The results of 2-year pollen monitoring of an urban network in Perugia,Central Italy

Continuous pollen monitoring of an urban network consisting of three stations has been undertaken for a period of 2 years in Perugia, central Italy. The aim has been to establish whether the Perugia pollen trap, active since 1983, is still representative of the area following recent urbanisation. Quantitative differences were found between the stations, reflecting different vegetational areas, but only slight differences were detected in relation to the timing of the principal period of pollination. Therefore, although individual pollen traps are necessary to characterize fully the different areas, one trap is sufficient to determine the key allergenic thresholds in the studied area.     

A Hydrogen-Donating Monohydroxamate Scavenges Ferryl Myoglobin Radicals

The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (Fe^IV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H₂O₂ addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.     

Purification and characterization of a vaterite-inducing peptide, pelovaterin, from the eggshells of Pelodiscus sinensis (Chinese soft-shelled turtle)

Proteins play a crucial role in the biomineralization of hard tissues such as eggshells. We report here the purification, characterization, and in vitro mineralization studies of a peptide, pelovaterin, extracted from eggshells of a soft-shelled turtle. It is a glycine-rich peptide with 42 amino acid residues and three disulfide bonds. When tested in vitro, the peptide induced the formation of a metastable vaterite phase. The floret-shaped morphology formed at a lower concentration ( approximately 1 microM) was transformed into spherical particles at higher concentrations (>500 microM). The solution properties of the peptide are investigated by circular dichroism (CD), fluorescence emission spectroscopy, and dynamic light scattering (DLS) experiments. The conformation of pelovaterin changed from an unordered state at a low concentration to a beta-sheet structure at high concentrations. Fluorescence emission studies indicated that the quantum yield is significantly decreased at higher concentrations, accompanied by a blue shift in the emission maximum. At higher concentrations a red-edge excitation shift was observed, indicating the restricted mobility of the peptide. On the basis of these observations, we discuss the presence of a peptide concentration-dependent monomer-multimer equilibrium in solution and its role in controlling the nucleation, growth, and morphology of CaCO(3) crystals. This is the first peptide known to induce the nucleation and stabilization of the vaterite phase in solution.     

How many conservation units are there for the endangered grassland earless dragons?

Species are the most commonly recognised unit for conservation management, yet significant variation can exist below the level of taxonomic recognition and there is a lack of consensus around how a species might be defined. This definition has particular relevance when species designations are used to apportion conservation effort and when definitions might be made through legislation. Here, we use microsatellite DNA analyses to test the proposition that the last remaining populations of the endangered grassland earless dragon (Tympanocryptis pinguicolla) harbour substantial cryptic genetic variation. Our study provides strong evidence that long historical isolation and the recent impacts of urbanization, have led to genetic differentiation in microsatellite DNA allele frequencies and high numbers of private alleles among three genetic clusters. This differentiation is partially concordant with previous mitochondrial DNA analyses, which show the two regions (Canberra and Monaro) where this species exists, to be reciprocally monophyletic, but differs through the identification of a third genetic cluster that splits a northern Canberra cluster from that of southern Canberra. Our data also identify a stark contrast in population genetic structure between clusters such that high levels of genetic structure are evident in the highly urbanised Canberra region but not in the largely rural Monaro region. We conclude that this species, like many reptiles, harbours considerable cryptic variation and currently comprises three distinct and discrete units. These units could be classified as separate species for the purpose of conservation under the relevant Australian and international Acts drawing management appropriate to that status.     

Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). Although this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment because its restricted linearity is incompatible with enhanced throughput microtiter plate screening. In this article, we report the development and validation of a nonenzymatically coupled screening assay in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate, and we describe its utility in supporting a structure-based design, small-molecule inhibitor campaign against Trypanosoma brucei PTR1 and DHFR-TS.     

Transgenic expression of dominant negative tuberin through a strong constitutive promoter results in a tissue-specific tuberous sclerosis phenotype in the skin and brain

Tuberous sclerosis (TS) is a common autosomal dominant disorder caused by loss or malfunction of hamartin (tsc1) or tuberin (tsc2). Many lesions in TS do not demonstrate loss of heterozygosity for these genes, implying that dominant negative forms of these genes may account for some hamartomas and neoplasms in TS. To test this hypothesis, we expressed a dominant negative allele of tuberin (DeltaRG) behind the cytomegalovirus promoter in NIH3T3 cells and transgenic mice. This allele binds hamartin but has a deletion in the C terminus of tuberin, leading to constitutive activation of rap1 and rab5/rabaptin. Expression of DeltaRG in NIH3T3 cells led to a strong induction of reactive oxygen species, induction of vascular endothelial growth factor, and malignant transformation in vivo. Expression of DeltaRG driven by the constitutive cytomegalovirus promoter led to high level expression in all murine tissues examined, including skin, kidney, liver, and brain. Surprisingly, mice expressing the DeltaRG transgene developed a fibrovascular collagenoma in the dermis, which closely resembles the Shagreen patch observed in human patients with TS. In addition, numerous small subpial collections of external granule cells in the cerebellum were observed, which may be the murine equivalent of subependymal giant cell astrocytomas or tubers commonly seen in TS patients. Thus, expression of a dominant negative tuberin in multiple tissues can lead to a tissue-specific phenotype resembling some of the findings in human TS. Our data are the first to demonstrate that specific signaling abnormalities underlie specific hamartomas in a model of a human genetic disorder.     

Can Amphipod Behavior Help to Predict Chronic Toxicity of Sediments?

Amphipods are widely used in both acute and chronic (sub-lethal) sediment tests. Acute sediment tests provide relatively rapid results, but may fail to detect moderately toxic contaminants that are bound to the sediment, whereas chronic life-cycle tests are rarely performed as they are time consuming and expensive. Observations during chronic testing of oil-contaminated sediment suggested that there may be a link between the behavior of the marine amphipod Corophium volutator and reduction in growth rate. Behavior tests were performed with six individual amphipods per treatment using sediment spiked with weathered Forties oil with burrowing time, re-emergence from sediment, and activity prior to burrowing as endpoints. Further behavior tests were used to predict the chronic toxicity of sediments spiked with three crude oils each with a dominant unresolved complex mixture of hydrocarbons (UCM). The effect of sediment type on behavior was also investigated. The results suggested that although the behavior test could not be used alone as a viable alternative to sediment toxicity tests, it could prove useful as an adjunct to acute tests, and help select sediments that deserve further investigation.     

Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR

EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)-polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18-21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.