A genetic polymorphism of matrix metalloproteinase 9 (MMP-9) affects the changes in circulating MMP-9 levels induced by highly active antiretroviral therapy in HIV patients

We examined whether two functional polymorphisms (g.-1562C>T and g.-90(CA)14-24) in the matrix metalloproteinase (MMP)-9 gene or MMP-9 haplotypes affect the circulating levels of pro-MMP-9 and pro-MMP-9/TIMP-1 (tissue inhibitor of metalloproteinase-1) ratios in AIDS patients, and modulate alterations in these biomarkers after highly active antiretroviral therapy (HAART). We studied 82 patients commencing HAART. Higher pro-MMP-9 concentrations and pro-MMP-9/TIMP-1 ratios were found in CT/TT patients compared with CC patients. HAART decreased pro-MMP-9 levels and pro-MMP-9/TIMP-1 ratios in CT/TT patients, it did not modify pro-MMP-9 levels and it increased pro-MMP-9/TIMP-1 ratios in CC patients. The g.-90(CA)14-24 polymorphism, however, produced no significant effects. Moreover, we found no significant differences in HAART-induced changes in plasma pro-MMP-9, TIMP-1 and pro-MMP-9/TIMP-1 ratios when different MMP-9 haplotypes were compared. These findings suggest that the g.-1562C>T polymorphism affects pro-MMP-9 levels in patients with AIDS and modulates the alterations in pro-MMP-9 levels caused by HAART, thus possibly affecting the risk of cardiovascular complications. The Pharmacogenomics Journal (2009) 9, 265-273; doi: 10.1038/tpj.2009.13; published online 21 April 2009

Unprecedented oxidation of a biologically active aroylhydrazone chelator catalysed by iron(III): serendipitous identification of diacylhydrazine ligands with high iron chelation efficacy

Ligands of the 2-pyridylcarbaldehyde isonicotinoylhydrazone class show high iron (Fe) sequestering efficacy and have potential as agents for the treatment of Fe overload disease. We have investigated the mechanisms responsible for their high activity. X-ray crystallography studies show that the tridentate chelate 2-pyridylcarbaldehyde isonicotinoylhydrazone undergoes an unexpected oxidation to isonicotinoyl(picolinoyl)hydrazine when complexed with Fe-III. In contrast, in the absence of Fel the parent hydrazone is not oxidized in aerobic aqueous solution. To examine whether the diacylhydrazine could be responsible for the biological effects of 2-pyridylcarbaldehyde isonicotinoylhydrazone, their Fe chelation efficacy was compared. In contrast to its parent hydrazone, the diacylhydrazine showed little Fe chelation activity. Potentiometric titrations suggested that this might be because the diacylhydrazine was charged at physiological pH, hindering its access across membranes to intracellular Fe pools. In contrast, the Fe complex of this diacylhydrazine was charge neutral, which may allow facile movement through membranes. These data allow a model of Fe chelation for this compound to be proposed: the parent aroylhydrazone diffuses through cell membranes to bind Fe and is subsequently oxidized to the diacylhydrazine complex which then diffuses from the cell. Other diacylhydrazine analogues that were charge neutral at physiological pH demonstrated high Fe chelation efficacy. Thus, for this class of ligands, the charge of the chelator appears to be an important factor for determining their ability to access intracellular Fe. The results of this study are significant for understanding the biological activity of 2-pyridylcarbaldehyde isonicotinoylhydrazone and for the design of novel diacylhydrazine chelators for clinical use.

Formation of cumulenes, triple-bonded, and related compounds by flash vacuum thermolysis of five-membered heterocycles

Flash vacuum thermolysis of a large variety of heterocyclic compounds is a useful means of production of ketenes, ketenimines, thioketenes, allenes, iminopropadienones, bis(imino)propadienes, iminopropadienethiones, carbodiimides, isothiocyanates, acetylenes, fulminic acid, nitrile imines and nitrile ylides, nitriles, cyanamides, cyanates, and other compounds, often in preparatively useful yields.

