Intraovarian activins are required for female fertility

Activins have diverse roles in multiple physiological processes including reproduction. Mutations and loss of heterozygosity at the human activin receptor ACVR1B and ACVR2 loci are observed in pituitary, pancreatic, and colorectal cancers. Functional studies support intraovarian roles for activins, although clarifying the in vivo roles has remained elusive due to the perinatal death of activin betaA knockout mice. To study the roles of activins in ovarian growth, differentiation, and cancer, a tissue-specific knockout system was designed to ablate ovarian production of activins. Mice lacking ovarian activin betaA were intercrossed to Inhbb homozygous null mice to produce double activin knockouts. Whereas ovarian betaA knockout females are subfertile, betaB/betaA double mutant females are infertile. Strikingly, the activin betaA and betaB/betaA-deficient ovaries contain increased numbers of functional corpora lutea but do not develop ovarian tumors. Microarray analysis of isolated granulosa cells identifies significant changes in expression for a number of genes with known reproductive roles, including Kitl, Taf4b, and Ghr, as well as loss of expression of the proto-oncogene, Myc. Thus, in contrast to the known tumor suppressor role of activins in some tissues, our data indicate that activin betaA and betaB function redundantly in a growth stimulatory pathway in the mammalian ovary.     

Expression and characterization of Acidothermus cellulolyticus E1 endoglucanase in transgenic duckweed Lemna minor 8627

Endoglucanase E1 from Acidothermus cellulolyticus was expressed cytosolically under control of the cauliflower mosaic virus 35S promoter in transgenic duckweed, Lemna minor 8627 without any obvious observable phenotypic effects on morphology or rate of growth. The recombinant enzyme co-migrated with the purified catalytic domain fraction of the native E1 protein on western blot analysis, revealing that the cellulose-binding domain was cleaved near or in the linker region. The duckweed-expressed enzyme was biologically active and the expression level was up to 0.24% of total soluble protein. The endoglucanase activity with carboxymethylcellulose averaged 0.2 units mg protein(-1) extracted from fresh duckweed. The optimal temperature and pH for E1 enzyme activity were about 80 degrees C and pH 5, respectively. While extraction with HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) buffer (pH 8) resulted in the highest recovery of total soluble proteins and E1 enzyme, extraction with citrate buffer (pH 4.8) at 65 degrees C enriched relative amounts of E1 enzyme in the extract. This study demonstrates that duckweed may offer new options for the expression of cellulolytic enzymes in transgenic plants.     

Isolation and characterization of Cr(VI) reducing Cellulomonas spp. from subsurface soils: implications for long-term chromate reduction

Microbial enrichments from Cr(VI) contaminated and uncontaminated US Department of Energy Hanford Site sediments produced Cr(VI) reducing consortia when grown in the presence of Cr(VI) with acetate, D-xylose or glycerol as a carbon and energy source. Eight of the nine isolates from the consortia were Gram positive and four of these were identified by 16S rRNA sequence homology and membrane fatty acid composition as belonging to the genus Cellulomonas. Two strains, ES6 and WS01, were further examined for their ability to reduce Cr(VI) under growth and non-growth conditions. During fermentative growth on D-xylose, ES6 and WS01 decreased aqueous Cr(VI) concentrations from 0.04 mM Cr(VI) to below the detection limit (0.002 mM Cr(VI)) in less than three days and retained their ability to reduce Cr(VI) even after four months of incubation. Washed ES6 and WS01 cells also reduced Cr(VI) under non-growth conditions for over four months, both with and without the presence of an exogenous electron donor. K-edge XANES spectroscopy confirmed the reduction of Cr(VI) to Cr(III). The ability to reduce Cr(VI) after growth had stopped and in the absence of an external electron donor, suggests that stimulation of these types of organisms may lead to effective long-term, in situ passive reactive barriers for Cr(VI) removal. Our results indicate that Cr(VI) reduction by indigenous Cellulomonas spp. may be a potential method of in situ bioremediation of Cr(VI) contaminated sediment and groundwater.     

