Display of cell surface sites for fibronectin assembly is modulated by cell adherence to (1)F3 and C-terminal modules of fibronectin.

BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C. CONCLUSIONS: These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.

The Haemophilus influenzae HMW1C protein is a glycosyltransferase that transfers hexose residues to asparagine sites in the HMW1 adhesin.

The Haemophilus influenzae HMW1 adhesin is a high-molecular weight protein that is secreted by the bacterial two-partner secretion pathway and mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. In recent work, we discovered that HMW1 is a glycoprotein and undergoes N-linked glycosylation at multiple asparagine residues with simple hexose units rather than N-acetylated hexose units, revealing an unusual N-glycosidic linkage and suggesting a new glycosyltransferase activity. Glycosylation protects HMW1 against premature degradation during the process of secretion and facilitates HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence. In the current study, we establish that the enzyme responsible for glycosylation of HMW1 is a protein called HMW1C, which is encoded by the hmw1 gene cluster and shares homology with a group of bacterial proteins that are generally associated with two-partner secretion systems. In addition, we demonstrate that HMW1C is capable of transferring glucose and galactose to HMW1 and is also able to generate hexose-hexose bonds. Our results define a new family of bacterial glycosyltransferases.

Identification and assessment of potential wind energy project sites in the State of Maryland

Gemstone Team Renewables

Role of DNA binding sites and slow unbinding kinetics in titration-based oscillators.

Genetic oscillators, such as circadian clocks, are constantly perturbed by molecular noise arising from the small number of molecules involved in gene regulation. One of the strongest sources of stochasticity is the binary noise that arises from the binding of a regulatory protein to a promoter in the chromosomal DNA. In this study, we focus on two minimal oscillators based on activator titration and repressor titration to understand the key parameters that are important for oscillations and for overcoming binary noise. We show that the rate of unbinding from the DNA, despite traditionally being considered a fast parameter, needs to be slow to broaden the space of oscillatory solutions. The addition of multiple, independent DNA binding sites further expands the oscillatory parameter space for the repressor-titration oscillator and lengthens the period of both oscillators. This effect is a combination of increased effective delay of the unbinding kinetics due to multiple binding sites and increased promoter ultrasensitivity that is specific for repression. We then use stochastic simulation to show that multiple binding sites increase the coherence of oscillations by mitigating the binary noise. Slow values of DNA unbinding rate are also effective in alleviating molecular noise due to the increased distance from the bifurcation point. Our work demonstrates how the number of DNA binding sites and slow unbinding kinetics, which are often omitted in biophysical models of gene circuits, can have a significant impact on the temporal and stochastic dynamics of genetic oscillators.

Evidence for distinct dehydrogenase and isomerase sites within a single 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase protein

Complementary DNA encoding human 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (30-HSD) has been expressed in transfected GH4C1 with use of the cytomegalovirus promoter. The activity of the expressed protein clearly shows that both dehydrogenase and isomerase enzymatic activities are present within a single protein. However, such findings do not indicate whether the two activities reside within one or two closely related catalytic sites. With use of [3H]-5-androstenedione, the intermediate compound in dehydroepiandrosterone (DHEA) transformation into 4-androstenedione by 3β-HSD, the present study shows that 4MA (N,N-diethyl-4-rnethyl-3-oxo-4-aza-5α-androstane-17β-carboxamide) and its analogues inhibit DHEA oxidation competitively while they exert a noncompetitive inhibition of the isomerization of 5-androstenedione to 4-androstenedione with an approximately 1000-fold higher Ki value. The present results thus strongly suggest that dehydrogenase and isomerase activities are present at separate sites on the 3β-HSD protein. In addition, using 5α-dihydrotestosterone (DHT) and 5α-androstane-3β,17β-diol as substrates for dehydrogenase activity only, we have found that dehydrogenase activity is reversibly and competitively inhibited by 4MA. Such data suggest that the irreversible step in the transformation of DHEA to 4-androstenedione is due to a separate site possessing isomerase activity that converts the 5-ene-3-keto to a much more stable 4-ene-3-keto configuration. © 1991 American Chemical Society.