Inhibition of proliferation of HT-29 colon adenocarcinorna cells by carboxylate NSAIDs and their acyl glucuronides

Many nonsteroidal anti-inflammatory drugs (NSAIDs) which have antiproliferative activity in colon cancer cells are carboxylate compounds forming acyl glucuronide metabolites. Acyl glucuronides are potentially reactive, able to hydrolyse, rearrange into isomers, and covalently modify proteins under physiological conditions. This study investigated whether the acyl glucuronides (and isomers) of the carboxylate NSAIDs diflunisal, zomepirac and diclofenac had antiproliferative activity on human adenocarcinoma. HT-29 cells in culture. Included as controls were the carboxylate NSAIDs themselves, the non-carboxylate NSAID piroxicam, and the carboxylate non-NSAID valproate, as well as its acyl glucuronide and isomers. The compounds were incubated at 1-3000 muM with HT-29 cells for 24 hr, with [H-3]-thymidine added for an additional 2 hr incubation. IC50 values were calculated from the concentration-inhibition response curves for thymidine uptake. The four NSAIDs inhibited thymidine uptake, with IC50 values about 200-500 muM. All of the NSAID acyl glucuronides (and isomers, tested in the case of diflunisal) showed antiproliferative activity broadly comparable to the parent drugs. This activity may stem from direct uptake of intact glucuronide/isomers followed by covalent modification of proteins critical in the cell replication process. However, hydrolysis during incubation and cellular uptake of liberated parent NSAID will play a role. In HT-29 cells incubated with zomepirac, covalently modified proteins in cytosol were detected by immunoblotting with a zomepirac antibody, suggesting that HT-29 cells do have the capacity to glucuronidate zomepirac. The anti-epileptic drug valproate had no effect on inhibition of thymidine uptake, though, surprisingly, its acyl glucuronide and isomers were active. The reasons for this are unclear at present. (C) 2001 Elsevier Science Inc. All rights reserved.

Perceived constraints and social support for active leisure among mothers with young children

Evidence suggests that women who are mothers of young children have lower levels of physical activity than women of similar age who do not have children (Brown, Lee, Mishra, & Bauman, 2000). The purposes of this study were to explore the factors that constrain mothers of young children from being more physically active, and the relationship between physical activity and levels of social support available to the women. The empirical basis for this examination was provided through a study of activity levels and barriers to physical activity experienced by a sample of 543 mothers of young children from differing socioeconomic backgrounds. The data indicate that: (a) more than two-thirds of the mothers were inadequately active in their leisure time for health benefit; (b) while the vast majority of mothers expressed a desire to be more active, they were inhibited in their ability to act out their leisure preferences by a combination of structural (e.g., lack of time, money, energy) and ideological influences (e.g., sense of commitment to others); (c) access to social support (from partners, family, and friends) was seen to place some women in a better position than others to negotiate constraints that inhibit leisure participation; and (d) within groups of varying socioeconomic status (SES) there was wide variation in the amount of time spent each week in active leisure.

Anthropogenic and Natural Organohalogen Compounds in Blubber of Dolphins and Dugongs (Dugong dugon) from Northeastern Australia

A range of organohalogen compounds (10 polychlorinated biphenyl [PCB] congeners, DDT and metabolites, chlordane-related compounds, the potential natural organochlorine compound Q1, toxaphene, hexachlorobenzene, hexachlorocyclohexanes, dieldrin, and several yet unidentified brominated compounds) were detected in the blubber of four bottlenose dolphins (Tursiops truncatus), one common dolphin (Delphinus delphis), and seven dugongs (Dugong dugon), as well as in adipose tissue of a green turtle (Chelonia mydas) and a python (Morelia spilota) from northeast Queensland (Australia). The green turtle and dugongs accumulated lower organohalogen levels than the dolphins. Lower levels in dugongs were expected because this species is exclusively herbivorous. Highest PCB and DDT levels recorded in dugongs were 209 and 173 mug/kg lipids, respectively. Levels of the nonanthropogenic heptachlorinated compound Q1 (highest level in dugongs was 160 mug/kg lipids) were estimated using the ECD response factor of trans-nonachlor. Highest organohalogen levels were found in blubber of dolphins for sumDDT (575-52,500 mug/kg) and PCBs (600-25,500 mug/kg lipids). Furthermore, Q1 was a major organohalogen detected in all samples analyzed, ranging from 450 -9,100 mug/kg lipids. The highest concentration of Q1 determined in this study represents the highest concentration reported to date in an environmental sample. Levels of chlordane-related compounds were also high (280-7,700 mug/kg, mainly derived from trans-nonachlor), but concentrations of hexachlorobenzene, hexachlorocyclohexanes, dieldrin, and toxaphene were relatively low and contributed little to the overall organohalogen contamination. Furthermore, a series of three major (BC-1, BC-2, and BC-3) and six minor (BC-4 through BC-9) unknown brominated compounds were observable by extracting m/z 79 and m/z 81 from the GC/ECNI-MS full scan run. Structural proposals were made for the two major recalcitrant compounds (referred to as BC-1 and BC-2). BC-2 appears to be a tetrabromo-methoxy-diphenylether (512 u) and BC-1 has 14 u (corresponding with an additional CH2 group) more relative to BC-1. In general the organohalogen pattern observed in blubber of dolphins was different compared to similar samples from other locations in the world, which is apparent from the fact that the four major abundant signals in the GC/ECD chromatogram. of D. delphis originated from the four unknown compounds Q1, BC-1, BC-2, and BC-3.

