The Impact of Seeding Method on Diversity and Plant Distribution in Two Restored Grasslands

Previous studies have compared grassland restoration techniques based on resulting species richness and composition. However, none have determined if different techniques generate different plant distributions in space, which may further impact restoration success. This study tests if there are quadrat‐scale (1 m²) differences between paired drilled and broadcast plantings in diversity, composition, and plant distributions. Higher competition intensity in and more contiguous spaces between rows in drill‐seeded restorations were hypothesized to result in larger patches of native grasses and exotic species. Two paired drill‐ and broadcast‐seeded plantings were sampled in June 2007 in Iowa, U.S.A. Within 10 quadrats in each planting, we measured species abundance with point intercept sampling and plant distributions by dividing the quadrat into 64 cells and recording the most abundant species in each cell. Drilled and broadcast plantings at both sites had similar Simpson’s diversity and evenness. However, the effect of planting type on species richness, composition, and plant distribution was site dependent. Native warm‐season grasses in one site, and exotic species in the second, occupied more space and were distributed in larger patches in drilled plantings. Furthermore, drilled canopies consistently captured more light than broadcast canopies. This suggests that initial differences in seed placement can affect resulting plant distributions, resource use, and potentially long‐term species turnover. Mechanisms structuring vegetation in these communities need to be further investigated to determine if this approach can provide more information on long‐term diversity maintenance in restorations than traditional measures.     

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.     

A structure-function study of ligand recognition by CD22beta

B-cell-specific CD22 is a member of a group of cell adhesion molecules within the immunoglobulin superfamily that display binding to glycans with terminal sialic acid residues. Binding of endogenous ligands to CD22 triggers B-cell activation and proliferation. It is therefore conceivable that high affinity ligands for CD22 may be of value as inhibitors of B-cell activation in allergy and chronic inflammation. In this study, we aimed to delineate the structural requirements for ligand binding to CD22. A library of 20 mono-, di-, and trisaccharide analogs of the basic binding motif Neu5Ac(alpha2,6)Lac was synthesized and screened for affinity for CD22beta. In general, CD22 ligand recognition appeared to be rather tolerant with respect to structural modifications of the anomeric sugar on a mono-, di-, and trisaccharide level, although affinity was increased by the presence of a nitro aromatic group at C-2. The most potent monovalent ligand, Neu5Ac-4-nitrobenzoyl-Glc, was selected to generate multivalent ligands based on either a glutamate or Tris cluster core. All multivalent ligands displayed at least a 10-fold increased affinity for CD22 compared with the corresponding monovalent glycoside. Interestingly, a maximal gain in affinity was already obtained for bivalent ligands, regardless of the terminal glycoside. A trivalent Tris-based cluster of Neu5Ac-4-nitrobenzoyl-Glc displayed a 300-fold higher affinity compared with the basic binding motif, which makes it, to our knowledge, the most potent antagonist for CD22 yet synthesized. As our in vitro fluorescence-activated cell sorting studies demonstrated efficient cellular uptake of a CD22 substrate, the most potent ligand in this study may hold promise as a homing device for immunomodulatory compounds and cytostatics.     

RERG is a novel ras-related, estrogen-regulated and growth-inhibitory gene in breast cancer

Using microarray analysis, we identified a unique ras superfamily gene, termed RERG (ras-related and estrogen-regulated growth inhibitor), whose expression was decreased or lost in a significant percentage of primary human breast tumors that show a poor clinical prognosis. Importantly, high RERG expression correlated with expression of a set of genes that define a breast tumor subtype that is estrogen receptor-positive and associated with a slow rate of tumor cell proliferation and a favorable prognosis for these cancer patients. RERG mRNA expression was induced rapidly in MCF-7 cells stimulated by beta-estradiol and repressed by tamoxifen treatment. Like Ras, RERG protein exhibited intrinsic GDP/GTP binding and GTP hydrolysis activity. Unlike Ras proteins, RERG lacks a known recognition signal for COOH-terminal prenylation and was localized primarily in the cytoplasm. Expression of RERG protein in MCF-7 breast carcinoma cells resulted in a significant inhibition of both anchorage-dependent and anchorage-independent growth in vitro and inhibited tumor formation in nude mice. These features of RERG are strikingly different from most Ras superfamily GTP-binding pro-teins and suggest that the loss of RERG expression may contribute to breast tumorigenesis.     

