Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae

Background

Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA) in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background.

Results

Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR)-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type.

Conclusions

The study presented here correlates structural differences in WTA to their functional characteristics, thereby providing important information aiding to improve our understanding of molecular host-microbe interactions and probiotic functionality.

     

Cephenniini with prothoracic glands and internal cavities: new taxa,enigmatic characters and phylogeny of the Cephennomicrus group of genera (Coleoptera,Staphylinidae, Scydmaeninae)

Genera of the Cephennomicrus group of the Cephenniini (Scydmaenitae) are revised, and the following new taxa are described: Trichokrater gen.n. , Trichokrater ekkentros sp.n. (type species of Trichokrater) (Borneo), Cephennococcus gen.n. , Cephennococcus kuchingensis sp.n. (type species of Cephennococcus) (Borneo), Cephennococcus kenyirensis sp.n. (West Malaysia), Cephennococcus crassus sp.n. (Borneo), Cephennococcus minutissimus sp.n. (West Malaysia), Pomphopsilla gen.n. , Pomphopsilla luhya sp.n. (type species of Pomphopsilla) (Kenya) and Pomphopsilla soror sp.n. (Kenya). Unique subcuticular pockets with setose openings on the pronotum of Trurlia and Trichokrater are identified as glandular structures. Enigmatic internal prothoracic cavities are described for the first time in Scydmaeninae (in Cephennococcus, Pomphopsilla and a female of an undescribed genus from Sulawesi); their fine structure and function remain unknown. Parsimony‐based cladistic analysis of the adult morphology of genera of Cephenniini provided robust evidence for a monophyly of the Cephennomicrus group, composed of Cephennomicrus, Cephennula, Lathomicrus, Pomphopsilla, Cephennococcus, Trurlia, Trichokrater and two undescribed Oriental genera known from females only; this distinct and well‐supported lineage is a sister group of Cephennodes + Hlavaciellus. The genus Cephennomicrus represented in the analysis by species belonging to three previously postulated species groups is not monophyletic, and a comprehensive study comprising more taxa is necessary to reclassify this heterogeneous group.     

Infants' exploration of scented toys: effects of prior experiences

To evaluate breastfed infants' responses to scented objects, we videotaped the facial and bodily reactions of sixty-three infants as they explored, in succession, three toys that were identical in appearance but different in their characteristic odor. Two of the toys were scented with odorants previously shown to be transmitted to human milk, one with ethanol and the other with vanilla, whereas the third toy was unscented. Each videotape was subjected to frame-by-frame analysis to measure a variety of behaviors that are considered either to be exploratory in nature in that they lead to perceptual information about the object or to reflect the infants' hedonic reaction. Analyses of these behaviors revealed that the infants looked more and vocalized less in the presence of the vanilla-scented toy and spent less time manipulating the ethanol-scented toy when compared with the unscented toy. Moreover, differential exposure to the odors of ethanol and vanilla, as indicated by differential consumption of alcohol by a parent or use of vanilla-scented product by the mother, was related to differential responses to these odors. These findings suggest that human infants are able to detect and retain information about the chemical features of their environment.      

Phylogeny of a new supertribe Cephenniitae with generic review of Eutheiini and description of a new tribe Marcepaniini (Coleoptera: Staphylinidae: Scydmaeninae)

Genera of Eutheiini are reviewed and Eutheimorphus is removed from this tribe of ant‐like stone beetles (Scydmaeninae) and transferred to Cephenniini. A monogeneric Marcepaniini trib.n. is described to accommodate Marcepania gen.n. from Malaysia, with five species: M. semengohensis sp.n. (the type species of Marcepania), M. tuberculata sp.n. , M. seramaensis sp.n. , M. minutissima sp.n. and M. elongata sp.n. A phylogenetic analysis of all genera of Cephenniini, Eutheiini and Marcepaniini based on adult morphological characters resulted in recovering a well‐supported monophyletic clade Eutheiini + (Marcepaniini + Cephenniini) and these tribes are included in Cephenniitae stat.n. (Eutheiini and Cephenniini are therefore removed from Scydmaenitae). Only a weak support for monophyly of Eutheiini was found, but morphological characters allow for maintaining this presumably relic group as a separate tribe. Previously proposed monophyletic groups within Cephenniini were recovered as such, but after inclusion of Eutheimorphus, a sister taxon to the ‘Cephennomicrus group’, the latter lineage gained weak statistical support. The evolutionary history of Cephenniitae is discussed, with focus on known northern hemisphere fossils classified in Scydmaenitae and Hapsomelitae, but possibly closely allied to Cephenniitae. Establishing the supertribe Cephenniitae is the first step toward a profound reclassification of Scydmaeninae on a robust phylogenetic basis. This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:B0E1B12D-9587-4C4F-A908-A12A0C424A8C .