Crystal structure of yeast acetohydroxyacid synthase: A target for herbicidal inhibitors

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides. (C) 2002 Elsevier Science Ltd.

Disability focus groups: A strategy for including people with disability in parishes

This paper reports the findings of an exploratory qualitative study examining parish awareness of the need to include individuals with disability in genuine and meaningful ways. Based on the interviews of over thirty persons, including people with disability, parents, parish workers, volunteers and human service workers within church agencies in the Catholic Archdiocese of Brisbane, Australia, the authors devised a process for including individuals in parishes. This process, known as a Disability Focus Group, was then implemented in one parish as a pilot study. The outcomes of the pilot are discussed.

Further observations on the Enenteridae Yamaguti, 1958 (Digenea, Lepocreadioidea) of the Indo-West Pacific region, including a new species from Western Australia

Measurements are given for all and full descriptions and illustrations for some of the following enenterid species: Enenterum aureum Linton, 1910 in Kyphosus bigibbus and K. sydneyanus? from Ningaloo Coral Reef, Western Australia, K. vaigiensis from off Heron Island, Queensland and K. vaigiensis from off Moorea, French Polynesia; E. mannarense Hafeezullah, 1980 in K. bigibbus and K. sydneyanus? from Ningaloo Coral Reef; E. elongatum Yamaguti, 1970 in K. vaigiensis from Heron Island, Queensland and K. bigibbus and K. sydneyanus? from Ningaloo Coral Reef; Koseiria alanwilliamsi sp. nov. in Kyphosus cornelii from off Kalbarri, Western Australia; Koseiria xishaense Gu et Shen, 1983 in K. vaigiensis from off Heron Island and K. bigibbus from off Palau, Micronesia; Proenenterum isocotylum Manter, 1954 in Aplodactylus arctidens from off Stanley, Tasmania; R ericotylum Manter, 1954 in A. arctidens from off Stanley; Cadenatella isuzumi Machida, 1993 from Kyphosus bigibbus and K. sydneyanus? from Ningaloo Coral Reef; Cadenatella pacifica (Yamaguti, 1970) from Kyphosus bigibbus from Ningaloo Coral Reef. Two recent cladistic studies of the Enenteridae are discussed and a further analysis has shown that Enenterum and Cadenatella are monophyletic, whilst Koseiria appears polyphyletic. The zoogeography and host-specificity of Kyphosus-inhabiting enenterids is discussed.

Catalytically active Dengue virus NS3 protease forms aggregates that are separable by size exclusion chromatography

An active form of the Dengue virus protease NS3 (CF40.Gly.NS3pro) was expressed in Escherichia coli. This construct consists of a critical 40 amino acid cofactor domain from NS2B fused to the N-terminal 184 amino acid protease domain of NS3 via a flexible, covalent linker (Gly(4)SerGly(4)). The recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the N-terminus for ease of purification using metal affinity chromatography. However, the presence of lower molecular weight impurities after affinity chromatography indicated the need for additional purification steps. The consistent appearance of these impurities suggested that they may be the products of proteolysis and/or auto-proteolysis. The latter possibility was subsequently excluded by the observation of the same impurities in a purified, catalytically inactive form of the recombinant protease (CF40.Gly.NS3pro.SA). Further analysis indicated that these impurities may represent premature translation termination products. Regardless of their origin, they were shown to form various sized aggregates with full-length CF40.Gly.NS3pro that can be separated by size exclusion chromatography, yielding fractions of active protease of sufficient purity for crystallisation trials. The ultimate goal of these studies is to obtain a crystal structure of a catalytically active form of the Dengue virus NS3 protease for structure-based drug design. (C) 2002 Elsevier Science (USA). All rights reserved.

Electrospray liquid chromatography/mass spectrometry fingerprinting of Acanthophis (death adder) venoms: taxonomic and toxinological implications

Death adders (genus Acanthophis) are unique among elapid snakes in both morphology and venom composition. Despite this genus being among the most divergent of all elapids, the venom has been historically regarded as relatively quite simple. In this study, liquid chromatography/mass spectrometry (LC/MS) analysis has revealed a. much greater diversity in venom composition, including the presence of molecules of novel molecular weights that may represent a new class of venom component. Furthermore, significant variation exists between species and populations,, which allow for the LC/MS fingerprinting of each species. Mass profiling of Acanthophis venoms clearly demonstrates the effectiveness of this technique which underpins fundamental studies ranging from chemotaxonomy to drug design. Copyright (C) 2002 John Wiley Sons, Ltd.

The use of ACE-inhibitors after myocardial infarction

Background: There is good evidence that angiotensin-converting enzyme (ACE) inhibitors are beneficial after myocardial infarction (MI). However, it is not known how widely this evidence is used in practice and whether all eligible patients receive this therapy. Aim: To assess the usage of ACE inhibitors in patients after MI in a large teaching hospital. Method: A one month prospective analysis, combined with a three month retrospective analysis, was conducted at the Royal Brisbane Hospital (RBH) in February-March 2000. Patients admitted with an MI or who had been diagnosed with an MI during admission from November 1999 to March 2000 were identified from the coronary care unit (CCU) records. Inpatient medication charts and outpatient records were then reviewed. Information collected included: ACE inhibitor use, doses, reasons for prescribing/not prescribing ACE inhibitors, and ACE inhibitor prescribers (cardiologists or general physicians). Results: Forty four patients with an MI were included in the study, 28 of whom were prescribed ACE-inhibitors (64%). Twenty four of the 28 patients on ACE inhibitors were prescribed perindopril. The major reason given for prescribing ACE inhibitors was signs of congestive cardiac failure. All ACE inhibitors initiated in patients after MI at RBH were ordered by cardiologists. Conclusion: ACE inhibitors were prescribed appropriately in 88% of patients who met criteria for their use. This high percentage of appropriate prescribing was encouraging. Reevaluation as part of an ongoing quality assurance activity could be used to ensure this is maintained.

