Diversification of the Structural Determinants of Fibroblast Growth Factor-Heparin Interactions: IMPLICATIONS FOR BINDING SPECIFICITY*

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K_D values varying between 38 nm (FGF-18) and 620 nm (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 m⁻¹ s⁻¹ and FGF-9, 130,000 m⁻¹ s⁻¹). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.     

Morphological and Inter Simple Sequence Repeat (ISSR) Markers Analyses of Corynespora cassiicola Isolates from Rubber Plantations in Malaysia

Morphological features and Inter Simple Sequence Repeat (ISSR) polymorphism were employed to analyse 21 Corynespora cassiicola isolates obtained from a number of Hevea clones grown in rubber plantations in Malaysia. The C. cassiicola isolates used in this study were collected from several states in Malaysia from 1998 to 2005. The morphology of the isolates was characteristic of that previously described for C. cassiicola. Variations in colony and conidial morphology were observed not only among isolates but also within a single isolate with no inclination to either clonal or geographical origin of the isolates. ISSR analysis delineated the isolates into two distinct clusters. The dendrogram created from UPGMA analysis based on Nei and Li's coefficient (calculated from the binary matrix data of 106 amplified DNA bands generated from 8 ISSR primers) showed that cluster 1 encompasses 12 isolates from the states of Johor and Selangor (this cluster was further split into 2 sub clusters (1A, 1B), sub cluster 1B consists of a unique isolate, CKT05D); while cluster 2 comprises of 9 isolates that were obtained from the other states. Detached leaf assay performed on selected Hevea clones showed that the pathogenicity of representative isolates from cluster 1 (with the exception of CKT05D) resembled that of race 1; and isolates in cluster 2 showed pathogenicity similar to race 2 of the fungus that was previously identified in Malaysia. The isolate CKT05D from sub cluster 1B showed pathogenicity dissimilar to either race 1 or race 2.     

C-2-aryl O-substituted HI-236 derivatives as non-nucleoside HIV-1 reverse-transcriptase inhibitors

Several novel thiourea derivatives of the NNRTI HI-236 substituted at the C-2 oxygen of the phenyl ring have been synthesized and evaluated for their inhibitory activity against HIV-1 (IIIB) replication in MT-2 cell cultures. The compounds were synthesized in order to fine-tune the activity of HI-236 as well as to gain insight into spatial characteristics in the pocket pertaining to the positional choice of tether in the design of [NRTI]-tether-[HI-236] bifunctional inhibitors. Two of the thiourea derivatives bearing a butynyl (6c) or hydroxyethyl tether (6n) were endowed with improved anti-HIV activity compared to HI-236. NNRTI activity was confirmed by a cell-free RT assay on six of the derivatives in which 6c returned an IC(50) of 3.8 nM compared to 28 nM for HI-236, establishing it as an improved lead for HI-236. The structure-activity profile is discussed in terms of potential interactions in the NNRTI pocket as suggested by a docking model using AutoDock, which have a bearing on the bifunctional drug design.     

Spatial and seasonal changes of dissolved and particulate organic C in the North Sea

Sampling of the central region of the North Sea was carried out to study the spatial and seasonal changes of dissolved and particulate organic C (DOC and POC, respectively). The surface waters were collected during four cruises over a year (Autumn 2004–Summer 2005). DOC and POC concentrations were measured using high temperature catalytic oxidation methods. The surface water concentrations of DOC and POC were spatially and temporally variable. There were significantly different concentrations of DOC and POC between the inshore and offshore waters in winter and summer only, with no clear trend in autumn and spring. Highest mean concentrations of DOC were measured in spring with lower and similar mean concentrations for other seasons. POC showed a clear seasonal cycle throughout the year with highest surface mean concentrations found in autumn and spring, but lowest in winter and summer. The DOC distributions during autumn and spring were strongly correlated with chlorophyll suggesting extracellular release from phytoplankton was an important DOC source during these two seasons. The lower concentrations of DOC in summer were probably due to the heterotrophic uptake of labile DOC. The seasonal changes in the C:N molar ratios of surface DOM (dissolved organic matter) resulted in higher mean C:N molar ratios in spring and lower ratios in winter. These high ratios may indicate nutrient limitation of heterotrophic uptake immediately after the spring bloom. There is limited data available for DOC cycling in these productive shelf seas and these results show that DOC is a major component of the C cycle with partial decoupling of the DOC and DON cycling in the central North Sea during the spring bloom. Handling editor: Luigi Naselli-Flores     

NH+/4-N assimilation by Rhodobacter capsulatus ATCC 23782 grown axenically and non-axenically in N and C rich media

Assimilation of NH+/4-N and formation of cell biomass in Rhodobacter capsulatus ATCC 23782 were studied in batch cultures as a function of N and C concentration and light intensity. Growth occurred satisfactorily up to N and C levels of 1.2 and 6.0g/1, respectively. The maximum biomass density achieved was 2.3 g biomass-C/l at 0.8 g N/l and 4.0g C/l. Media containing initial C/N ratios of 5 provided good growth and almost complete assimilation and recovery of NH+/4-N and lactate-C, respectively. A light intensity of about 120 μE/m²/s was adequate for efficient growth. At low levels of NH+/4-N (<0.05 g N/l), the photobacterium could not maintain dominance under non-axenic growth conditions. Chloroxuron was necessary to prevent algal overgrowth. At concentrations of 0.2 and 0.4 g NH+/4-N/l, the photo-bacterium maintained dominance over several months under the appropriate conditions of temperature (30°C), light intensity (120μE/m²/s), carbon supply (C/N = 5) and cell residence time (5.5d). The protein of Rhb. capsulatus ATCC 23782 was rich in essential amino acids.     

NH + 4 -N recovery from ammonia-treated straw-whey ensilage and cell biomass formation by Rhodobacter capsulatus ATCC 23782 on silage filtrates

Summary Growth of Rhodobacter capsulatus ATCC 23782 on silage filtrates under both axenic and non-axenic conditions was evaluated. Under both conditions, 96% assimilation of NH ₊ ⁴ -N at rates of about 0.1 g/l·d was attained. Assimilation was complete when the C/N ratio was adjusted to ca. 5. Thus assimilation rates of 0.2 g/l·d were achieved for an acid-C and NH ₊ ⁴ -N concentration of 2.0 and 0.4 g/l respectively. The corresponding biomass production amounted about 104 mg/g silage, dry matter.