Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(−)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (−)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

1,1,1-Trichloro-2,2-bis(4-iodophenyl)ethane

In the structure of the title compound C14H9Cl3I2, which is the p-iodophenyl analogue of the insecticide DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane], isomorphism between the two compounds has been confirmed. In the molecule the dihedral angle between the planes of the two phenyl rings is 65.8(4)deg. which compares with 64.7(7)deg. in DDT.

2,2-Bis(4-butoxyphenyl)-1,1,1-trichloroethane

In the structure of the title compound C22H27Cl302, which is the p-butoxyphenyl analogue of the insecticidally active p-methoxyphenyl compound methoxychlor, the dihedral angle between the two phenyl rings is 79.61(11)deg. Present also in the structure is an intramolecular aromatic C-H...Cl interaction [3.361(2)Ang].

Hydrogen-bonding in cyclic imides and amide carboxylic acid derivatives from the facile reaction of cis-cyclohexane-1,2-carboxylic anhydride with o- and p-anisidine and m- and p-aminobenzoic acids

The structures of the open chain amide carboxylic acid rac-cis-[2-(2-methoxyphenyl)carbamoyl]cyclohexane-1-carboxylic acid, C15H19NO4, (I) and the cyclic imides rac-cis-2-(4-methoxyphenyl)-3a,4,5,6,7,7-hexahydroisoindole-1,3-dione,C15H17NO3, (II), chiral cis-2-(3-carboxyphenyl)-3a,4,5,6,7,7a-hexahydroisoindole-1,3-dione, C15H15NO4,(III) and rac-cis-2-(4-carboxyphenyl)- 3a,4,5,6,7,7a-hexahydroisoindole-1,3-dione monohydrate, C15H15NO4. H2O) (IV), are reported. In the amide acid (I), the phenylcarbamoyl group is essentially planar [maximum deviation from the least-squares plane = 0.060(1)Ang. for the amide O atom], the molecules form discrete centrosymmetric dimers through intermolecular cyclic carboxy-carboxy O-H...O hydrogen-bonding interactions [graph set notation R2/2(8)]. The cyclic imides (II)--(IV) are conformationally similar, with comparable phenyl ring rotations about the imide N-C(aromatic) bond [dihedral angles between the benzene and isoindole rings = 51.55(7)deg. in (II), 59.22(12)deg. in (III) and 51.99(14)deg. in (IV). Unlike (II) in which only weak intermolecular C-H...O(imide) hydrogen bonding is present, the crystal packing of imides (III) and (IV) shows strong intermolecular carboxylic acid O-H...O hydrogen-bonding associations. With (III), these involve imide O-atom acceptors, giving one-dimensional zigzag chains [graph set C(9)], while with the monohydrate (IV), the hydrogen bond involves the partially disordered water molecule which also bridges molecules through both imide and carboxyl O-atom acceptors in a cyclic R4/4(12) association, giving a two-dimensional sheet structure. The structures reported here expand the structural data base for compounds of this series formed from the facile reaction of cis-cyclohexane-1,2-dicarboxylic anhydride with substituted anilines, in which there is a much larger incidence of cyclic imides compared to amide carboxylic acids.

Formal complaints of workplace sexual harassment lodged with Australian Human Rights and Equal Opportunity Commissions - 1 July 2009 - 31 December 2009

This report presents an analysis of quantitative data collected from the Australian Human Rights Commission, the Anti-Discrimination Commission of Queensland, the Victorian Equal Opportunity and Human Rights Commission, the Anti-Discrimination Board of New South Wales, the Equal Opportunity Commission of South Australia, the Australian Capital Territory Human Rights Commission, the Equal Opportunity Commission Western Australia, the Northern Territory Anti-Discrimination Commission, and the Office of the Anti-Discrimination Commissioner (Tasmania) (hereafter referred to as the Commissions). The data comprise formal complaints lodged under the various federal, state and territory anti-discrimination laws in the period 1 July 2009 to 31 December 2009 where a complainant had alleged sexual harassment in the area of employment.

