Design, synthesis, and biological activity of novel factor Xa inhibitors: 4-aryloxy substituents of 2,6-diphenoxypyridines

A novel series of triaryloxypyridines have been designed to inhibit factor Xa, a serine protease strategically located in the coagulation cascade. Inhibitor 5e has a K(I) against factor Xa of 0.12nM and is greater than 8000- and 2000-fold selective over two related serine proteases, thrombin and trypsin, respectively. The 4-position of the central pyridine has been identified as a site that tolerates various substitutions without deleterious effects on potency and selectivity. This suggests that the 4-position of the pyridine ring is an ideal site for chemical modifications to identify inhibitors with improved pharmacokinetic characteristics. This investigation has resulted in inhibitor 5d, which has an oral availability of 6% in dogs. The synthesis, in vitro activity, and in vivo profile of this class of inhibitors is outlined.     

Swim exercise training and adaptations in the antioxidant defense system of myocardium of old rats: relationship to swim intensity and duration

We examined a suitable swim program of different intensities and durations that could evoke changes in the myocardial antioxidant capacity in 22-month-old rats. Male rats (Rattus norvegicus) were assigned to either a sedentary control (SE-C) group or one of six trainee groups. Animals were swim-exercised for 4 weeks with either 20 min or 40 min/day, and three intensities, low, moderate and high. Low-intensity at 20 min/day elicited maximum swim velocity (Sv) and endurance capacity (P<0.05). While serum total cholesterol, triglyceride and low-density lipoprotein (LDL-C) levels were significantly reduced, high-density lipoprotein (HDL-C) showed an increase (P<0.05) in low-intensity trained rats (20 min/day) over SE-C. Notable reduction in blood lactate was also evident. Exercise training significantly increased superoxide dismutase (Mn-SOD), decreased lipid peroxidation products, malondialdehyde and lipofuscin in the left and right ventricles. Increased Mn-SOD with concomitant decrease in lipofuscin in left ventricle was significantly greater than in right ventricle. Moderate- to high-intensity exercise was not effective in either reducing lipid peroxidation products or elevating Mn-SOD activity. These data suggest that swim training at low-intensity of 20 min/day is beneficial as a major protective adaptation against oxidative stress in old myocardium.     

Novel indolo[2,1-b]quinazoline analogues as cytostatic agents: synthesis,biological evaluation and structure-activity relationship

In our endeavor to design and synthesize novel anticancer agents, a new series of indoloquinazoline compounds were prepared and tested initially for anticancer activity in vitro against a panel of human cancer cell lines. Most of these compounds exhibited cytotoxic activity in in vitro screens. Compounds were selected and further evaluated using a modified Hollow Fiber Assay for their preliminary in vivo activity against 12 cell lines implanted in the subcutaneous and intraperitoneal compartments in mice. The results indicate that these compounds may constitute a new class of anticancer agents.     

Calmodulin mediated activation of acetyl-CoA carboxylase during aflatoxin production by Aspergillus parasiticus

The relevance of Ca2+-calmodulin-mediated processes in channelling acetate for aflatoxin formation was investigated by studying the influence of trifluoperazine (an anticalmodulin agent) on [14C]-acetate incorporation and activity of acetyl-CoA carboxylase in Aspergillus parasiticus NRRL 2999. Culturing the organism in presence of 0.14 mmol l-1 trifluoperazine resulted in 55% decrease of [14C]-acetate incorporation into aflatoxin B1, along with an 80% decrease in acetyl-CoA carboxylase activity at periods corresponding to maximal aflatoxin production. Concomitant decrement (35%) in the activity of glucose-6-phosphate dehydrogenase indicated decreased availability of reduction potential (NADPH) required for aflatoxin biosynthesis. The ability of calmodulin to activate and trifluoperazine to inhibit acetyl-CoA carboxylase activity in a dose-dependent manner was also noted under in vitro conditions. The combined results suggest calmodulin-mediated activation of acetyl-CoA carboxylase as an important event for aflatoxin production.     

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.     

Persistence and efficacy of spinosad residues in farm stored wheat

Degradation and insecticidal effectiveness of spinosad residues were evaluated in Kansas during November 2000 to November 2001 in farm bins holding wheat (34-metric ton capacity). About 50 kg of hard red winter wheat from each of three bins were brought to the laboratory and treated separately with 1-ml aqueous suspensions of spinosad to provide rates of 0.1, 0.5,1, 3, 6 mg (AI)/kg of wheat. Wheat treated with distilled water served as the control treatment. Untreated and spinosad-treated wheat samples (250 g each) were placed in three plastic pouches of two different mesh sizes, and buried 2.5 cm below the grain surface. Pouches with large mesh openings were used to monitor insect infestations and kernel damage in untreated and spinosad-treated samples. Pouches with small mesh were used for extracting spinosad residues and for conducting laboratory bioassays with adults of the lesser grain borer, Rhyzopertha dominica (F.) and red flour beetle, Tribolium castaneum (Herbst) at 28 degrees C and 65% RH. Wheat temperature and relative humidity near the pouches during the 1 yr of storage ranged from -10 to 32 degrees C and 50 to 70%, respectively. Moisture of wheat samples varied from 12.4 to 13%. Observed spinosad residues on wheat samples were 25% less than the calculated rates of 0.1 to 6 mg/kg. However, these residues were stable during the 1 yr of storage, and killed all R. dominica adults exposed for 14 d in the laboratory. Mortality of T. castaneum adults increased with an increase in spinosad rate. The linear regression slope of LD50s (0.3-2.7 mg/kg) against storage time was not significantly different from zero, indicating no loss in spinosad toxicity to T. castaneum adults. Insect species, insect numbers, and kernel damage over time in wheat samples inside pouches with large mesh openings were highly inconsistent, and failed to accurately characterize spinosad performance. Laboratory bioassays with R. dominica and T castaneum adults using grain from pouches with small mesh openings accurately gauged spinosad persistence and insecticidal activity under the field conditions.     