Species diversity structure analysis at two sites in the tropical rain forest of Sumatra

Data from a hilly forest study site at Batang Ule, Sumatra, are organized into 30 100-m × 10-m subplots lying perpendicular to the line of maximal topographic gradient, from the valley to the plateau/ridge. The following methodological question is addressed: what species diversity measures are best used in order to reveal the ecologically distinct regions in the site. The main tool used to answer this question is the α-diversity curve (Hα). Graphical examination of tree and species densities, and α-diversity curves identifies an anomalous species diversity behaviour of the ‘ridge above the slope’ subplots which may have implications on land-facet class definitions. Factor analysis of the α-diversity curves indicates that the diversity space is two-dimensional: i.e. two diversity measures are sufficient to characterize the site; the species density (H0), and the Berger-Parker index (H[infty infinity]). In the two-dimensional diversity-space three distinct species diversity groups are found which relate to the topographic gradient at the Batang Ule site. The results are compared with those for a flat homogeneous site at Pasirmayang, Sumatra. The implications of the results on land-classifications in species-diversity mapping and conservation strategy are discussed.

Tree species-area and species-diameter relationships at three lowland rain forest sites in Sumatra

Data from three forest sites in Sumatra (Batang Ule, Pasirmayang and Tebopandak) have been analysed and compared for the effects of sample area cut-off, and tree diameter cut-off. An 'extended inverted exponential model' is shown to be well suited to fitting tree-species-area curves. The model yields species carrying capacities of 680 for Batang Ule, 380 species for Pasirmayang, and 35 for Tebopandak (tree diameter >10cm). It would seem that in terms of species carrying capacity, Tebopandak and Pasirmayang are rather similar, and both less diverse than the hilly Batang Ule site. In terms of conservation policy, this would mean that rather more emphasis should be put on conserving hilly sites on a granite substratum. For Pasirmayang with tree diameter >3cm, the asymptotic species number estimate is 567, considerably higher than the estimate of 387 species for trees with diameter >10cm. It is clear that the diameter cut-off has a major impact on the estimate of the species carrying capacity. A conservative estimate of the total number of tree species in the Pasirmayang region is 632 species! In sampling exercises, the diameter cut-off should not be chosen lightly, and it may be worth adopting field sampling procedures which involve some subsampling of the primary sample area, where the diameter cut-off is set much lower than in the primary plots.

Characterisation of the South West European Marine Sites: Summary Report. Occasional Publication of the Marine Biological Association 14

The UK and EU have recently committed to an ecosystem-based approach to the management of our marine environment. In line with the requirements of the Habitats regulations, all consents likely to significantly affect Special Areas of Conservation (SACs) and Special Protection Areas (SPAs) are to be reviewed. As part of this process, 'site characterisation' is seen as an important first step towards the improved management of designated sites. This characterisation series, undertaken by the Marine Biological Association of the United Kingdom and funded by the Environment Agency and English Nature, sets out to determine the current status of designated marine sites in South West England, and how vulnerable (or robust) they are to contaminants (metals, organics, nutrients) and other anthropogenic pressures. Using published information and unpublished data-sets from regulatory agencies, conservation bodies and research institutes (particularly those of the PMPS*), evidence is compiled on the links between potentially harmful 'activities', environmental quality, and resultant biological consequences. This includes an evaluation of long-term change. The focus is the effect of water and sediment quality on the key interest features of European Marine sites in the South West of England, namely: - Fal and Helford cSAC (MBA Occasional Publication 8) - Plymouth Sound and Estuaries cSAC/ SPA (MBA Occasional Publication 9) - Exe Estuary SPA (MBA Occasional Publication 10) - Chesil and the Fleet cSAC/ SPA (MBA Occasional Publication 11) - Poole Harbour SPA (MBA Occasional Publication 12) - Severn Estuary pSAC/SPA (MBA Occasional Publication 13) Detailed analysis for each of these sites is provided individually. The summary report contains an overview of physical properties, uses and vulnerability for each of these sites, together with brief comparisons of pollution sources, chemical exposure (via sediment and water) and evidence of biological impact (from bioaccumulation to community-level response). Limitations of the data, and gaps in our understanding of these systems are highlighted and suggestions are put forward as to where future research and surveillance is most needed. Hopefully this may assist the statutory authorities in targeting future monitoring and remedial activities. * PMSP: Plymouth Marine Sciences Partnership, comprising the Marine Biological Association (MBA), University of Plymouth (UoP), the Sir Alister Hardy Foundation for Ocean Science, and Plymouth Marine Laboratories (PML)

Characterisation of European Marine Sites: Essex Estuaries European Marine Site Occasional Publication of the Marine Biological Association 17

This report provides an overview of water and sediment quality within the Essex Estuaries European Marine Site (EMS) and examines evidence for their influence on biological condition. Site characterisation has been accomplished by review of published literature and unpublished reports, together with interrogation of summary data sets for tidal waters provided by EA.