Dipyridamole is neuroprotective for cultured rat embryonic cortical neurons

The effects of a clinically useful cardiovascular agent, dipyridamole, were examined in a rodent tissue culture model of neuroprotection. Dipyridamole effectively protected rat embryonic day 18 (E18) cortical neurons from either 48 h trophic deprivation or 48 h exposure to the glutathione synthesis inhibitor, L-buthionine (R,S) sulfoximine. The neuron sparing actions of dipyridamole were time- and concentration-dependent and mimicked the actions of exogenously applied glutathione. These results demonstrate that dipyridamole protects primary neuronal cultures against either trophic or chemically mediated insults, and suggest that dipyridamole has a potent antioxidant ability that compensates for glutathione depletion in neuronal cultures.     

The future of interdisciplinary research and training: how to conquer the silo guardians

We have entered a new era in biomedical research in which large interdisciplinary teams are being established to answer important scientific questions. Scientists of multidisciplinary backgrounds within universities are combining forces and inter-institutional consortia that include alliances between academia and industry are springing up around the country to generate breakthrough advances. A number of driving forces are at work to establish these collaborative research approaches. By contrast, there also are barriers to be surmounted by institutions with silo mentalities for effective partnerships to be established. In order for this new era of research to reach maximal effectiveness, new approaches to education of the young and retraining of established administrators and scientists must take place. These issues were explored thoroughly at the 2006 annual meeting of the Association of Anatomy, Cell Biology and Neurobiology Chairpersons (AACBNC) that was held in Aruba from January 18 to 21. The theme of this historic meeting was the Future of Interdisciplinary Research and Training: Breaking Down the Barriers. In this introductory article, we discuss the formation of a trendsetting Institute of Biomedical Sciences and Technology, the concept of the AACBNC meeting, and the influence of the Institute on the content of the meeting. The proceedings of this meeting, including Nobel Laureate Papers and Nobel Round-Table Discussions on the future of interdisciplinary research and training, are contained in this special issue of Experimental Biology and Medicine, a journal dedicated to the publication of multidisciplinary and interdisciplinary research in the biomedical sciences.     

A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736–1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.     

Segmental motions of rat thymidylate synthase leading to half‐the‐sites behavior

Thymidylate synthase (TS) is a homodimeric enzyme with two equivalent active sites composed of residues from both subunits. Despite the structural symmetry of the enzyme, certain experimental results are consistent with half‐the‐sites activity, suggesting negative cooperativity between the active sites. To gain insight into the mechanism behind this phenomenon, we explore segmental motions of rat TS in the absence of ligands, with normal mode analysis as a tool. Using solvent accessible surface area of the active site pocket as a monitor of the degree of opening of the active sites, we classified the first 25 nontrivial normal modes, obtained from the web server of the program ElNémo, according to the behavior of the active sites. We found seven modes that open and close both sites symmetrically and nine that do so in an anticorrelated fashion. We characterized the motions of these modes by visual inspection and through measurement of distances between selected atoms lining the active site pockets. The segments that regulate access to the active site correspond to the loop containing R44, helix K, and a long loop containing residues 103–125, in agreement with a large body of crystallographic studies. These elements can be activated together or in isolation. There are more asymmetric modes than symmetric ones in the set we analyzed, probably accounting for the half‐the‐sites behavior of the enzyme. Three of the asymmetric modes result in changes at the dimer interface and indicate the endpoints of possible communication pathways between the active sites. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 549–559, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

The primary in vivo steroidal alkaloid glucosyltransferase from potato

To provide tools for breeders to control the steroidal glycoalkaloid (SGA) pathway in potato, we have investigated the steroidal alkaloid glycosyltransferase (Sgt) gene family. The committed step in the SGA pathway is the glycosylation of solanidine by either UDP-glucose or UDP-galactose leading to α-chaconine or α-solanine, respectively. The Sgt2 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. SGT1 has glucosyltransferase activity in vitro, but in vivo serves as the UDP-galactose:solanidine galactosyltransferase. Two alleles of the Sgt2 gene were isolated and its function was established with antisense transgenic lines and in vitro assays of recombinant protein. In tubers of transgenic potato (Solanum tuberosum) cvs. Lenape and Desirée expressing an antisense Sgt2 gene construct, accumulation of α-solanine was increased and α-chaconine was reduced. Studies with recombinant SGT2 protein purified from yeast show that SGT2 glycosylation activity is highly specific for UDP-glucose as a sugar donor. This data establishes the function of the gene product (SGT2), as the primary UDP-glucose:solanidine glucosyltransferase in vivo.     

Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody

Human IgG4 subtype antibodies have often been reported to have a significant portion (5-50%) of a heavy chain-light chain dimer ("half-antibody") on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS-PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating. Inclusion of N-ethylmaleimide in the sample buffer prevented the disulfide scrambling. Presumably, cyclization of the flexible IgG4 hinge during this disulfide scrambling leads to the preferential separation of heavy chains. A second condition producing half-antibody was reoxidation after exposure to reductant, where 46% of the antibody was trapped in the intrachain disulfide form. The amount of half-antibody was reduced to 4% by reoxidation in the presence of a mixture of oxidized and reduced glutathione. When the improved sample preparation conditions were used, IgG4 mAb freshly isolated from cells contained 4.5-15% half-antibody, indicating that equilibration of the interchain and intrachain hinge disulfide pairing was not always attained in cells.     

The challenge of enzyme cost in the production of lignocellulosic biofuels

With the aim of understanding the contribution of enzymes to the cost of lignocellulosic biofuels, we constructed a techno-economic model for the production of fungal cellulases. We found that the cost of producing enzymes was much higher than that commonly assumed in the literature. For example, the cost contribution of enzymes to ethanol produced by the conversion of corn stover was found to be $0.68/gal if the sugars in the biomass could be converted at maximum theoretical yields, and $1.47/gal if the yields were based on saccharification and fermentation yields that have been previously reported in the scientific literature. We performed a sensitivity analysis to study the effect of feedstock prices and fermentation times on the cost contribution of enzymes to ethanol price. We conclude that a significant effort is still required to lower the contribution of enzymes to biofuel production costs.     

Roles of DCL4 and DCL3b in rice phased small RNA biogenesis

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.