Chemical reactions and applications of the reductive surface of porous silicon.

The chemical reactivity of freshly prepared porous silicon is similar to that of a reducing agent on the surface of the nanocrystallites. Ag+ spontaneously reduces to form Ag0 granular coatings on the surface of porous silicon at the expense of the oxidation of silicon hydride and silicon. Atomic Force Microscopy shows that the thickness and topography of the Ag0 coating depend on the concentration of Ag+ with the porous silicon surface being the limiting reagent. In-situ Raman Spectroscopy shows an Ag layer on the silicon and Si:O layer immediately after etching and exposure to Ag+ and O2 respectively. Ag0 coated on the surface and in the pores of the porous silicon proves to be an excellent material for Surface Enhanced Raman Spectroscopy and the natural low electron affinity on the surface of porous silicon replaces the need for a negative bias to prepare very stable diamond coatings on the surface of silicon.

Prevalence of serum antibodies to canine adenovirus and canine herpesvirus in the European red fox (Vulpes vulpes) in Australia.

OBJECTIVES: To determine the seroprevalence and aspects of the epidemiology of canine adenovirus (CAdV) and canine herpesvirus (CaHV-1) in European red foxes (Vulpes vulpes) in Australia. DESIGN: Serum samples were collected opportunistically from foxes in 1991-1994 in Western Australia (WA) and South Australia (SA) and in 1980-1984 and 1990-1994 in New South Wales (NSW) and the Australian Capital Territory (ACT). The sera were examined for antibody to CAdV and CaHV-1 using ELISAs. Seroprevalence in the different regions was determined for both viruses and the CAdV data were analysed for interactions between decade of collection, age, season, region and gender using logistic regression. RESULTS: The overall prevalence of antibody to CAdV was 23.2% (308/1326) but was significantly higher in sera collected in the eastern states of Australia (47%: 233/498) than in WA (9%: 75/828). Overall, in NSW and the ACT, there was a significantly lower prevalence in juveniles than in adults and the prevalence in juveniles in the 1990s was significantly lower than in the 1980s. The prevalence was also significantly lower in the autumn than in the winter for juveniles but the reverse held for adults. The NSW and ACT data were subdivided into eastern (including the ACT) and western regions. This revealed a significantly higher prevalence in the winter than in the autumn for the west and the reverse in the east. In WA, the northern rangeland regions of WA had lower prevalence (1.9%) than the southern agriculture regions (10.7%). Seasonally, there was a peak prevalence in the spring dropping through the summer and autumn and rising again in the winter. This seasonal pattern was also found in the combined data for all sites in the 1990s. There was no gender difference in prevalence of CAdV either overall or in different regions. The overall prevalence of antibody to CaHV-1 was 2.2% (28/1300). The small number of positives allowed only limited statistical analysis that did not reveal any differences in decade of collection, age, season or region. CONCLUSIONS: CAdV infection is common in the Australian fox population whereas CaHV-1 infection is rare. For CAdV, the age and seasonal patterns of seroprevalence were generally consistent with the recruitment of young susceptible foxes into the population in the spring and the accumulation of infections with age. The differences in regional prevalences correlated with fox density. The low prevalence of antibody to CaHV-1 suggests that CaHV-1 may be a more suitable vector than CAdV for bait delivery of immunocontraceptive antigens to foxes in Australia.

Vaccines for fertility regulation of wild and domestic species.

The application of fertility control technologies for the management of animal populations is still a relatively new concept and remains to be proven on a large scale. While there have been demonstrations of its utility using crude antigenic preparations of porcine zona pellucida, refined, cheap and easily delivered vaccines are still in the early phases of development, especially those required for remote delivery to free-ranging wild animal populations. The relative slow emergence of such technologies for practical application clearly reflects the complexity of the science and the requirement for a multidisciplinary research approach when attempting to develop such technologies for wild animal management. In many cases, one of the areas where further information is required is the immunobiology of such target species. Effective immunocontraceptive vaccines will also be critically dependent upon the design of the delivery systems. These will be unique to each species under study. A major challenge for free-ranging species will be to ensure that the vaccine can induce a long-lasting immune response to a high percentage of the target population, thus reducing the frequency and, hence, the cost of vaccine application. Many of the delivery systems being considered will utilise recombinant organisms. Hence, considerations on the use of such organisms needs to take into account the political, ethical and safety constraints prior to any environmental release. Also of prime importance is the issue of species specificity. In some instances, this will be a major challenge and careful thought will need to be given to ensure that specificity can be built into the vaccine at several levels. These might include the target antigen or epitopes, the microbial or other delivery vector, and, where applicable, the bait matrix and any target-specific attractive properties it may require. If these caveats can be satisfied and the public is able to accept the use of such vaccines, the potential for their application to manage animal populations and their impact is most certainly assured.

Antigen-specific systemic and reproductive tract antibodies in foxes immunized with Salmonella typhimurium expressing bacterial and sperm proteins.

Attenuated Salmonella typhimurium strains are potential 'safe' delivery vectors of an oral immunocontraceptive vaccine for the European red fox (Vulpes vulpes). In the present study, model bacterial (Escherichia coli heat-labile enterotoxin B subunit, LTB) and fox sperm (fSP10) antigens were expressed in S. typhimurium SL3261 (delta aroA) under the control of the trc promoter. Adult female foxes were given three oral immunizations with SL3261 containing either LTB (SL3261/pLTB), fSP10 (SL3261/pFSP10) or a control plasmid (pKK233-2 or pTrc99A). All foxes raised serum (IgG) and vaginal (IgG and IgA) antibodies against S. typhimurium lipopolysaccharide (LPS). Each fox that received SL3261/pLTB raised high titre LTB-specific serum and vaginal IgG antibodies. However, only one of four foxes immunized with SL3261/pFSP10 raised an anti-fSP10 immune response, in the form of low titre serum and vaginal IgG antibodies. No vaginal IgA antibodies were raised against either LTB or fSP10 in these experiments. The immune responses against recombinant LTB and fSP10 resulted chiefly from the initial dose of antigen in the inocula and were minimally influenced by continued in vivo antigen expression. This study demonstrates for the first time in the female red fox that oral Salmonella can elicit specific systemic and reproductive tract antibodies against heterologous, recombinant proteins.

Cloning and characterization of the cDNA encoding the PH20 protein in the European red fox Vulpes vulpes.

The PH20 protein is thought to play a crucial role in mammalian fertilization. The fox PH20 homologue has been cloned from a testis cDNA library and the deduced protein sequence shows high levels of homology to PH20 proteins isolated from other species. Unlike other PH20 proteins the fox protein does not appear to be membrane associated through a GPI-linkage nor does it show the presence of a transmembrane domain at the C-terminus of the protein. It is in this region that the proteins appear to be least conserved. Immunolocalization studies on fox sperm show that the PH20 protein is located on the inner acrosomal membrane. Transcription of PH20 in the fox is seasonally regulated, with the mRNA expressed during those months when spermatogenesis is at its peak. The PH20 sequence described in this paper has been submitted to the Genbank database and has the accession number U41412.

Lessons learned from an Internet GP information system.

We describe the prototype of an application that in actual use would allow GPs to find out more information about consultants at hospitals. This would aid the GP in making the decision about which consultant a patient should be referred to. The requirements of the application from the GP's perspective are described, together with some of the issues that have to be resolved before hospitals can provide the necessary information in a standard format. The application is implemented as a client--server system using standard Internet technologies such as Java and HTML. This architecture has a number of advantages but also revealed some issues concerning security and the format of data, among other things. The project showed that there is a desire for such a system and that that desire can be fulfilled at a relatively low cost.

Effectiveness of ambulance paramedics versus ambulance technicians in managing out of hospital cardiac arrest.

