Aplysamine-1 and related analogs as histamine H3 receptor antagonists

Aplysamine-1 (1), a marine natural product, was synthesized and screened for in vitro activity at the human and rat histamine H3 receptors. Aplysamine-1 (1) was found to possess a high binding affinity for the human H3 receptor (Ki = 30+/-4 nM). Synthetic analogs of 1, including des-bromoaplysamine-1 (10) and dimethyl-{2-[4-(3-piperidin-1-yl-propoxy)-phenyl]-ethyl}-amine (13), were potent H3 antagonists.     

Effects of bilayer cholesterol on human erythrocyte hexose transport protein activity in synthetic lecithin bilayers

In this study, we describe the effects of altered bilayer cholesterol content on reconstituted, protein-mediated sugar transport. The system used was the human erythrocyte sugar transporter (band 4.5) reconstituted into the bilayers of large unilamellar vesicles. Vesicle preparations were formed from synthetic lecithins whose bilayer cholesterol content ranged from 0 to 50 mol %. Transport was measured by microturbidimetric analysis over the temperature range of 0-65 degrees C while bilayer physical state was characterized by differential scanning calorimetry. Reconstituted transport activity was irreversibly lost between 62 and 65 degrees C. The Km for reconstituted transport was found to increase only slightly with increasing temperature and was not systematically affected by bilayer cholesterol content. The most striking observation of this study is that over certain critical cholesterol concentrations, as little as a 2.5% change in bilayer cholesterol can result in as much as a 100-fold change in Vmax per reconstituted protein. Our findings run counter to the view that increasing bilayer cholesterol content monotonically transforms a membrane into a state of "intermediate fluidity". Abrupt, cholesterol-induced bilayer reorganizations occurring at 15-20 and 30 mol % bilayer cholesterol are markedly reflected in altered sugar transport rates. Increasing the cholesterol content of crystalline distearoyllecithin bilayers inhibits the activity of the reconstituted transporter. It is apparent from these studies that bilayer "fluidity" is neither the sole nor a major determinant of the Indeed, we find the effect of cholesterol on transport activity is independent of its ability to fluidize membranes.     

Effect of bilayer cholesterol content on reconstituted human erythrocyte sugar transporter activity

The influence of altered bilayer cholesterol content on the catalytic activity of the human red cell hexose transporter was examined by reconstitution of the transport protein (band 4.5) into bilayers of large unilamellar vesicles formed from dipalmitoyl lecithin and varying amounts of cholesterol. The physical state of the bilayers was characterized by differential scanning calorimetry. The major findings are as follows: changes in bilayer phase behavior occur at membrane cholesterol levels of 15 to 20 mol % and 30 to 40 mol %; and the catalytic activity of the reconstituted transporter (Vmax/transporter) correlates with bilayer phase behavior. In crystalline bilayers, this is seen as an abrupt, stimulation of activity at 15 mol % cholesterol (which is reversed at 17.5 mol %) and a gradual acceleration of activity between 30 to 40 mol % cholesterol. In fluid bilayers (where activity is high), activity is unaffected by 10, 20, and 30 mol % cholesterol. However, 12.5 and 17.5 mol % cholesterol reduce activity by 100-fold. These studies demonstrate that small changes in bilayer cholesterol content result in drastic alterations in transporter activity. Transporter sensitivity to cholesterol is a complex rather than monotonic function of bilayer cholesterol content and appears to be primarily determined by bilayer composition rather than by bilayer "fluidity."     

Subunit molecular mass assignment of 14,654 Da to the soluble beta-galactoside-binding lectin from bovine heart muscle and demonstration of intramolecular disulfide bonding associated with oxidative inactivation.

