Influence of concomitant injuries on post-concussion symptoms after a mild traumatic brain injury - a prospective multicentre cohort study.

Objectives: To compare post-concussion symptoms (PCS) and return to normal activities between mild Traumatic Brain Injury (mTBI) patients with or without concomitant injuries at 7-and 90 days post-mTBI.Methods: Design: Sub-analysis of a multicentre prospective cohort study. PARTICIPANTS AND SETTING: patients with mTBI from 7 Canadian Emergency Departments. PROCEDURE: Research assistants conducted telephone follow-ups using the Rivermead Postconcussion Symptoms Questionnaire (RPQ) at 7-, 30- and 90 days post-mTBI. MAIN OUTCOME: Presence of PCS (RPQ: ≥3 symptoms) at 90 days. SECONDARY OUTCOMES: RPQ score ≥21, prevalence of individual RPQ symptoms and patients' return to normal activities, at 7- and 90-days. Adjusted risk ratios (RR) were calculated.Results: 1725 mTBI patients were included and 1055 (61.1%) had concomitant injuries. Patients with concomitant injuries were at higher risk of having ≥3 symptoms on the RPQ (RR:1.26 [95% CI 1.01-1.58]) at 90 days. They were also at higher risk of experiencing specific symptoms (dizziness, fatigue, headaches and taking longer to think) and of non-return to their normal activities (RR:2.11 [95% CI 1.30-3.45]).Conclusion: Patients with concomitant injuries have slightly more PCS and seemed to be at higher risk of non-return to their normal activities 90 days, compared to patients without concomitant injuries.

Vector-apodizing phase plate coronagraph: design, current performance, and future development [Invited].

Over the last decade, the vector-apodizing phase plate (vAPP) coronagraph has been developed from concept to on-sky application in many high-contrast imaging systems on 8 m class telescopes. The vAPP is a geometric-phase patterned coronagraph that is inherently broadband, and its manufacturing is enabled only by direct-write technology for liquid-crystal patterns. The vAPP generates two coronagraphic point spread functions (PSFs) that cancel starlight on opposite sides of the PSF and have opposite circular polarization states. The efficiency, that is, the amount of light in these PSFs, depends on the retardance offset from a half-wave of the liquid-crystal retarder. Using different liquid-crystal recipes to tune the retardance, different vAPPs operate with high efficiencies (${\gt}96\%$) in the visible and thermal infrared (0.55 µm to 5 µm). Since 2015, seven vAPPs have been installed in a total of six different instruments, including Magellan/MagAO, Magellan/MagAO-X, Subaru/SCExAO, and LBT/LMIRcam. Using two integral field spectrographs installed on the latter two instruments, these vAPPs can provide low-resolution spectra (${\rm{R}} \sim 30$) between 1 µm and 5 µm. We review the design process, development, commissioning, on-sky performance, and first scientific results of all commissioned vAPPs. We report on the lessons learned and conclude with perspectives for future developments and applications.

Generalized schemes for high-throughput manipulation of the Desulfovibrio vulgaris genome.

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

Effect of pimobendan on echocardiographic values in dogs with asymptomatic mitral valve disease.

BACKGROUND: Pimobendan (PIMO) is a novel inodilator that has shown promising results in the treatment of advanced mitral valve disease (MVD), but little is known about its hemodynamic effects, especially regarding the mitral regurgitant volume in naturally occurring MVD. HYPOTHESIS: The addition of pimobendan to treatment decreases the regurgitant fraction (RF) in dogs with asymptomatic MVD. ANIMALS: Twenty-four client-owned dogs affected by International Small Animal Cardiac Health Council class Ib MVD. METHODS: Prospective, blinded, and controlled clinical trial. Dogs were assigned to a PIMO treatment group (n = 19) (0.2-0.3 mg/kg q12h) or a control group (n = 5). Echocardiographic evaluations were performed over a 6-month period. RESULTS: The addition of PIMO to treatment did not decrease the RF of dogs affected by asymptomatic class 1b MVD over the study period (P= .85). There was a significant increase in the ejection fraction of the PIMO treated dogs at 30 days (80.8 +/- 1.42 versus 69.0 +/- 2.76, corrected P= .0064), and a decrease in systolic left ventricular diameter (corrected P= .011) within the PIMO group compared with baseline. However, this improvement in systolic function was not sustained over the 6-month trial period. CONCLUSION AND CLINICAL IMPORTANCE: This study did not identify beneficial long-term changes in the severity of mitral regurgitation after addition of PIMO to angiotensin converting enzyme inhibitor treatment of dogs with asymptomatic MVD.

