Spatial and temporal clusters of Barmah Forest virus disease in Queensland, Australia.

OBJECTIVE: To identify the spatial and temporal clusters of Barmah Forest virus (BFV) disease in Queensland in Australia, using geographical information systems and spatial scan statistic (SaTScan). METHODS: We obtained BFV disease cases, population and statistical local areas (SLAs) boundary data from Queensland Health and Australian Bureau of Statistics, respectively, during 1992-2008 for Queensland. A retrospective Poisson-based analysis using SaTScan software and method was conducted to identify both purely spatial and space-time BFV disease high-rate clusters. A spatial cluster size of a proportion of the population and a 200 km radius and varying time windows from 1 to 12 months were chosen (for the space-time analysis). RESULTS: The spatial scan statistic detected a most likely significant purely spatial cluster (including 23 SLAs) and a most likely significant space-time cluster (including 24 SLAs) in approximately the same location. Significant secondary clusters were also identified from both the analyses in several locations. CONCLUSIONS: This study provides evidence of the existence of statistically significant BFV disease clusters in Queensland, Australia. The study also demonstrated the relevance and applicability of SaTScan in analysing ongoing surveillance data to identify clusters to facilitate the development of effective BFV disease prevention and control strategies in Queensland, Australia.

Prevalence, intensity and risk factors for soil-transmitted helminth infection in a South Indian fishing village.

A study of the prevalence, intensity and risk factors for soil-transmitted helminth infection was undertaken among school children aged 5-9 years attending a primary school in the fishing village in Peda Jalaripet, Visakhapatnam, South India. One hundred and eighty nine (92.6%) of 204 children were infected with one or more soil transmitted helminth parasites. The predominant parasite was Ascaris lumbricoides (prevalence of 91%), followed by Trichuris trichiura (72%) and hookworm (54%). Study of age-specific prevalence and intensity of infection revealed that the prevalence and intensity of A. lumbricoides infection was higher among younger children than older children. While aggregation of parasite infection was observed, hookworm infection was more highly aggregated than either A. lumbricoides or T. trichiura. Multivariate analysis identified parental occupation, child's age and mother's education as the potential risk factors contributing to the high intensity of A. lumbricoides infection. Children from fishing families with low levels of education of the mother had the highest intensity of A. lumbricoides infection. As the outcome of chemotherapy programs to control soil transmitted helminth infection is dependant on the dynamics of their transmission, there is a need for further studies to better define the role of specific factors that determine their prevalence, intensity and aggregation in different epidemiological settings.

Studies on the kinetics of oxidation of 4-hydroxyanisole by tyrosinase.

The potential use of 4-hydroxyanisole as a chemotherapeutic agent in the treatment of malignant melanoma led us to investigate the kinetics of oxidation of this tyrosine analogue by tyrosinase. We found that addition of amino acids accelerated the reaction, resulting in a reduction in length of the characteristic lag period of monohydric phenol oxidation. The lag period was abolished completely by an aliquot of exhausted 4-hydroxyanisole/tyrosinase reaction mixture and by very low concentrations of thiol-containing compounds. We conclude that the reaction-accelerating property of non-thiol amino acids is due to the reductive addition of the ortho-quinone reaction product to nucleophilic groups of the amino acids. The dihydric phenol product which results is capable of met-tyrosinase recruitment by electron donation to the cupric active site generating the cuprous form of the enzyme which binds oxygen and is able to oxidise monohydric phenols. Abolition of the lag period by an aliquot of exhausted reaction mixture is probably due to recruitment of the met-enzyme by catecholic oligomers of the quinone product. Thiol containing compounds are able to abolish the lag period due to the ability of these compounds to reduce met-tyrosinase directly.

Initial mushroom tyrosinase-catalysed oxidation product of 4-hydroxyanisole is 4-methoxy-ortho-benzoquinone.

Evidence is presented that the first and major product of the oxidation of 4-hydroxyanisole (4HA) by tyrosinase is 4-methoxy ortho benzoquinone (4-MOB). 4-MOB was synthesized by oxidation of 4HA by potassium nitrodisulphonate and comparisons made between the synthetic quinone and an extract of a reaction mixture in which 4HA had been completely oxidized by mushroom tyrosinase. The chemical species were found to be identical in UV/visible absorption spectrum, 1H-NMR spectrum, and by thin-layer chromatography.

Major primary cytotoxic product of 4-hydroxyanisole oxidation by mushroom tyrosinase is 4-methoxy ortho benzoquinone.

