Characterization of follistatin-type domains and their contribution to myostatin and activin a antagonism

Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-β family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-β family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists.     

Aerobic Biotransformation of N-Nitrosodimethylamine and N-Nitrodimethylamine by Benzene-, Butane-, Methane-, Propane-, and Toluene-Fed Cultures

N-Nitrosodimethylamine (NDMA) is an emerging contaminant of concern. N-nitrodimethylamine (DMNA) is a structural analog to NDMA. NDMA and DMNA have been found in drinking water, groundwater, and other media and are of concern due their toxicity. The authors evaluated biotransformation of NDMA and DMNA by cultures enriched from contaminated groundwater growing on benzene, butane, methane, propane, or toluene. Maximum specific growth rates of enriched cultures on butane (μ_max = 1.1 h^?1) and propane (μ_max = 0.65 h^?1) were 1 to 2 orders of magnitude higher than those presented in the literature. Growth rates of mixed cultures grown on benzene (μ_max = 1.3 h^?1), methane (μ_max = 0.09 h^?1), and toluene (μ_max = 0.99 h^?1) in these studies were similar to those presented in the literature. NDMA biotransformation rates for methane oxidizers (υ_max = 1.4 ng min^?1 mg^?1) and toluene oxidizers (υ_max = 2.3 ng min^?1 mg^?1) were comparable to those presented in the literature, whereas the biotransformation rate for propane oxidizers (υ_max = 0.37 ng min^?1 mg^?1) was lower. NDMA biotransformation rates for benzene oxidizers (υ_max = 1.02 ng min^?1 mg^?1) and butane oxidizers (υ_max = 1.2 ng min^?1 mg^?1) were comparable to those reported for other primary substrates. These studies showed that DMNA biotransformation rates for benzene (υ_max = 0.79 ng min^?1 mg^?1), butane (υ_max = 1.0 ng min^?1 mg^?1), methane (υ_max = 2.1 ng min^?1 mg^?1), propane (υ_max = 1.46 ng min^?1 mg^?1), and toluene (υ_max = 0.52 ng min^?1 mg^?1) oxidizers were all comparable. These studies highlight potential bioremediation methods for NDMA and DMNA in contaminated groundwater.     

Identification of the lipid droplet targeting domain of the Cidea protein

Cidea, the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) domain-containing protein, is targeted to lipid droplets in mouse adipocytes, where it inhibits triglyceride hydrolysis and promotes lipid storage. In mice, Cidea may prevent lipolysis by binding and shielding lipid droplets from lipase association. Here we demonstrate that human Cidea localizes with lipid droplets in both adipocyte and nonadipocyte cell lines, and we ascribe specific functions to its protein domains. Expression of full-length Cidea in undifferentiated 3T3-L1 cells or COS-1 cells increases total cellular triglyceride and strikingly alters the morphology of lipid droplets by enhancing their size and reducing their number. Remarkably, both lipid droplet binding and increased triglyceride accumulation are also elicited by expression of only the carboxy-terminal 104 amino acids, indicating this small domain directs lipid droplet targeting and triglyceride shielding. However, unlike the full-length protein, expression of the carboxy-terminus causes clustering of small lipid droplets but not the formation of large droplets, identifying a novel function of the N terminus. Furthermore, human Cidea promotes lipid storage via lipolysis inhibition, as the expression of human Cidea in fully differentiated 3T3-L1 adipocytes causes a significant decrease in basal glycerol release. Taken together, these data indicate that the carboxy-terminal domain of Cidea directs lipid droplet targeting, lipid droplet clustering, and triglyceride accumulation, whereas the amino terminal domain is required for Cidea-mediated development of enlarged lipid droplets.     

High-level secretion of biologically active aprotinin from the yeastPichia pastoris

Summary A synthetic gene encoding aprotinin (bovine pancreatic trypsin, inhibitor) was fused to theSaccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site.Pichia pastoris strains were developed to'express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion.P. pastoris containing a single copy of the vector secreed approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipoten with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.     

