Chemical composition of the attachment pad secretion of the locust Locusta migratoria

This study is the first attempt to characterise the chemical composition of the secretion of the smooth pads of the locust Locusta migratoria and to relate this to the composition of the cuticle coverage of the pads and the wings. Gas-chromatography and mass-spectrometry (GC-MS) were the principal techniques used for the characterization of these materials. Secretion droplets were visualised and quantified with the aid of diverse microscopic techniques. The chemical composition of prints is shown to differ from the cuticle coverage, in particular, with respect to the fatty acid distribution: in the secretion, saturated and unsaturated fatty acids with chain lengths between C₁₆ and C₂₀ in both the free form and as glycerides predominate, whereas cuticle coverage contains waxes of long-chained fatty-acids bound to long-chain primary alcohols. The second important difference is the significant amount of glucose and other saccharides found in methanolyzates of the pad fluid. A considerable amount of the amino acids (up to 53%) was detected in the non-volatile portion of the fluid. Data obtained from the shock-freezing, carbon-platinum coating and replica preparation show that the secretory droplets contain nano-droplets on their surfaces. The results lead us to suggest that the pad secretion is an emulsion consisting of lipidic nano-droplets dispersed in an aqueous liquid. According to the chemical composition of the secretion, a high-viscosity of the fluid may be suggested. Presumably, the fluid is a kind of a coupling agent, promoting and strengthening adhesion between otherwise incompatible materials by providing the proximity of contact for intermolecular forces.     

Subtracted restriction fingerprinting--a tool for bacterial genome typing

Reproducible, discriminative, high-throughput methods are required for the identification of bacterial strains and isolates in a clinical environment. A new molecular typing method for bacteria was developed and tested on Salmonella and E. coli species. The technique is called subtracted restriction fingerprinting and is based on double restriction enzyme digestion of genomic DNA followed by end labeling. The "detection" enzyme produces TTAA overhangs that are filled in with digoxigenated nucleotides for subsequent detection, while the "subtraction" enzyme produces GCGC overhangs that are filled in with biotinylated nucleotides that permit the removal of this subset of fragments with either streptavidin-coated magnetic particles or AffiniTip streptavidin columns. The two restriction enzymes are selected to produce a fragment size profile suitable for a specific analytical system. In this demonstration of the principle of subtracted restriction fingerprinting, analysis of Salmonella enterica subsp. enterica serovar Dublin and E. coli on a 30-cm 1.2% agarose gel revealed up to 50 sharp evenly spaced bands, which were sufficient for the discrimination between various isolates and substrains. The restriction enzyme combinations suitable for the analysis of Salmonella and E. coli are presented. The method requires fewer enzymatic steps than amplified fragment length polymorphism, does not need the specialized DNA preparation essential for pulsed field gel electrophoresis, and has a higher reproducibility than PCR-based methods.     

Synthesis and conformation of four 16,17-diols in the 3-methoxy-13alpha-estra-1,3,5(10)-triene series

All four diasteromeric 16,17-diols in the 3-methoxy-13alpha-estra-1,3,5(10)-triene series have been synthesized. The trans-diols 1 and 2 can be obtained by hydroborating the 17-enol acetate 6 (61%, ratio 27:73, preferred alpha attack). OsO(4) dihydroxylation of the olefin 7 yielded the cis-diols 3 and 4 (ratio 13:87). The dihydroxylation proceeds with preference for beta attack caused by a C-ring twist-boat form of 7. The conformations of the diols 2 and 4, the 17-benzyl-17-hydroxy compounds 9 and 10 (obtained by Grignard reaction), and the 16alpha-bromo-17beta-hydroxy compound 8 were determined by X-ray analysis and by 1H NMR spectroscopy in solution. Some compounds, in spite of a 17beta-hydroxy group, had a conformation with a ring C chair form (4, 8, 9) caused by intermolecular interaction in the solid state. The rest of the compounds studied here (2, 10) possessed a conformation with a ring C twist-boat form, which has been also found for all 17beta-substituted compounds in solution. The preferred conformation of the D-ring with 17beta-substituents seems to be the 16beta-envelope form or near this form, but the existence of the 16alpha-envelope form (inversion of the ring D) for some compounds showed great variance in the conformation of ring D, which is substituent dependent.     

Regulation of the Chk2 protein kinase by oligomerization-mediated cis- and trans-phosphorylation

Chk2 is a serine/threonine protein kinase found mutated in certain hereditary and sporadic cancers. Ionizing radiation (IR) activates the kinase activity of Chk2 in a phosphorylation-dependent manner. ATM phosphorylates Chk2 on threonine 68, which promotes oligomerization and phosphorylation on threonines 383 and 387 within the activation loop of the catalytic domain. In this study, threonines 68, 383, and 387 were confirmed as sites of Chk2 phosphorylation both in vitro and in vivo. In addition, serine 516 was identified as a novel IR-inducible phosphorylation site in vivo and as a site of autophosphorylation in vitro. Interestingly, Chk2 was capable of autoactivation in the absence of IR when overproduced in bacteria, in 293 cells, and in murine embryonic fibroblasts lacking Chk2. A kinase-inactive mutant of Chk2 was phosphorylated on T68 and T383/T387 but not on S516 in cells containing Chk2 and on T68 but not T383/T387 or S516 in cells lacking Chk2. This establishes a dependency on Chk2 kinase activity for phosphorylation of T383/T387 and S516 but not for T68 in vivo. We demonstrate that T68 phosphorylation is regulated by kinases in addition to ATM and Chk2. Taken together, our data indicate that autophosphorylation of Chk2 can occur both in cis and in trans and suggest that oligomerization may regulate Chk2 activation by promoting these cis- and trans-phosphorylation events. The importance of oligomerization is underscored by the observation that substitution of isoleucine for threonine at position 157, a mutation found in a subset of patients with Li-Fraumeni syndrome, impairs both Chk2 oligomerization and autophosphorylation.     

