The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

Kinetics of the acyl transfer catalyzed by Xanthomonas α-amino acid ester hydrolase was studied. The enzyme hydrolyzed d-α-phenylglycine methyl ester (d-PG-OMe) to give equimolar amounts of d-α-phenylglycine and methanol. With d-PG-OMe as an acyl donor and 7-amino-3-deacetoxy-cephalosporanic acid (7-ADCA) as an acyl acceptor, the enzyme transferred the acyl group from d-PG-OMe to 7-ADCA in competition with water. The addition of amine nucleophiles (7-ADCA and 6-aminopenicillanic acid) decreased the molecular activity (k_o) of the enzyme-catalyzed hydrolysis of d-PG-OMe, whereas it did not alter the Michaelis constant (K_M), and plots of l/k_o against the initial concentration of a nucleophile (n_o) gave a straight line. These results support the assumptions that the overall process for hydrolysis and acyl transfer proceeds through a common acyl-enzyme intermediate, that the acylation step of the enzyme is rate-limiting, and that the transfer competes with the hydrolysis of the acyl donor.     

An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.     

Dock8 interacts with Nck1 in mediating Schwann cell precursor migration

During embryonic development of the peripheral nervous system (PNS), Schwann cell precursors migrate along neuronal axons to their final destinations, where they will myelinate the axons after birth. While the intercellular signals controlling Schwann cell precursor migration are well studied, the intracellular signals controlling Schwann cell precursor migration remain elusive. Here, using a rat primary cell culture system, we show that Dock8, an atypical Dock180-related guanine-nucleotide exchange factor (GEF) for small GTPases of the Rho family, specifically interacts with Nck1, an adaptor protein composed only of Src homology (SH) domains, to promote Schwann cell precursor migration induced by platelet-derived growth factor (PDGF). Knockdown of Dock8 or Nck1 with its respective siRNA markedly decreases PDGF-induced cell migration, as well as Rho GTPase activation, in precursors. Dock8, through its unique N-terminal proline-rich motif, interacts with the SH3 domain of Nck1, but not with other adaptor proteins composed only of SH domains, e.g. Grb2 and CrkII, and not with the adaptor protein Elmo1. Reintroduction of the proline-rich motif mutant of Dock8 in Dock8 siRNA-transfected Schwann cell precursors fails to restore their migratory abilities, whereas that of wild-type Dock8 does restore these abilities. These results suggest that Nck1 interaction with Dock8 mediates PDGF-induced Schwann cell precursor migration, demonstrating not only that Nck1 and Dock8 are previously unanticipated intracellular signaling molecules involved in the regulation of Schwann cell precursor migration but also that Dock8 is among the genetically-conservative common interaction subset of Dock family proteins consisting only of SH domain adaptor proteins.     

Stability of cytochromes c′ from psychrophilic and piezophilic Shewanella species: implications for complex multiple adaptation to low temperature and high hydrostatic pressure

Extremophiles - The stability of dimeric cytochrome c′ from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the...     

Acoustic emissions of Sorex unguiculatus (Mammalia: Soricidae): Assessing the echo‐based orientation hypothesis

Shrew species have been proposed to utilize an echo‐based orientation system to obtain additional acoustic information while surveying their environments. This system has been supported by changes in vocal emission rates when shrews encounter different habitats of varying complexity, although detailed acoustic features in this system have not been reported. In this study, behavioral experiments were conducted using the long‐clawed shrew (Sorex unguiculatus) to assess this orientation system. Three experimental conditions were set, two of which contained obstacles. Short‐click, noisy, and different types of tonal calls in the audible‐to‐ultrasonic frequency range were recorded under all experimental conditions. The results indicated that shrews emit calls more frequently when they are facing obstacles or exploring the experimental environment. Shrews emitted clicks and several different types of tonal calls while exploring, and modified the use of different types of calls for varying behavior. Furthermore, shrews modified the dominant frequency and duration of squeak calls for different types of obstacles, that is, plants and acrylic barriers. The vocalizations emitted at short inter‐pulse intervals could not be observed when shrews approached these obstacles. These results are consistent with the echo‐based orientation hypothesis according to which shrews use a simple echo‐orientation system to obtain information from their surrounding environments, although further studies are needed to confirm this hypothesis.     

Phylogeography and population genetics of pine butterflies: Sky islands increase genetic divergence

The sky islands of southeastern Arizona (AZ) mark a major transition zone between tropical and temperate biota and are considered a neglected biodiversity hotspot. Dispersal ability and host plant specificity are thought to impact the history and diversity of insect populations across the sky islands. We aimed to investigate the population structure and phylogeography of two pine‐feeding pierid butterflies, the pine white (Neophasia menapia) and the Mexican pine white (Neophasia terlooii), restricted to these “islands” at this transition zone. Given their dependence on pines as the larval hosts, we hypothesized that habitat connectivity affects population structure and is at least in part responsible for their allopatry. We sampled DNA from freshly collected butterflies from 17 sites in the sky islands and adjacent high‐elevation habitats and sequenced these samples using ddRADSeq. Up to 15,399 SNPs were discovered and analyzed in population genetic and phylogenetic contexts with Stacks and pyRAD pipelines. Low genetic differentiation in N. menapia suggests that it is panmictic. Conversely, there is strong evidence for population structure within N. terlooii. Each sky island likely contains a population of N. terlooii, and clustering is hierarchical, with populations on proximal mountains being more related to each other. The N. menapia habitat, which is largely contiguous, facilitates panmixia, while the N. terlooii habitat, restricted to the higher elevations on each sky island, creates distinct population structure. Phylogenetic results corroborate those from population genetic analyses. The historical climate‐driven fluxes in forest habitat connectivity have implications for understanding the biodiversity of fragmented habitats.     

A single amino acid in the PSPG-box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

Curcumin glucosyltransferase (CaUGT2) isolated from cell cultures of Catharanthus roseus exhibits unique substrate specificity. To identify amino acids involved in substrate recognition and catalytic activity of CaUGT2, a combination of domain swapping and site-directed mutagenesis was carried out. Exchange of the PSPG-box of CaUGT2 with that of NtGT1b (a phenolic glucosyltransferase from tobacco) led to complete loss of enzyme activity in the resulting recombinant protein. However, replacement of Arg378 of the NtGT1b PSPG-box with cysteine, the corresponding amino acid in CaUGT2, restored the catalytic activity of the chimeric enzyme. Further site-directed mutagenesis revealed that the size of the amino acid side-chain in that particular site is critical to the catalytic activity of CaUGT2.