Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.     

Generation of highly potent DYRK1A-dependent inducers of human β-Cell replication via Multi-Dimensional compound optimization

Small molecule stimulation of β-cell regeneration has emerged as a promising therapeutic strategy for diabetes. Although chemical inhibition of dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is sufficient to enhance β-cell replication, current lead compounds have inadequate cellular potency for in vivo application. Herein, we report the clinical stage anti-cancer kinase inhibitor OTS167 as a structurally novel, remarkably potent DYRK1A inhibitor and inducer of human β-cell replication. Unfortunately, OTS167’s target promiscuity and cytotoxicity curtails utility. To tailor kinase selectivity towards DYRK1A and reduce cytotoxicity we designed a library of fifty-one OTS167 derivatives based upon a modeled structure of the DYRK1A-OTS167 complex. Indeed, derivative characterization yielded several leads with exceptional DYRK1A inhibition and human β-cell replication promoting potencies but substantially reduced cytotoxicity. These compounds are the most potent human β-cell replication-promoting compounds yet described and exemplify the potential to purposefully leverage off-target activities of advanced stage compounds for a desired application.     

Sex pheromones in three Bradysia species (Dipt., Sciaridae): novel bioassays with female body extracts and fractions

Females of the fungus gnat species Bradysia difformis , Bradysia optata and Bradysia tilicola are able to produce sexual attractants. In males, the female extracts elicit species-specific courtship behaviour and attraction in an olfactometer bioassay. Attractiveness of the sex pheromone extracts seems to differ in the investigated species, being highly attractive for males in B. difformis and only moderately attractive in B. tilicola . Female B. difformis are attractive from late pupal stages until death of the imago. In contrast, female B. optata and B. tilicola are not attractive to males before emergence from the pupa and attractiveness decreases in older females. The pheromones seem to be located on the surface of several body compartments of freshly emerged females. Sexual pheromones of all three species represent multicomponent blends and can be separated in several fractions via thin layer chromatography (TLC). Males of all species show courtship behaviour when placed on developed TLC sheets, indicating the position of the biologically active compounds. In B. difformis and B. optata , the most non-polar TLC fraction is attractive to males, whereas the single fractions of B. tilicola -pheromone elicited almost no attraction of males.     

A comprehensive proteome map of glandular trichomes of hop (Humulus lupulus L.) female cones: Identification of biosynthetic pathways of the major terpenoid‐related compounds and possible transport proteins

Female flowers of hop (Humulus lupulus) are an essential source of terpenoid‐related compounds, which are mainly used as flavoring in the beer brewing process. The compounds involved are bitter acids, terpenophenolics, as well as mono‐ and sesquiterpenoids. In this work, we analyzed the proteome of purified glandular trichomes (lupulin glands) from female flowers, which produce and accumulate these compounds. An extensive 2D‐LC‐MS/MS analysis identified 1015 proteins. Of these, most correspond to housekeeping and primary metabolism‐related proteins, albeit predominantly including amino acid and lipid metabolism, which feeds the specialized (secondary) metabolism. Indeed, 75 proteins belong to the specialized metabolism. No less than 40 enzymes are involved in the synthesis of terpenoid‐derived compounds and 21 are predicted transporters, some of which might be involved in the transport of specialized metabolites. We discuss the possible routes involved in the intra‐ and intercellular translocation of terpenoids and their precursors. This comprehensive proteomic map of the glandular trichomes of hop female flowers represents a valuable resource to improve our knowledge on the function of glandular trichomes.     

Priming biotic factors for optimal protocorm-like body and callus induction in hybrid Cymbidium (Orchidaceae), and assessment of cytogenetic stability in regenerated plantlets

Protocorm-like bodies (PLBs) and callus were induced in epiphytic hybrid Cymbidium Twilight Moon ‘Day Light’, where induction capacity was strongly explant dependent. Following the use of various explant sources (PLB, leaf tip or base, root tip or base, cell and tissue ‘suspension’), highest PLB formation and callus induction occurred when we used whole PLBs, PLB segments or PLB transverse thin cell layers (tTCLs) or longitudinal TCLs (lTCLs). Plantlet growth and photosynthetic state from whole or bisected PLBs, as well as from tTCLs were not significantly different, after analysis of chlorophyll content. However plantlets generated from lTCLs showed lower values for growth and photosynthetic parameters. All resultant plants were shown to be cytogenetically identical using RAPD and mtDNA analysis despite cytological variation and endopolyploidy occuring between different plant parts. Acclimatization and survival rate was shown to be 100% in the generated plants.     

Developmental changes in gangliosides during myogenesis of a rat myoblast cell line and its drug resistant variants.

Cloned cells of a myoblast line show the presence of GM₃, GM₂, GM₁ and GD_1a gangliosides. The amount of GM₃, GM₂ and GM₁ gangliosides does not vary significantly during the differentiation of myoblasts to myotubes. However, the concentration of GD_1a transiently increases almost 3-fold just prior to the fusion of myoblasts and returns to the basal levels in the myotubes. Mutant myoblasts selected for 5-azacytidine resistance and unable to fuse produce only GM₃ and traces of GM₂. We conclude that GD_1a probably participates in the fusion process through yet unknown mechanism.     

The geographic distribution of pubescence in the sea daisy, Borrichia aborescens, on Bahamian Islands

Borrichia aborescens (L.) DC. is a salt‐tolerant perennial herb that is common on small islands in the central Bahamas. Two morphotypes are present: one with densely pubescent leaves and one with glabrous leaves. I conducted surveys in three archipelagos to document the geographical distribution of pubescence in this species and to infer the underlying mechanisms. B. aborescens was also grown from seed in a greenhouse. The pubescent form of B. aborescens was relatively more abundant on small islands than on nearby large ‘mainland’ islands. In two of the three archipelagoes, pubescence increased with distance on small islands. The pubescent form was relatively more abundant on small islands exposed to the open ocean compared to small islands that were protected by mainland islands or reefs on all sides. On a large mainland island, the pubescent form decreased in relative abundance inland from the coast. B. aborescens cultivated in a greenhouse revealed the effect of a genetic factor on the expression of pubescence. The observed patterns of variation are consistent with a physiological explanation for the adaptive benefit of pubescence. Trichomes may prevent physical blockage of the stomata by accumulation of salt in areas near breaking waves and salt spray.     

Microinjected interferon does not promote an antiviral response in Hela cells

Human fibroblast interferon (Hu IFN beta) was directly introduced with glass micropipets into the cytoplasm of Hela cells. Such an injection of more than 10(4) molecules per cell failed to induce any antiviral state when challenged with vesicular stomatitis virus (VSV). These findings are discussed in relation to the possible role of internalization in the mechanism of antiviral action of interferon.     

Density gradient enrichment of Escherichia coli lpxL mutants

We previously described enrichment of conditional Escherichia coli msbA mutants defective in lipopolysaccharide export using Ludox density gradients (Doerrler WT (2007) Appl Environ Microbiol 73; 7992–7996). Here, we use this approach to isolate and characterize temperature-sensitive lpxL mutants. LpxL is a late acyltransferase of the pathway of lipid A biosynthesis (The Raetz Pathway). Sequencing the lpxL gene from the mutants revealed the presence of both missense and nonsense mutations. The missense mutations include several in close proximity to the enzyme's active site or conserved residues (E137K, H132Y, G168D). These data demonstrate that Ludox gradients can be used to efficiently isolate conditional E. coli mutants with defects in lipopolysaccharide biosynthesis and provide insight into the enzymatic mechanism of LpxL.