Suppression of coatomer mutants by a new protein family with COPI and COPII binding motifs in Saccharomyces cerevisiae

Protein trafficking is achieved by a bidirectional vesicle flow between the various compartments of the eukaryotic cell. COPII coated vesicles mediate anterograde protein transport from the endoplasmic reticulum to the Golgi apparatus, whereas retrograde Golgi-to-endoplasmic reticulum vesicles use the COPI coat. Inactivation of COPI vesicle formation in conditional sec21 (gamma-COP) mutants rapidly blocks transport of certain proteins along the early secretory pathway. We have identified the integral membrane protein Mst27p as a strong suppressor of sec21-3 and ret1-1 mutants. A C-terminal KKXX motif of Mst27p that allows direct binding to the COPI complex is crucial for its suppression ability. Mst27p and its homolog Yar033w (Mst28p) are part of the same complex. Both proteins contain cytoplasmic exposed C termini that have the ability to interact directly with COPI and COPII coat complexes. Site-specific mutations of the COPI binding domain abolished suppression of the sec21 mutants. Our results indicate that overexpression of MST27 provides an increased number of coat binding sites on membranes of the early secretory pathway and thereby promotes vesicle formation. As a consequence, the amount of cargo that can bind COPI might be important for the regulation of the vesicle flow in the early secretory pathway.     

Molecular phylogenetics of allodapine bees,with implications for the evolution of sociality and progressive rearing

Allodapine bees have long been regarded as providing useful material for examining the origins of social behavior. Previous researchers have assumed that sociality arose within the Allodapini and have linked the evolution of sociality to a transition from mass provisioning to progressive provisioning of brood. Early phylogenetic studies of allodapines were based on morphological and life-history data, but critical aspects of these studies relied on small character sets, where the polarity and coding of characters is problematic. We used nucleotide sequence data from one nuclear and two mitochondrial gene fragments to examine phylogenetic structure among nine allodapine genera. Our data set comprised 1506 nucleotide positions, of which 402 were parsimony informative. Maximum parsimony, log determinant, and maximum likelihood analyses produced highly similar phylogenetic topologies, and all analyses indicated that the tropical African genus Macrogalea was the sister group to all other allodapines. This finding conflicts with that of previous studies, in which Compsomelissa + Halterapis formed the most basal group. Changing the basal node of the Allodapini has major consequences for understanding evolution in this tribe. Our results cast doubt on the previous hypotheses that progressive provisioning and castelike social behavior evolved among lineages leading to the extant allodapine taxa. Instead, our results suggest that mass provisioning in Halterapis is a derived feature and that social behavior is an ancestral trait for all allodapine lineages. The forms of social behavior present in extant allodapines are likely to have resulted from a long evolutionary history, which may help explain the complexity of social traits found in many allodapine bees.     

Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts

Pseudomonas aeruginosa infection is a serious complication in patients with cystic fibrosis and in immunocompromised individuals. Here we show that P. aeruginosa infection triggers activation of the acid sphingomyelinase and the release of ceramide in sphingolipid-rich rafts. Ceramide reorganizes these rafts into larger signaling platforms that are required to internalize P. aeruginosa, induce apoptosis and regulate the cytokine response in infected cells. Failure to generate ceramide-enriched membrane platforms in infected cells results in an unabated inflammatory response, massive release of interleukin (IL)-1 and septic death of mice. Our findings show that ceramide-enriched membrane platforms are central to the host defense against this potentially lethal pathogen.     

Expression of truncated PrP targeted to Purkinje cells of PrP knockout mice causes Purkinje cell death and ataxia

PrP knockout mice with disruption of only the PrP-encoding region (Zürich I-type) remain healthy, whereas mice with deletions extending upstream of the PrP-encoding exon (Nagasaki-type) suffer Purkinje cell loss and ataxia, associated with ectopic expression of Doppel in brain, particularly in Purkinje cells. The phenotype is abrogated by co-expression of full-length PrP. Doppel is 25% similar to PrP, has the same globular fold, but lacks the flexible N-terminal tail. We now show that in Zürich I-type PrP-null mice, expression of N-terminally truncated PrP targeted to Purkinje cells also leads to Purkinje cell loss and ataxia, which are reversed by PrP. Doppel and truncated PrP probably cause Purkinje cell degeneration by the same mechanism.     

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes

The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood. Here, we show that a group of WD40 beta-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state. TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p. In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co-chaperone GimC/prefoldin for efficient transfer to the chaperonin. GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability. These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC-assisted folding.