An update on genetic, structural and functional studies of arylamine N-acetyltransferases in eucaryotes and procaryotes

Arylamine N-acetyltransferase (NAT) was first identified as the inactivator of the anti-tubercular drug isoniazid, The enzyme was shown to catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of the hydrazine drug. The rate of inactivation of isoniazid was polymorphically distributed in the population and was one of the first examples of pharmacogenetic variation, NAT was identified recently in Mycobacterium tuberculosis and is a candidate for; modulating the response to isoniazid, Genome sequences have revealed many homologous members of this unique family of enzymes. The first three-dimensional structure of a member of the NAT family identifies a catalytic triad consisting of aspartate, histidine and cysteine proposed to form the activation mechanism. So far, all procaryotic NATs resemble the human enzyme which acetylates isoniazid (NAT2), Human NAT2 is characteristic of drug-metabolizing enzymes: it is found in liver and intestine, In humans and other mammals, there are up to three different isoenzymes. If only one isoenzyme is present, it is like human NAT1. Human NAT1 and its murine equivalent specifically acetylate the folate catabolite p-amino-benzoylglutamate. NAT1 and its murine homologue each have a ubiquitous tissue distribution and are expressed early in development at the blastocyst stage, During murine embryonic development, NAT is expressed in the developing neural tube. The proposed endogenous role of NAT in folate metabolism, and its multi-allelic nature, indicate that its role in development should be assessed further.

Functional analysis, using in vitro mutagenesis, of amino acids located in the phenylalanine hydroxylase active site

The 3-dimensionaI structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the K-m for substrate or cofactor. me conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. in contrast the PAH mutant, S349T, showed an 18-fold increase in K-m for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate K-m, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different. (C) 2000 Academic Press.

Delayed onset of electromyographic activity of vastus medialis obliquus relative to vastus lateralis in subjects with patellofemoral pain syndrome

Objective: To determine whether electromyographic (EMG) onsets of vastus medialis obliquus (VMO) and vastus lateralis (VL) are altered in the presence of patellofemoral pain syndrome (PFPS) during the functional task of stair stepping. Design: Cross-sectional. Setting: University laboratory. Patients: Thirty-three subjects with PFPS and 33 asymptomatic controls. Interventions: Subjects ascended and descended a set of stairs-2 steps, each 20-cm high-at usual stair-stepping pace. EMG readings of VMO and VL taken on middle stair during step up (concentric contraction) and step down (eccentric contraction). Main Outcome Measures: Relative difference in onset of surface EMG activity of VMO compared with VL during a stair-stepping task. EMG onsets were determined by using a computer algorithm and were verified visually. Results: In the PFPS population, the EMG onset of VL occurred before that of VMO in both the step up and step down phases of the stair-stepping task (p < .05). In contrast, no such differences occurred in the onsets of EMG activity of VMO and VL in either phase of the task for the control subjects. Conclusion: This finding supports the hypothesized relationship between changes in the timings of activity of the vastimuscles and PFPS. This finding provides theoretical rationale to support physiotherapy treatment commonly used in the management of PFPs.

In vivo measurement of the effect of intra-abdominal pressure on the human spine

In humans, intra-abdominal pressure (IAP) is elevated during many everyday activities. This experiment aimed to investigate the extent to which increased IAP-without concurrent activity of the abdominal or back extensor muscles-produces an extensor torque. With subjects positioned in side lying on a swivel table with its axis at L3, moments about this vertebral level were measured when IAP was transiently increased by electrical stimulation of the diaphragm via the phrenic nerve. There was no electromyographic activity in abdominal and back extensor muscles. When IAP was increased artificially to similar to 15% of the maximum IAP amplitude that could be generated voluntarily with the trunk positioned in flexion, a trunk extensor moment (similar to6 Nm) was recorded. The size of the effect was proportional to the increase in pressure. The extensor moment was consistent with that predicted from a model based on measurements of abdominal cross-sectional area and IAP moment arm. When IAP was momentarily increased while the trunk was flexed passively at a constant velocity, the external torque required to maintain the velocity was increased. These results provide the first in vivo data of the amplitude of extensor moment that is produced by increased IAP. Although the net effect of this extensor torque in functional tasks would be dependent on the muscles used to increase the IAP and their associated flexion torque, the data do provide evidence that IAP contributes, at least in part, to spinal stability. (C) 2001 Elsevier Science Ltd. All rights reserved.