OBJECTIVE: To determine the effectiveness of extended trained ambulance personnel (paramedics) for the management of out of hospital cardiac arrest. METHODS: A retrospective cohort study of patients who suffered a cardiac arrest between 1 January 1992 and 31 July 1994, and who were transported to their local accident and emergency (A&E) department. Data were collected on basic demography, operational time intervals, and ambulance crew status. Further clinical data were collected, and outcome measures included status on arrival at A&E, status on leaving A&E (hospital admission), and status on leaving hospital. The data were analysed using univariate and multivariate techniques. RESULTS: Univariate analysis showed the likelihood of arriving in A&E with a return of spontaneous circulation was more than doubled among patients attended by a paramedic crew compared with those attended by technicians (relative risk = 2.48, 95% confidence interval 1.34 to 4.60). The likelihood of successful hospital admission was also significantly increased (RR = 1.92, 95% CI 1.13 to 3.27); however, beyond this point, further survival benefits appeared to be much smaller. Similar findings were revealed using multivariate analysis. Second level modelling revealed further possible differences between paramedic and technician crews according to type of incident. Patients successfully admitted to hospital who died before discharge remained severely disabled between admission and death. CONCLUSIONS: There are marked short term survival advantages after cardiac arrest associated with paramedic care, but these probably diminish rapidly over time.

A bait-delivered immunocontraceptive vaccine for the European red fox (Vulpes vulpes) by the year 2002?

An orally-delivered immunocontraceptive vaccine is being developed for the control of fox populations. A number of genes (PH-20, LDH-C4, ZP3) encoding gamete proteins have been cloned, produced in recombinant expression systems and used in fertility trials to test the efficacy of these antigens. As the immunocontraceptive vaccine will be delivered in a bait, there is a requirement for a greater understanding of the immune responses of the reproductive mucosa in canids, and the assessment of the best vaccine delivery system that will evoke a mucosal antibody response. Several vaccine delivery systems including microencapsulated antigens, and both vaccinia virus and bacterial vectors are being investigated. Oral administration of Salmonella typhimurium recombinants expressing different fox sperm antigens stimulates both systemic IgG responses to the antigen and a mucosal immune response within the female reproductive tract in the fox, indicating that salmonella may have potential with respect to the oral delivery of antigen. The enhancement of mucosal immune responses to orally-delivered vaccines is also being examined, research focussing on the possible use of fox-specific cytokines or the beta-subunit of cholera toxin in forming part of the vaccine construct.

Regulation of reproductive tract immunoglobulins by oestradiol-17beta in the European Red Fox.

The effect of the ovarian hormone, oestradiol-17beta, on reproductive tract immunity in the female fox was investigated. Reproductive tract antibody responses were induced by either Peyer's patch immunization with a recombinant fox sperm protein, or by oral immunization with live, attenuated Salmonella typhimurium. The effect of exogenous oestradiol-17beta or the stage of the oestrous cycle on reproductive tract immunity was assessed. The secretion of specific vaginal IgA, but not vaginal IgG, antibodies was reduced by exogenous treatment with oestradiol-17beta, while both specific vaginal IgA and vaginal IgG levels declined during the period of natural oestrus. It is concluded that oestradiol-17beta, and probably other reproductive hormones, are involved in the regulation of antibody-secretion in the fox reproductive tract, and that reproductive status is an important factor to consider in the design and application of vaccines which aim to induce immunity within the female reproductive tract.

Cloning, sequencing, and characterization of LDH-C4 from a fox testis cDNA library.

A full-length cDNA encoding the sperm-specific enzyme lactate dehydrogenase-C4 was isolated from a fox testis cDNA expression library and sequenced. The deduced translated protein sequence was shown to be 86% identical to that of human LDH-C4. In the fox testis, mRNA encoding LDH-C4 was first detected in pachytene spermatocytes. The LDH-C4 protein monomer was identified in Western blots of sperm membrane extracts as having a molecular weight of approximately 35,000, consistent with the monomeric size of this subunit previously identified in sperm from other species. The LDH-C4 protein is localized on the sperm plasma membrane overlying the principal piece of the tail. Based on the available sequence data, we were able to identify an epitope within the N-terminal region of the LDH-C4 amino-acid sequence which when administered to female foxes is antigenic and produces antibodies capable of recognizing the native protein.

Cloning and partial characterization of the cDNA encoding the fox sperm protein FSA-Acr.1 with similarities to the SP-10 antigen.

