Extrapolation of in situ data from 1-km squares to adjacent squares using remote sensed imagery and airborne lidar data for the assessment of habitat diversity and extent.

Habitat surveillance and subsequent monitoring at a national level is usually carried out by recording data from in situ sample sites located according to predefined strata. This paper describes the application of remote sensing to the extension of such field data recorded in 1-km squares to adjacent squares, in order to increase sample number without further field visits. Habitats were mapped in eight central squares in northeast Estonia in 2010 using a standardized recording procedure. Around one of the squares, a special study site was established which consisted of the central square and eight surrounding squares. A Landsat-7 Enhanced Thematic Mapper Plus (ETM+) image was used for correlation with in situ data. An airborne light detection and ranging (lidar) vegetation height map was also included in the classification. A series of tests were carried out by including the lidar data and contrasting analytical techniques, which are described in detail in the paper. Training accuracy in the central square varied from 75 to 100 %. In the extrapolation procedure to the surrounding squares, accuracy varied from 53.1 to 63.1 %, which improved by 10 % with the inclusion of lidar data. The reasons for this relatively low classification accuracy were mainly inherent variability in the spectral signatures of habitats but also differences between the dates of imagery acquisition and field sampling. Improvements could therefore be made by better synchronization of the field survey and image acquisition as well as by dividing general habitat categories (GHCs) into units which are more likely to have similar spectral signatures. However, the increase in the number of sample kilometre squares compensates for the loss of accuracy in the measurements of individual squares. The methodology can be applied in other studies as the procedures used are readily available.

Inhibition of antibiotic-resistant Staphylococcus aureus by the broad-spectrum dihydrofolate reductase inhibitor RAB1.

The bacterial burden on human health is quickly outweighing available therapeutics. Our long-term goal is the development of antimicrobials with the potential for broad-spectrum activity. We previously reported phthalazine-based inhibitors of dihydrofolate reductase (DHFR) with potent activity against Bacillus anthracis, a major component of Project BioShield. The most active molecule, named RAB1, performs well in vitro and, in a cocrystal structure, was found deep within the active site of B. anthracis DHFR. We have now examined the activity of RAB1 against a panel of bacteria relevant to human health and found broad-spectrum applicability, particularly with regard to gram-positive organisms. RAB1 was most effective against Staphylococcus aureus, including methicillin- and vancomycin-resistant (MRSA/VRSA) strains. We have determined the cocrystal structure of the wild-type and trimethoprim-resistant (Phe 98 Tyr) DHFR enzyme from S. aureus with RAB1, and we found that rotational freedom of the acryloyl linker region allows the phthalazine moiety to occupy two conformations. This freedom in placement also allows either enantiomer of RAB1 to bind to S. aureus, in contrast to the specificity of B. anthracis for the S-enantiomer. Additionally, one of the conformations of RAB1 defines a unique surface cavity that increases the strength of interaction with S. aureus. These observations provide insights into the binding capacity of S. aureus DHFR and highlight atypical features critical for future exploitation in drug development.

An assessment of long term ecosystem research activities across European socio-ecological gradients.

Integration of European long term ecosystem research (LTER) would provide important support for the management of the pan-European environment and ecosystems, as well as international policy commitments. This does require appropriate coverage of Europe and standardised frameworks and research methods between countries. Emerging interest in socio-ecological systems prompted the present assessment of the distribution of LTER activities across European socio-ecological gradients. This paper presents a European stratification with a 1 km(2) resolution, delineating 48 broad socio-ecological regions. The dataset is based on an existing biogeophysical stratification constructed using multivariate clustering of mainly climatic variables and a newly developed socio-economic stratification based on an economic density indicator. The coverage of European LTER facilities across the socio-ecological gradients is tested using this dataset. The analysis shows two strong biases in the present LTER effort. Firstly, urban and disturbed regions are consistently under-represented, illustrating a bias for traditional ecological research away from human activity. Secondly, the Mediterranean, for which some of the most extreme global change impacts are projected, is receiving comparatively little attention. Both findings can help guide future investment in the European LTER network - and especially in a Long Term Socio-Ecological Research (LTSER) component- to provide a more balanced coverage. This will provide better scientific understanding of pan-European environmental concerns and support the management of natural resources and international policy commitments in the European Union.

