Biomedical subjects
P Rice
Publications and source records attributed to P Rice.
Protamines of reptiles.
We have characterized for the first time the complete primary structure of the main protamine components of the sperm from four reptiles: Chrysemys picta (turtle), Elaphe obsoleta (snake), Anolis carolinensis (lizard), and Alligator mississipiensis (crocodilian). These species were chosen to represent one of each of the main phylogenetic branches of this taxonomic group. Comparison of these protamine sequences with those already available from other vertebrate groups allows us to define properly the chemical consensus composition of protamines and provides a unique insight into their molecular evolution and classification.
Detection of human papillomavirus type-16 DNA utilising microtitre-plate based amplification reactions and a solid-phase enzyme-immunoassay detection system.
The development of a nested polymerase chain reaction (PCR) assay to detect low concentrations of human papillomavirus type-16 (HPV-16) DNA for epidemiological studies is described. The PCR utilises primers located in the E5 open reading frame, has an analytical sensitivity of 4 HPV-16 genomes and does not produce amplicons from other common genital HPVs (types-6, -11, -18, -31 and 33). This assay was carried out in 96-well plates utilising internal primers labelled with dinitrophenol (DNP) and biotin so that amplicons can be captured onto streptavidincoated plates and detected using an alkaline phosphatase-labelled monoclonal antibody to DNP. The assay was effective for detecting HPV-16 DNA in plasmids, cell-lines and, both freshly collected or archival (formalin-fixed/paraffin embedded) clinical specimens. This system is therefore suitable for epidemiological studies to identify individuals infected with HPV-16 DNA in episomal form who may be at increased risk of developing anogenital carcinomas.
Analytic sensitivities of hybrid-capture, consensus and type-specific polymerase chain reactions for the detection of human papillomavirus type 16 DNA.
Human papillomavirus type 16 (HPV-16) DNA is detected commonly in cervical carcinomas; in this study, we have determined the analytical sensitivities of Hybrid Capture, HPV-consensus PCR, and three HPV-16-specific polymerase chain reactions (PCRs) for the detection of HPV-16 DNA. Samples investigated included a cervical cancer cell line, cervical scrapes from 20 patients attending colposcopy clinics, and buccal swabs from eight immunosuppressed children. HPV-16 E7 and E5-nested PCRs [Cavuslu et al. (1996): Journal of Virological Methods, in press] produced positive signals from samples containing fewer than ten HPV-16 genomes per reaction. HPV-consensus PCR [Manos et al. (1989): Cancer Cells 7:209-214] and HPV-16 PCR using primers of van den Brule et al. [(1990): Journal of Clinical Microbiology 25:2739-2743] were of intermediate sensitivity (i.e., produced positive signals from samples containing 250 and 2,500 HPV-16 genoms/reaction, respectively) and Hybrid Capture could detect just 50,000 HPV-16 genomes/reaction. Highest rates of positivity for cervical samples were detected with HPV-16 E7 or E5-nested PCRs [50% (10 of 20 samples) and 60% (12 of 20 samples) positive, respectively], intermediate rates with HPV-consensus PCR and PCRs using the primers of van den Brule et al. [both 35% (7 of 20 samples)], and lowest rates of positivity [25% (5 of 20 samples)] with Hybrid Capture. None of eight buccal swab samples from immunosuppressed children were positive by Hybrid Capture, yet three (37.5%) were positive by HPV-16 E5-nested PCR. These data indicate that HPV-16 type-specific PCRs should be used for the investigation of specimens that may contain low amounts of HPV-16 DNA.
Ki-ras and p53 mutations are early and late events, respectively, in urethane-induced pulmonary carcinogenesis in A/J mice.
In the A/J strain of mice, urethane (ethyl carbamate) induces lung hyperplasia, adenoma, and adenocarcinoma in a time-dependent manner. These distinct morphological stages may correlate with sequential molecular genetic changes in this mouse model. To test this hypothesis, we investigated the presence of mutations involving Ki-ras and p53 in urethane-induced lung lesions in A/J mice at early and late stages of tumorigenesis. We precisely microdissected 40 lung lesions from paraffin-embedded sections. Ki-ras mutations around codon 61 and p53 mutations in exons 5-8 were identified by polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing techniques. In 29 early-stage lung lesions classified as hyperplasias (seven) or adenomas (22), we observed 19 Ki-ras mutations (66%), including three silent mutations and one double mutation at different codons, and one silent p53 mutation (3.5%). In 11 late-stage adenomas, we identified nine activating Ki-ras mutations (82%) and four missense p53 mutations (36%). These results indicate that Ki-ras mutations arise early, whereas p53 mutations occur relatively late during the benign stages of urethane-induced lung carcinogenesis in A/J mice.
Retroviral integrases and their cousins.