A novel form of ataxia oculomotor apraxia characterized by oxidative stress and apoptosis resistance.

Several different autosomal recessive genetic disorders characterized by ataxia with oculomotor apraxia (AOA) have been identified with the unifying feature of defective DNA damage recognition and/or repair. We describe here the characterization of a novel form of AOA showing increased sensitivity to agents that cause single-strand breaks (SSBs) in DNA but having no gross defect in the repair of these breaks. Evidence for the presence of residual SSBs in DNA was provided by dramatically increased levels of poly (ADP-ribose)polymerase (PARP-1) auto-poly (ADP-ribosyl)ation, the detection of increased levels of reactive oxygen/nitrogen species (ROS/RNS) and oxidative damage to DNA in the patient cells. There was also evidence for oxidative damage to proteins and lipids. Although these cells were hypersensitive to DNA damaging agents, the mode of death was not by apoptosis. These cells were also resistant to TRAIL-induced death. Consistent with these observations, failure to observe a decrease in mitochondrial membrane potential, reduced cytochrome c release and defective apoptosis-inducing factor translocation to the nucleus was observed. Apoptosis resistance and PARP-1 hyperactivation were overcome by incubating the patient's cells with antioxidants. These results provide evidence for a novel form of AOA characterized by sensitivity to DNA damaging agents, oxidative stress, PARP-1 hyperactivation but resistance to apoptosis.

PARP-3 associates with polycomb group bodies and with components of the DNA damage repair machinery.

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.

A high level of cyclooxygenase-2 inhibitor selectivity is associated with a reduced interference of platelet cyclooxygenase-1 inactivation by aspirin.

Both nonsteroidal anti-inflammatory drugs, such as ibuprofen, and the prototypical selective cyclooxygenase (Cox)-2 inhibitors DuP-697 and NS-398 block the inhibition of Cox-1 by aspirin in vitro. However, clinical studies have shown that the Cox-2 selective drugs (or coxibs) rofecoxib and etoricoxib, at therapeutic doses, do not interfere with the antiplatelet effect of aspirin, in contrast to ibuprofen. Here, we have evaluated the relative potential of ibuprofen and various coxibs to interfere with the inactivation of Cox-1 by aspirin by using purified enzyme and calcium ionophore-activated human platelets. The irreversible inactivation of Cox-1 by aspirin can be antagonized by ibuprofen and coxibs, albeit with widely different potencies. The rank order of potencies for this process (ibuprofen > celecoxib > valdecoxib > rofecoxib > etoricoxib) parallels that obtained for the inhibition of Cox-1-mediated thromboxane B(2) production by calcium ionophore-stimulated platelets. The antagonism of aspirin therefore likely involves a competition at the enzyme active site. The EC(50) value for the antagonism against 10 microM aspirin for each drug is approximately 10- to 40-fold lower than the corresponding IC(50) value for inhibition of platelet Cox-1 activity, consistent with the much weaker initial binding of aspirin to Cox-1 as compared with arachidonic acid. These results show that a low affinity for Cox-1 and a high degree of Cox-2 selectivity confers a low potential to block aspirin inhibition of platelet Cox-1, consistent with the results of clinical studies.

Mechanism of acetaminophen inhibition of cyclooxygenase isoforms.