Discovery of N-phenyl nicotinamides as potent inhibitors of Kdr

Inhibition of tumor-induced angiogenesis is a promising strategy in anticancer research. Neovascularization is a process required for both tumor growth and metastasis. Enhanced understanding of the underlying molecular mechanisms has led to the discovery of a variety of pharmaceutically attractive targets. Decades of investigation suggest that vascular endothelial growth factor (VEGF) and its receptors, in particular VEGFR2 or kinase insert-domain-containing receptor (Kdr), play a critical role in the growth and survival of endothelial cells in newly forming vasculature. The clinical utility of inhibitors of this receptor tyrosine kinase is currently under intense investigation. Herein we report our efforts in this arena.     

Design, synthesis, and biological testing of pyrazoline derivatives of combretastatin-A4

Fourteen N-acetylated and non-acetylated 3,4,5-tri- or 2,5-dimethoxypyrazoline analogs of combretastatin-A4 (1) were synthesized. A non-acetylated derivative (5a) with the same substituents as CA-4 (1) was the most active compound in the series, with IC(50) values of 2.1 and 0.5 microM in B16 and L1210 cell lines, respectively. In contrast, a similar compound with an acetyl group at N1 of the pyrazoline ring (6g) showed poor activity in the cell lines studied. A cell-based assay indicated that compound 5a caused extensive microtubule depolymerization with an EC(50) value of 7.1 microM in A-10 cells while no activity was seen with the acetylated compound. Molecular modeling studies showed that these compounds possess a twisted conformation similar to CA-4 (1).     

Nearest neighbor parameters for inosine x uridine pairs in RNA duplexes

An enzyme family known as adenosine deaminases that act on RNA (ADARs) catalyzes adenosine deamination in RNA. ADARs act on RNA that is largely double-stranded and convert adenosine to inosine, resulting, in many cases, in an I x U pair. Thermodynamic parameters derived from optical melting studies are reported for a series of 14 oligoribonucleotides containing single I x U pairs adjacent to Watson-Crick pairs. In order to determine unique linearly independent nearest neighbor parameters for I x U pairs, four duplexes containing 3'-terminal I x U pairs and four duplexes containing 5'-terminal I x U pairs have also been thermodynamically characterized. This data was combined with previously published data of seven duplexes containing internal, terminal, or tandem I x U pairs from Strobel et al. [Strobel, S. A., Cech, T. R., Usman, N., and Beigelman, L. (1994) Biochemistry 33, 13824-13838] and Serra et al. [Serra, M. J., Smolter, P. E., and Westhof, E. (2004) Nucleic Acids Res. 32, 1824-1828]. On average, a duplex with an internal I x U pair is 2.3 kcal/mol less stable than the same duplex with an A-U pair, however, a duplex with a terminal I x U pair is 0.8 kcal/mol more stable than the same duplex with an A-U pair. Although isosteric with a G-U pair, on average, a duplex with an internal I x U pair is 1.9 kcal/mol less stable than the same duplex with a G-U pair, however, a duplex with a terminal I x U pair is 0.9 kcal/mol more stable than the same duplex with a G-U pair. Duplexes with tandem I x U pairs are on average 5.9 and 3.8 kcal/mol less stable than the same duplex with tandem A-U or tandem G-U pairs, respectively. Using the combined thermodynamic data and a complete linear least-squares fitting routine, nearest neighbor parameters for all nearest neighbor combinations of I x U pairs and an additional parameter for terminal I x U pairs have been derived.     

Broad HIV-1 neutralization mediated by CD4-binding site antibodies

We have identified several patient sera showing potent and broad HIV-1 neutralization. Using antibody adsorption and elution from selected gp120 variants, the neutralizing specificities of the two most broadly reactive sera were mapped to the primary receptor CD4-binding region of HIV-1 gp120. Novel antibodies to the CD4-binding site are elicited in some HIV-1-infected individuals, and new approaches to present this conserved region of gp120 to the immune system may result in improved vaccine immunogens.     

Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli

We describe facile isolation of full-length IgG antibodies from combinatorial libraries expressed in E. coli. Full-length heavy and light chains are secreted into the periplasm, where they assemble into aglycosylated IgGs that are captured by an Fc-binding protein that is tethered to the inner membrane. After permeabilizing the outer membrane, spheroplast clones expressing so-called E-clonal antibodies, which specifically recognize fluorescently labeled antigen, are selected using flow cytometry. Screening of a library constructed from an immunized animal yielded several antibodies with nanomolar affinities toward the protective antigen of Bacillus anthracis.