The recently determined structures of the catalytic domains of HIV integrase, avian sarcoma virus integrase and the Mu transposase are strikingly similar to each other and also exhibit significant similarity to several nucleases. All these enzymes of cut polynucleotides, leaving 3'OH and 5'PO4 groups. The integrase and transposase also possess a strand-transfer activity that splices DNA. The structural similarities among members of this superfamily of polynucleotidyl transferases suggest that they share a similar mechanism of catalysis.
Small fragment wounds: biophysics and pathophysiology.
This paper considers the wounding effects of small fragments in modern warfare. Small fragment wounds may be expected to predominate on a future conventional battlefield; however, studies and models of "military" wounds have tended to focus on bullets as the wounding projectile. This paper discusses briefly the types of fragment projectile expected from modern munitions. It goes on to define a model for such projectiles, and describes the interaction with soft tissue simulants. The extent of penetration, temporary cavitation, and contamination by foreign material are all considered. This work with simulants is validated by experimental shots against animal tissue. A wound model in an experimental animal is described. This model was used to investigate the hematologic, biochemical, and histologic effects of a small fragment wound. The effects on skin and skeletal muscle are described. By sampling at various times (up to 1 week) after wounding, the natural progress of these wounds has been ascertained. The results from 28 experimental animals, with untreated fragment wounds, are reported. The most important findings are that the skin damage is very localized and that the muscle damage is limited, with little necrotic tissue in the track. Furthermore, the extent of the muscle damage, peripheral to the wound track, improves with time, healing within a few days, provided the wound remains free from infection. There was no clinical or microbiologic evidence of infection in those animals followed for up to 3 days. However, of eight animals followed to 1 week, three developed infected wounds. This work has implications for the management of soft tissue wounds caused by fragmentation munitions. The conventional military approach has been to treat penetrating war wounds by exploration, debridement, excision of dead tissue, and delayed primary closure; conservative treatment has largely been regarded as inappropriate. The work presented here shows that the potential culture medium within the wound is small and can be removed by the normal bodily responses. There is no need for surgery, provided that infection can be prevented. It may be inferred that if bacterial colonization can be prevented in the early stages by the timely use of antibiotics, surgery may be unnecessary. Further studies are planned to investigate this possibility.
Hepatitis B vaccination schedules in genitourinary medicine clinics.
OBJECTIVES: To compare two vaccination schedules in delivering hepatitis B vaccine to at-risk genitourinary medicine clinic attenders. SETTING: Genitourinary medicine clinic of St Thomas' Hospital, London, UK. METHODS: Two vaccination protocols were compared. Between January 1991 and December 1992, individuals had doses of recombinant hepatitis B vaccine at 0, 1 and 6 months (standard). From January until October 1993 doses of vaccine were administered at 0, 1 and 2 months (accelerated), following which timing of a booster dose was made on the basis of hepatitis B surface antibody (anti-HBs) assessment. Case notes were reviewed with regard to compliance rates and anti-HBs levels. RESULTS: Two hundred and fourteen individuals were included (standard 104, accelerated 110). Of the standard group 80.8% and 61.5% attended for the 2nd and 3rd doses respectively compared with 80.0% and 75.5% of the accelerated group (attendance for the 3rd dose chi 2 = 4.19, p < 0.05). For both of these groups compliance was significantly better in those who requested vaccination rather than being offered it opportunistically (chi 2 = 4.86, p < 0.05). Seroconversion rates were not significantly different between the two groups (87.5% versus 83.1%). A significantly higher proportion of the standard group, however, achieved anti-HBs levels greater than 100 i.u./l. CONCLUSIONS: Completion of hepatitis B vaccination was improved by changing to a 0, 1 and 2 month protocol. Levels of anti-HBs achieved in the accelerated group, however, were lower. If it is confirmed that maintaining anti-HBs levels is not important in retaining protection against hepatitis B then the accelerated schedule has clear advantage. If not, the advantages may be nullified by the need, in some, for an early booster.
An in vitro and an in vivo unscheduled DNA synthesis assay with a zinc oxide/hexachloroethane (Zn/HCE) smoke.
1. Since Zn/HCE smoke has been shown previously to be weakly positive in the Ames test, and negative in the bone marrow micronucleus assay, other assays including a second in vivo assay examining unscheduled DNA synthesis (UDS) in rat hepatocytes has been carried out, as recommended by the UK Department of Health guidelines. 2. Zn/HCE smoke was assessed for its ability to induce DNA repair in an UDS assay both in vitro in cultured rat hepatocytes and in rat hepatocytes after in vivo treatment by inhalation. 3. For the in vitro investigation, two studies were carried out assessing media exposed to Zn/HCE smoke using at least seven concentrations up to a toxic level. At the highest concentration of Zn/HCE smoke, where some viable cells were seen, an increase in UDS was observed in both experiments. However this was not statistically significant, was only seen at a level where toxicity was observed and was therefore considered not to be biologically significant. 4. In the in vivo investigation, one study was carried out in three separate parts, assessing two doses of Zn/HCE smoke characterised by their zinc content as approximately 20 and 56 micrograms l-1 air. A dose-related increase in UDS was observed which was not statistically significant. The positive control behaved as anticipated, showing a highly statistically significant response. 5. It was concluded that Zn/HCE smoke did not induce unscheduled DNA repair in the in vitro or in vivo UDS assays under the conditions used in the studies. The overall lack of genotoxic effect of this smoke in this and previous studies in this laboratory would not suggest a major health hazard.