Acetaminophen has similar analgesic and antipyretic properties to nonsteroidal antiinflammatory drugs (NSAIDs), which act via inhibition of cyclooxygenase enzymes. However, unlike NSAIDs, acetaminophen is at best weakly antiinflammatory. The mechanism by which acetaminophen exerts its therapeutic action has yet to be fully determined, as under most circumstances, acetaminophen is a very weak cyclooxygenase inhibitor. The potency of acetaminophen against both purified ovine cyclooxygenase-1 (oCOX-1) and human cyclooxygenase-2 (hCOX-2) was increased approximately 30-fold by the presence of glutathione peroxidase and glutathione to give IC50 values of 33 microM and 980 microM, respectively. Acetaminophen was found to be a good reducing agent of both oCOX-1 and hCOX-2. The results are consistent with a mechanism of inhibition of acetaminophen in which it acts to reduce the active oxidized form of COX to the resting form. Inhibition would therefore be more effective under conditions of low peroxide concentration, consistent with the known tissue selectivity of acetaminophen.

Etoricoxib (MK-0663): preclinical profile and comparison with other agents that selectively inhibit cyclooxygenase-2.

We report here the preclinical profile of etoricoxib (MK-0663) [5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonylphenyl) pyridine], a novel orally active agent that selectively inhibits cyclooxygenase-2 (COX-2), that has been developed for high selectivity in vitro using whole blood assays and sensitive COX-1 enzyme assays at low substrate concentration. Etoricoxib selectively inhibited COX-2 in human whole blood assays in vitro, with an IC(50) value of 1.1 +/- 0.1 microM for COX-2 (LPS-induced prostaglandin E2 synthesis), compared with an IC(50) value of 116 +/- 8 microM for COX-1 (serum thromboxane B2 generation after clotting of the blood). Using the ratio of IC(50) values (COX-1/COX-2), the selectivity ratio for the inhibition of COX-2 by etoricoxib in the human whole blood assay was 106, compared with values of 35, 30, 7.6, 7.3, 2.4, and 2.0 for rofecoxib, valdecoxib, celecoxib, nimesulide, etodolac, and meloxicam, respectively. Etoricoxib did not inhibit platelet or human recombinant COX-1 under most assay conditions (IC(50) > 100 microM). In a highly sensitive assay for COX-1 with U937 microsomes where the arachidonic acid concentration was lowered to 0.1 microM, IC(50) values of 12, 2, 0.25, and 0.05 microM were obtained for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively. These differences in potency were in agreement with the dissociation constants (K(i)) for binding to COX-1 as estimated from an assay based on the ability of the compounds to delay the time-dependent inhibition by indomethacin. Etoricoxib was a potent inhibitor in models of carrageenan-induced paw edema (ID(50) = 0.64 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 0.34 mg/kg), LPS-induced pyresis (ID(50) = 0.88 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.6 mg/kg/day) in rats, without effects on gastrointestinal permeability up to a dose of 200 mg/kg/day for 10 days. In squirrel monkeys, etoricoxib reversed LPS-induced pyresis by 81% within 2 h of administration at a dose of 3 mg/kg and showed no effect in a fecal 51Cr excretion model of gastropathy at 100 mg/kg/day for 5 days, in contrast to lower doses of diclofenac or naproxen. In summary, etoricoxib represents a novel agent that selectively inhibits COX-2 with 106-fold selectivity in human whole blood assays in vitro and with the lowest potency of inhibition of COX-1 compared with other reported selective agents.

Ichthyophonus-like infection in wild amphibians from Québec, Canada.

Myositis associated with infection by Ichthyophonus-like organisms was diagnosed in 35 of 260 (13%) wild amphibians collected in Quebec, Canada, from 1959 to 1964 (n = 30), and 1992 to 1999 (n = 230). Infection was diagnosed in 17 green frogs Rana clamitans, 9 wood frogs R. sylvatica, 4 red-spotted newts Notophthalmus viridescens, 3 bullfrogs R. catesbeiana, 1 spring peeper Pseudacris crucifer, and 1 pickerel frog R. palustris. The spring peeper and one of the bullfrogs were collected in 1964 from the Mont Saint-Hilaire Biosphere Reserve, indicating long-term presence of the organism. Spores of the organisms invaded striated muscle fibers and were associated with variable degrees of granulomatous and eosinophilic inflammation. Infection was considered fatal in 2 green frogs, 1 wood frog, and 1 red-spotted newt. It was considered potentially significant in 3 additional green frogs in which up to 100% of the fibers of some muscles were replaced by spores associated with a severe granulomatous reaction. Ultrastructural features of Ichthyophonus-like spores included a thick trilaminated wall, a paramural cytoplasm, multiple nuclei, oval mitochondria with short tubulo-vesicular cristae and numerous ribosomes. This report represents 4 new host records and shows that ichthyophonosis is enzootic in amphibians from Quebec.