It has been shown previously that the initial product of mushroom tyrosinase-catalysed oxidation of the monophenol 4-hydroxyanisole (4HA) is 4-methoxy ortho benzoquinone (4-MOB). This study presents evidence that 4-MOB is primarily responsible for the cytotoxicity of 4HA oxidation products in vitro. Equivalent toxicity in a model system was produced by products of tyrosinase catalysed oxidation of 4HA and by synthetic 4-MOB. Cytotoxicity was estimated both by a blebbing assay and by plating efficiency of exposed cells. HPLC analysis of the reaction mixture revealed a positive correlation between cytotoxicity and 4-MOB concentration.

DNA synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes.

We have investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in 3H-thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. We conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.

The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content.

The relationship between the timing of both sperm nuclear decondensation and male pronucleus formation in the oocyte and the relative level of disulfide bonds within the sperm nucleus was evaluated. Since reduction of sperm nuclear disulfide (S-S) bonds is a prerequisite for sperm nuclear decondensation in vitro and in vivo, we hypothesized that sperm nuclei with relatively few S-S bonds would require less time to decondense in the oocyte than sperm nuclei with higher numbers of S-S bonds, and that male pronucleus formation would occur more rapidly as well. Four types of hamster sperm nuclei, in which the extent of S-S bonding differed, were microinjected into hamster oocytes, and the time course of sperm nuclear decondensation and male pronucleus formation was charted. Cauda epididymal sperm nuclei, which are rich in S-S bonds, required 45-60 min to decondense. In contrast, nuclei containing few S-S bonds (namely sonication-resistant spermatid nuclei and cauda epididymal sperm nuclei treated in vitro with the S-S bond-reducing agent dithiothreitol) decondensed within 5-10 min of microinjection. Caput epididymal sperm nuclei, with intermediate S-S bond content, decondensed in 10-20 min. Regardless of when decondensation occurred, formation of the male pronucleus never preceded that of the female pronucleus, which occurred 1.25-1.5 h after microinjection. However, sperm nuclei with few S-S bonds were more likely than S-S rich nuclei to transform into male pronuclei in synchrony with the formation of the female pronucleus. We conclude that the timing sperm nuclear decondensation and pronucleus formation depends in part upon the S-S bond content of the sperm nucleus.

DNA synthesis in the fertilizing hamster sperm nucleus: sperm template availability and egg cytoplasmic control.

To assess the role of the availability of sperm nuclear templates in the regulation of DNA synthesis, we correlated the morphological status of the fertilizing hamster sperm nucleus with its ability to synthesize DNA after in vivo and in vitro fertilization. Fertilized hamster eggs were incubated in 3H-thymidine for varying periods before autoradiography. None of the decondensed sperm nuclei nor early (Stage I) male pronuclei present after in vivo or in vitro fertilization showed incorporation of label, even in polyspermic eggs in which more advanced pronuclei were labeled. In contrast, medium-to-large pronuclei (mature Stage II pronuclei) consistently incorporated 3H-thymidine. To investigate the contribution of egg cytoplasmic factors to the regulation of DNA synthesis, we examined the timing of DNA synthesis by microinjected sperm nuclei in eggs in which sperm nuclear decondensation and male pronucleus formation were accelerated experimentally by manipulation of sperm nuclear disulfide bond content. Although sperm nuclei with few or no disulfide bonds decondense and form male pronuclei faster than nuclei rich in disulfide bonds, the onset of DNA synthesis was not advanced. We conclude the the fertilizing sperm nucleus does not become available to serve as a template for DNA synthesis until it has developed into a mature Stage II pronucleus, and that, as with decondensation and pronucleus formation, DNA synthesis also depends upon egg cytoplasmic factors.

Metabolism of 1-naphthol by tyrosinase.

1-Naphthol was metabolized by the polyphenol oxidase, tyrosinase, primarily to 1,2-naphthoquinone and to small amounts of 1,4-naphthoquinone as well as to covalently bound products. The inhibition of covalent binding by ethylenediamine, which reacts specifically with 1,2-naphthoquinone but not 1,4-naphthoquinone, suggested that most of the covalent binding was due to 1,2-naphthoquinone or a metabolite of similar structure. The activation by tyrosinase of 1-naphthol to covalently bound products suggested that it may alter the reaction kinetics of the enzyme. This was investigated by studying the effects of 1-naphthol on the tyrosinase-catalysed oxidation of 4-hydroxyanisole. Preincubation of tyrosinase with 1-naphthol increased the lag period of the oxidation of 4-hydroxyanisole, which may be due to a decrease in the amount of active enzyme, as well as to a reaction of 1-naphthol with 3,4-anisylquinone, an oxidation product of 4-hydroxyanisole. The metabolic activation of 1-naphthol by tyrosinase to covalently bound species suggests that 1-naphthol or a structurally related derivative may be of potential therapeutic application in the treatment of cells high in tyrosinase activity, such as certain melanomas.

The production and properties of a tissue plasminogen activator from normal epithelial cells grown in microcarrier culture.

A plasminogen activator with different biochemical and physical properties to the one obtained from Bowes melanoma cells has been isolated from a normal epithelial cell line derived from guinea-pig keratocytes (GPK). Cell growth and enzyme production is carried out in microcarrier cultures using up to 15 g Cytodex 3 per litre. Cells are maintained in optimum conditions by the use of a closed perfusion loop and the system has been scaled up to 20 litres. Initially enzyme production was in serum-free medium following a growth phase in serum supplemented medium. However, the discovery that the cells produce the enzyme mainly during the replicative phase has led to investigating the use of serum-substitutes and growth factors on cell growth and enzyme production. The aim is to harvest the enzyme after the growth phase which precludes the use of high (greater than 1%) serum concentrations. The biochemical and biophysical properties of the enzyme are described.

Actions of catechol oestrogens on concentrations of serum luteinizing hormone in the adult castrated rat: various effects of 4-hydroxyoestradiol and 2-hydroxyoestradiol.

The pharmacological effect of 2-hydroxyoestradiol (2-OHE2) and 4-OHE2 on concentrations of LH in the chronically castrated rat have been compared with that of oestradiol in order to determine whether the in-vivo activity is altered by insertion of a hydroxyl group at position 2 or 4 of the aromatic A ring; these derivatives are naturally occurring oestrogen metabolites. Four groups of six adult male rats were used 4 weeks after bilateral orchidectomy. The right jugular vein was exposed under ether anaesthesia and a basal blood sample taken (10.00 h) immediately before an intravenous injection of vehicle alone (0.1 ml ethanol with 0.01% ascorbic acid), oestradiol, 2-OHE2 or 4-OHE2 (10 micrograms of each in 0.1 ml vehicle). Blood was taken from each animal at 2, 4, 6, 8 and 24 h after treatment and serum assayed for LH. Baseline LH levels were similar in the four groups. At 2 h there was no change in 2-OHE2-treated rats but there was a significant decrease of serum levels of LH in rats treated with oestradiol and 4-OHE2 compared with vehicle-treated controls. The decrease in LH was quantitatively similar in oestradiol- and 4-OHE2-treated groups and was sustained at 4, 6 and 8 h, returning to control values at 24 h. In subsequent experiments the effects of lower doses of these two steroids were compared and the potency of 4-OHE2 was estimated to be about 25% that of oestradiol. In a further experiment, 2-OHE2 (100 micrograms) had no effect when given alone, but when injected i.v. immediately before treatment with 1 microgram oestradiol, it was able to inhibit the suppression of LH by oestradiol. In conclusion, 4-OHE2 had a potent effect in lowering plasma LH levels whereas 2-OHE2, even at a high dose (100 micrograms), did not suppress LH but it was able to inhibit the effect of oestradiol. These differences in biological activity may reflect more rapid metabolism of 2-OHE2 or differences in binding properties of these catechol oestrogens to the oestrogen receptor.

Catecholestrogens and induction of sexual behavior in the ovariectomized rat.

The ability of the catecholestrogens, 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol (2-OHE2) to induce lordosis in ovariectomized rats primed with progesterone was assessed following either short- or long-term exposure to the steroids. 4-OHE2 successfully induced lordosis whether administered as two bolus intrajugular injections separated by a 3-hour interval or continuously for 7 days via subcutaneously implanted osmotic minipumps. 2-OHE2, however, was ineffective under both conditions. When administered subcutaneously in conjunction with 3 days of estradiol benzoate (E2B) treatment, neither of the catecholestrogens exhibited any antiestrogenic effect as compared with the lordosis response to E2B alone. Likewise, simultaneous administration of 2-OHE2 did not significantly alter the response to intrajugular injections of estradiol (E2) or 4-OHE2. The results indicate that 4-OHE2 (but not 2-OHE2) mimicks the lordosis-inducing action of E2 and that neither catecholestrogen has any antiestrogenic effect in this behavioral test system.