Residue 225 determines the Na(+)-induced allosteric regulation of catalytic activity in serine proteases.

Residue 225 in serine proteases is typically Pro or Tyr and specifies an important and unanticipated functional aspect of this class of enzymes. Proteases with Y225, like thrombin, are involved in highly specialized functions like blood coagulation and complement that are exclusively found in vertebrates. In these proteases, the catalytic activity is enhanced allosterically by Na+ binding. Proteases with P225, like trypsin, are typically involved in digestive functions and are also found in organisms as primitive as eubacteria. These proteases have no requirement for Na+ or other monovalent cations. The molecular origin of this physiologically important difference is remarkably simple and is revealed by a comparison of the Na+ binding loop of thrombin with the homologous region of trypsin. The carbonyl O atom of residue 224 makes a key contribution to the coordination shell of the bound Na+ in thrombin, but is oriented in a manner incompatible with Na+ binding in trypsin because of constraints imposed by P225 on the protein backbone. Pro at position 225 is therefore incompatible with Na+ binding and is a direct predictor of the lack of allosteric regulation in serine proteases. To directly test this hypothesis, we have engineered the thrombin mutant Y225P. This mutant has lost the ability to bind Na+ and behaves like the allosteric slow (Na(+)-free) form. The Na(+)-induced allosteric regulation also bears on the molecular evolution of serine proteases. A strong correlation exists between residue 225 and the codon used for the active site S195. Proteases with P225 typically use a TCN codon for S195, whereas proteases with Y225 use an AGY codon. It is proposed that serine proteases evolved from two main lineages: (i) TCN/P225 with a trypsin-like ancestor and (ii) AGY/Y225 with a thrombin-like ancestor. We predict that the Na(+)-induced allosteric regulation of catalytic activity can be introduced in the TCN/P225 lineage using the P225Y replacement.

Crystal structure of the T state of allosteric yeast chorismate mutase and comparison with the R state.

The crystal structure of the tyrosine-bound T state of allosteric yeast Saccharomyces cerevisiae chorismate mutase was solved by molecular replacement at a resolution of 2.8 angstroms using a monomer of the R-state structure as the search model. The allosteric inhibitor tyrosine was found to bind in the T state at the same binding site as the allosteric activator tryptophan binds in the R state, thus defining one regulatory binding site for each monomer. Activation by tryptophan is caused by the larger steric size of its side chain, thereby pushing apart the allosteric domain of one monomer and helix H8 of the catalytic domain of the other monomer. Inhibition is caused by polar contacts of tyrosine with Arg-75 and Arg-76 of one monomer and with Gly-141, Ser-142, and Thr-145 of the other monomer, thereby bringing the allosteric and catalytic domains closer together. The allosteric transition includes an 8 degree rotation of each of the two catalytic domains relative to the allosteric domains of each monomer (domain closure). Alternatively, this transition can be described as a 15 degree rotation of the catalytic domains of the dimer relative to each other.

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

Final report : offsite manufacture in Australia Final report off-site manufacture in Australia

Off-site Manufacture (OSM) has long been recognised, both in Australia and internationally, as offering numerous benefits to all parties in the construction process. More importantly, it is recognised as a key vehicle for driving improvement within the construction industry. The uptake of OSM in construction is however limited, despite well documented benefits. The research aims to determine the ‘state-of-the-art’ of OSM in Australia. It confirms the benefits and identifies the real and perceived barriers to the widespread adoption of OSM. Further the project identifies opportunities for future investment and research. Although numerous reports have been produced in the UK on the state of OSM adoption within that region, no prominent studies exist for the Australian context. This scoping study is an essential component upon which to build any initiatives that can take advantage of the benefits of OSM in construction. The Construction 2020 report predicted that OSM is set to increase in use over the next 5-15 years, further justifying the need for such a study. The long-term goal of this study is to contribute to the improvement of the Australian construction industry through a realisation of the potential benefits of OSM.

Off-site Manufacture in Australia : Current State and Future Directions

Off-site manufacture (OSM) offers numerous benefits to all parties in the construction process. The uptake of OSM in Australia has, however, been limited. This limited uptake corresponds to similar trends in the UK and US, although the level of OSM there appears to be increasing. This project undertook three workshops — one each in Victoria, Queensland and Western Australia — and 18 interviews with key stakeholders to assist in identifying the general benefits and barriers to OSM uptake in the Australian construction industry. Seven case studies were also undertaken, involving construction projects that used OSM, ranging from civil projects through to residential. Each of these case studies has been analysed to identify what worked and what didn’t, and suggest the lessons to be learned from each project.