p56(lck), ZAP-70, SLP-76, and calcium-regulated effectors are involved in NF-kappaB activation by bisperoxovanadium phosphotyrosyl phosphatase inhibitors in human T cells.

This study investigates the second messengers involved in NF-kappaB activation by the bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors. We first initiated a time course analysis of bpV-mediated activation of the human immunodeficiency virus type-1 long terminal repeat- and NF-kappaB-driven reporter gene. Our results showed a slower and more transient activation of both kappaB-regulated luciferase-encoding vectors by bpV compounds when compared with the action of tumor necrosis factor-alpha (TNF). Time course analyses of NF-kappaB translocation by shift assay experiments further confirmed these results, hence implying distinct pathways of NF-kappaB activation for bpV compounds and TNF. Attempts to characterize the bpV-dependent signaling cascade revealed that the src family protein tyrosine kinase p56(lck) was critical for NF-kappaB induction by bpV. Furthermore, p56(lck) interaction with the intracytoplasmic tail of CD4 markedly enhanced such induction. Optimal activation of NF-kappaB following bpV treatment necessitated downstream effectors of p56(lck) such as the syk family protein tyrosine kinase ZAP-70 and the molecular adaptor SLP-76. Importantly, reduced NF-kappaB activation was observed when capacitative calcium entry was deficient but also upon pharmacological inhibition of calmodulin and calcineurin. Altogether, these results suggest that induction of NF-kappaB by phosphotyrosyl phosphatase bpV inhibitors necessitates both proximal and distal effectors of T cell activation.

Seminal vesicle production and secretion of growth hormone into seminal fluid.

Production of foreign proteins in the tissues of transgenic animals represents an efficient and economical method of producing therapeutic and pharmaceutical proteins. In this study, we demonstrate that the mouse P12 gene promoter specific to the male accessory sex gland can be used to generate transgenic mice that express human growth hormone (hGH) in their seminal vesicle epithelium. The hGH is secreted into the ejaculated seminal fluids with the seminal vesicle lumen contents containing concentrations of up to 0.5 mg/ml. As semen is a body fluid that can be collected easily on a continuous basis, the production of transgenic animals expressing pharmaceutical proteins into their seminal fluid could prove to be a viable alternative to use of the mammary gland as a bioreactor.

Inhibition of cathepsin K by nitric oxide donors: evidence for the formation of mixed disulfides and a sulfenic acid.

The cysteine protease cathepsin K is believed to play a key role in bone resorption as it has collagenolytic activity and is expressed predominantly and in high levels in bone resorbing osteoclast cells. The addition of nitric oxide (NO) and NO donors to osteoclasts in vitro results in a reduction of bone resorption, although the mechanism of this effect is not fully understood. The S-nitroso derivatives of glutathione (GSNO) and N-acetylpenicillamine (SNAP) and the non-thiol NO donors NOR-1 and NOR-3 all inhibited the activity of purified cathepsin K in a time- and concentration-dependent manner (IC(50) values after 15 min of preincubation at pH 7.5 of 28, 105, 0.4, and 10 microM, respectively). Cathepsin K activity in Chinese hamster ovary cells stably transfected with cathepsin K was also inhibited by the above NO donors with similar potencies. GSNO at 100 microM also completely inhibited the autocatalytic maturation at pH 4.0 of procathepsin K to cathepsin K. The inhibition of cathepsin K by GSNO was rapidly reversed by DTT, but inhibition by NOR-1 was not reversed by DTT, and analysis of the inhibited cathepsin K for S-nitrosylation using the Greiss reaction gave negative results in both cases. Analysis of the protein by electrospray liquid chromatography/mass spectrometry showed that the inhibition of cathepsin K by GSNO resulted in a mass increase of 306 +/- 2 Da, consistent with the formation of a glutathione adduct. Prior inhibition of cathepsin K by the active site thiol-modifying inhibitor E-64 blocked the modification by GSNO, indicating that the glutathione adduct is likely formed at the active site cysteine. Treatment of cathepsin K with NOR-1 resulted in a mass increase of between 30 and 50 Da, corresponding to the oxidation of a cysteine to sulfinic and sulfonic acids. Cotreatment of cathepsin K with NOR-1 plus the sulfenic acid reagent dimedone resulted in a mass increase of approximately 141 Da, which is consistent with the formation of a dimedone adduct. This result demonstrates that the NOR-1-dependent formation of cathepsin K sulfinic and sulfonic acids occurs via a sulfenic acid. These results show that inhibition of cathepsin K activity and its autocatalytic maturation represent two potential mechanisms by which NO can exert its inhibitory effect on bone resorption. This work also shows that oxidative thiol modifications besides S-nitrosylation should be considered when the effects of NO and NO donors on critical thiol-containing proteins are investigated.