A simple near-patient test for nicotine and its metabolites in urine to assess smoking habit.

We describe a disposable, near-patient urine test to monitor cigarette smoking. A plastic device contains the sealed dried reagents to measure nicotine and its metabolites, by a colorimetric assay. The device can be used to give a qualitative assessment of tobacco consumption, simply by observing a colour change. Alternatively, the test can be quantified by measuring the light absorbance with a simple colorimeter, and a concentration of nicotinic metabolites obtained with reference to a cotinine standard. A correction factor for the concentration of the urine sample, based on light absorbance, allows the result to be expressed as a ratio to urine concentration. This method correlates with reported daily cigarette consumption (r = 0.69, p < 0.0001) and compares favourably with cotinine, as measured by gas chromatography (r = 0.89, p < 0.0001). The method provides a simple-to-use, inexpensive way to monitoring tobacco consumption in extralaboratory situations.

The countryside information system: A strategic-level decision support system.

The Institute of Terrestrial Ecology (ITE) has monitored ecological change in Great Britain (GB) since 1978. The task has been undertaken using a stratified sampling scheme working with a 1 km square as the sample unit. In more recent years, scientific researchers at ITE have been working closely with the policy-makers of the United Kingdom Department of the Environment. The presentation of information to policy advisors and planners was a component within a large project investigating the ecological consequences of land-use change. A simple PC-based decision support system was developed during the project and subsequently has been expanded to produce a marketable product. The system, called the Countryside Information System (CIS), presents and links information at national, regional and thematic levels along with qualifying data describing accuracy and appropriateness of use (i.e., metadata). An integral part of the CIS is the ITE Land Classification, which divides GB into 32 environmental land classes; all 250 000 squares have been classified. The classification allows sampled data to be presented and, as the co-ordinate system is widely used in GB, it allows census datasets to be linked and compared. CIS has been described as a Geographical Information System, but the classification, data held within the system, and the use of metadata to assist in interpretation of results make the system much more decision-support oriented. Indeed, government departments have been involved in directing the development and are now starting to use the system to answer parliamentary questions and formulate, assess and monitor environmental policy. The CIS is an open system, running on a standard PC in Microsoft Windows. Tools for loading and editing new datasets (both sample and census) are incorporated in the suite of programs. The Windows environment and users comments during development have produced a system with an intuitive feel, removing some of the overhead of acquiring specialised technical skills before being able to operate a system. This paper describes the CIS and presents examples of its applications.

An electrochemical study of the factors responsible for modulating the reduction potential of putidaredoxin.

The gene coding for putidaredoxin has been synthesized using a combination of chemical and enzymatic methods and subsequently expressed in Escherichia coli. The recombinant protein characterized by electronic spectroscopy, mass spectrometry, and electrochemistry was found to be identical to putidaredoxin obtained from Pseudomonas putida. Polylysine was found to promote the fast and reversible electrochemistry of putidaredoxin at negatively charged electrodes such as indium-doped tin oxide or gold surfaces modified with mercaptoalkanoate groups. The value of the heterogeneous electron transfer rate constant obtained from solutions containing a mixture of putidaredoxin and polylysine (k(s) = 1.3 x 10(-3) cm/s) is one order of magnitude larger than the values reported previously at gold electrodes modified with mercaptoethylamine or at antimony-doped tin oxide semiconductor electrodes. It was observed that when the reduction potential of putidaredoxin is measured by cyclic voltammetry, the resultant value is consistently more positive (64 mV) than the reduction potential measured with potentiometric titrations. A comparison between the electrochemical responses of putidaredoxin and spinach ferredoxin, combined with the examination of their corresponding three-dimensional structures, indicates that the positive shift in the reduction potential of putidaredoxin originates from the formation of a transient complex between putidaredoxin and polylysine at the electrode surface. The formation of this transient complex modulates the reduction potential of putidaredoxin by lowering the value of the dielectric constant around its iron-sulfur cluster microenvironment, specifically by neutralizing negative charges surrounding the active site and by excluding water from the solvent exposed iron sulfur cluster. The observed positive shift in E degrees ', which is induced by complexation with polylysine at the electrode-surface, suggests that similar factors are likely to contribute to the anodic shift in the E degrees ' of cytochrome P450(cam)-bound putidaredoxin (+44 mV) with respect to the E degrees ' measured for free putidaredoxin.