In vitro reconstitution and preparative purification of complexes between the chemokine receptor CXCR4 and its ligands SDF-1alpha, gp120-CD4 and AMD3100

CXCR4 belongs to the family of G protein-coupled receptors and mediates the various developmental and regulatory effects of the chemokine SDF-1alpha. In addition, CXCR4 acts as a co-receptor along with CD4 for the HIV-1 viral glycoprotein gp120. Recently, there has also been a small molecule described that antagonizes both SDF-1 and gp120 binding to CXCR4. The structural and mechanistic basis for this dual recognition ability of CXCR4 is unknown largely due to the technical challenges of biochemically producing the components of the various complexes. We expressed the human CXCR4 receptor using a modified baculovirus expression vector that facilitates a single step antibody affinity purification of CXCR4 to >80% purity from Hi5 cells. The recombinant receptor undergoes N-linked glycosylation, tyrosine sulfation and is recognized by the 12G5 conformation specific antibody against human CXCR4. We are able to purify CXCR4 alone as well as complexed with its endogenous ligand SDF-1, its viral ligand gp120, and a small molecule antagonist AMD3100 by ion-exchange chromatography. We anticipate that the expression and purification scheme described in this paper will facilitate structure-function studies aimed at elucidating the molecular basis for CXCR4 recognition of its endogenous chemokine and viral ligands.     

Thermo-optically Induced Reorganizations in the Main Light Harvesting Antenna of Plants. I. Non-Arrhenius Type of Temperature Dependence and Linear Light-intensity Dependencies

Thermo-optically induced structural reorganizations have earlier been identified in isolated LHCII, the main chlorophyll a/b light harvesting complexes of Photosystem II, and in granal thylakoid membranes [Cseh et al. (2000) Biochemistry 39: 15250–15257; Garab et al. (2002) Biochemistry 41: 15121–15129]. According to the thermo-optic mechanism, structural changes can be induced by fast, local thermal transients due to the dissipation of excess excitation energy. In this paper, we analyze the temperature and light-intensity dependencies of thermo-optically induced reversible and irreversible reorganizations in the chiral macrodomains of lamellar aggregates of isolated LHCII and of granal thylakoid membranes. We show that these structural changes exhibit non-Arrhenius type of temperature dependencies, which originate from the ‘combination’ of the ambient temperature and the local thermal transient. The experimental data can satisfactorily be simulated with the aid of a simple mathematical model based on the thermo-optic effect. The model also predicts, in good accordance with experimental data published earlier and presented in this paper, that the reorganizations depend linearly on the intensity of the excess light, a unique property that is probably important in light adaptation and photoprotection of plants.     

Cell Wall Composition of Neurospora crassa Under Conditions of Copper Toxicity

The mycelia of Neurospora crassa grown in the presence of high concentrations of copper were blue in color, but only on a medium containing inorganic nitrate and phosphate as the nitrogen and phosphate sources, respectively. The cell wall isolate of the blue mycelia contained large amounts (12%) of copper and higher amounts of chitosan, phosphate, and amino groups, with a 42% decrease in the chitin content. Although all the glucosamine of the cell wall of control cultures could be released within 6 h of hydrolysis with acid, that of the blue mycelium required prolonged hydrolysis for 24 h. On removal of copper, the cell wall of the blue mycelium could quantitatively bind again to copper as well as to zinc. Although zinc binding was fivefold greater, copper alone was preferentially bound from a mixture of the two metal ions. Supplementation of iron along with copper in the culture medium resulted in the disappearance of the blue color of the mycelium and restoration of normal growth and composition of the cell wall, probably by limiting the uptake of copper from the medium. The possibility of the cell wall being a specific site of lesion in copper toxicity in the mold is discussed.     

Reversible changes in structure and function of photosynthetic apparatus of pea (Pisum sativum) leaves under drought stress

The effects of drought on photosynthesis have been extensively studied, whereas those on thylakoid organization are limited. We observed a significant decline in gas exchange parameters of pea (Pisum sativum) leaves under progressive drought stress. Chl a fluorescence kinetics revealed the reduction of photochemical efficiency of photosystem (PS)II and PSI. The non-photochemical quenching (NPQ) and the levels of PSII subunit PSBS increased. Furthermore, the light-harvesting complexes (LHCs) and some of the PSI and PSII core proteins were disassembled in drought conditions, whereas these complexes were reassociated during recovery. By contrast, the abundance of supercomplexes of PSII-LHCII and PSII dimer were reduced, whereas LHCII monomers increased following the change in the macro-organization of thylakoids. The stacks of thylakoids were loosely arranged in drought-affected plants, which could be attributed to changes in the supercomplexes of thylakoids. Severe drought stress caused a reduction of both LHCI and LHCII and a few reaction center proteins of PSI and PSII, indicating significant disorganization of the photosynthetic machinery. After 7 days of rewatering, plants recovered well, with restored chloroplast thylakoid structure and photosynthetic efficiency. The correlation of structural changes with leaf reactive oxygen species levels indicated that these changes were associated with the production of reactive oxygen species.