We have isolated and characterized a cDNA, cFSA-Acr.1, encoding a testis-specific fox sperm antigen. The antigen is located on the inner acrosomal compartment, and is expressed during spermatogenesis on the developing acrosome of round and elongating spermatids. Database searches with the deduced amino acid sequence of cFSA-Acr.1 revealed that the clone has high homology to both human and baboon sperm protein SP-10, and the mouse sperm protein, MSA-63. The region of highest homology is within the carboxyl terminus. In the middle of the open reading frame, the fox sequence shows unique sequences absent from both the human, baboon SP-10, and mouse MSA-63 sequences. In addition to cFSA-Acr.1, two other clones were also isolated from the same fox testis cDNA library, and sequence analysis shows that they may represent alternatively spliced mRNAs coding for other FSA-Acr proteins.

Induction of specific IgA responses in rats after oral vaccination with biodegradable microspheres containing a recombinant protein.

Diseases which affect mucosal surfaces cause considerable mortality and morbidity. New vaccine technologies are now available which justify a reappraisal of oral delivery not only for infectious disease control but also to control mucosal physiological processes such as fertility. Biodegradable microspheres have been investigated for their use as an oral delivery vehicle in rats using a recombinant antigen derived from fox sperm. Unencapsulated antigen administered in saline by the oral route produced a negligible response although an improved response was obtained if administered directly into the duodenum. This response was considerably enhanced if Peyer's patch (PP) priming was performed by direct injection of antigen in Freund's complete adjuvant (FCA) prior to intraduodenal (ID) delivery. In contrast, microencapsulated antigen given orally produced a substantial response, which was predominantly IgA specific, and almost equal in magnitude to that obtained by PP priming and ID boosting with native antigen. Direct ID delivery produced a similar response but when PP were primed with microencapsulated antigen in FCA the response to ID boosting was greater than with any of the other protocols investigated. These data demonstrate the efficacy of biodegradable microspheres in producing an IgA antibody response following oral vaccination.

Cloning and characterization of a fox sperm protein FSA-1.

A monoclonal antibody was raised to a fox sperm protein (FSA-1) which was found to be localized to the inner acrosomal compartment of sperm fixed in methanol. Western blots of testicular germ cell membrane extracts probed with this antibody identified a major protein band with a molecular weight of 36,000. Immunofluorescent studies on fox testis sections showed that the antigen is expressed on round and elongating spermatids on a crescent-shaped structure, which probably represents the developing acrosome. An antibody specific for FSA-1 was used to screen a fox testis cDNA library for its cognate gene. An 875-bp cDNA clone was isolated and sequenced revealing an open reading frame. Searches of the GenBank and EMBL databases with the nucleic acid sequence revealed significant homology (86%) of FSA-1 with 406 bases of an unidentified RNA transcript from human fetal brain (EST02625). Northern blot analysis of fox testis RNA samples identified an RNA transcript of approximately 0.9 kb during the months when spermatogenesis is active. Zoo Northern blots (at high stringency) reveal an RNA transcript of a similar size present in testis RNA from dogs and mice. Zoo Southern analysis (high stringency) reveal genomic sequences present in dogs, mice, cattle and sheep. At present, the function of the FSA-1 gene product remains unknown, but it may play a role as a structural protein component of the acrosome.

Experimental strategies for the development of an immunocontraceptive vaccine for the European red fox, Vulpes vulpes.

The development of an immunocontraceptive vaccine to control fox populations in Australia would confer considerable advantages in controlling the long-term impact of this predator on native and endangered species. Studies are currently under way to identify sperm antigens that might be used in such a vaccine, and some of these studies are described. It is proposed that such a vaccine would be delivered orally in a bait, thereby stimulating a mucosal immune response to the foreign antigen(s). Such a vaccine requires a detailed understanding of reproductive-tract mucosal immunity in foxes, and selection of the most effective form of antigen delivery. Those under consideration include viral or bacterial vectors and microencapsulated antigens.

Voronoi modeling: the binding of triazines and pyrimidines to L. casei dihydrofolate reductase.

Vorom is a computer-aided method of drug design which can model a biological receptor given only binding data of known ligands. Using the binding energies of known competitive, reversible ligands of a biological macromolecule, vorom can make predictions about the binding energies and conformations of other small molecules binding to that receptor as well as provide information about the geometry and physicochemical characteristics of the binding site. One such model of L. casei dihydrofolate reductase was made. The model was able to predict the binding energies of 31 pyrimidine and triazine inhibitors out of a total set of 47, using only eight of the molecules (four pyrimidines and four triazines) as input. The binding energy of methotrexate, which is neither a pyrimidine nor a triazine, was correctly predicted. The binding mode of methotrexate predicted by vorom is entirely consistent with known X-ray data. The predicted binding modes of the pyrimidine inhibitors and the geometry of the site model are also consistent with published NMR and crystallographic data.

