical segments of nine base-pairs with an additional base stacked at each 3' end, plus a "free" nucleotide, not belonging to the helical segments, but firmly bound by the protein. Sulfate ions are located exactly on 5-fold axes and each is coordinated by ten asparagine side-chains. Of the 10,080 structural waters, 168 per asymmetric unit, about 20% serve to bridge the macromolecular components at protein-protein and protein-nucleic acid interfaces. Binding of RNA to the protein involves some salt linkages, particularly to the phosphate of the free nucleotide, but the major contribution is from an intricate network of hydrogen bonds. There are numerous water molecules in the RNA-protein interface, many serving as intermediate hydrogen bond bridges. The sugar-phosphate backbone contributes most of the donors and acceptors for the RNA. The helical RNA conformation is nearest that of A form DNA. The central region of a helical segment is most extensively involved in contacts with protein, and exhibits low thermal parameters which increase dramatically toward the ends. The visible RNA represents approximately 59% of the total nucleic acid in the virion and is derived from the single-stranded genome, which has folded upon itself to form helical segments. Linking of the helices and the free nucleotides in a contiguous and efficient manner severely restricts the disposition of the remaining, unseen nucleic acid. Using the remaining nucleotides it is possible to fold the RNA according to motifs that provide a periodic distribution of RNA structural elements compatible with the icosahedrally symmetrical arrangement seen in the crystallographic structure. The intimate relationship between protein and nucleic acid in STMV suggests an assembly pathway based on the cooperative and coordinated co-condensation of RNA with capsid protein dimers.
Little is known about birth or mortality rates of twins in The Gambia. There are no reports of the causes of death among twins in sub-Saharan Africa. We identified twin births and deaths from a community-based study which had been carried out in a large rural region of The Gambia over a 5-year period from 1989 to 1993. The overall twinning rate excluding stillbirths was 14.3 (95% CI 12.9, 15.8) per 1000 live deliveries. This was significantly lower among the Mandinka ethnic group (8.8 per 1000) than among the Serahulis (15.1 per 1000) or Fulas (18.3 per 1000). The early-neonatal, late-neonatal and post-neonatal twin mortality rates were 114.0, 45.9 and 64.2 per 1000 live twin births, respectively. In comparison, the early-neonatal, late-neonatal and post-neonatal mortality rates of singletons were 18.6, 16.0 and 41.1 per 1000, respectively. In the post-neonatal period, malnutrition was more frequently a cause of death among twins than among singletons (7.8 per 1000 twin births vs 2.0 per 1000 singleton births; p = 0.0008). Appropriate strategies for preventing malnutrition are required for this high-risk group.
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The protein thaumatin was studied as a model macromolecule for crystallization in microgravity-environment experiments conducted on two US Space Shuttle missions (USML-2 and LMS). In this investigation, we have evaluated and compared the quality of space- and earth-grown thaumatin crystals using X-ray diffraction analyses, and characterized them according to crystal size, diffraction resolution limit and mosaicity. Two different approaches for growing thaumatin crystals in the microgravity environment, dialysis and liquid-liquid diffusion, were employed as a joint experiment by our two investigative teams. Thaumatin crystals grown in a microgravity environment were generally larger in volume and the total number of crystals was less, relative to crystals grown on earth. They diffracted to significantly higher resolution and with improved diffraction properties, as judged by relative plots of I/sigma versus resolution. The mosaicity of space-grown crystals was significantly less than that of crystals grown on earth. Increased concentrations of protein in the crystallization chambers in microgravity led to larger crystals. The data presented here lend further support to the idea that protein crystals of improved quality can be obtained in a microgravity environment.
The protein thaumatin was studied as a model macro-molecule for crystallization in microgravity-environment experiments conducted on two US Space Shuttle missions (USML-2 and LMS). In this investigation, we have evaluated and compared the quality of space- and earth-grown thaumatin crystals using X-ray diffraction analyses, and characterized them according to crystal size, diffraction resolution limit and mosaicity. Two different approaches for growing thaumatin crystals in the microgravity environment, dialysis and liquid-liquid diffusion, were employed as a joint experiment by our two investigative teams. Thaumatin crystals grown in a microgravity environment were generally larger in volume and the total number of crystals was less, relative to crystals grown on earth. They diffracted to significantly higher resolution and with improved diffraction properties, as judged by relative plots of I/sigma versus resolution. The mosaicity of space-grown crystals was significantly less than that of crystals grown on earth. Increased concentrations of protein in the crystallization chambers in microgravity led to larger crystals. The data presented here lend further support to the idea that protein crystals of improved quality can be obtained in a microgravity environment.