Cyclic Imide and Open-Chain Amide Carboxylic Acid Derivatives from the Facile Reaction of cis-Cyclohexane-1,2-dicarboxylic Anhydride with Three Substituted Anilines

The structures of the compounds from the reaction of cis-cyclohexane-1,2-dicarboxylic anhydride with 4-chloroaniline [rac-N-(4-chlorophenyl)-2-carboxycycloclohexane-1-carboxamide] (1), 4-bromoaniline [2-(4-bromophenyl)-perhydroisoindolyl-1,3-dione] (2) and 3-hydroxy-4-carboxyaniline (5-aminosalicylic acid) [2-(3-hydroxy-4-carboxyphenyl)-perhydroisoindolyl-1,3-dione] (3) have been determined at 200 K. Crystals of the open-chain amide carboxylic acid 1 are orthorhombic, space group Pbcn, with unit cell dimensions a = 20.1753(10), b = 8.6267(4), c = 15.9940(9) Å, and Z = 8. Compounds 2 and 3 are cyclic imides, with 1 monoclinic having space group P21 and cell dimensions a = 11.5321(3), b = 6.7095(2), c = 17.2040(5) Å, β = 102.527(3)o. Compound 3 is orthorhombic with cell dimensions a = 6.4642(3), b = 12.8196(5), c = 16.4197(7) Å. Molecules of 1 form hydrogen-bonded cyclic dimers which are extended into a two-dimensional layered structure through amide-group associations: 3 forms into one-dimensional zigzag chains through carboxylic acid…imide O-atom hydrogen bonds, while compound 2 is essentially unassociated. With both cyclic imides 2 and 3, disorder is found which involves the presence of partial enantiomeric replacement of the cis-cyclohexane-1,2-substituted ring systems.

Hydrogen-bonding in the structures of the 1:1 salts of isonipecotamide with a set of four polyfunctional monocyclic heteroaromatic carboxylic acids

The crystal structures of the 1:1 proton-transfer compounds of isonipecotamide (piperidine-4-carboxamide) with the monocyclic heteroaromatic carboxylic acids, isonicotinic acid, picolinic acid, dipicolinic acid and pyrazine-2,3-dicarboxylic acid have been determined at 200 K and their hydrogen-bonding patterns examined. The compounds are respectively anhydrous 4-carbamoylpiperidinium pyridine-4-carboxylate (1), the partial hydrate 4-carbamoylpiperidinium pyridine-2-carboxylate 0.25 water (2), the solvate 4-carbamoylpiperidinium 6-carboxypyridine-2-carboxylate methanol monosolvate (3), and anhydrous 4-carbamoylpiperidinium 3-carboxypyrazine-2-carboxylate (4). In compounds 1 and 3, hydrogen-bonding interactions give two-dimensional sheet structures which feature enlarged cyclic ring systems, while in compounds 2 and 4, three-dimensional structures are found. The previously described cyclic R2/2(8) hydrogen-bonded amide-amide dimer is present in 2 and 3. The hydrogen-bonding in 2 involves the partial-occupancy water molecule while the structure of 4 is based on inter-linked homomolecular hydrogen-bonded cation-cation and anion-anion associated chains comprising head-to-tail interactions. This work further demonstrates the utility of the isonipecotamide cation in the generation of chemically stable hydrogen-bonded systems, particularly with aromatic carboxylate anions, providing crystalline solids.

Origins of Australian Football: Victoria's Early History. Volume 1 Amateur Heroes and the Rise of the Clubs, 1858 to 1876

This is volume 1 in a series of four volumes about the origins of Australian football as it evolved in Victoria between 1858 and 1896. This volume addresses its very beginnings as an amateur sport and the rise of the first clubs. Invented by a group of Melbourne cricketers and sports enthusiasts, Australian Rules football was developed through games played on Melbourne's park lands and was originally known as "Melbourne Football Club Rules". This formative period of the game saw the birth of the first 'amateur heroes' of the game. Players such as T.W. Wills, H.C.A. Harrison, Jack Conway, George O'Mullane and Robert Murray Smith emerged as warriors engaged in individual rugby-type scrimmages. The introduction of Challenge Cups was an important spur for this burgeoning sport. Intense competition and growing rivalries between clubs such as Melbourne, South Yarra, Royal Park, and Geelong began to flourish and the game developed as a result. By the 1870s the game "Victorian Rules" had become the most popular outdoor winter sport across the state. In subsequent decades, rapid growth in club football occurred and the game attracted increasing media attention.