The discovery of rofecoxib, [MK 966, Vioxx, 4-(4'-methylsulfonylphenyl)-3-phenyl-2(5H)-furanone], an orally active cyclooxygenase-2-inhibitor.

The development of a COX-2 inhibitor rofecoxib (MK 966, Vioxx) is described. It is essentially equipotent to indomethacin both in vitro and in vivo but without the ulcerogenic side effect due to COX-1 inhibition.

Rofecoxib [Vioxx, MK-0966; 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone]: a potent and orally active cyclooxygenase-2 inhibitor. Pharmacological and biochemical profiles.

The discoveries that cyclooxygenase (COX)-2 is an inducible form of COX involved in inflammation and that COX-1 is the major isoform responsible for the production of prostaglandins (PGs) in the gastrointestinal tract have provided a rationale for the development of specific COX-2 inhibitors as a new class of anti-inflammatory agents with improved gastrointestinal tolerability. In the present study, the preclinical pharmacological and biochemical profiles of rofecoxib [Vioxx, also known as MK-0966, 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone], an orally active COX-2 inhibitor, are described. Rofecoxib is a potent inhibitor of the COX-2-dependent production of PGE(2) in human osteosarcoma cells (IC(50) = 26 +/- 10 nM) and Chinese hamster ovary cells expressing human COX-2 (IC(50) = 18 +/- 7 nM) with a 1000-fold selectivity for the inhibition of COX-2 compared with the inhibition of COX-1 activity (IC(50) > 50 microM in U937 cells and IC(50) > 15 microM in Chinese hamster ovary cells expressing human COX-1). Rofecoxib is a time-dependent inhibitor of purified human recombinant COX-2 (IC(50) = 0.34 microM) but caused inhibition of purified human COX-1 in a non-time-dependent manner that could only be observed at a very low substrate concentration (IC(50) = 26 microM at 0.1 microM arachidonic acid concentration). In an in vitro human whole blood assay, rofecoxib selectively inhibited lipopolysaccharide-induced, COX-2-derived PGE(2) synthesis with an IC(50) value of 0.53 +/- 0.02 microM compared with an IC(50) value of 18.8 +/- 0.9 microM for the inhibition of COX-1-derived thromboxane B(2) synthesis after blood coagulation. Using the ratio of the COX-1 IC(50) values over the COX-2 IC(50) values in the human whole blood assay, selectivity ratios for the inhibition of COX-2 of 36, 6.6, 2, 3, and 0.4 were obtained for rofecoxib, celecoxib, meloxicam, diclofenac, and indomethacin, respectively. In several in vivo rodent models, rofecoxib is a potent inhibitor of carrageenan-induced paw edema (ID(50) = 1.5 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 1.0 mg/kg), lipopolysaccharide-induced pyresis (ID(50) = 0.24 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.74 mg/kg/day). Rofecoxib also has a protective effect on adjuvant-induced destruction of cartilage and bone structures in rats. In a (51)Cr excretion assay for detection of gastrointestinal integrity in either rats or squirrel monkeys, rofecoxib has no effect at doses up to 200 mg/kg/day for 5 days. Rofecoxib is a novel COX-2 inhibitor with a biochemical and pharmacological profile clearly distinct from that of current nonsteroidal anti-inflammatory drugs and represents a new therapeutic class of anti-inflammatory agents for the treatment of the symptoms of osteoarthritis and rheumatoid arthritis with improved gastrointestinal tolerability.