The major extracellular protease from the fungus Pencillium cyclopium was crystallized in the presence of p-phenylmethanesulfonyl fluoride (PMSF) and investigated by X-ray diffraction analysis. It was subsequently cloned and the amino acid sequence deduced from its cDNA. Although the sequence is only 49% identical to that of proteinase K of Tritirachium album, the three-dimensional structures of the two proteases are virtually identical. The model for P. cyclopium protease was refined by simulated annealing to an R of 18% at 1.7 A resolution. The greatest variation from the proteinase K polypeptide is in loop 114-134 and is due to the absence of a disulfide bridge in the P. cyclopium protease that is present in proteinase K. A difference was also observed in the orientation of the histidine in the catalytic triad, though this could be due to the presence of PMSF at the active site. The coordination geometry of the strongly bound calcium in the P. cyclopium protease is octahedral and uses some different protein ligands than does proteinase K. In the protease from P. cyclopium there is no cysteine near the active site, nor is there a second calcium binding site as is found in proteinase K, suggesting that neither is important to catalytic activity.
Sensory neurons are a major derivative of the neural crest for which there have been no definitive molecular markers in mammals. We have developed a method that combines differential hybridization with degenerate RT-PCR to rapidly screen gene families for members exhibiting differential expression among tissues or cell types. We used this approach to search for transcription factor-encoding genes specifically expressed in mammalian sensory neurons. A novel paired homeodomain protein, called DRG11, was identified. DRG11 is expressed in most sensory neurons, including trkA-expressing neurons, but not in glia or sympathetic neurons. Unexpectedly, it is also expressed in the dorsal horn of the spinal cord, a region to which NGF-dependent sensory neurons project. These data suggest that DRG11 is not only a useful marker for sensory neurons, but may also function in the establishment or maintenance of connectivity between some of these neurons and their central nervous system targets.
Monoclonal antibodies were produced that are specific for the three major pertussis toxin-sensitive G protein alpha-subunits present in mammalian brain--alpha o, alpha i1, and alpha i2--using purified bovine brain G proteins, purified rat brain G proteins, and purified recombinant alpha i2, respectively. These monoclonal antibodies were used to monitor changes in the concentrations of the three G protein alpha-subunits during differentiation of PC12 cells and human neuroblastoma LA-N-5 cells. In PC12 cells, levels of alpha i1 but not alpha i2 increased during nerve growth factor-induced differentiation. In contrast, alpha i2 but not alpha i1 increased when LA-N-5 cells were differentiated with retinoic acid. The concentration of alpha o increased in both cell lines during differentiation. Electrophoretic resolution of alpha o subtypes revealed that although alpha o2 was the major subtype in undifferentiated cells, only the concentration of alpha o1 increased during differentiation of both PC12 and LA-N-5 cells. The level of 43-kDa growth-associated protein, a protein known to associate with alpha o, increased similarly to that of alpha o1. ADP-ribosylation of alpha o, alpha i1, and alpha i2 with pertussis toxin did not alter the reactivities of the monoclonal antibodies, but toxin treatment of cells reduced the concentrations of each protein after 24 h. There was no change in the concentration of alpha q, which is not ADP-ribosylated by pertussis toxin. Thus, these new monoclonal antibodies enabled the detection of differential increases in subtypes of alpha i and alpha o associated with neuronal differentiation.
The crystal structure of anti-idiotopic Fab 409.5.3, made against an E2 specific feline infectious peritonitis virus-neutralizing antibody 730.1.4, has been determined in its free from, at 2.9 A resolution by molecular replacement. This antibody, used as an immmunogen, elicits the production of anti-anti-idiotypic antibodies that in turn neutralize the virus. The structure of the uncomplexed Fab was refined using constrained-restrained least squares minimization and simulated annealing in combination with conjugate gradient techniques to a crystallographic R of 0.22 based on 16,482 unique reflections between 20.0 and 2.9 A. The free antiidiotypic Fab shows, when compared to its complexed form, a 5 degrees rotation of its variable light with respect to its variable heavy domain and rearrangement of complementarity determining region loops, which permits optimization of the stereocomplementarity between interacting molecules. This finding supports the induced fit hypothesis for antibody antigen interaction.