Hx, a novel fluorescent, minor groove and sequence specific recognition element: design, synthesis, and DNA binding properties of p-anisylbenzimidazole-imidazole/pyrrole-containing polyamides

With the aim of incorporating a recognition element that acts as a fluorescent probe upon binding to DNA, three novel pyrrole (P) and imidazole (I)-containing polyamides were synthesized. The compounds contain a p-anisylbenzimidazolecarboxamido (Hx) moiety attached to a PP, IP, or PI unit, giving compounds HxPP (2), HxIP (3), and HxPI (4), respectively. These fluorescent hybrids were tested against their complementary nonfluorescent, non-formamido tetraamide counterparts, namely, PPPP (5), PPIP (6), and PPPI (7) (cognate sequences 5'-AAATTT-3', 5'-ATCGAT-3', and 5'-ACATGT-3', respectively). The binding affinities for both series of polyamides for their cognate and noncognate sequences were ascertained by surface plasmon resonance (SPR) studies, which revealed that the Hx-containing polyamides gave binding constants in the 10(6) M(-1) range while little binding was observed for the noncognates. The binding data were further compared to the corresponding and previously reported formamido-triamides f-PPP (8), f-PIP (9), and f-PPI (10). DNase I footprinting studies provided additional evidence that the Hx moiety behaved similarly to two consecutive pyrroles (PP found in 5-7), which also behaved like a formamido-pyrrole (f-P) unit found in distamycin and many formamido-triamides, including 8-10. The biophysical characterization of polyamides 2-7 on their binding to the abovementioned DNA sequences was determined using thermal melts (ΔT(M)), circular dichroism (CD), and isothermal titration calorimetry (ITC) studies. Density functional calculations (B3LYP) provided a theoretical framework that explains the similarity between PP and Hx on the basis of molecular electrostatic surfaces and dipole moments. Furthermore, emission studies on polyamides 2 and 3 showed that upon excitation at 322 nm binding to their respective cognate sequences resulted in an increase in fluorescence at 370 nm. These low molecular weight polyamides show promise for use as probes for monitoring DNA recognition processes in cells.     

Passive sound localization of prey by the pallid bat (Antrozous p. pallidus)

Summary The pallid bat (Antrozous p. pallidus) uses passive sound localization to capture terrestrial prey. This study of captive pallid bats examined the roles of echolocation and passive sound localization in prey capture, and focused on their spectral requirements for accurate passive sound localization.Crickets were used as prey throughout these studies. All tests were conducted in dim, red light in an effort to preclude the use of vision. Hunting performance did not differ significantly in red light and total darkness, nor did it differ when visual contrast between the terrestrial prey and the substrate was varied, demonstrating that the bats did not use vision to locate prey.Our bats apparently used echolocation for general orientation, but not to locate prey. They did not increase their pulse emission rate prior to prey capture, suggesting that they were not actively scanning prey. Instead, they required prey-generated sounds for localization. The bats attended to the sound of walking crickets for localization, and also attacked small, inanimate objects dragged across the floor. Stationary and/or anesthetized crickets were ignored, as were crickets walking on substrates that greatly attenuated walking sounds. Cricket communication sounds were not used in prey localization; the bats never captured stationary, calling crickets.The accuracy of their passive sound localization was tested with an open-loop passive sound localization task that required them to land upon an anesthetized cricket tossed on the floor. The impact of a cricket produced a single 10–20 ms duration sound, yet with this information, the bats were able to land within 7.6 cm of the cricket from a maximum distance of 4.9 m. This performance suggests a sound localization accuracy of approximately ±1° in the horizontal and vertical dimensions of auditory space. The lower frequency limit for accurate sound localization was between 3–8 kHz. A physiological survey of frequency representation in the pallid bat inferior colliculus suggests that this lower frequency limit is around 5 kHz.