Leukemia-associated Rho guanine nucleotide exchange factor, a Dbl family protein found mutated in leukemia, causes transformation by activation of RhoA

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) was originally identified as a fusion partner with mixed-lineage leukemia in a patient with acute myeloid leukemia. LARG possesses a tandem Dbl homology and pleckstrin homology domain structure and, consequently, may function as an activator of Rho GTPases. In this study, we demonstrate that LARG is a functional Dbl protein. Expression of LARG in cells caused activation of the serum response factor, a known downstream target of Rho-mediated signaling pathways. Transient overexpression of LARG did not activate the extracellular signal-regulated kinase or c-Jun NH(2)-terminal kinase mitogen-activated protein kinase cascade, suggesting LARG is not an activator of Ras, Rac, or Cdc42. We performed in vitro exchange assays where the isolated Dbl homology (DH) or DH/pleckstrin homology domains of LARG functioned as a strong activator of RhoA, but exhibited no activity toward Rac1 or Cdc42. We found that LARG could complex with RhoA, but not Rac or Cdc42, in vitro, and that expression of LARG caused an increase in the levels of the activated GTP-bound form of RhoA, but not Rac1 or Cdc42, in vivo. Thus, we conclude that LARG is a RhoA-specific guanine nucleotide exchange factor. Finally, like activated RhoA, we determined that LARG cooperated with activated Raf-1 to transform NIH3T3 cells. These data demonstrate that LARG is the first functional Dbl protein mutated in cancer and indicate LARG-mediated activation of RhoA may play a role in the development of human leukemias.     

Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane

Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908-8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.     

Nuclear domains enriched in RNA 3'-processing factors associate with coiled bodies and histone genes in a cell cycle-dependent manner

Nuclear domains, called cleavage bodies, are enriched in the RNA 3'-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle-dependent expression of histone genes.     

Studies of cytochrome c oxidase-driven H(+)-coupled phosphate transport catalyzed by the Saccharomyces cerevisiae Pho84 permease in coreconstituted vesicles

The proton-coupled Pho84 phosphate permease of Saccharomyces cerevisiae, overexpressed as a histidine-tagged chimera in Escherichia coli, was detergent-solubilized, purified, and reconstituted into proteoliposomes. Proteoliposomes containing the Pho84 protein were fused with proteoliposomes containing purified cytochrome c oxidase from beef heart mitochondria. Both components of the coreconstituted system were functionally incorporated in tightly sealed membrane vesicles in which the cytochrome c oxidase-generated electrochemical proton gradient could drive phosphate transport via the proton-coupled Pho84 permease. The metal dependency of transport indicates that a metal-phosphate complex is the translocated substrate.     

Mutations linked to leukoencephalopathy with vanishing white matter impair the function of the eukaryotic initiation factor 2B complex in diverse ways

Leukoencephalopathy with vanishing white matter (VWM) is a severe inherited human neurodegenerative disorder that is caused by mutations in the genes for the subunits of eukaryotic initiation factor 2B (eIF2B), a heteropentameric guanine nucleotide exchange factor that regulates both global and mRNA-specific translation. Marked variability is evident in the clinical severity and time course of VWM in patients. Here we have studied the effects of VWM mutations on the function of human eIF2B. All the mutations tested cause partial loss of activity. Frameshift mutations in genes for eIF2Bepsilon or eIF2Bbeta lead to truncated polypeptides that fail to form complexes with the other subunits and are effectively null mutations. Certain point mutations also impair the ability of eIF2Bbeta or -epsilon to form eIF2B holocomplexes and also diminish the intrinsic nucleotide exchange activity of eIF2B. A point mutation in the catalytic domain of eIF2Bepsilon impairs its ability to bind the substrate, while two mutations in eIF2Bbeta actually enhance eIF2 binding. We provide evidence that expression of VWM mutant eIF2B may enhance the translation of specific mRNAs. The variability of the clinical phenotype in VWM may reflect the multiple ways in which VWM mutations affect eIF2B function.     

Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid

The present results demonstrate that pyridoxal, pyridoxal 5′-phosphate (PLP) and pyridoxal 5′-diphospho-5′-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the ε-amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (K_i = 40 μM) that blocks the cleavable complex formation. Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein. The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK^*DSIR). Targeted lysine (K^*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K₅₃₂), Candida albicans (K₄₆₈), Saccharomyces cerevisiae (K₄₅₈) and Schizosaccharomyces pombe (K₅₀₅). In the human enzyme, K₅₃₂, belonging to the active site acts as a general acid catalyst and is therefore essential for activity. The spatial orientation of K₅₃₂–PLP within the active site was approached by molecular modeling using available crystallographic data. The PLP moiety was found at close proximity of several active residues. PLP could be involved in the cellular control of topoisomerases IB. It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid.