Molecular cloning and physiological effects of brushtail possum interleukin-1beta

Interleukin-1beta (IL-1beta) was isolated from LPS-stimulated brushtail possum alveolar macrophages using PCR primers based on conserved regions of mammalian IL-1beta. The complete cDNA was cloned by 5' and 3' rapid amplification of cDNA ends (RACE). The predicted protein of 269 amino acids shared 4346% identity with several mammalian IL-1beta proteins. Constructs were made to express the mature IL-1beta in Escherichia coli and two recombinant IL-1beta proteins, rpIL-1beta1 and rpIL-1beta2, which differed in length by four amino acids at the N-terminus, were produced. Both proteins induced a weak proliferative response in a possum thymocyte assay. Possums injected intravenously with 100 microg of rpIL-1beta1 or rpIL-1beta2 showed profound changes in body temperature and numbers of circulating leukocytes. A sharp decrease in temperature occurred within 2 h of administration followed by an elevation of temperature peaking at 24 h. The smaller rpIL-1beta1 protein had a greater effect on temperature than rpIL-1beta2. Both rpIL-1beta proteins caused a marked decrease in number of neutrophils and lymphocytes at 2-6 h after injection. At 24 h after injection, neutrophil and lymphocyte numbers were elevated 6.0-fold and 2.6-fold, respectively in the possums injected with rpIL-1beta1 and 3.9-fold and 1.5-fold, respectively in the possums injected with rpIL-1beta2. Fibrinogen levels were elevated at 24 and 72 h after injection with both proteins. In comparison, neither recombinant bovine IL-1beta (rbIL-1beta) nor PBS had significant effects on body temperature or blood haematology. The studies have shown that the two recombinant forms of IL-1beta were biologically active in possums and that the IL-1beta with four fewer amino acids at the N-terminus was the more active.     

Radiocarbon dating techniques for tephras in central North Island,New Zealand

Tephra samples from Whakatane Ash and Rotoma Ash in central North Island of New Zealand were subject to a wide range of chemical treatments in order to isolate different components of soil organic matter for radiocarbon dating. These samples were radiocarbon-dated before and after treatments and were then compared with radiocarbon dates derived from charcoal in the same tephra formation.Tephra deposits when dated as whole samples without prior chemical treatments, yielded unreliable radiocarbon dates. The reliability was improved by chemical treatments, particularly those involving acid hydrolysis with 6 M HCl or 72% H₂SO₄. Improvement, however, was less using the classical humus extraction and fractionation procedure.     

Can giant clam (Tridacna squamosa) populations be restored on Singapore's heavily impacted coral reefs?

• 1. Giant clams have been a sustainable resource for millennia, but unregulated harvesting has led to local extinctions within the Indo‐Pacific region. Giant clam mariculture can produce large numbers of juveniles for restocking wild populations where natural recruitment is low or absent.
• 2. Singapore is surrounded by more than 60 small islands, many with fringing reefs. These reefs, however, experience increased turbidity and sedimentation resulting from massive coastal development projects and regular dredging of shipping lanes.
• 3. Seven reefs off Singapore's southern islands were surveyed (9670 m²) for giant clams. Also, an experiment was conducted to determine the growth of Tridacna squamosa reared in aquaria under three light treatments: ～50% ambient photosynthetically active radiation (PAR); ～25% ambient PAR; and ～12% ambient PAR. Finally, 144 clams (T. squamosa) were transplanted to four reefs around Singapore to study survival and growth in a heavily impacted environment.
• 4. A total of 23 adult clams from three species were found during the survey, representing a mean density of 0.24 per 100 m². Most clams were found at Raffles Lighthouse, Singapore's ‘best’ reef. No juvenile clams were encountered. In the aquarium experiment, clam growth was significantly different among the three light treatments, with growth greatest in the ～50% ambient PAR treatment. Of the 144 transplanted clams, 116 (80.6%) were recovered after 7 months. All specimens had increased in size, with growth rates among reefs ranging from 3.3 mm month^?1 (SD=1.3 mm) to 4.8 mm month^?1 (SD=1.6 mm).
• 5. Results suggest that, despite high levels of sedimentation and turbidity on Singapore's reefs, giant clams can survive and grow well. Restocking efforts using maricultured clams may be effective in enhancing the dwindling local populations. It is not clear, however, whether a self‐sustaining community can be established as high sedimentation may hinder larval settlement and survival.

Copyright © 2007 John Wiley & Sons, Ltd.     

Distribution and partial characterisation of acid hydrolysable organic nitrogen in six New Zealand soils

Six New Zealand topsoils of widely different origins and properties were subjected to 6m HC1 hydrolysis and the distribution of N fractions and amino acids were determined qualitatively and quantitatively.Of the total-N in the soils studied 83–91%, was hydrolysable with 6m HCl. The largest proportion of the hydrolysable N was α-amino acid N (38– 42%). followed by hydrolysable-unknown N (HUN) (14–24%), and NH₄⁺-N (14–22%). A significant proportion (25–50%) of the HUN fraction was accounted for by the non α-amino acid-N. Oxidative (3% H₂O₂) hydrolysis released N-phenoxy amino acid-N and possibly N-compounds which were complexed with phenols and sugars. All soils had a similar amino-acid composition with a predominance of acidic amino-acids.     

Biological nitrogen fixing capacity and biomass production of different understorey pastures in a Pinus radiata-pasture agroforestry system in New Zealand

Quantitative field measurements of biological nitrogen fixation (BNF) and biomass production by four different understorey pastures in a Pinus radiata-pasture agroforestry system were determined over a period of one year. The trees were two years old at the beginning of this study and the understorey pastures were being cut and removed for silage. The BNF was determined using the ¹⁵N dilution technique. Pastures of ryegrass+clover, cocksfoot+clover, phalaris+clover and lucerne were used. Substantial amounts of BNF were found (71 to 230 kg N ha^–1 year^–1) with lucerne showing the highest N fixation. However, lucerne derived only 71 to 72% of its N from the atmosphere (%Ndfa) during the spring/summer period compared to 83–97% with clovers, thus the net N demand from the soil was substantially higher with lucerne. This caused increased N stress to the trees. Clover in ryegrass+clover pasture fixed more N than the other grass+clover pastures. Although pasture position in relation to trees did not affect annual pasture total DMY and %Ndfa, pastures north of tree row grew better than those in other positions. Trees significantly affected the BNF of legumes and the botanical composition of pastures with highest BNF and legume production occurring in pastures midway between two rows of trees. These results suggest that it would be advantageous to evaluate different legumes and grasses for tolerance of shade and moisture stress in future studies. As the trees studied were only 1.5 to 3 m in height, their effects on BNF, seasonal pasture biomass production and botanical composition are expected to increase with tree dominance in the ecosystem with time. Amounts of N fixed were related to the productivity (i.e. dry matter and N yield) and seasonal persistence of the legumes. The productivity was high in spring and summer and low in autumn and winter.     

Performance assessment and validation of a paramagnetic particle-based enzyme-linked immunosorbent assay for chlorpyrifos in agricultural runoff waters

A commercial magnetic particle-based enzyme-linked immunosorbent assay (ELISA) kit for the insecticide chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate] was evaluated for its specificity, precision, and accuracy, its susceptibility to matrix interferences in agricultural and environmental surface waters, and its comparability to a gas chromatographic/flame photometric (GC/FPD) method for the determination of organophosphorus pesticides in natural waters. Repeatability, reproducibility, and accuracy studies show that the kit satisfies current U.S. Environmental Protection Agency criteria for the assessment of analytical methods. Observable matrix effects were found to be present in all of the environmental test waters, with the slopes of calibration curves generated in each of the test matrices deviating from that of the control matrix by as much as 16%. Specificity studies indicate that the chlorpyrifos polyclonal antibody adequately differentiates the target compound from other structurally similar organophosphorus pesticides, with the exception of its methyl analogue. Cross-reactivity with chlorpyrifos-methyl was approximately 37%, while reactivity with diazinon, pyridaphenthion, diclofenthion, bromiphos-ethyl, bromiphos-methyl, pirimiphos-ethyl, and chlorpyrifos oxon ranged from 1.6 to 10.7%. Cross-reactivity with pirimiphos-methyl, 3,5,6-trichloro-2-pyridinol, diethyl phosphate, and diethyl thiophosphate was negligible (<1%). Validation of the paramagnetic particle ELISA format was accomplished using water samples from two monitoring studies that were collected, split, and analyzed directly by ELISA and by GC/FPD. Results of the two analytical methods were then compared using standard t tests, regression analysis, and differences against mean measurement (bias) plots. While the agreement between the two methods was determined to be satisfactory, ELISA exhibits consistent positive bias in environmental matrices. Several preanalysis mitigation steps were suggested that may help moderate bias, but additional study is recommended to explicate the exact factors responsible for its consistent overestimation of results.     

Duration and method of fixation affects the sensitivity of a digoxygenin-labelled DNA probe in detecting Kudoa thyrsites in Atlantic salmon skeletal muscle

The effect of fixative and duration of fixation on the sensitivity of a non-radioactive in situ hybridisation (ISH) protocol to detect Kudoa thyrsites small subunit ribosomal deoxyribonucleic acid (DNA) was investigated. Strong ISH reactions were detected in 5-μm sections of paraffin-embedded Atlantic salmon muscle after fixation for 1 day in Davidson's solution (DS). Reactions were weak following 3 or 5 days fixation and absent after 17 or 28 days fixation. Strong ISH reactions were observed after 1, 3 or 5 days fixation in neutral buffered formalin (NBF). The reactions were weak after 17 days and weak to nonexistent after 28 days of fixation. Reactions were consistently strong after fixation in 95% ethanol for up to 28 days. Some mature spores reacted weakly or not at all by ISH. Parasite DNA was weakly amplified by polymerase chain reaction (PCR) from paraffin-embedded muscle after 1 day of fixation in DS but not after fixation for 3, 5, 17 or 28 days. Amplified DNA was detected after fixation in NBF for 1, 3 and 5 days, but not after 17 or 28 days. In contrast, PCR consistently amplified DNA from paraffin-embedded, ethanol-fixed muscle. Caution should be used in the choice of fixative and duration of fixation when preserving Atlantic salmon tissues for molecular diagnosis of K. thyrsites.     

Replicative and cytopathic potential of HTLV-III/LAV with sor gene deletions

The genome of the human T-lymphotropic virus type III (HTLV-III/LAV) has the potential to encode at least three polypeptides in addition to those encoded by the gag, pol, and env genes. In this study, the product of the sor (short open reading frame) region, which overlaps the 3' end of the pol gene, was found to be a protein with a molecular weight of 23,000. An assay was developed for testing the ability of cloned HTLV-III proviruses to produce viruses cytopathic for T4+ lymphocytes. In the cell line used, C8166, neither the HTLV-III sor gene product nor the complete 3'-orf gene product were necessary for the replication or cytopathic effects of the HTLV-III.     

Vaccination of Poultry Workers: Delivery and Uptake of Seasonal Influenza Immunization

Avian influenza is a highly infectious disease in poultry and although the risk of human infection is low, concerns exist that it could evolve into a new human strain of pandemic potential if reassortment with a human influenza virus occurs. In January 2007, the UK government introduced a programme to vaccinate poultry workers to reduce the potential of such an event. This study evaluates the delivery, uptake and costs of the programme in three counties of England. A questionnaire survey was completed by consultants in public health in all the Primary Care Trusts in Norfolk, Suffolk and Cambridgeshire in May 2007. The delivery of the programme varied between Primary Care Trusts, including being delivered in some cases by clinics in primary care, by general practitioners and occupational health services in others. The uptake of vaccination was low ranging from 7% to 29% at a cost of £29 to £132 per person vaccinated. Vaccination of poultry workers as a public health measure to prevent an influenza pandemic is likely to be ineffective with the level of coverage found in this evaluation in our region.