Testes-specific transgene expression in insulin-like growth factor-I transgenic mice.

Insulin-like growth factor-I (IGF-I) is a low molecular weight peptide that mediates the cell proliferating actions of growth hormone. Evidence exists indicating that IGF-I is produced by various cell types and this growth factor has been implicated in a variety of reproductive processes. To investigate the effect of IGF-I over-expression on reproductive systems, we generated three independent lines of transgenic mice harbouring a human IGF-I cDNA (hIGF-I) under the control of a Cytomegalovirus immediate early (CMV) promoter. The CMV promoter was used in an attempt to direct expression of IGF-I into a variety of tissues both reproductive and non-reproductive. Yet expression of the foreign hIGF-I gene, determined by Northern blot, was found to occur only in the testicular tissues of the male mice, apparently due to methylation of the transgene in all the tissues tested except the testes, which demonstrate transgene hypomethylation. Evaluation of the transgene expression during testicular development revealed that expression begins between 10 and 15 days of development, coinciding with the appearance of the zygotene and pachytene primary spermatocytes during early spermatogenesis, therefore indicating germ line expression of the transgene. Extensive study of the CMV-hIGF-I transgenic lines of mice has revealed that the effects of the transgene expression do not extend beyond the testicular tissues. No significant differences (P > 0.05) in the IGF-I serum levels, growth rates, or testicular histology have been observed between transgenic and non-transgenic male siblings. The ability of transgenic males to produce offspring also appears unaffected. Evaluation of the IGF binding protein (IGFBP) levels in the testicular tissues of CMV-hIGF-I transgenic mice by Western ligand blot revealed an increase in the concentration of testicular proteins with molecular weights corresponding to IGFBP-2 and IGFBP-3. These results suggest that the testicular over-expression of IGF-I induces increased IGFBP localization in this tissue. Inhibition of IGF activity by the IGFBPs would explain the lack of a dramatic physiological effect in the CMV-hIGF-I transgenic mice, despite the presence of elevated testicular IGF-I. The observation that testis specific IGF-I overexpression induces localization of IGFBPs in this tissue confirms the existence of a well regulated testicular IGF system and supports the convention that this growth factor plays an important role in testicular function.

Structure-based design of COX-2 selectivity into flurbiprofen.

Comparative computer modeling of the X-ray crystal structures of cyclooxygenase isoforms COX-1 and COX-2 has led to the design of COX-2 selectivity into the nonselective inhibitor flurbiprofen. The COX-2 modeling was based on a postulated binding mode for flurbiprofen and took advantage of a small alcove in the COX-2 active site created by different positions of the Leu384 sidechain between COX-1 and COX-2. The design hypothesis was tested by synthesis and biological assay of a series of flurbiprofen analogs, culminating in the discovery of several inhibitors having up to 78-fold selectivity for COX-2 over COX-1.

Substituted heterocyclic analogs as selective COX-2 inhibitors in the flosulide class.

Substituted heterocyclic analogs in the Flosulide class were investigated as potential selective cyclooxygenase-2 inhibitors. 6-(4-Ethyl-2-thiazolylthio)-5-methanesulfonamido-3H-isobe nzofuran-1-one 14 was found to be the optimal compound in the series with superior in vitro and in vivo activities.

Analysis of prostaglandin G/H synthase-2 inhibition using peroxidase-induced luminol luminescence.

