Enzyme adsorption in porous supports: local thermodynamic equilibrium model

Enzyme adsorption from a finite bath (batch adsorption) onto porous spherical supports is investigated both experimentally and theoretically using beta-galactosidase and Duolite ion-exchange resin as a model system. Efficient numerical techniques are presented that have been used in conjunction with a parameter estimation routine to evaluate adsorption isotherm constants. Results show that even for adsorption processes lasting almost 10 h, the majority of the enzyme is confined to the outer half of the support and, for high initial enzyme concentrations in the bath, this loading takes place as a slowly moving front. Information on the enzyme distribution has practical importance in the design of immobilized enzyme reactors that in previous works have almost always been analyzed assuming a uniform catalyst distribution.     

Steady-state and transient behavior in microbial methanification: II. Mathematical modeling and verification

It has been shown that the Upflow Anaerobic Sludge Bed (UASB) system data reported earlier(1) cannot be explained by simple Monod-type substrate consumption patterns. An autoinhibition model was also ruled out because the substrate concentration range over which hysteresis was observed was much larger than such a model would predict. However, propionic and acetic acids were found to inhibit each other's conversion machineries. Since in the UASB system the biocatalyst is flocculated, it was found that a model additionally incorporating this facet of the reactor set-up could explain the steady-state data very well. Using the parameters generated from steady-state data and data from butyric acid step change,(1) i.e., the entire set of parameters (Table I), a very good agreement between predicted and observed data was found. International Mathematical and Statistical Libraries (IMSL) and Upjohn's NONLIN library combined with various root-finding and integrating subroutines were used for parameter estimation. The model thus described was used to predict the response of the UASB system when acetic acid and propionic acid influent concentrations were stepped-up/down. The agreement between the predicted and observed data was found to be excellent in each case during the step-up schedule. During the step-down the data seemed to indicate that the UASB system, like any other chemostat, responded faster than predicted. This could be due to the fact that when the culture has to "gear up" part of the lag time is the time required for the cell to produce the requisite amount of enzymes. In the case of "gearing down" this time is not required and the system responds faster.     

Steady-state and transient behavior in microbial methanification: I. Experimental results

The process of methanification of volatile fatty acids (VFA) was studied to elucidate its kinetics. An upflow anaerobic sludge bed (UASB) system was used to perform the experiments. At residence times of less than 2.5 h the UASB system was found to exhibit hysteresis with respect to acetic and propionic acid consumption but not with respect to butyric acid consumption. These hysteretic effects could be attributed to the manner in which the various VFA-consuming cultures were structured inside the flocculated biomass in light of the cross-inhibitory effects of the acetic- and propionic-acid-consuming fractions of the total culture. (Butyric acid proved to be non-interactive.) Production of methane was found to respond almost instantaneously to changes in the inlet conditions of the UASB system. This indicated that methane is not primarily growth associated, as has often been assumed, but is related to changes in the culture's maintenance energy requirements. Reactor operation was found to be stable even when the concentration of each VFA in the feed was simultaneously changed by 50%. Even at very high organic throughput rates (35 kg COD/day m(3)-reactor) conversions of 82% were observed.     

Magnetic resonance imaging of self-assembled biomaterial scaffolds

Current interest in biomaterials for tissue engineering and drug delivery applications have spurred research into self-assembling peptide amphiphiles (PAs). Nanofiber networks formed from self-assembling PAs can be used as biomaterial scaffolds with the advantage of specificity by the incorporation of peptide-epitopes. Imaging the materials noninvasively will give information as to their fate in vivo. We report here the synthesis and in vitro MR images of self-assembling peptide amphiphile contrast agents (PACAs) that form nanofibers. At 400 MHz using a 0.1 mM Gd(III) conjugate of the PA we observed a T(1) three times that of a control gel. The PA derivative was doped into various epitope bearing PA solutions and upon gelling resulted in a homogeneous biomaterial as imaged by MRI.     

Design,synthesis, and evaluation of simple phenol amides as ERRγ agonists

Herein we report the design and synthesis of a series of simple phenol amide ERRγ agonists based on a hydrazone lead molecule. Our structure activity relationship studies in this series revealed the phenol portion of the molecule to be required for activity. Attempts to replace the hydrazone with more suitable chemotypes led to a simple amide as a viable alternative. Differential hydrogen-deuterium exchange experiments were used to help understand the structural basis for binding to ERRγ and aid in the development of more potent ligands.     

Insight into the Roles of Helicase Motif Ia by Characterizing Fanconi Anemia Group J Protein (FANCJ) Patient Mutations

Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding and remodeling of structured DNA or RNA, which is coordinated by conserved helicase motifs. FANCJ is a DNA helicase that is genetically linked to Fanconi anemia, breast cancer, and ovarian cancer. Here, we characterized two Fanconi anemia patient mutations, R251C and Q255H, that are localized in helicase motif Ia. Our genetic complementation analysis revealed that both the R251C and Q255H alleles failed to rescue cisplatin sensitivity of a FANCJ null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, our biochemical assays demonstrated that both purified recombinant proteins abolished DNA helicase activity and failed to disrupt the DNA-protein complex. Intriguingly, R251C impaired DNA binding ability to single-strand DNA and double-strand DNA, whereas Q255H retained higher binding activity to these DNA substrates compared with wild-type FANCJ protein. Consequently, R251C abolished its DNA-dependent ATP hydrolysis activity, whereas Q255H retained normal ATPase activity. Physically, R251C had reduced ATP binding ability, whereas Q255H had normal ATP binding ability and could translocate on single-strand DNA. Although both proteins were recruited to damage sites in our laser-activated confocal assays, they lost their DNA repair function, which explains why they exerted a domain negative effect when expressed in a wild-type background. Taken together, our work not only reveals the structural function of helicase motif Ia but also provides the molecular pathology of FANCJ in related diseases.     

Genetic relationship of Porteresia coarctata Tateoka using molecular markers

ABSTRACT

Twenty-one species belonging to Oryza, including wild rices, were compared with a tetraploid (2n=48) halophytic wild rice relative, Porteresia coarctata Tateoka (=Oryza coarctata) for the genetic relatedness using AFLP and RAPD markers. Diploid and tetraploid groups were clearly separated except in the case of a few species where the clustering was unique and different. The molecular analysis has helped in positioning Porteresia in the vicinity of other wild rice species, and to better understand the pattern of species differentiation in Oryza. From our study, O. australiensis seems to be related to P. coarctata; thus, O. australiensis may be an effective “bridge” species in transferring genetic traits from P. coarctata to O. sativa. The usefulness of molecular marker systems for studying polymorphism and classification, and in clarifying genetic relationships between wild species has been confirmed.     

Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration

Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.     

An eye on the head: the development and evolution of craniofacial muscles

Skeletal muscles exert diverse functions, enabling both crushing with great force and movement with exquisite precision. A remarkably distinct repertoire of genes and ontological features characterise this tissue, and recent evidence has shown that skeletal muscles of the head, the craniofacial muscles, are evolutionarily, morphologically and molecularly distinct from those of the trunk. Here, we review the molecular basis of craniofacial muscle development and discuss how this process is different to trunk and limb muscle development. Through evolutionary comparisons of primitive chordates (such as amphioxus) and jawless vertebrates (such as lampreys) with jawed vertebrates, we also provide some clues as to how this dichotomy arose.