Alcohol, tobacco and breast cancer - collaborative reanalysis of individual data from 53 epidemiological studies, including 58 515 women with breast cancer and 95 067 women without the disease

Alcohol and tobacco consumption are closely correlated and published results on their association with breast cancer have not always allowed adequately for confounding between these exposures. Over 80% of the relevant information worldwide on alcohol and tobacco consumption and breast cancer were collated, checked and analysed centrally. Analyses included 58515 women with invasive breast cancer and 95067 controls from 53 studies. Relative risks of breast cancer were estimated, after stratifying by study, age, parity and, where appropriate, women's age when their first child was born and consumption of alcohol and tobacco. The average consumption of alcohol reported by controls from developed countries was 6.0 g per day, i.e. about half a unit/drink of alcohol per day, and was greater in ever-smokers than never-smokers, (8.4 g per day and 5.0 g per day, respectively). Compared with women who reported drinking no alcohol, the relative risk of breast cancer was 1.32 (1.19 - 1.45, P < 0.00001) for an intake of 35 - 44 g per day alcohol, and 1.46 (1.33 - 1.61, P < 0.00001) for greater than or equal to 45 g per day alcohol. The relative risk of breast cancer increased by 7.1% (95% CI 5.5-8.7%; P

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is preorganized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

Inhibitors of COP-mediated Transport and Cholera Toxin Action Inhibit Simian Virus 40 Infection

Simian virus 40 (SV40) is a nonenveloped virus that has been shown to pass from surface caveolae to the endoplasmic reticulum in an apparently novel infectious entry pathway. We now show that the initial entry step is blocked by brefeldin A and by incubation at 20degreesC. Subsequent to the entry step, the virus reaches a domain of the rough endoplasmic reticulum by an unknown pathway. This intracellular trafficking pathway is also brefeldin A sensitive. Infection is strongly inhibited by expression of GTP-restricted ADP-ribosylation factor 1 (Arf1) and Sar1 mutants and by microinjection of antibodies to betaCOP. In addition, we demonstrate a potent inhibition of SV40 infection by the dipeptide N-benzoyl-oxycarbonyl-Gly-Phe-amide, which also inhibits late events in cholera toxin action. Our results identify novel inhibitors of SV40 infection and show that SV40 requires COPI- and COPII-dependent transport steps for successful infection.

Cellular responses to Schistosoma japonicum cathepsin D aspartic protease

Lymphocyte proliferation and cytokine production were measured in groups of mice vaccinated (but not subsequently challenge infected) with recombinant forms of Schistosoma japonicum cathepsin D aspartic protease, rSjASP1 (expressed in bacteria; enzymatically inactive) and rSjASP2 (expressed in insect cells; enzymatically active). Both forms of the schistosome enzyme induced significant proliferation of splenocytes recovered from vaccinated mice, and expression of interferon (IFN)-gamma, interleukin (IL)-4 and IL-10 mRNA in these cells was detected using reverse transcriptase-polymerase chain reaction. Secretion of IFN-gamma, IL-4 and IL-10 by splenocytes from vaccinated mice was confirmed and quantified using enzyme-linked immunosorbent assay. IFN-gamma was the most abundant cytokine produced, followed by IL-4 and IL-10 in rank order. These findings indicated that vaccination of mice with the schistosome protease induces a mixed Th1/Th2 cytokine response, which may explain the modest level of protection after challenge infection in cathepsin d-vaccinated mice, reported previously.

Osteonectin/SPARC induction by ectopic beta(3) integrin in human radial growth phase primary melanoma cells

Expression of the beta(3) integrin subunit in melanoma in situ has been found to correlate with tumor thickness, the ability to invade and metastasize, and poor prognosis. Transition from the radial growth phase (RGP) to the vertical growth phase (VGP) is a critical step in melanoma progression and survival and is distinguished by the expression of beta(3), integrin. The molecular pathways that operate in melanoma cells associated with invasion and metastasis were examined by ectopic induction of the beta(3), integrin subunit in RGP SBcl2 and WM1552C melanoma cells, which converts these cells to a VGP phenotype. We used cDNA representational difference analysis subtractive hybridization between beta(3)-Positive and -negative melanoma cells to assess gene expression profile changes accompanying RGP to VGP transition. Fourteen fragments from known genes including osteonectin (also known as SPARC and BM-40) were identified after three rounds of representational difference analysis. Induction of osteonectin was confirmed by Northern and Western blot analysis and immunohistochemistry and correlated in organotypic cultures with the beta(3)-induced progression from RGP to VGP melanoma. Expression of osteonectin was also associated with reduced adhesion to vitronectin, but not to fibronectin. Osteonectin expression was not blocked when melanoma cells were cultured with anti-alpha(v)beta(3) LM609 mAb, mitogen-activated protein kinase, or protein kinase C inhibitors, indicating that other signaling pathway(s) operate through a(v)beta(3) integrin during conversion from RGP to VGP.