The inducible form of the heme-protein prostaglandin G/H synthase (PGHS-2 or COX-2) has been established as a pivotal enzyme in the cascade of events leading to inflammation, hyperalgesia, and pyresis and represents a major therapeutic target in inflammatory disease. Accordingly, we have exploited the heme-catalyzed hydroperoxidase activity of recombinant hCOX-2 to generate luminescence in the presence of luminol, or a cyclic naphthalene hydrazide, and the substrate arachidonic acid. Arachidonate-induced luminescence was shown to be an index of real-time catalytic activity and demonstrated the turnover inactivation of the enzyme. Luminol luminescence was proportional to hCOX-2 concentration and gave accurate Km determinations for arachidonate. Inhibition of hCOX-2 activity, measured by luminescence, by a variety of selective (for COX-2) and nonselective inhibitors showed rank orders of potency similar to those observed with other in vitro and whole cell methods using the recombinant protein. The sensitivity of the luminescence assay also allowed determination of inhibitor potency at substrate concentrations below Km, distinguishing competitive inhibitors such as ibuprofen from time-dependent inhibitors such as DuP-697. Finally the use of higher quantum-yielding luminol analogues allowed measurement of cyclooxygenase activity at extremely low substrate and protein concentrations, enabling a variety of novel assay formats.

The interaction of arginine 106 of human prostaglandin G/H synthase-2 with inhibitors is not a universal component of inhibition mediated by nonsteroidal anti-inflammatory drugs.

The three-dimensional cocrystal structures of ovine prostaglandin G/H synthase-1 (PGHS-1) with S-flurbiprofen and murine PGHS-2 with S-flurbiprofen and indomethacin reveal that the carboxylate acid groups of these nonsteroidal anti-inflammatory drugs (NSAIDs) form a salt bridge with the guanidinium group of Arg120 in PGHS-1 and Arg106 in PGHS-2. Mutagenesis studies confirmed that the Arg120 residue of PGHS-1 is critical for binding of substrate and inhibitors through ionic interactions of its guanidinium group with the carboxylate moieties of arachidonic acid and certain NSAIDs. We report here that the analogous R106E substitution in human PGHS-2 results in a catalytically active enzyme with a 30-fold higher Km value for arachidonic acid. Comparison of the inhibition of hPGHS-2(R106E) with wild-type hPGHS-2 by 11 structurally diverse selective and nonselective PGHS inhibitors revealed a 0-1000-fold decrease in inhibitory potency on the mutant enzyme. The loss of inhibitory potency of NSAIDs on hPGHS-2(R106E) could not be correlated with the presence or absence of a carboxylate functional group in the inhibitor, as was demonstrated previously for the PGHS-1(R120E) mutant, or with the selective or nonselective nature of the PGHS inhibitor. The decreases in the inhibitory potencies on hPGHS-2(R106E) by the carboxylate-containing NSAIDs flurbiprofen, indomethacin, meclofenamic acid, and diclofenac on hPGHS-2(R106E) were 965-, 48-, 5.5-, and 4.5-fold, respectively. The nonuniversal requirement for interaction of the carboxylate group of certain NSAIDs with the Arg106 residue in hPGHS-2 is supported by the observation that the methyl ester derivative of indomethacin was a more potent inhibitor than indomethacin on both hPGHS-2 and hPGHS-2(R106E). The greatest loss of potency for inhibition of hPGHS-2(R106E) was observed with the hPGHS-2-selective sulfonamide-containing inhibitors NS-398 and flosulide. The PGHS-2-selective inhibitor DuP697 and a desbromo-sulfonamide analogue of DuP697 displayed equivalent potency on hPGHS-2(R106E) and hPGHS-2. The change in inhibitory potency of NS-398 on hPGHS-2(R106E) was due to a difference in the kinetics of inhibition, with NS-398 displaying time-dependent inhibition of hPGHS-2 but time-independent inhibition of PGHS-2(R106E). The time-dependent inhibition of hPGHS-2 by DuP697 was not affected by the presence of the R106E mutation. We conclude that the Arg106 residue of hPGHS-2 is involved in binding arachidonic acid and certain NSAIDs, but interactions with Arg106 are not a universal requirement for inhibition by either carboxylate-containing NSAIDs or PGHS-2-selective inhibitors.