Rapid Isolation of the 7S-Nerve Growth Factor Complex and Its Subunits from Murine Submaxillary Glands and Saliva

7S-Nerve growth factor (NGF) and its alpha, beta-NGF, and gamma subunits have been purified from murine submaxillary glands and saliva by a combination of gel filtration on rigid polyvinyl gels, reversed-phase liquid chromatography on short alkyl chain supports (C4 columns), and ion-exchange chromatography on silica-based carboxymethyl columns. This technique is superior to previously used methods in that it is much more rapid and allows the purification of larger quantities of polypeptide from the same amount of starting material. Beta-NGF prepared with this method elicits the outgrowth of fibers of cells of a pheochromocytoma cell line (PC 12) in vitro, indicating that the biological activity is not impaired by the organic solvents and strong acids utilized for its isolation.     

Design and synthesis of orally available MEK inhibitors with potent in vivo antitumor efficacy

The structure-based design, synthesis, and biological evaluation of two novel series of potent and selective MEK kinase inhibitors are described herein. The elaboration of a lead pyrrole derivative to a bicyclic dihydroindolone core provided compounds with high potency and good drug-like pharmaceutical properties. Further scaffold modification afforded a series of dihydroindolizinone inhibitors, including an orally available advanced preclinical MEK inhibitor with potent in vivo antitumor efficacy.     

Effect of the H, K-ATPase inhibitor, esomeprazole magnesium, on gut total antioxidant capacity in mice

Antioxidant depletion is believed to be a mechanism involved in the pathophysiology of several upper gastrointestinal disorders, and H, K-ATPase inhibitors can alter free radical production by neutrophils. We hypothesized that the H, K-ATPase inhibitor esomeprazole magnesium would decrease gut free radical production with a concomitant increase in gut total antioxidant capacity. A/J mice (n = 10/group) received either vehicle (control) or one of three concentrations of esomeprazole magnesium in vehicle by once-daily gavage for 10 days. Using tissue extracts from stomach and colon, total antioxidant capacity, lipid peroxide levels, and constitutive Cu/Zn-superoxide dismutase were measured using validated assays. There was a dose-related increase in total antioxidant capacity (analysis of variance, P < 0.001) in stomach, but there was no change in the colon. In the assessment of free radical production, there was a trend toward decreased lipid peroxide levels in stomach from mice receiving esomeprazole. In stomach, Cu/Zn-superoxide dismutase activity was increased (ANOVA: p=.03) in mice receiving esomeprazole. In conclusion, gastric total antioxidant capacity and Cu/Zn-superoxide dismutase activity are increased by esomeprazole, and these changes may result in part from decreased free radical production. The present results support the notion that the pharmacological effects of this agent on upper intestinal tissue are more complex than previously thought, and appear to involve both enzymatic and nonenzymatic tissue antioxidants.     

Membrane Raft Disruption Promotes Axonogenesis in N2a Neuroblastoma Cells

Membrane rafts are discrete microdomains found in cell membranes that contain cholesterol and glycosphingolipids such as gangliosides. As cholesterol is a major component of membrane rafts, its sequestration by the polyene filipin can be used to disrupt them. In previous work we observed that membrane raft disruption by filipin treatment of murine neuroblastoma N2a cells led to changes in expression of cell processes. In this study, we determined the type of process formation induced by filipin treatment as well as whether their expression was accompanied by changes in ganglioside content or subcellular distribution. The results indicate that the processes formed were axonal in nature and their expression was accompanied by changes in both ganglioside content as well as the subcellular localization of GM1. Special issue article in honor of Dr. George DeVries.     

Biological threat characterization research: a critical component of national biodefense

Biological warfare (BW) threat assessments identify and prioritize BW threats to civilian and military populations. In an ideal world, they provide policymakers with clear and compelling guidance to prioritize biodefense research, development, testing, evaluation, and acquisition of countermeasures. Unfortunately, the biodefense community does not exist in an ideal world. National security professionals responsible for crafting BW threat assessments often are challenged by factors that limit the clarity and/or timeliness of those assessments. Moreover, the potential for life science advances to enhance threats enabled by state programs and the possibility that non-state actors may pursue crude but effective BW methodologies will drastically expand the scope of the perceived threat. Appropriate investment of federal biodefense funds will require some mechanism for validating and prioritizing present and future threats. Ideally, such a mechanism will incorporate empirical data targeted to elucidate actual hazards. In this regard, the Department of Homeland Security's creation of a Biological Threat Characterization Program for the technical validation of threat agents will be a valuable addition to the nation's overall biodefense strategy. This article articulates the need for a coordinated national biological threat characterization program, discusses some of the principal challenges associated with such research, and suggests a few options for their resolution.     

Species abundance distribution and dynamics in two locally coupled communities

This study considered a model for species abundance dynamics in two local community (or islands) connected to a regional metacommunity. The model was analyzed using continuous probabilistic technique that employs Kolmogorov-Fokker-Planck forward equation to derive the probability density of the species abundance in the two local communities. Using this technique, we proposed a classification for the species abundance dynamics in the local communities. This classification was made based on such characteristics as immigration intensity, species representation in the metacommunity and the size of local communities. We further distinguished several different scenarios for species abundance dynamics using different ecological characteristics such as species persistence, extinction and monodominance in one or both local communities. The similarity of the species abundance distributions between the two local communities was studied using the correlation coefficient between species abundances in two local communities. The correlation is a function of migration rates between local communities and between local and metacommunity. Immigration between local communities drives the homogenization of the local communities, while immigration from the metacommunity will differentiate them. This community subdivision model provides useful insights for studying the effect of landscape fragmentation on species diversity.     

ANALYSIS OF STARCH CONTENT IN CHLORELLA PYRENOIDOSA (CHLOROPHYTA) BY MORPHOLOGICAL AND QUANTITATIVE TECHNIQUES

Chlorella pyrenoidosa (Chick) was grown heterotrophically in batch culture on defined medium with glucose. Morphometric analysis of cells in the exponential growth phase showed that starch accounted for 57% of the volume of the chloroplast and 36% of the total cell volume. During the stationary growth phase, the amount of starch accounted for only 36% of the chloroplast volume and 13% of the total cell volume. This represented a 36% decrease in the amount of starch/cell between the exponential and stationary phase. Determination of starch as grams/cell using quantitative techniques on cell extracts showed a comparable decrease in the amount of starch during this same transition. Based on these results, morphometric techniques provided an accurate assay of starch and have the added advantage of visualization of cellular structures not available when quantitative techniques are used.     

Structure-based optimization of 1H-imidazole-2-carboxamides as Axl kinase inhibitors utilizing a Mer mutant surrogate

Axl has been a target of interest in the oncology field for several years based on its role in various oncogenic processes. To date, no wild-type Axl crystal structure has been reported. Herein, we describe the structure-based optimization of a novel chemotype of Axl inhibitors, 1H-imidazole-2-carboxamide, using a mutated kinase homolog, Mer(I650M), as a crystallographic surrogate. Iterative optimization of the initial lead compound (1) led to compound (21), a selective and potent inhibitor of wild-type Axl. Compound (21) will serve as a useful compound for further in vivo studies.