Functional significance of the beta-hairpin in the insecticidal neurotoxin omega-atracotoxin-Hv1a

omega -Atracotoxin-Hv1a is an insect-specific neurotoxin whose phylogenetic specificity derives from its ability to antagonize insect, but not vertebrate, voltage-gated calcium channels. In order to help understand its mechanism of action and to enhance its utility as a lead compound for insecticide development, we used a combination of protein engineering and site-directed mutagenesis to probe the toxin for key functional regions. First, we constructed a Hairpinless mutant in which the C-terminal beta -hairpin, which is highly conserved in this family of neurotoxins, was excised without affecting the fold of the residual disulfide-rich core of the toxin. The Hairpinless mutant was devoid of insecticidal activity, indicating the functional importance of the hairpin. We subsequently developed a highly efficient system for production of recombinant toxin and then probed the hairpin for key functional residues using alanine-scanning mutagenesis followed by a second round of mutagenesis based on initial hits from the alanine scan. This revealed that two spatially proximal residues, Asn(27) and Arg(35), form a contiguous molecular surface that is essential for toxin activity. We propose that this surface of the beta -hairpin is a key site for interaction of the toxin with insect calcium channels.

Processing of urinary pheromones in Antechinus stuartii (Marsupialia: Dasyuridae): Functional magnetic resonance imaging of the brain

Little is known of the neural mechanisms of marsupial olfaction. However, functional magnetic resonance imaging (fMRI) has made it possible to visualize dynamic brain function in mammals without invasion. In this study, central processing of urinary pheromones was investigated in the brown antechinus, Antechinus stuartii, using fMRI. Images were obtained from 18 subjects (11 males, 7 females) in response to conspecific urinary olfactory stimuli. Significant indiscriminate activation occurred in the accessory olfactory bulb, entorhinal, frontal, and parietal cortices in response to both male and female urine. The paraventricular nucleus of hypothalamus, ventrolateral thalamic nucleus, and medial preoptic area were only activated in response to male urine. Results of this MRI study indicate that projections of accessory olfactory system are activated by chemo-sensory cues. Furthermore, it appears that, based on these experiments, urinary pheromones may act on the hypothalamo-pituitary-adrenocortical axis via the paraventricular nucleus of the hypothalamus and may play an important role in the unique life history pattern of A. stuartii. Finally, this study has demonstrated that fMRI may be a powerful tool for investigations of olfactory processes in mammals.

Cytokine-related genes identified from the RIKEN full-length mouse cDNA data set

To identify novel cytokine-related genes, we searched the set of 60,770 annotated RIKEN mouse cDNA clones (FANTOM2 clones), using keywords such as cytokine itself or cytokine names (such as interferon, interleukin, epidermal growth factor, fibroblast growth factor, and transforming growth factor). This search produced 108 known cytokines and cytokine-related products such as cytokine receptors, cytokine-associated genes, or their products (enhancers, accessory proteins, cytokine-induced genes). We found 15 clusters of FANTOM2 clones that are candidates for novel cytokine-related genes. These encoded products with strong sequence similarity to guanylate-binding protein (GBP-5), interleukin-1 receptor-associated kinase 2 (IRAK-2), interleukin 20 receptor alpha isoform 3, a member of the interferon-inducible proteins of the Ifi 200 cluster, four members of the membrane-associated family 1-8 of interferon-inducible proteins, one p27-like protein, and a hypothetical protein containing a Toll/Interleukin receptor domain. All four clones representing novel candidates of gene products from the family contain a novel highly conserved cross-species domain. Clones similar to growth factor-related products included transforming growth factor beta-inducible early growth response protein 2 (TIEG-2), TGFbeta-induced factor 2, integrin beta-like 1, latent TGF-binding protein 4S, and FGF receptor 4B. We performed a detailed sequence analysis of the candidate novel genes to elucidate their likely functional properties.

Functional expression cloning reveals proapoptotic role for protein phosphatase 4

Functional expression cloning strategies are highly suitable for the analysis of the molecular control of apoptosis. This approach has two critical advantages. Firstly, it eliminates prior assumptions about the properties of the proteins involved, and, secondly, it selectively targets proteins that are causally involved in apoptosis control and which affect the crucial cellular decision between survival and death. The application of this strategy to the isolation of cDNAs conferring resistance to dexamethasone and gamma-irradiation resulted in the isolation of a partial cDNA for the catalytic subunit of protein phosphatase 4 (PP4). Cells transfected with this partial cDNA in an expression vector downregulated PP4 and were resistant to both dexamethasone and UV radiation, as demonstrated by both membrane integrity and colony-forming assays. These observations suggest that PP4 plays an important proapoptotic role in T lymphocytes.

Designing teams for first-of-a-kind, complex systems using the initial phases of cognitive work analysis: Case study

We present a technique for team design based on cognitive work analysis (CWA). We first develop a rationale for this technique by discussing the limitations of conventional approaches for team design in light of the special characteristics of first-of-a-kind, complex systems. We then introduce the CWA-based technique for team design and provide a case study of how we used this technique to design a team for a first-of-a-kind, complex military system during the early stages of its development. In addition to illustrating the CWA-based technique by example, the case study allows us to evaluate the technique. This case study demonstrates that the CWA-based technique for team design is both feasible and useful, although empirical validation of the technique is still necessary. Applications of this work include the design of teams for first-of-a-kind, complex systems in military, medical, and industrial domains.

