Approaches to functional genomics in filamentous fungi

The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.     

LONGTMR3IGF-I as a more potent alternative to insulin in serum-free culture of HEK293 cells

LONG^TMR³IGF-I, an analogue of insulin-like growth factor (IGF)-I, was specifically engineered for use in biopharmaceutical protein production in mammalian cells. LONG^TMR³IGF-I is capable of supporting the growth and survival of Chinese hamster ovary cells in serum-free media at concentrations at least 200-fold lower than required for insulin. LONG^TMR³IGF-I also acts as a more potent growth and survival factor than either insulin or native IGF-I in SF culture of human embryonic kidney (HEK293) cells. To investigate the basis of the enhanced potency of LONG^TMR³IGF-I we have examined the mechanism of action of these mitogens in HEK293 cells. All mitogens tested were found to activate the TypeI IGF receptor (IGF-IR) and insulin receptor (IR) in a dose-responsive manner. However, the level of activation of both receptors after stimulation with LONG^TMR³IGF-I, at lower concentrations, was greater than with either insulin or IGF-I. The greater potency of LONG^TMR³IGF-I in activating the IR, despite having a low affinity for IRs, suggests the presence of heterotetrameric IGF-IR/IR dimers. Interestingly, the decrease in IGF-IR activation at higher concentrations of LONG^TMR³IGF-I suggests that the dose-response curve may be bell-shaped.     

Development of an in-stream migration model for Gammarus pulex L. (Crustacea, Amphipoda) as a tool in river restoration management

Ecological models can act as interesting tools to support decision-making in river restoration management. In particular models which are capable of predicting the habitat requirements of species are of considerable importance to ensure that the planned actions have the desired effects on the aquatic ecosystem. To this end, Artificial Neural Network (ANN) models were tested and optimized for the prediction of the habitat suitability for Gammarus pulex, a relevant indicator species in water quality assessment. Although ANN models are in general quite robust with a rather high predictive reliability, the model performance had to be increased with regard to simulations for river restoration management. In particular, it has been shown that spatial and temporal expert-rules could possibly be included. Migration dynamics of downstream drift and upstream migration of the organisms and migration barriers along the river (weirs, culverted river sections,␣...) might indeed deliver important additional information on the effectiveness of the restoration plans, and also on the timing of the expected effects. In this context, an additional in-stream migration model for Gammarus pulex was developed. This migration model, implemented in a Geographical Information System (GIS), has been used to simulate a practical river restoration scenario for a river in Flanders, Belgium. The case study illustrated that the removal of a weir, at a particular site, resulted in the improvement of the habitat suitability for Gammarus pulex. The ANN models predicted that after restoration the habitat was suitable again for Gammarus pulex. The migration model indicated that the restored parts of the river would be recolonized within about 2 months. In this way, decision makers can have an idea whether and when a restoration option will have a desired effect.     

Obesity indices and self-reported functional health in men and women in the EPIC-Norfolk

Objective: To investigate the association between two indices of obesity, BMI and waist‐to‐hip ratio (WHR), and self‐reported physical and mental functional health. Research Methods and Procedures: We examined the relationship between obesity indices and self‐reported physical and mental functional health measured by the Anglicized version of the Short‐Form 36‐item questionnaire in a population‐based cross sectional study of 16, 806 men and women 40 to 79 years old living in the general community in Norfolk, United Kingdom. Results: Higher BMI and WHR were both independently associated with poorer self‐reported physical functional health in men and women. The effect of BMI was greater in women compared with men, and the effect of WHR was greater in men compared with women, for poor physical functional health. Higher WHR but not BMI was associated with lower mental functional health in men and women. Discussion: High BMI and WHR seem to be adversely related to self‐perceived functional health in both men and women, although their relative impacts seem to differ by sex. Our findings also highlight the importance of using WHR in addition to BMI in assessing the impact of obesity on health outcome.     

Recognition of culture state using two-dimensional gel electrophoresis with an artificial neural network

Proteomic technologies were applied to the examination of nutrient components in culture broth. In bioprocesses, many types of media have been proposed and used on the commercial scale. Natural nutrients, the chemical components of which cannot be identified completely, are often used in fermentation processes such as in the production of baker's yeast, alcoholic beverages, amino acids, and pharmaceuticals. The catabolic activities of the microorganisms in these processes vary with the species used. We used an artificial neural network (ANN) to recognize the sufficiency of chemical elements based on the protein spots resolved in 2-DE, and we evaluated this technique using the leave-one-out method. We also attempted to reduce the number of input data for spot selection based on sensitivity analysis of the ANN, and the selected data were used to improve accuracy.     