Histidine decarboxylase activity in trauma-resistant rats.

Studies have been carried out to determine the possible role of nascent histamine in the development of traumatic shock. This was done by examining histidine decarboxylase (HD) activity of the lung, spleen, and plasma following exposure to trauma in normal and trauma-resistant rats. In normal rats, there was a significant increase in lung HD activity at 15 min and 4 h; and in the spleen the HD activity increased significantly at 4 h. In trauma-resistant rats exposed to trauma, there were no changes in enzyme activity in the lung and less pronounced changes in the spleen. The plasma HD activity remained stable in normal and resistant rats following episodes of trauma. Changes in total erythrocyte hemoglobin were observed in both normal and trauma-adapted rats following exposure to this stress, increasing significantly in normal rats, but decreasing in trauma-resistant rats. Blood volume decreased significantly at 4 h after trauma in normal animals; whereas only a slight decrease was noted in resistant animals. The data support the concept that newly formed histamine contributes to the pathogenesis of shock. It is also proposed that the increased resistance, characteristic of trauma-adapted rats, could be partly due to an inhibition of enzyme activation following trauma.

A portable silicon photodiode luminometer.

A simple, inexpensive, battery-powered, portable luminometer which is based on a silicon photodiode is described. The instrument is intended to measure the light produced by chemiluminescent and bioluminescent reactions. The device shows a good detection limit and, in a bioluminescent reaction for adenosine 5'-triphosphate (ATP), detected 0.5 pmol in 1 ml of aqueous solution. The instrument measures irradiance from 10(-13) to 10(-11) W cm-2 at the sensor, within the range 300 to 900 nm.

The ITE Land classification: Providing an environmental stratification of Great Britain.

The surface of Great Britain (GB) varies continuously in land cover from one area to another. The objective of any environmentally based land classification is to produce classes that match the patterns that are present by helping to define clear boundaries. The more appropriate the analysis and data used, the better the classes will fit the natural patterns. The observation of inter-correlations between ecological factors is the basis for interpreting ecological patterns in the field, and the Institute of Terrestrial Ecology (ITE) Land Classification formalises such subjective ideas. The data inevitably comprise a large number of factors in order to describe the environment adequately. Single factors, such as altitude, would only be useful on a national basis if they were the only dominant causative agent of ecological variation.The ITE Land Classification has defined 32 environmental categories called 'land classes', initially based on a sample of 1-km squares in Great Britain but subsequently extended to all 240 000 1-km squares. The original classification was produced using multivariate analysis of 75 environmental variables. The extension to all squares in GB was performed using a combination of logistic discrimination and discriminant functions. The classes have provided a stratification for successive ecological surveys, the results of which have characterised the classes in terms of botanical, zoological and landscape features.The classification has also been applied to integrate diverse datasets including satellite imagery, soils and socio-economic information. A variety of models have used the structure of the classification, for example to show potential land use change under different economic conditions. The principal data sets relevant for planning purposes have been incorporated into a user-friendly computer package, called the 'Countryside Information System'.

Complete isomer-specific 1H and 13C NMR assignments of the heme resonances of rat liver outer mitochondrial membrane cytochrome b5.

Singly and doubly labeled delta-aminolevulinic acid derivatives were used to prepare rat liver outer mitochondrial membrane (OM) cytochrome b5 containing a 13C-labeled heme active site. A variety of NMR experiments, including HMBC and INADEQUATE in conjunction with the more commonly used HMQC, NOESY, and COSY, were conducted to make unambiguous assignments of protonated carbons and the quaternary pyrrole-alpha and -beta carbons in both isomeric forms of the paramagnetic active center of OM cytochrome b5. Because the long interpulse delays in the HMBC experiment have a detrimental effect on the detectability of fast relaxing paramagnetically affected resonances. INADEQUATE is proposed as the experiment of choice for assigning quaternary carbons in paramagnetic hemes with carefully chosen macrocycle labeling patterns. Furthermore, the applicability of the INADEQUATE experiment to paramagnetic heme active sites should be facilitated greatly by the availability of biosynthetic methods for producing isotopically labeled b-hemes and, more recently, isotopically labeled c-hemes.

A mercuric iodide detector unit implantable and externally powered for use in radionuclide tracer studies in small animals.

A small externally powered implantable gamma-radiation detector unit (35 X 12 mm diameter containing a mercuric iodide detector (HgI2), charge amplifier, radiotransmitter and an inductive power supply has been constructed. Since the total weight of the device is 5 g, it is suitable for implantation in laboratory animals such as rats or hamsters. A comparison between the implanted HgI2 detector unit and a conventional stationary 2 X 2 inch NaI(TI) detector has been performed by simultaneous measurements of the 133Xe clearance from a depot. Using 125I-iothalamate a kidney function study of an unrestrained hamster is shown as an example of measurements in the unrestrained animal.

Diastereoselective synthesis of substituted tetrahydroquinoline-4-carboxylic esters by a tandem reduction-reductive amination reaction.

A diastereoselective synthesis of 1-methyl-2-alkyl- and 2-alkyl-1,2,3,4-tetrahydroquinoline-4-carboxylic esters has been developed from methyl (2-nitrophenyl)acetate (1). The method involves alkylation of 1 with an allylic halide, ozonolysis of the double bond, and catalytic hydrogenation. The final hydrogenation initiates a tandem sequence involving (1) reduction of the aromatic nitro group, (2) condensation of the aniline or hydroxylamine(8) nitrogen with the side chain carbonyl, (3) reduction of the resulting nitrogen intermediate, and (4) reductive amination of the tetrahydroquinoline with formaldehyde produced in the ozonolysis to give a methyl (+/-)-1-methyl-2-alkyl-1,2,3,4-tetrahydroquinoline-4-carboxylate. Removal of the formaldehyde prior to hydrogenation gives the simple (+/-)-2-alkyl derivatives. The products are isolated in high yield as single diastereomers having the C-2 alkyl group cis to the C-4 carboxylic ester. The reaction has been extended to the synthesis of tricyclic structures with similar high diastereoselection.

Application of robotics In the clinical laboratory.

The basic types of robot are explained, and the performances and costs of some commercial examples are given. The potential advantages and problems of introducing robots into clinical laboratories are identified and the specifcation of a suitable robot is developed. None of the commercially available robots meets all aspects of the specificalion, and currently the purchase of a robot is considered premature for most clinical laboratories.

A comparison of different biogeographical classifications of Europe, Great Britain and Spain.

Various biogeographical and bioclimatic classifications of a number of regions, countries and continents have been created to meet different objectives. A policy maker might ask the question 'why is there no single accepted classification and how do the different classifications compare with one another?' In order to answer these two questions three classifications created by different methods for Great Britain and two for Spain are compared using the Kappa statistic. All of the classifications were created from data on cellular grids with a set window size. Further non-statistical comparisons are made with other classifications. The biogeographic classifications studied in this paper produced three different types of zone: those that were always identified whatever the method; those that were broadly similar but where the boundaries differed; and those that were unique to a particular classification. These different types of zone are likely to exist for any comparison between classifications of a particular region. The extent of the geographic window from which data were obtained had a major effect on the classification of grid cells at the edges of the window. For example, the few grid cells in the south of England, with characteristics of continental Europe, are not detected if data from Great Britain alone are used for the classification. We conclude that the data window should always be larger than the area for which the classification is being made. The objective Kappa statistic, although useful, was not capable of discerning similarities and dissimilarities that appear obvious to the subjective human eye. Although the details of the classifications differed there were broad similarities between the classifications and these differences reflect important divisions along major environmental axes that have been inferred by earlier biogeographers. As the divisions are real there is a sound basis for their use in future land use or environmental policy.

National-scale vegetation change across Britain; an analysis of sample-based surveillance data from the Countryside Surveys of 1990 and 1998.

Patterns of vegetation across Great Britain (GB) between 1990 and 1998 were quantified based on an analysis of plant species data from a total of 9596 fixed plots. Plots were established on a stratified random basis within 501 1 km sample squares located as part of the Countryside Survey of GB. Results are primarily conveyed in terms of a classification of national land-cover into 22 mutually exclusive Broad Habitat types. Each of the fixed vegetation plots could be assigned to the Broad Habitat in which they were located in either year. Two types of analysis are reported, both based on changes in plant species composition within monitoring plots. The first examined turnover and net change between Broad Habitat types. The second quantified more subtle changes that had occurred within each Broad Habitat using a series of condition measures that summarized multivariate plant species data as a single scalar value for each plot at each time. There are major difficulties in using uncontrolled, large-scale surveillance data to unravel causal linkages and no attempt was made to quantitatively partition variation among competing causes. However, it was clear that results were broadly consistent with environmental drivers known to have operated prior to and during the survey interval. Large-scale vegetation changes could be summarized in terms of shifts along gradients of substrate fertility and disturbance. Changes implied increased nutrient availability across upland and lowland ecosystems while, in lowland landscapes, linear features and small biotope fragments saw a marked shift to species compositions associated with greater shade and less disturbance.

Assessing stock and change in land cover and biodiversity in GB: an introduction to Countryside Survey 2000.

Countryside Survey 2000 (CS2000) is the latest in a series of surveys designed to measure and evaluate stock and change of land cover, landscape features, freshwaters, habitats and the vegetation of Great Britain. The ideas behind CS2000 developed during the 1960s and 1970s and culminated in the first survey of vegetation and land cover in 1978. One kilometer sample squares were selected at random using an environmental stratification. Subsequent surveys took place in 1984, 1990 and 1998, revisiting the original sample locations, whilst progressively expanding in scope and sample size; CS2000 included soils, breeding birds, remotely sensed imagery, freshwater biota and hydromorphology. Countryside Survey data may be interpreted using the pressure-state-response model, by selecting indicators of process and quality, and by identifying models of expected responses to different pressures. Thus, results showing losses of hedgerows between 1984 and 1990 stimulated new protection for these features. Ideally, CS2000 data should be used to stimulate experiments to distinguish between different pressures, in order to ensure that policy and management responses are both appropriate and achievable.The experience from CS2000 may prove helpful for the design and management of other large scale monitoring programmes of ecosystems. In particular, the scope of the survey, and the use to which the data are applied, have evolved through time, and yet continuity was essential for change to be detected efficiently. These objectives were reconciled by collecting the data in a disaggregated form, allowing a high degree of flexibility in both analysis and reporting.

13C NMR spectroscopic and X-ray crystallographic study of the role played by mitochondrial cytochrome b5 heme propionates in the electrostatic binding to cytochrome c.

The role played by the outer mitochondrial membrane (OM) cytochrome b5 heme propionate groups in the electrostatic binding between OM cytochrome b5 and horse heart cytochrome c was investigated by 13C NMR spectroscopy and X-ray crystallography. To achieve these aims, 13C-labeled heme OM cytochrome b5 was expressed in Escherichia coli as previously described [Rivera M., Walker, F.A. (1995) Anal. Biochem. 230, 295-302]. Assignment of the resonances arising from the heme propionate carbons in ferricytochrome b5 was carried out by a combination of one- and two-dimensional NMR experiments. Titrations of [13C]heme-labeled OM cytochrome b5 with horse heart cytochrome c were carried out in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b5. The results from these titrations clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b5 participates in the electrostatic stabilization of the complex between OM cytochrome b5 and horse heart cytochrome c. Similar experiments carried out monitoring 13C resonances arising from several other heme substituents demonstrated that the stoichiometry of the complex is 1:1. A conditional binding constant, K which equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was obtained for the formation of the complex by fitting the binding curves obtained experimentally to a model based on this stoichiometry. The X-ray crystal structure of rat liver OM cytochrome b5 solved to 2.7 A resolution shows that the structures of bovine liver microsomal cytochrome b5 and rat liver OM cytochrome b5 are almost identical when compared at medium resolution. The similarity between the two structures, combined with the findings that only the heme propionate located on the exposed heme edge of OM cytochrome b5 participates in the electrostatic binding to cytochrome c and that the stability of this complex is similar to that measured for the association between microsomal cytochrome b5 and cytochrome c, clearly indicates that the site of interaction on OM cytochrome b5 is almost identical to the one elucidated for microsomal cytochrome b5. It is therefore possible to conclude that the large body of information gathered by many investigators for the nonphysiological interaction between microsomal cytochrome b5 and cytochrome c (recently reviewed) [Mauk, A. G. Mauk, M. R., Moore, G. R., & Northrup, S. H. (1995) Bioenerg. Biomembr. 27, 311-330] has indeed biological as well as pedagogical validity.