The soluble dimeric beta-galactoside-binding lectin (subunit molecular mass, approximately 14 kDa) of bovine heart muscle, in common with the 14-kDa lectins of several other animal species, displays carbohydrate-binding activity when it is in the reduced state, but the purified lectin loses this activity upon oxidation. In the present study, the presence of any post-translational modification and the mechanism of the oxidative inactivation have been investigated by analyses of the reduced and oxidized forms of the purified bovine lectin by electrospray ionization-mass spectrometry (ESI-MS) and by liquid secondary ion mass spectrometry (LSIMS) of tryptic and peptic peptides. By ESI-MS, the molecular mass of the reduced lectin is determined to be 14,654.6 +/- 0.9 Da, and that of the oxidized lectin is 14,649.3 +/- 1.1 Da. These masses correspond to the amino acid sequence of the protein with the cysteines having free sulfhydryl groups in the reduced state and forming disulfide bonds in the oxidized state. There is no evidence of post-translational modification in either lectin form except for monoacetylation already predicted for alanine at the blocked N-terminal end. Pronounced differences in charge distribution in the electrospray ionization mass spectra of the reduced and oxidized lectin, reflecting a change in the number of accessible protonation sites in the oxidized protein, are consistent with the protein being held in an altered conformation by covalent bonding. The results of LSIMS analyses of tryptic and peptic peptides in conjunction with Edman sequencing indicate that disulfide bonding occurs predominantly between Cys2 and Cys130, Cys16 and Cys88, and Cys42 and Cys60. There is no evidence of oxidation of Trp68. These results, taken together with observations that almost the complete polypeptide chain is necessary for the functional integrity of the carbohydrate recognition domain (Abbott, W. M., and Feizi, T. (1991) J. Biol. Chem. 266, 5552-5557) point to intramolecular disulfide bonding with a change in protein folding and conformation as the mechanism of oxidative inactivation of the purified bovine lectin.     

Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2?μM to as low as 110?nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5?brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.     

The significance of antibiotic production by Pseudomonas aureofaciens PA 147-2 for biological control of Phytophthora megasperma root rot of asparagus

Pseudomonas aureofaciens PA147-2 produces an antibiotic (Af⁺) which inhibits the growth of fungal phytopathogens on phosphate buffered potato dextrose agar (PBPDA). To determine the role of the antibiotic in disease suppression in vivo, PA147-2 and an antibiotic-deficient Tn5 mutant (Af^-) PA109, were tested for their ability to suppress root rot of Asparagus officinalis seedlings caused by Phytophthora megasperma var sojae, in the glasshouse. Seedlings coinoculated with the pathogen and the wildtype strain PA147-2, showed a significantly reduced level of infection and disease severity compared to seedlings inoculated with the pathogen alone. However, 100% of seedlings treated with Af^- mutant PA109 were diseased. Furthermore, a strain derived from mutant PA109, restored to Af⁺ through allele replacement of Tn5 by homologous recombination, gave similar levels of disease suppression as the wildtype. This suggests the antibiotic produced by PA147-2 is important for the control of P. megasperma in vitro as well as in planta. Treatment of seedlings with PA147-2 and derived strains including the Tn5 mutant (Af^-) strain improved plant weight by 40–100% in the presence and absence of the pathogen suggesting PA147-2 also has a direct growth stimulatory effect independent of antibiotic production.     

Entomophthora muscae (Entomophthorales: Entomophthoracae) mycosis in the onion fly,Delia antiqua (Diptera: Anthomyiidae)

Entomophthora muscae was identified as a common fungal pathogen of the onion fly, Delia antiqua, and the adult seed corn maggot, D. platura. Low infection levels also were found in populations of the cluster fly, Pollenia rudis (Diptera: Muscidae), and the tiger fly, Coenosia tigrina (Diptera: Muscidae). The disease cycle, as it affects D. antiqua in the onion agroecosystem, is described, including the etiology, symptomatology, and phenology. Natural infection levels approaching 100% were noted early in the spring and in late fall, impacting the 1st and 3rd generations of the D. antiqua population significantly. A lagged density-dependent disease response was noted at the gross population level, although more specific biological interactions may be involved in regulating the disease intensity.