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Biomedical subjects

H Field

Publications and source records attributed to H Field.

At least 19 recordsLinked to original sources
PubMed2005

Collection, seminal characteristics and chilled storage of spermatozoa from three species of free-range flying fox (Pteropus spp.).

This study reports observations on the collection and characteristics of semen from free-range populations of flying fox in Brisbane, Australia. Semen was successfully recovered by electroejaculation from 107 of 115 wild flying foxes (Pteropus alecto, Pteropus poliocephalus and Pteropus scapulatus). A proportion of ejaculates collected from all three species contained seminal vesicle secretions, the incidence of which appeared related to breeding season. Ejaculate volume was small (5--160 microL), requiring a specialised collection vessel and immediate extension to avoid desiccation. Sperm morphological abnormalities and characteristics are described for the first time. In two species (P. scapulatus and P. alecto), sperm quality varied with breeding season. Dilution in Tris-citrate-fructose buffer and subsequent incubation (37 degrees C) of Pteropus semen for 2-3h appeared to have a negative impact on sperm motility and the percentage of sperm with intact plasma membranes and acrosomes and represents a concern for the potential development and use of assisted breeding technology in these species. Preliminary attempts to develop a short-term chilled preservation protocol for flying fox semen revealed that sperm viability (percentage motility and percentage live sperm with intact acrosomes) was significantly reduced after 102 h chilled storage at 5 degrees C; nevertheless, approximately 40% of the spermatozoa were still motile and contained intact acrosomes. Glycerol was neither protective nor detrimental to sperm survival during chilled storage. Microbial flora of the prepuce, urethra and semen of all species were isolated and their antibiotic susceptibility tested. Tetracycline, penicillin, ciprofloxacin, and ceftazidime were the most effective antibiotics in preventing growth of all identified bacteria; however, their effects on sperm survival were not investigated.

Acrosome↗
PubMed2005

Efficacy of topical aciclovir for the treatment of feline herpetic keratitis: results of a prospective clinical trial and data from in vitro investigations.

This study aimed to evaluate the efficacy of topical ophthalmic aciclovir applied five times daily as a treatment for feline herpesvirus type 1 (FHV-1) keratitis in a group of cats in a first-opinion practice setting. Cats with ocular signs indicative of FHV-1 or Chlamydophila species infection, predominantly conjunctivitis and keratitis, were tested for FHV-1 antigen using an immunofluorescent technique on air-dried conjunctival swabs. They were first treated with topical chlortetracycline with efficacy against Chlamydophila species and then, in cases positive for FHV-1, with topical aciclovir. The time to recovery was determined and illustrated using a Kaplan-Meier plot. Three cats were infected with Chlamydophila species and showed a median time to recovery of 14 days (95 per cent confidence interval [CI] 10 to 18 days), while 30 cats infected with FHV-1 showed a median time to recovery of 12 days (95 per cent CI 10 to 14 days). The drug dose at which 50 per cent plaque reduction (ED50) occurred in a standard plaque reduction assay was determined in an in vitro study. This showed a mean (SD) ED50 of aciclovir of 25 (3.5) mg/ml compared with 0.4 (0.05) mg/ml for trifluorothymidine, a drug known to be efficacious against FHV-1. The study shows that even though aciclovir is generally considered to lack efficacy against ocular FHV-1 infection, when used frequently it can have a beneficial effect in FHV-1 conjunctivitis and keratitis.

Acyclovir↗
PubMed2004

Novel viral encephalitides associated with bats (Chiroptera)--host management strategies.

Several novel viruses recently described in bats of the genus Pteropus (sub-order Megachiroptera) in Australia and southeast Asia cause encephalitic disease in animals and humans. These viruses include Hendra virus and Nipah virus (genus Henipavirus, family Paramyxoviridae) and Australian bat lyssavirus (ABLV; genus Lyssavirus, family Rhabdoviridae). Broadly, strategies for disease prevention and control in the spillover host are directed at minimising direct or indirect contact with the natural host, improving farm-gate and on-farm biosecurity, and better disease recognition and diagnosis. Additional strategies for ABLV include the use of rabies vaccine for effective pre- and post-exposure prophylaxis in humans. Effective management strategies in the natural host are predicated on an understanding of the ecology of the disease in the natural host, and the identification and avoidance of factors putatively associated with emergence, such as habitat loss, land use change and demographic shifts. A possible future management strategy for ABLV in reservoir populations is immunisation using bait or plant-derived vaccination.

Animals↗
PubMed2001

The natural history of Hendra and Nipah viruses.

Pteropid bats (flying foxes), species of which are the probable natural host of both Hendra and Nipah viruses, occur in overlapping populations from India to Australia. Ecological changes associated with land use and with animal husbandry practices appear most likely to be associated with the emergence of these two agents.

Animals↗
PubMed2000

Nipah virus: a recently emergent deadly paramyxovirus.

A paramyxovirus virus termed Nipah virus has been identified as the etiologic agent of an outbreak of severe encephalitis in people with close contact exposure to pigs in Malaysia and Singapore. The outbreak was first noted in late September 1998 and by mid-June 1999, more than 265 encephalitis cases, including 105 deaths, had been reported in Malaysia, and 11 cases of encephalitis or respiratory illness with one death had been reported in Singapore. Electron microscopic, serologic, and genetic studies indicate that this virus belongs to the family Paramyxoviridae and is most closely related to the recently discovered Hendra virus. We suggest that these two viruses are representative of a new genus within the family Paramyxoviridae. Like Hendra virus, Nipah virus is unusual among the paramyxoviruses in its ability to infect and cause potentially fatal disease in a number of host species, including humans.

Animals↗
PubMed2000

Cell-cycle and developmental regulation of TbRAB31 localisation, a GTP-locked Rab protein from Trypanosoma brucei.

Rab proteins are small GTPases that control the direction and timing of vesicle fusion during intracellular trafficking between membraneous compartments. Genome sequencing and EST analysis of Trypanosoma brucei indicates that the trypanosome Rab (TbRAB) gene family, and hence complexity of intracellular transport pathways, is intermediate between Saccharomyces cerevisiae and mammals. TbRAB31 is a constitutively expressed T. brucei Rab protein (formerly Trab7p) and is the product of one of two closely linked TbRAB genes, the other being TbRAB2 (TbRab2p, in: Field H, Ali BRS, Sherwin T, Gull K, Croft SL, Field MC. TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells. J Cell Sci 1999; 112:147-156), involved in ER to Golgi transport. TbRAB31 has high homology to members of the Sec4/Ypt1 subfamily of Rab proteins from S. cerevisiae and to Rab13 and Rab11 from higher eukaryotes. Recombinant TbRAB31 binds GTP but, unusually for a Rab protein, has undetectable GTPase activity resulting in a constitutively GTP-bound protein. Antibodies against TbRAB31 recognise a discrete structure located between the kinetoplast and nucleus in interphase procyclic cells; by contrast the structure is morphologically more complex in bloodstream form (BSF) parasites, consisting of at least two foci. TbRAB31 behaviour was also studied during the cell cycle; TbRAB31 always localised to a discrete structure that duplicated very early in mitosis and relocated to daughter cells in a coordinate manner with the basal body and kinetoplast, suggesting the involvement of microtubules. Additional evidence suggests that TbRAB31 localises to the trypanosome Golgi complex. Firstly, the interphase position of TbRAB31 is consistent with a Golgi location. Secondly, the TbRAB31 structure is also recognised by cross-reacting antibodies to mammalian beta-coatomer protein (beta-COP), which localises to the Golgi in mammalian cells. Thirdly, the fluorescent ceramide analogue, BODIPY-TR-ceramide, a reliable marker of the mammalian Golgi apparatus, exhibited overlapping distribution with TbRAB31. The location of BODIPY-TR-ceramide was confirmed at the trypanosome Golgi by histochemistry with diaminobenzidine and electron microscopy.

Animals↗
PubMed2000

Toxicological screening in trauma.

OBJECTIVES: To determine the prevalence and patterns of alcohol and drug use in patients with major trauma. METHODS: Consecutive trauma patient enrollment, 24 hours a day, was envisaged with anonymised patient data on gender, age band, and mechanism of injury collected. The study group had surplus plasma quantitatively analysed for ethanol concentration, and urine samples were initially screened, via immunoassay, for opiates, cannabinoids, amphetamines, benzodiazepines, cocaine, and methadone. Confirmation and specification of individual positive results was then performed using thin layer or gas-liquid chromatography. Drugs of treatment given in the resuscitation room, if subsequently detected in the urine samples, were excluded from the final results. RESULTS: There were 116 eligible trauma patients assessed and treated in the resuscitation room over a six month period, of which 93 (80%) were enrolled. Altogether 27% of this trauma population had plasma ethanol concentrations greater than 80 mg/dl. There was a significantly higher prevalence of alcohol intoxication in the group not involved in a road traffic accident (RTA) compared with the group who were involved in a RTA. Initial screening of urine for drugs revealed a prevalence of 51%. After 12 exclusions due to iatrogenic administration of opiates, the final confirmed prevalence was 35% in this trauma population. The individual drug prevalence was 13% for cannabinoids, 11% for codeine, 8% for morphine, 6% for amphetamine, 6% for benzodiazepines, 3% for cocaine, 1% for dihydrocodeine, and 1% for methadone. CONCLUSIONS: There is a notable prevalence of drug and alcohol use in this British accident and emergency trauma population. A significantly higher prevalence for alcohol intoxication was found in the non-RTA group compared with the RTA group. The patterns of drug usage detected reflect local influences and less cocaine use is seen compared with American studies. The association between alcohol, drugs, and trauma, together with ethically acceptable methods of screening, are discussed.

Accidental Falls↗
PubMed1999

Newly discovered viruses of flying foxes.

Flying foxes have been the focus of research into three newly described viruses from the order Mononegavirales, namely Hendra virus (HeV), Menangle virus and Australian Bat Lyssavirus (ABL). Early investigations indicate that flying foxes are the reservoir host for these viruses. In 1994, two outbreaks of a new zoonotic disease affecting horses and humans occurred in Queensland. The virus which was found to be responsible was called equine morbillivirus (EMV) and has since been renamed HeV. Investigation into the reservoir of HeV has produced evidence that antibodies capable of neutralising HeV have only been detected in flying foxes. Over 20% of flying foxes in eastern Australia have been identified as being seropositive. Additionally six species of flying foxes in Papua New Guinea have tested positive for antibodies to HeV. In 1996 a virus from the family Paramyxoviridae was isolated from the uterine fluid of a female flying fox. Sequencing of 10000 of the 18000 base pairs (bp) has shown that the sequence is identical to the HeV sequence. As part of investigations into HeV, a virus was isolated from a juvenile flying fox which presented with neurological signs in 1996. This virus was characterised as belonging to the family Rhabdoviridae, and was named ABL. Since then four flying fox species and one insectivorous species have tested positive for ABL. The third virus to be detected in flying foxes is Menangle virus, belonging to the family Paramyxoviridae. This virus was responsible for a zoonotic disease affecting pigs and humans in New South Wales in 1997. Antibodies capable of neutralising Menangle virus, were detected in flying foxes.

Animals↗
PubMed1999

GTPases in protozoan parasites: tools for cell biology and chemotherapy.

Small G proteins belong to a superfamily of GTPases related to the protooncogene ras, and function as master control elements for a range of cellular functions. This ability is related to their low rate of substrate turnover; GTPases catalyse the conversion of GTP to GDP, but with a rate in the order of one substrate per second, orders of magnitude slower than 'good' enzyme catalysis, but placing the reaction into the temporal frame of many cellular processes including signal transduction, cytoskeletal reorganization and vesicle trafficking. In this article, Mark Field, Bassam Ali and Helen Field describe some recent advances in G-protein studies in the parasite field, concentrating on the protozoan parasites. Because of their numerous roles in cell biology, understanding parasite G proteins has great potential for increasing our knowledge of parasite cellular physiology, as well as providing important inroads into vital processes for potential therapeutic exploitation.

Animals↗
PubMed1999

TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells.

The Rab family of small GTPases is a subset of the Ras superfamily. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway, such as ER-to-Golgi transport, probably by controlling SNARE complex assembly. In Trypanosoma brucei a number of Rab proteins have been isolated by EST analysis; here we characterise one of these, TbRab2p (originally designated Trab1p), which is a member of the Ypt1p subfamily of Rab proteins. Recombinant TbRab2p is capable of hydrolysing GTP and is post-translationally modified in vitro by addition of a geranylgeranyl prenyl group, properties of an authentic Rab GTPase. Antibodies against recombinant TbRab2p show that in trypanosomes TbRab2p is localised primarily to the endoplasmic reticulum (ER) and colocalises with BiP in wild-type trypanosomes. Over expression of TbRab2p in procyclic form T. brucei results in a cell population having a 40-fold increase in TbRab2p expression. In these cells biosynthesis of procyclin, a secretory pathway glycoprotein, is decreased, accompanied by an increase in general protein biosynthesis, suggesting that excess TbRab2p affects ER function. Heterologous expression of TbRab2p in COS cells resulted in targeting to the pre-Golgi transport intermediate (ERGIC), indicating that the targeting information is conserved between mammals and trypanosomes. Clustal and phylogenetic analyses support assignment of TbRab2p as a Rab2 homologue. In addition, over expression of TbRab2p in trypanosomes results in membrane reorganisation and formation of opaque vesicular structures visible by phase contrast microscopy, consistent with accumulation of ER-derived vesicular structures in cells highly overexpressing TbRab2p. Ultrastructural examination by electron microscopy confirmed the presence of a tubulo-vesicular membrane bound compartment in close proximity to the cis-Golgi, probably equivalent to the ERGIC. TbRab2p is therefore a new ER/ERGIC marker for T. brucei.

Amino Acid Sequence↗
PubMed1998

Complexity of trypanosomatid endocytosis pathways revealed by Rab4 and Rab5 isoforms in Trypanosoma brucei.

Small G proteins of the Rab family are responsible for vesicle fusion and control flux during intracellular transport. Rab5 is important in endosome maturation and Rab4 in recycling of endocytic material. Three Rab5 isoforms identified so far in mammals and three in the yeast genome suggest that conservation of multiple Rab5 isoforms is required for sophisticated regulation of endocytosis. Trypanosoma brucei homologues of Rab5 and Rab4 (TbRab5A and TbRab4) have been identified. Here we report cloning of a second Rab5 homologue, TbRab5Bp. The TbRAB5A and -5B genes are not linked in the genome, and phylogenetic reconstruction indicates that multiple Rab5 isoforms in yeast, mammals, and trypanosomes evolved independently. Northern blots demonstrate that TbRab5A, -5B, and TbRab4 messages are expressed in bloodstream form (BSF) and procyclic forms of the parasite even though endocytosis is not very active in the latter form. mRNA levels of TbRab5A and -4 are constitutive. Multiple-sized TbRab5B messages at very low abundance are detected, with greater expression in BSF. Also, the TbRab5B mRNA has a large 3'-untranslated region suggestive of potentially complex regulation, and therefore TbRab5Bp may be an important regulator of differential endocytosis levels between BSF and procyclic stage parasites. Affinity purified antibodies raised to C-terminal peptide sequences of all three TbRab proteins recognized small vesicular cytoplasmic structures, which for TbRab5Ap and -5Bp are predominantly near the flagellar pocket. TbRab5Bp colocalizes with invariant surface glycoprotein 100 (ISG100), a protein entering the endocytotic pathway in BSF parasites, whereas in procyclic cells populations of vesicles stained with both TbRab5Ap and -5Bp substantially overlap; TbRab5 proteins are therefore components of the endocytotic pathway. TbRab4p localizes to vesicular structures throughout the cytoplasm, with some overlap with TbRab5Bp, but the majority occupying a different compartment to the TbRab5s. Therefore the trypanosome endosomal system has been functionally dissected for the first time; these reagents provide a unique opportunity for manipulation of the protozoan endosomal system to further our understanding of drug uptake mechanisms and virulence.