Assessment of the biochemical effects of percutaneous exposure of sulphur mustard in an in vitro human skin system.
1. Sulphur mustard (HD) is a potent chemical warfare agent which causes incapacitating blisters on human skin. There is no specific pretreatment nor therapy against this agent and the mechanism of dermo-epidermal cleavage is unclear. The aim of this study was to use a human skin explant system to determine the consequences of percutaneous exposure to HD. 2. Increased activities of serine proteases associated with blistering disorders in humans were detected from human skin explants after exposure to HD. The most consistent response and the highest protease activities measured were found for trypsin. This class of enzyme is therefore implicated in the dermo-epidermal separation which is associated with blistering in humans following exposure to HD. 3. An inflammatory response was observed in the skin explants exposed to HD. At low doses of HD it was characterised by the presence of neutrophils in the papillary dermis, culminating in the infiltration of the epidermis by these inflammatory cells at higher concentrations of HD. A variety of other histopathological changes in the explants was found such as focal dermo-epidermal separation, nuclear pyknosis and perinuclear vacuolation. 4. The study indicates that full thickness human skin explants can be used to investigate various aspects of the possible pathogenesis of HD-induced skin damage, including the associated inflammatory response.
Effects of furosemide, torasemide and controlled fluid intake on perfluoroisobutene induced lung oedema and mortality.
Permeability type lung oedema has been induced in rats by exposing them to the lung oedemagen perfluoroisobutene (PFIB). The loop diuretic furosemide (CAS 54-31-9) reduces the lung oedema and the pattern and severity of the pathological changes associated with inhalation of PFIB (mean Ct 945 mg.min.m-3) and delays the time to death following inhalation of an LCt65 equivalent of the gas. Combined administration of the loop diuretic torasemide (CAS 56211-40-6) and water deprivation reduces PFIB induced oedema and mortality during the treatment period (24 h). Controlling the intake of fluids after treatment prevents the rapid increase in oedema and mortality which occurs when water is returned ad libitum. Torasemide and water deprivation followed by controlled fluid intake delays but does not reduce the overall mortality due to an LCt70 of PFIB.
Compared toxicity of the potassium channel blockers, apamin and dendrotoxin.
The central toxicities of two potassium ion channel blockers, apamin and alpha-dendrotoxin (DTx), have been compared. Both apamin and dendrotoxin injected intracerebroventricularly produced signs of poisoning, including tremor and ataxia; however, only DTx produced changes in brain electrical activity, with high voltage spikes and epileptiform activity and subsequent brain damage. DTx, but not apamin, increased the amplitude of evoked field potentials and caused repetitive firing of neurones in hippocampal slices. Signs of poisoning following peripheral (intraperitoneal) administration of apamin were similar to those following central administration, including dramatic haemorrhagic effects on the lungs of decedent animals. These results are consistent with dendrotoxin being a centrally-active neurotoxin producing epileptiform activity and brain damage, whilst apamin produces its most significant pathology in the lung, possibly involving a neurogenic mechanism.
Structure of the bacteriophage Mu transposase core: a common structural motif for DNA transposition and retroviral integration.
The crystal structure of the core domain of bacteriophage Mu transposase, MuA, has been determined at 2.4 A resolution. The first of two subdomains contains the active site and, despite very limited sequence homology, exhibits a striking similarity to the core domain of HIV-1 integrase, which carries out a similar set of biochemical reactions. It also exhibits more limited similarity to other nucleases, RNase H and RuvC. The second, a beta barrel, connects to the first subdomain through several contacts. Three independent determinations of the monomer structure from two crystal forms all show the active site held in a similar, apparently inactive configuration. The enzymatic activity of MuA is known to be activated by formation of a DNA-bound tetramer of the protein. We propose that the connections between the two subdomains may be involved in the cross-talk between the active site and the other domains of the transposase that controls the activity of the protein.
Toxicity of sulphur mustard in adult rat lung organ culture.