Structural and expression analysis of hepatic vitellogenin gene during ovarian maturation in Anguilla japonica

Vitellogenin (Vtg), the precursor molecule for yolk, is synthesized in the liver under estrogenic control. In all oviparous species, including fish, the process of vitellogenesis is crucial to subsequent embryonic development. This study attempted to obtain the cDNA encoding for Vtg from female Japanese eels, Anguilla japonica. Rapid amplification of cDNA ends (RACE) and polymerase chain reaction (PCR) were used to amplify Vtg cDNA prepared from liver extracts. Obtained PCR products were subcloned and sequenced. The overall sequence of eel Vtg cDNA isolated in this study contained 5395 bp nucleotides. This Vtg sequence encodes 1743 amino acids of the precursor molecule, and is entirely composed of the characteristic N-terminal lipovitellin-I region, an internal polyserine domain region, and a c-terminal lipovitellin-II region. The deduced amino acid sequence from these clones shares 34–61% identity with other teleost Vtgs. Northern blot assays of Vtg gene expression following hormonal treatment demonstrated that this Vtg is synthesized in the liver under stimulation by estradiol injection. However, Vtg synthesis may not be enhanced by salmon pituitary homogenate (SPH) induction for the developing ovarian follicles. Notably, the effect of methyltestosterone, following SPH injection, may be more appropriate for the uptake of Vtg by ovarian follicle maturation during the artificial maturation of Japanese female eels.     

Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis

The crystal structure of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis was determined. Prolyl tripeptidyl aminopeptidase consists of beta-propeller and catalytic domains, and a large cavity between the domains; this structure is similar to dipeptidyl aminopeptidase IV. A catalytic triad (Ser603, His710, and Asp678) was located in the catalytic domain; this triad was virtually identical to that of the enzymes belonging to the prolyl oligopeptidase family. The structure of an inactive S603A mutant enzyme complexed with a substrate was also determined. The pyrrolidine ring of the proline residue appeared to fit into a hydrophobic pocket composed of Tyr604, Val629, Trp632, Tyr635, Tyr639, Val680, and Val681. There were characteristic differences in the residues of the beta-propeller domain, and these differences were related to the substrate specificity of tripeptidyl activity. The N-terminal amino group was recognized by salt bridges, with two carboxyl groups of Glu205 and Glu206 from a helix in dipeptidyl aminopeptidase IV. In prolyl tripeptidyl aminopeptidase, however, the Glu205 (located in the loop) and Glu636 were found to carry out this function. The loop structure provides sufficient space to accommodate three N-terminal residues (Xaa-Xaa-Pro) of substrates. This is the first report of the structure and substrate recognition mechanism of tripeptidyl peptidase.     

Design and DNA-binding of metallo-supramolecular cylinders conjugated to peptides

Di-nuclear metallo-supramolecular “cylinders”, based on bis-pyridylimine ligands, are end-functionalised with short peptides. The design and synthesis of one tetra-cationic triple-stranded (iron(II)) and three di-cationic double-stranded (copper(I) or silver(I)) cylinder-peptide conjugates are described. DNA-binding experiments, using circular and linear dichroism spectroscopies, confirm the binding and indicate that the iron(II) complexes cause DNA to bend or coil. Artificial nuclease activity by the copper(I) complexes is demonstrated by gel electrophoresis studies.     

Crystal structures of the complexes between vancomycin and cell-wall precursor analogs

The crystal structures of three vancomycin complexes with two vancomycin-sensitive cell-wall precursor analogs (diacetyl-Lys-D-Ala-D-Ala and acetyl-D-Ala-D-Ala) and a vancomycin-resistant cell-wall precursor analog (diacetyl-Lys-D-Ala-D-lactate) were determined at atomic resolutions of 1.80 A, 1.07 A, and 0.93 A, respectively. These structures not only reconfirm the "back-to-back" dimerization of vancomycin monomers and the ligand-binding scheme proposed by previous experiments but also show important structural features of strategies for the generation of new glycopeptide antibiotics. These structural features involve a water-mediated antibiotic-ligand interaction and supramolecular structures such as "side-by-side" arranged dimer-to-dimer structures, in addition to ligand-mediated and "face-to-face" arranged dimer-to-dimer structures. In the diacetyl-Lys-D-Ala-D-lactate complex, the interatomic O...O distance between the carbonyl oxygen of the fourth residue of the antibiotic backbone and the ester oxygen of the D-lactate moiety of the ligand is clearly longer than the corresponding N-H...O hydrogen-bonding distance observed in the two other complexes due to electrostatic repulsion. In addition, two neighboring hydrogen bonds are concomitantly lengthened. These observations provide, at least in part, a molecular basis for the reduced antibacterial activity of vancomycin toward vancomycin-resistant bacteria with cell-wall precursors terminating in -D-Ala-D-lactate.