Amino Acid Sequence↗
PubMed1997

Tandem duplication of rab genes followed by sequence divergence and acquisition of distinct functions in Trypanosoma brucei.

The Ras superfamily of small G proteins governs unidirectional cellular processes by virtue of GTP hydrolysis and concomitant conformational changes, which are in turn regulated by a number of accessory factors. Members of the Rab subfamily are important for correct targeting and fusion of intra-organellar vesicles loaded with trafficking proteins and lipids. During evolution from a prototype gene, novel functions may be acquired by duplicated daughter genes; for Rab proteins, this can be tested by location, which is specifically related to the function of each Rab. We have found an example of two rab genes in Trypanosoma brucei (trab genes) that clearly arose by tandem duplication, being highly related to each other and remaining juxtaposed in the genome, whose products have dramatically different subcellular locations, indicative of discrete functions. These two trab genes, isolated on a single genomic clone, are separated by a short intervening sequence and are in a head-to-tail orientation. The nucleotide sequences of the open reading frames and intervening sequence were determined and show that the genes are paralogues, probably arising from an ancient tandem duplication. Both genes are most homologous to ypt1 and sec4 in the Saccharomyces cerevisiae genome, while phylogenetic reconstruction indicates that although they have clearly diverged, the proteins are more closely related to each other than to other Rab protein sequences available in the data base. Immunofluorescence microscopy, using antibodies raised against the recombinant Trab proteins, clearly demonstrates that the native Trab proteins have completely distinct subcellular locations in the trypanosome. Trab1p is present in a widespread reticular location similar to BiP, suggesting an endoplasmic reticulum location, while Trab7p is observed in a discrete structure adjacent to the kinetoplast. Most interestingly, the Trab7p-positive compartment also appears to divide at the same time, or just prior to, the kinetoplast, i.e. early in mitosis, suggestive of association with structures in the flagellar pocket region. An estimate of the divergence time indicates that the trab1/trab7 duplication occurred approximately 100 million years ago, and therefore, the persistence of this pair suggests an essential role in the survival of T. brucei.

Algorithms↗
PubMed1997

Collection and short-term preservation of semen from free-ranging eastern grey kangaroos (Macropus giganteus: Macropodidae).

OBJECTIVES: To evaluate electro-ejaculation of free-range eastern grey kangaroos in the field and assess the efficacy of four diluents to preserve sperm motility over a 48-h period at 5 degrees C. PROCEDURE AND DESIGN: Under gaseous anaesthesia, 25 free-range kangaroos were electro-ejaculated and characteristics of the ejaculate noted. Spermatozoa obtained from eight ejaculates were diluted in phosphate buffered saline containing various combinations of egg yolk and glucose and refrigerated at 5 degrees C for 48 h. RESULTS: Spermatozoa were recovered from 24 of 28 ejaculates. Mean (+/- SEM) semen volume (mL) and pH were 25.0 +/- 1.9 and 7.1 +/- 0.1 respectively. The forward motility (%), rate of movement of sperm (0 to 5) and sperm concentration (x 10(6)/mL) were 77.4 +/- 1.5, 3.8 +/- 0.9 and 31.2 +/- 7.3 respectively. There was no significant difference between the four diluents in their ability to maintain forward motility of spermatozoa over 48 h. However, rate of movement over the same period was significantly (P < 0.01) improved when sperm were diluted in phosphate buffered saline containing 10% egg yolk. CONCLUSIONS: Electro-ejaculation is a safe and reliable method for collecting semen from free-ranging eastern grey kangaroos. Preliminary attempts at short-term preservation showed that the motility of kangaroo spermatozoa could be adequately stored for 24 h and that the addition of egg yolk to the semen diluent was beneficial for improving the rate of sperm movement.