Analytical validation for a series of marker compounds used to assess renal drug elimination processes

Renal drug elimination is determined by glomerular filtration, tubular secretion, and tubular reabsorption. Changes in the integrity of these processes influence renal drug clearance, and these changes may not be detected by conventional measures of renal function such as creatinine clearance. The aim of the current study was to examine the analytic issues needed to develop a cocktail of marker drugs (fluconazole, rac-pindolol, para-aminohippuric acid, sinistrin) to measure simultaneously the mechanisms contributing to renal clearance. High-performance liquid chromatographic methods of analysis for fluconazole, pindolol, para-aminohippuric acid, and creatinine and an enzymatic assay for sinistrin were developed or modified and then validated to allow determination of each of the compounds in both plasma and urine in the presence of all other marker drugs. A pilot clinical study in one volunteer was conducted to ensure that the assays were suitable for quantitating all the marker drugs to the sensitivity and specificity needed to allow accurate determination of individual renal clearances. The performance of all assays (plasma and urine) complied with published validation criteria. All standard curves displayed linearity over the concentration ranges required, with coefficients of correlation greater than 0.99. The precision of the interday and intraday variabilities of quality controls for each marker in plasma and urine were all less than 11.9% for each marker. Recoveries of markers (and internal standards) in plasma and urine were all at least 90%. All markers investigated were shown to be stable when plasma or urine was frozen and thawed. For all the assays developed, there were no interferences from other markers or endogenous substances. In a pilot clinical study, concentrations of all markers could be accurately and reproducibly determined for a sufficient duration of time after administration to calculate accurate renal clearance for each marker. This article presents details of the analytic techniques developed for measuring concentrations of marker drugs for different renal elimination processes administered as a single dose to define the processes contributing to renal drug elimination.

Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobacter capsulatus revealed by raman spectroscopy

Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSORmodD, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other studies. This oxo ligand is found to be exchangeable directly with (DMSO)-O-18 or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm(-1) band, an extra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'as prepared' enzyme which is not present in the 'redox cycled' enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSORmodD form, identified by its characteristic Raman spectrum, is also present in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase.

Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD -> GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity. (C) 2001 Elsevier Science B.V. All rights reserved.

Characterization of pore structure and coordination of titanium in TiO2 and SiO2-TiO2 sol-pillared clays

Titania sol-pillared clay (TiO2 PILC) and silica-titania sol-pillared clay (SiO2-TiO2 PILC) were synthesized by the sol-gel method. Supercritical drying (SCD) and treatment with quaternary ammonium surfactants were used to tailor the pore structure of the resulting clay. It was found that SCD approach increased the external surface area of the PILCs dramatically and that treatment with surfactants could be used to tailor pore size because the mesopore formation in the galleries between the clay layers follows the templating mechanism as observed in the synthesis of MCM-41 materials. Highly mesoporous solids were thus obtained. In calcined TiO2 PILC, ultrafine crystallites in anatase phase, which are active for photocatalytic oxidation of organics, were observed. In SiO2-TiO2 PILCs and their derivatives, titanium was highly dispersed in the matrix of silica and no crystal phase was observed. The highly dispersed titanium sites are good catalytic centers for selective oxidation of organic compounds. (C) 2001 Academic Press.

A Ku-band active transmit-array module with a horn or patch array as a signal launching/receiving device

This paper is concerned with the design of a Ku-band active transmit-array module of transistor amplifiers excited by either a pyramidal horn or a patch array Optimal distances between the active transmit array and the signal-launching:receiving device, which is either a passive corporate-fed array or a horn, are determined to maximise the power gain at a design frequency: Having established these conditions, the complete structure is investigated in terms of operational bandwidth and near-field and far-field distributions measured at the output side of the transmit array, The experimental results show that the use of a corporate-fed array as an illuminating/receiving device gives higher gain and significantly larger operational bandwidth, An explanation for this behavior is sought.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.