Resprouting of <Emphasis Type="Italic">Echinacea angustifolia</Emphasis> Augments Sustainability of Wild Medicinal Plant Populations

Resprouting of Echinacea angustifolia Augments Sustainability of Wild Medicinal Plant Populations. Overharvest of wild Echinacea species root has been a significant concern to the herbal industry. Harvesters of wild Echinacea angustifolia showed us that even after harvesting the top 15 to 20 cm of root, some plants resprout. We marked locations of harvested plants at sites in Kansas and Montana and reexamined them two years later to see if they resprouted from remaining root reserves. Approximately 50% of the roots resprouted at both Kansas and Montana sampling sites, despite droughty weather conditions in Montana. The length of root harvested significantly affected the ability of the plant to resprout. Those plants that were more shallowly harvested and had less root length removed were more likely to resprout. These data indicate that echinacea stands can recover over time from intensive harvest if periods of nonharvest occur. Our echinacea harvest study emphasizes that the entire biology of medicinal plants must be considered when evaluating their conservation status.     

Mutation of the arginine finger in the active site of Escherichia coli DbpA abolishes ATPase and helicase activity and confers a dominant slow growth phenotype

Escherichia coli DEAD-box protein A (DbpA) is an ATP-dependent RNA helicase with specificity for 23S ribosomal RNA. Although DbpA has been extensively characterized biochemically, its biological function remains unknown. Previous work has shown that a DbpA deletion strain is viable with little or no effect on growth rate. In attempt to elucidate a phenotype for DbpA, point mutations were made at eleven conserved residues in the ATPase active site, which have exhibited dominant-negative phenotypes in other DExD/H proteins. Biochemical analysis of these DbpA mutants shows the expected decrease in RNA-dependent ATPase activity and helix unwinding activity. Only the least biochemically active mutation, R331A, produces small colony phenotype and a reduced growth rate. This dominant slow growth mutant will be valuable to determine the cellular function of DbpA.     

Physical association of the K3 protein of gamma-2 herpesvirus 68 with major histocompatibility complex class I molecules with impaired peptide and beta(2)-microglobulin assembly

To persist in the presence of an active immune system, viruses encode proteins that decrease expression of major histocompatibility complex class I molecules by using a variety of mechanisms. For example, murine gamma-2 herpesvirus 68 expresses the K3 protein, which causes the rapid turnover of nascent class I molecules. In this report we show that certain mouse class I alleles are more susceptible than others to K3-mediated down regulation. Prior to their rapid degradation, class I molecules in K3-expressing cells exhibit impaired assembly with beta(2)-microglobulin. Furthermore, K3 is detected predominantly in association with class I molecules lacking assembly with high-affinity peptides, including class I molecules associated with the peptide loading complex TAP/tapasin/calreticulin. The detection of K3 with class I assembly intermediates raises the possibility that molecular chaperones involved in class I assembly are involved in K3-mediated class I regulation.     

Complete functional rescue of the ABCA1-/- mouse by human BAC transgenesis

Humanized mouse models are useful tools to explore the functional and regulatory differences between human and murine orthologous genes. We have combined a bioinformatics approach and an in vivo approach to assess the functional and regulatory differences between the human and mouse ABCA1 genes. Computational analysis identified significant differences in potential regulatory sites between the human and mouse genes. The effect of these differences was assessed in vivo, using a bacterial artificial chromosome transgenic humanized ABCA1 mouse model that expresses the human gene in the absence of mouse ABCA1. Humanized mice expressed human ABCA1 protein at levels similar to wild-type mice and fully compensated for cholesterol efflux activity and lipid levels seen in ABCA1-deficient mice. Liver X receptor agonist administration resulted in significant increases in HDL values associated with parallel increases in the hepatic ABCA1 protein and mRNA levels in the humanized ABCA1 mice, as seen in the wild-type animals. Our studies indicate that despite differences in potential regulatory regions, the human ABCA1 gene is able to functionally fully compensate for the mouse gene. Our humanized ABCA1 mice can serve as a useful model system for functional analysis of the human ABCA1 gene in vivo and can be used for the generation of potential new therapeutics that target HDL metabolism.     

High genetic susceptibility to ethanol withdrawal predicts low ethanol consumption

C57BL/6J (B6) inbred mice are well known to drink large amounts of alcohol (ethanol) voluntarily and to have only modest ethanol-induced withdrawal under fixed dose conditions. In contrast, DBA/2J (D2) mice are ``teetotallers' and exhibit severe ethanol withdrawal. Speculation that an inverse genetic relationship existed between these two traits was substantiated by meta-analysis of existing data collected in multiple genetic models, including large panels of standard and recombinant inbred strains, their crosses, and selectively bred mouse lines. Despite methodological differences among laboratories in measurement of both preference drinking and withdrawal, a nearly universal finding was that genotypes consuming large amounts of 10% ethanol (calculated as g/kg/day) during two-bottle choice preference drinking were genetically predisposed to low withdrawal scores in independent studies after either acute or chronic ethanol treatment. Conversely, low-drinking genotypes had higher withdrawal severity scores. The genetic relationship appears to be strongest in populations derived from B6 and D2, where data from more genotypes (BXD RIs, B6D2F₂s, BXD RI F₁s, and B6D2F₂-derived selectively bred lines) were available for analysis. Gene mapping studies in these populations identified four chromosome regions [on Chromosomes (Chrs) 1, 2, 4, and 15] where genes might potentially influence both traits. Among genotypes with greater genetic diversity (for example, a panel of standard inbred strains or selectively bred lines), the relationship was less pronounced. Thus, reduced susceptibility to the development of high alcohol use may be supported by increased genetic susceptibility to ethanol withdrawal symptoms. Received: 15 September 1998 / Accepted: 8 October 1998     

Sequence and domain arrangements influence mechanical properties of elastin‐like polymeric elastomers

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self‐assembly that aligns lysine residues for crosslinking. As a result, both the full‐length monomer as well as elastin‐like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full‐length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self‐assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full‐length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 392–407, 2013.     

Nephroblastoma overexpressed (Nov) inhibits osteoblastogenesis and causes osteopenia

Nephroblastoma overexpressed (Nov), a member of the Cyr 61, connective tissue growth factor, Nov (CCN) family of proteins, is expressed by osteoblasts, but its function in cells of the osteoblastic lineage is not known. We investigated the effects of Nov overexpression by transducing murine ST-2 stromal and MC3T3 osteoblastic cells with a retroviral vector where Nov is under the control of the cytomegalovirus promoter. We also examined the skeletal phenotype of transgenic mice expressing Nov under the control of the human osteocalcin promoter. Overexpression of Nov in ST-2 cells inhibited the appearance of mineralized nodules and decreased alkaline phosphatase activity and osteocalcin mRNA levels. Nov overexpression inhibited the effect of bone morphogenetic protein (BMP)-2 on the phosphorylation of Smad 1/5/8; on the transactivation of 12xSBE-Oc-pGL3, a BMP/Smad signaling reporter construct, and of Wnt 3 on cytoplasmic beta-catenin levels; and on the transactivation of the Wnt/beta-catenin signaling reporter construct 16xTCF-Luc. Nov overexpression did not activate Notch or transforming growth factor beta signaling. Glutathione S-transferase pulldown assays demonstrated direct Nov-BMP interactions. Nov transgenic mice exhibited osteopenia. In conclusion, Nov binds BMP-2 and antagonizes BMP-2 and Wnt activity, and its overexpression inhibits osteoblastogenesis and causes osteopenia.