Stable strong order 1.0 schemes for solving stochastic ordinary differential equations

The Balanced method was introduced as a class of quasi-implicit methods, based upon the Euler-Maruyama scheme, for solving stiff stochastic differential equations. We extend the Balanced method to introduce a class of stable strong order 1. 0 numerical schemes for solving stochastic ordinary differential equations. We derive convergence results for this class of numerical schemes. We illustrate the asymptotic stability of this class of schemes is illustrated and is compared with contemporary schemes of strong order 1. 0. We present some evidence on parametric selection with respect to minimising the error convergence terms. Furthermore we provide a convergence result for general Balanced style schemes of higher orders.

Differential expression and distribution of syndecan-1 and -2 in periodontal wound healing of the rat

Cell-surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell–cell and cell–matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan-1 and -2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan-1, or -2 core proteins. Both syndecan-1 and -2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan-1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cell infiltration. During granulation tissue formation it was noted in fibroblast-like cells and newly formed blood vessels. Syndecan-1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan-1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan-2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan-2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan-2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan-1 and -2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan-1 may involve both cell–cell and cell–matrix interactions, while syndecan-2 showed a predilection to associate with cell–matrix interactions during hard tissue formation.

National Institute for Clinical Studies Report for Phase 1, Evidence Uptake Network : Best Practice Community Care for Clients with Chronic Venous Leg Ulcers

Chronic leg ulcers cause significant pain, cost, decreased quality of life and morbidity for a considerable segment of the older population (Graham et al., 2003a). At any given time the prevalence of patients with open leg ulcers receiving treatment is between 0.11% - 0.18% (Briggs & Closs 2003). Chronic leg ulcers occur in approximately 1 - 2% of the over 60 population in the US, UK, Europe and Australia (Baker & Stacey 1994; Johnson 1995; Lees & Lambert 1992; Margolis et al. 2002). Considerable research has been undertaken to determine the best treatment practices that will aid in the management and the healing of these ulcers, and practical and effective strategies and techniques for healing venous leg ulcers have been trialled to demonstrate their beneficial effects (Nelson et al. 2004; Cullum et al. 2001)...

In vitro and in vivo studies on protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) that is activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. Cleavage of this receptor exposes a neoepitope, termed the tethered ligand (TL), which binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptides (agonist peptides; APs) that are homologous to the TL sequence. Due to the irreversible nature of PAR2 proteolysis, downstream signal transduction is tightly regulated. Following activation, PAR2 is rapidly uncoupled from downstream signalling by the post-translational modifications phosphorylation and ubiquination which facilitate interactions with â- arrestin. This scaffolding protein couples PAR2 to the internalisation machinery initiating its desensitisation and trafficking through the early and late endosomes followed by receptor degradation. PAR2 is widely expressed in mammalian tissues with key roles for this receptor in cardiovascular, respiratory, nervous and musculoskeletal systems. This receptor has also been linked to pathological states with aberrant expression and signalling noted in several cancers. In prostate cancer, PAR2 signalling induces migration and proliferation of tumour derived cell lines, while elevated receptor expression has been noted in malignant tissues. Importantly, a role for this receptor has also been suggested in prostate cancer bone metastasis as coexpression of PAR2 and a proteolytic activator has been demonstrated by immunohistochemical analysis. Based on these data, the primary focus of this project has been on two aspects of PAR2 biology. The first is characterisation of cellular mechanisms that regulate PAR2 signalling and trafficking. The second aspect is the role of this receptor in prostate cancer bone metastasis. In addition, to permit these studies, it was first necessary to evaluate the specificity of the commercially available anti-PAR2 antibodies SAM11, C17, N19 and H99. The evaluation of the four commercially available antibodies was assessed using four techniques: immunoprecipitation; Western blot analysis; immunofluorescence; and flow cytometry. These approaches demonstrated that three of the antibodies efficiently detect ectopically expressed PAR2 by each of these techniques. A significant finding from this study was that N19 was the only antibody able to specifically detect N-glycosylated endogenous PAR2 by Western blot analysis. This analysis was performed on lysates from prostate cancer derived cell lines and tissue derived from wildtype and PAR2 knockout mice. Importantly, further evaluation demonstrated that this antibody also efficiently detects endogenous PAR2 at the cell surface by flow cytometry. The anti-PAR2 antibody N19 was used to explore the in vitro role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Significantly, use of the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling experiments using two approaches which showed that PAR2 stably expressed by CHO cells is palmitoylated and that palmitoylation occurs on cysteine 361. Another key finding from this study is that palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ~9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. Importantly, this study also identified that palmitoylation of this receptor within the Golgi apparatus is required for efficient agonist-induced rab11amediated trafficking of PAR2 to the cell surface. Interestingly, palmitoylation is also required for receptor desensitization, as agonist-induced â-arrestin recruitment and receptor degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. Collectively, these data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. This project also evaluated PAR2 residues involved in ligand docking. Although the extracellular loop (ECL)2 of PAR2 is known to be required for agonist-induced signal transduction, the binding pocket for receptor agonists remains to be determined. In silico homology modelling, based on a crystal structure for the prototypical GPCR rhodopsin, and ligand docking were performed to identify PAR2 transmembrane (TM) amino acids potentially involved in agonist binding. These methods identified 12 candidate residues that were mutated to examine the binding site of the PAR2 TL, revealed by trypsin cleavage, as well as of the soluble ligands 2f-LIGRLO-NH2 and GB110, which are both structurally based on the AP SLIGRLNH2. Ligand binding was evaluated from the impact of the mutated residues on PAR2-mediated calcium mobilisation. An important finding from these experiments was that mutation of residues Y156 and Y326 significantly reduced 2f-LIGRLO-NH2 and GB110 agonist activity. L307 was also important for GB110 activity. Intriguingly, mutation of PAR2 residues did not alter trypsin-induced signalling to the same extent as for the soluble agonists. The reason for this difference remains to be further examined by in silico and in vitro experimentation and, potentially, crystal structure studies. However, these findings identified the importance of TM domains in PAR2 ligand docking and will enhance the design of both PAR2 agonists and potentially agents to inhibit signalling (antagonists). The potential importance of PAR2 in prostate cancer bone metastasis was examined using a mouse model. In patients, prostate cancer bone metastases cause bone growth by disrupting bone homeostasis. In an attempt to mimic prostate cancer growth in bone, PAR2 responsive 22Rv1 prostate cancer cells, which form mixed osteoblastic and osteolytic lesions, were injected into the proximal aspect of mouse tibiae. A role for PAR2 was assessed by treating these mice with the recently developed PAR2 antagonist GB88. As controls, animals bearing intra-tibial tumours were also treated with vehicle (olive oil) or the prostate cancer chemotherapeutic docetaxel. The effect of these treatments on bone was examined radiographically and by micro-CT. Consistent with previous studies, 22Rv1 tumours caused osteoblastic periosteal spicule formation and concurrent osteolytic bone loss. Significantly, blockade of PAR2 signalling reduced the osteoblastic and osteolytic phenotype of 22Rv1 tumours in bone. No bone defects were detected in mice treated with docetaxel. These qualitative data will be followed in the future by quantitative micro-CT analysis as well as histology and histomorphometry analysis of already collected tissues. Nonetheless, these preliminary experiments highlight a potential role for PAR2 in prostate cancer growth in bone. In summary, in vitro studies have defined mechanisms regulating PAR2 activation, downstream signalling and trafficking and in vivo studies point to a potential role for this receptor in prostate cancer bone metastasis. The outcomes of this project are that a greater understanding of the biology of PAR2 may lead to the development of strategies to modulate the function of this receptor in disease.

1,1,1-Trichloro-2,2-bis(4-ethoxyphenyl)ethane

In the title compound, C18H19Cl3O2, which is the 4-ethoxyphenyl analogue of the insecticidally active 4-methoxyphenyl compound methoxychlor, the dihedral angle between the two benzene rings is 60.38(13)deg. An intramolecular aromatic C-H...Cl interaction is present.

Ca2+ regulates the Drosophila Stoned-A and Stoned-B proteins interaction with the C2B domain of Synaptotagmin-1.

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca(2+). The Ca(2+)-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca(2+) binding loop region that modulate the Ca(2+)-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca(2+)-binding loop region of C2B domain. The results indicate that Ca(2+)-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca(2+)-bound Synaptotagmin-1 associated synaptic vesicles.