Drivers constraints and the future of off-site manufacture in Australia.

Much has been written on Off-site Manufacture (OSM) in construction, particularly regarding the perceived benefits and barriers to implementation. However, very little understanding of the state of OSM in the Australian construction industry exists. A ‘scoping study' has recently been undertaken to determine the ‘state-of-the-art’ of OSM in Australia. This involved several industry workshops, interviews and case studies across four major states of Australia. The study surveyed a range of suppliers across the construction supply-chain, incorporating the civil, commercial and housing segments of the market. This revealed that skills shortages and lack of adequate OSM knowledge are generally the greatest issues facing OSM in Australia. The drivers and constraints that emerged from the research were, in large measure, consistent with those found in the US and UK, although some Australian anomalies are evident, such as the geographical disparity of markets. A comparative analysis with similar studies in the UK and US is reported, illustrating both the drivers and constraints confronting the industry in Australia. OSM uptake into the future is however dependent on many factors, not least of which is a better understanding of the construction process and its associated costs.

Site survey and analysis of highway trucks overloading status quo in Anhui

In 2004, with the increasing overloading restriction requirements of society in Anhui, a provincial comprehensive overloading transportation survey has been developed to take evaluations on overloading actuality and enforcement efficiency with the support of the World Bank. A total of six site surveys were conducted at Hefei, Fuyang, Luan, Wuhu, Huainan and Huangshan Areas with four main contents respectively: traffic volume, axle load, freight information and registration information. Via statistical analysis on the survey data, conclusions were gained that: vehicle overloading are very universal and serious problems at arterial highways in Anhui now. The traffic loads have far exceeded the designed endure capacity of highways and have caused prevalent premature pavement damage, especially for rigid pavement. The overloading trucks are unimpeded engaged in highway freight transportation actually due to the disordered overloading enforcement strategies and the deficient inspecting technologies.

The role of sensation seeking and need for cognition on web-site evaluations : a resource-matching perspective

The Internet theoretically enables marketers to personalize a Website to an individual consumer. This article examines optimal Website design from the perspective of personality trait theory and resource-matching theory. The influence of two traits relevant to Internet Web-site processing—sensation seeking and need for cognition— were studied in the context of resource matching and different levels of Web-site complexity. Data were collected at two points of time: personality-trait data and a laboratory experiment using constructed Web sites. Results reveal that (a) subjects prefer Web sites of a medium level of complexity, rather than high or low complexity; (b)high sensation seekers prefer complex visual designs, and low sensation seekers simple visual designs, both in Web sites of medium complexity; and (c) high need-for-cognition subjects evaluated Web sites with high verbal and low visual complexity more favourably.

Spectrometric and voltammetric studies of the interaction between quercetin and bovine serum albumin using warfarin as site marker with the aid of chemometrics

The interaction of quercetin, which is a bioflavonoid, with bovine serum albumin (BSA) was investigated under pseudo-physiological conditions by the application of UV–vis spectrometry, spectrofluorimetry and cyclic voltammetry (CV). These studies indicated a cooperative interaction between the quercetin–BSA complex and warfarin, which produced a ternary complex, quercetin–BSA–warfarin. It was found that both quercetin and warfarin were located in site I. However, the spectra of these three components overlapped and the chemometrics method – multivariate curve resolution-alternating least squares (MCR-ALS) was applied to resolve the spectra. The resolved spectra of quercetin–BSA and warfarin agreed well with their measured spectra, and importantly, the spectrum of the quercetin–BSA–warfarin complex was extracted. These results allowed the rationalization of the behaviour of the overlapping spectra. At lower concentrations ([warfarin] < 1 × 10−5 mol L−1), most of the site marker reacted with the quercetin–BSA, but free warfarin was present at higher concentrations. Interestingly, the ratio between quercetin–BSA and warfarin was found to be 1:2, suggesting a quercetin–BSA–(warfarin)2 complex, and the estimated equilibrium constant was 1.4 × 1011 M−2. The results suggest that at low concentrations, warfarin binds at the high-affinity sites (HAS), while low-affinity binding sites (LAS) are occupied at higher concentrations.