Animals↗
PubMed1996

Characterisation of protein isoprenylation in procyclic form Trypanosoma brucei.

Protein modification by isoprenylation is essential in mammals and other eukaryotes, but has not been demonstrated in the parasitic protozoa of the order kinetoplastida. A key regulatory enzyme of the mevalonate pathway, hydroxymethylglutaryl-coenzyme A reductase (HMG-R), and end products of the path, including dolichols, are present in Trypanosoma brucei. By metabolical labelling of procyclic form trypanosomes in the presence of compactin, an efficient inhibitor of HMG-R, followed by one-dimensional gel electrophoresis, we demonstrate that protein isoprenylation indeed takes place in this organism and at least 14 polypeptides bear the modification. Further characterization of labelled isoprenyl groups by methyl iodide cleavage and high pressure liquid chromatography identified both the farnesyl and geranylgeranyl moieties found covalently attached to proteins in other eukaryotes. The latter moiety was more abundant, as found in mammalian systems. Prolonged incubation with compactin grossly affected cell morphology and altered a number of subcellular structures as seen by electron microscopy. High concentrations of compactin were toxic, whilst lower concentrations were cytostatic. The primary morphological lesion is distinct from that of synvinolin, another inhibitor of HMG-R. The morphological changes correlated with a complete inhibition of HMG-R activity by compactin. Surprisingly there was a complete lack of HMG-R activity in procyclic cells grown for 1 or several days in 100 microM compactin, suggesting that degradation of the enzyme had occurred and compensatory upregulation mechanisms could not be successfully exploited by the parasite to overcome HMG-R inhibition. Subsequent alterations to the overall cell shape are seen after 3 days of compactin exposure. Overall these data indicate that T. brucei has an essential protein isoprenylation pathway that is conserved with the higher eukaryotes. Additionally, products of the MVA pathway are implicated in maintenance of cell architecture.

Animals↗
PubMed1996

Leptomonas seymouri, Trypanosoma brucei: a method for isolating trypanosomatid nuclear factors which bind T. brucei single-stranded g-rich telomere sequence.

Sequential expression of variant surface glycoproteins (VSG) in Trypanosoma brucei is the basis of antigenic variation which is essential for parasite survival. Telomere distal copies of VSG genes, so-called basic copies, provide a repository of VSG sequence information for variability, but actively expressed copies are found only at subtelomeric regions of chromosomes. Of eight or so expression sites (ES) in the T. brucei genome, only one is active at one time. Movement of a basic copy VSG gene to an ES requires a recombination event of unknown mechanism. The properties of telomeres have been speculated to be important for control of VSG expression or basic copy mobilization, prompting us to begin to investigate telomere-binding proteins in trypanosomatids. The T. brucei telomere sequence is known, facilitating design of synthetic telomeric DNAs. Here we describe a method for preparation of active trypanosomatid nuclear extracts. We show that in T. brucei and Leptomonas seymouri, factors can be detected which bind a g-rich single-strand telomere sequence based on the T. brucei telomere. The L. seymouri telomere-binding factor, LST-1, dissociates in the presence of high salt to produce a core factor, LST-2, migrating similarly to the T. brucei telomere-binding factor TBT-1. The affinity of LST-2 and TBT-1 for DNA under high salt conditions is characteristic of telomere proteins.

Animals↗