Transcriptional regulation in the testis: a role for transcription factor AP-1 complexes at various stages of spermatogenesis.

The products of two proto-oncogenes, c-fos and c-jun, have been implicated in signal transduction pathways as regulators of gene expression. Both proto-oncogenes are members of gene families encoding closely related proteins that together make up transcription factor AP-1. The expression of members of this transcription factor has been associated with cellular pathways that result in both mitosis and differentiation. We have been studying the process of spermatogenesis, which is a complex, continual cycle of cell renewal, proliferation and differentiation. Using a seasonal breeder, the European red fox (Vulpes vulpes), as our model, we have examined the expression of five AP-1 family members (c-fos, fra-1, fra-2, c-jun and junB) with a view to elucidating their role in the regulation of spermatogenesis. Unique patterns of expression, falling into three broad categories, were observed for the five genes: (i) continuous expression throughout the spermatogenic cycle (c-fos); (ii) expression only at times corresponding to the onset and shutdown of spermatogenesis (fra-1, fra-2 and c-jun); and (iii) expression only at the onset of the cycle (junB). Furthermore, the proteins were expressed in both premeiotic and post-meiotic cell types, suggesting a role in haploid, as well as diploid, gene expression in this tissue. The data suggest distinct, although not necessarily unrelated, roles for the different components of transcription factor AP-1 in the regulation of spermatogenesis.

Association of exogenous DNA with cattle and insect spermatozoa in vitro.

Spermatozoa isolated from domestic cattle (Bos taurus), the Australian sheep blowfly (Lucilia cuprina), and the honeybee (Apis mellifera) are capable of binding exogenous radiolabeled linear DNA. Both motile and nonmotile bovine sperm exhibit four distinct patterns of DNA association. Following treatment with DNase I, the relative proportion of one of these patterns increases specifically in living sperm, suggesting that a small proportion of DNA that associates with bovine sperm may be sequestered within the sperm head.

Immunological sexing of mammalian semen: current status and future options.

Although numerous schemes have been devised and promoted for the putative separation of X- and Y-chromosome-bearing spermatozoa, none has gained widespread acceptance. We have reexamined the possibility that Y-chromosome-bearing spermatozoa express a male-specific antigen on their plasma membrane, but that the same antigen is absent from X-chromosome-bearing spermatozoa. Immunohistochemical studies with high titer serologically detectable, male-specific antiserum revealed that serologically detectable, male-specific antigen is present on approximately 50% of spermatozoa and is located on both the postacrosomal region of the head and the midpiece of the flagellum. Similar results have also been described by other investigators working with bull spermatozoa. These data renew the question of whether the expression of serologically detectable, male-specific antigen on spermatozoa is a result of Y-chromosome-specific haploid gene expression. If so, then serologically detectable, male-specific antigen could possibly be targeted as a means of immunologically separating X- and Y-chromosome-bearing spermatozoa.

A proposed growth regulatory function for the serologically detectable sex-specific antigen H-Ys.

It is widely believed that the serologically detectable sex-specific antigen H-Ys plays a major role in the primary determination of sex. The cellular distribution of the antigen, however, seems to be at odds with its postulated function. Consideration of this apparent paradox has prompted the suggestion that the H-Ys antigen functions as a growth regulator, and that its role in the primary determination of sex can be accounted for on this basis. Circumstantial evidence is adduced that H-Ys is a growth regulator in the embryo, and this is supported by evidence from several sources not immediately related to embryonic growth or development. Genes coding for growth regulators can function as oncogenes in situations involving disordered regulation, and it is suggested that this accounts for the high incidence of ovarian neoplasms in H-Ys positive, but not in H-Ys negative, female patients with 46,XY gonadal dysgenesis (Swyer's syndrome). A postulated growth regulatory function of H-Ys lends weight to the contention of others, not only that the direction of differentiation of the indifferent gonad in the embryo is determined by its growth rate, but also that a common mechanism underlies genotypic sex determination and environmental sex determination.