The toxicity of the chemical warfare agent sulphur mustard, (bis-(2-chloroethyl)sulphide, HD), was examined in adult rat lung organ cultures. Assessment of HD-induced damage by the MTT cytotoxicity assay indicated that the median lethal concentration (LC50) of HD in these cultures was reproducible, and in the microM range. Damage to the lung slices was expressed only after a latent period of 48 h and did not increase significantly with longer expression times. Histopathological examination of HD-treated lung cultures showed that the structural changes in the lung tissue paralleled the toxicity measured biochemically, and were also similar to the damage found in animals and man exposed to HD in vivo. This in vitro model offers a useful tool with which to study the toxicity and mechanism of action of sulphur mustard.
Nucleoplasmin-mediated decondensation of Mytilus sperm chromatin. Identification and partial characterization of a nucleoplasmin-like protein with sperm-nuclei decondensing activity in Mytilus californianus.
We have been able to induce sperm nuclear decondensation in the mussel Mytilus californianus (mollusc) using either egg extracts or pure nucleoplasmin from Xenopus (amphibian). The nuclear decondensation involves removal of the sperm nuclear basic proteins (SNBPs) which bind to nucleoplasmin. An attempt has been made to isolate an ooplasmic factor from Mytilus with a similar sperm-chromatin decondensing activity. An acidic, thermostable protein with a molecular mass of 58,000 has been purified and partially characterized.
Complete sequence and characterization of the major sperm nuclear basic protein from Mytilus trossulus.
We have characterized the major protamine-like protein (PL-III) from the sperm of the mussel Mytilus trossulus. The molecular mass of the main protein component of PL-III, determined by mass spectrometry using fast atom bombardment and matrix-assisted laser desorption ionization was 11304 +/- 6 Da. The complete protein sequence was established by Edman degradation and the molecular mass derived from this sequence coincides with that experimentally determined. The protein has a sedimentation coefficient s20,w = 1.15 +/- 0.5 S which is consistent with a random coil of radius of gyration 34.3 +/- 1.4 A.
Protection against inhalation toxicity of ricin and abrin by immunisation.
1. Abrin and ricin are highly toxic plant proteins which are very similar in structure and function and inhibit protein synthesis in eukaryotes. 2. Rats have been immunised against either toxin using formaldehyde-toxoids by three subcutaneous injections at intervals of 3 weeks. For abrin, serum titres in 14 out of 15 rats were raised to between 1:12800 and 1:51200 after two injections, 6 weeks from the start of the experiment. Titres of between 1:256 and 1:1024 were also measured in lung washes after challenge with active abrin toxin. 3. The three major antibody classes, IgG, IgM and IgA were present in the immune sera but IgG and IgA only were detected in lung washes. The proportion of IgA to IgG was higher in the lung fluid than in sera. Rats immunised by abrin toxoid were protected against 5 LCt50's of abrin by inhalation but others exposed to ricin were not. 4. For ricin, serum titres ranged from 1:800 to 1:25600 after two injections and after a third injection the titre range was the same but population samples were weighted towards the higher titres. All rats immunised with ricin toxoid survived the challenge of 5 LCt50's of ricin toxin by inhalation over the observation period of 28 days post-challenge. 5. Representative immunised rats (abrin toxoid) were taken at various times post-exposure, humanely killed and tissues were examined for pathological changes. It was concluded that an apparently severe lung lesion occurred at a later time than in non-immunised, toxin challenged rats. This damage was not lethal over the experimental observation periods. 6. Immunisation by the sub-cutaneous route therefore protects against lethality from challenge by inhalation of ricin or abrin toxins but does not prevent significant lung damage.
Changes in connective tissue macromolecular components of Yucatan mini-pig skin following application of sulphur mustard vapour.
1. The aim of this study was to determine the nature of the macromolecular alterations in Yucatan mini-pig skin which occur following application of sulphur mustard vapour, with particular reference to laminin and type IV collagen. 2. The immunostaining of transfer blots from skin extracts run on SDS-PAGE gels revealed no evidence of cross-linking of type IV collagen or laminin. Laminin was, however, found to be partially degraded as determined by the resolution of 132 and 143 kDa fragments, possibly by the activation of proteases, following the application of sulphur mustard to pig skin. Type IV collagen was not subject to this form of degradation in the skin samples exposed to sulphur mustard. 3. Yucatan mini-pig skin was found to develop microblisters after exposure to sulphur mustard vapour. The immunohistochemical studies of sulphur mustard exposed skin revealed that separation of the epidermis from the dermis was found to occur within the lamina lucida of the subepidermal basement membrane, supporting the contention that cleavage of laminin networks occurs following mustard challenge. Immunohistochemical staining with anti-type IV collagen antibodies was restricted to the floor of the micro-blister lesions. 4. The results suggest that laminin may be a target for protease activation at the dermo-epidermal junction. This may account for the tendency of certain skin models to develop sulphur mustard-induced blistering. The Yucatan mini-pig may be valuable as a model to determine the efficacy of prophylactic and therapeutic regimes.