Palliative care at home: general practitioners working with palliative care teams

Home care is the preferred option for most people with a terminal illness. Providing home care relies on good community-based services, and a general practice workforce competent in palliative care practice and willing to accommodate patients' needs. Structured palliative care training of general practitioners is needed at undergraduate and postgraduate level, with attention to barriers to teamwork and communication. Good palliative care-can be delivered to patients at home by GPs (supported by specialist palliative care teams) and community nurses, with access to an inpatient facility when required. To optimise patient care, careful planning and good communication between all members of the healthcare team is crucial.

Combined in-fermenter extraction and cross-flow microfiltration for improved inclusion body processing

In this study we demonstrate a new in-fermenter chemical extraction procedure that degrades the cell wall of Escherichia coli and releases inclusion bodies (IBs) into the fermentation medium. We then prove that cross-flow microfiltration can be used to remove 91% of soluble contaminants from the released IBs. The extraction protocol, based on a combination of Triton X-100, EDTA, and intracellular T7 lysozyme, effectively released most of the intracellular soluble content without solubilising the IBs. Cross-flow microfiltration using a 0.2 mum ceramic membrane successfully recovered the granulocyte macrophagecolony stimulating factor (GM-CSF) IBs with removal of 91% of the soluble contaminants and virtually no loss of IBs to the permeate. The filtration efficiency, in terms of both flux and transmission, was significantly enhanced by infermenter Benzonase(R) digestion of nucleic acids following chemical extraction. Both the extraction and filtration methods exerted their efficacy directly on a crude fermentation broth, eliminating the need for cell recovery and re-suspension in buffer. The processes demonstrated here can all be performed using just a fermenter and a single cross-flow filtration unit, demonstrating a high level of process intensification. Furthermore, there is considerable scope to also use the microfiltration system to subsequently solubilise the IBs, to separate the denatured protein from cell debris, and to refold the protein using diafiltration. In this way refolded protein can potentially be obtained, in a relatively pure state, using only two unit operations. (C) 2004 Wiley Periodicals Inc.

Formal methods within a totally functional approach to programming

Taking functional programming to its extremities in search of simplicity still requires integration with other development (e.g. formal) methods. Induction is the key to deriving and verifying functional programs, but can be simplified through packaging proofs with functions, particularly folds, on data (structures). Totally Functional Programming avoids the complexities of interpretation by directly representing data (structures) as platonic combinators - the functions characteristic to the data. The link between the two simplifications is that platonic combinators are a kind of partially-applied fold, which means that platonic combinators inherit fold-theoretic properties, but with some apparent simplifications due to the platonic combinator representation. However, despite observable behaviour within functional programming that suggests that TFP is widely-applicable, significant work remains before TFP as such could be widely adopted.

Mediatizing educational policy: The journalistic field, science policy, and cross-field effects

This paper is concerned to demonstrate the usefulness of the theory of Bourdieu, including the concepts of field, logics of practice and habitus, to understanding relationships between media and policy, what Fairclough has called the 'mediatization' of policy. Specifically, the paper draws upon Bourdieu's accessible account of the journalistic field as outlined in On television and journalism. The usefulness of this work is illustrated through a case study of a recent Australian science policy, The chance to change. As this policy went through various iterations and media representations, its naming and structure became more aphoristic. This is the mediatization of contemporary policy, which often results in policy as sound bite. The case study also shows the cross-field effects of this policy in education, illustrating how today educational policy can be spawned from developments in other public policy fields.

Identification of a juvenile hormone esterase-like gene in the honey bee, Apis mellifera L. - Expression analysis and functional assays

Tight control over circulating juvenile hormone (JH) levels is of prime importance in an insect`s life cycle. Consequently, enzymes involved in JH metabolism, especially juvenile hormone esterases (JHEs), play major roles during metamorphosis and reproduction. In the highly eusocial Hymenoptera, JH has been co-opted into additional functions, primarily in the development of the queen and worker castes and in age-related behavioral development of workers. Within a set of 21 carboxylesterases predicted in the honey bee genome we identified one gene (Amjhe-like) that contained the main functional motifs of insect JHEs. Its transcript levels during larval development showed a maximum at the switch from feeding to spinning behavior, coinciding with a JH titer minimum. In adult workers, the highest levels were observed in nurse bees, where a low JH titer is required to prevent the switch to foraging. Functional assays showed that Amjhe-like expression is induced by JH-III and suppressed by 20-hydroxyecdysone. RNAi-mediated silencing of Amjhe-like gene function resulted in a six-fold increase in the JH titer in adult worker bees. The temporal profile of Amjhe-like expression in larval and adult workers, the pattern of hormonal regulation and the knockdown phenotype are consistent with the function of this gene as an authentic JHE. (C) 2008 Elsevier Inc. All rights reserved.

Functional and Evolutionary Insights from the Genomes of Three Parasitoid Nasonia Species

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.