Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild beta-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using beta-elimination/Michael addition methods.     

Changes in performance, muscle metabolites, enzymes and fibre types after short sprint training

In contrast to endurance training, little research has been carried out to investigate the effects of short (<10 s) sprint training on performance, muscle metabolism and fibre types. Nine fit male subjects performed a mean of 16 outdoor sprint running training sessions over 6 weeks. Distances sprinted were 30–80 m at 90–100% maximum speed and between 20 and 40 sprints were performed in each session. Endurance (maximal oxygen consumption; V˙O_{2 max}), sprint (10 m and 40 m times), sustained sprint (supramaximal treadmill run) and repeated sprint (6 × 40 m sprints, 24 s recovery between each) performance tests were performed before and after training. Muscle biopsy samples (vastus lateralis) were also taken to examine changes in metabolites, enzyme activities and fibre types. After training, significant improvements were seen in 40 m time (P < 0.01), supramaximal treadmill run time (P < 0.05), repeated sprint performance (P < 0.05) and V˙O_{2 max} (P < 0.01). Resting muscle concentrations of ATP and phosphocreatine did not change. Phosphorylase activity increased (P < 0.025), citrate synthase activity decreased (P < 0.01), but no significant changes were recorded in myokinase and phosphofructokinase activities. The proportion of type II muscle fibres increased significantly (P < 0.05). These results demonstrate that 6 weeks of short sprint training can improve endurance, sprint and repeated sprint ability in fit subjects. Increases in the proportion of type II muscle fibres are also possible with this type of training. Accepted: 5 January 1998     

Dbp5p/Rat8p is a yeast nuclear pore-associated DEAD-box protein essential for RNA export.

To identify Saccharomyces cerevisiae genes important for nucleocytoplasmic export of messenger RNA, we screened mutant strains to identify those in which poly(A)+ RNA accumulated in nuclei under nonpermissive conditions. We describe the identification of DBP5 as the gene defective in the strain carrying the rat8-1 allele (RAT = ribonucleic acid trafficking). Dbp5p/Rat8p, a previously uncharacterized member of the DEAD-box family of proteins, is closely related to eukaryotic initiation factor 4A(eIF4A) an RNA helicase essential for protein synthesis initiation. Analysis of protein databases suggests most eukaryotic genomes encode a DEAD-box protein that is probably a homolog of yeast Dbp5p/Rat8p. Temperature-sensitive alleles of DBP5/RAT8 were prepared. In rat8 mutant strains, cells displayed rapid, synchronous accumulation of poly(A)+ RNA in nuclei when shifted to the non-permissive temperature. Dbp5p/Rat8p is located within the cytoplasm and concentrated in the perinuclear region. Analysis of the distribution of Dbp5p/Rat8p in yeast strains where nuclear pore complexes are tightly clustered indicated that a fraction of this protein associates with nuclear pore complexes (NPCs). The strong mutant phenotype, association of the protein with NPCs and genetic interaction with factors involved in RNA export provide strong evidence that Dbp5p/Rat8p plays a direct role in RNA export.     

Characteristics of Airborne Actinomycete Spores

Airborne actinomycete spores, important contaminants in occupational and residential environments, were studied with respect to their (i) release into the air, (ii) aerodynamic and physical size while airborne, and (iii) survival after collection onto agar with an impactor. Three actinomycete species were selected for the tests to exemplify the three main spore types: Streptomyces albus for arthrospores, Micromonospora halophytica for aleuriospores, and Thermoactinomyces vulgaris for endospores. The results show that the incubation conditions (temperature, time, and nutrients) needed for the development of spores for their release into air are different from the conditions that are needed for colony growth only. Additional drying of M. halophytica and T. vulgaris cultures was needed before spores could be released from the culture. The aerodynamic sizes of the spores, measured with an aerodynamic particle sizer, ranged from 0.57 (T. vulgaris) to 1.28 μm (M. halophytica). The physical sizes of the spores, when measured with a microscope and an image analysis system, were found to be smaller than previously reported in the literature. The relative recovery of the spores on agar media ranged from 0.5 (T. vulgaris) to 35% (S. albus). The results indicate that the culturability of the collected airborne actinomycete spores varies widely and is affected by several variables, such as the species and the sampling flow rate. Therefore, alternatives to commonly used cultivation methods need to be developed for the enumeration of actinomycete spores.     

Opening the Social Gateway: Early Vocal and Social Sensitivities in Brown-Headed Cowbirds (Molothrus ater)

The organization of cowbird ( Molothrus ater ) social groups affords individuals living in the groups different opportunities for learning and also structures trajectories of social development. Here, we studied the influence of adults on social organization of very young cowbirds. In three experiments, we housed juvenile birds in large, seminatural environments that either contained or did not contain adult conspecifics. We then observed the social associations and vocalizations of juveniles in each environment. The presence of adults in the social environment influenced the assortment and singing patterns of juveniles, although throughout the three experiments adults rarely interacted physically with juveniles. Juveniles housed with adults interacted with other juveniles more often and sang significantly less often than juveniles housed without adults. Effects of adult presence or absence on social organization and singing patterns emerged extremely rapidly and could be reversed quickly. Taken as a whole, the experiments revealed that very young cowbirds, in the first days of independence from their hosts, were sensitive to, and reacted rapidly to, the composition of their social environment. Specifically, presence of other age classes of individuals within the group increased juvenile associations and delayed production of vocalizations by juvenile males. Self-organization within the social group produced different social environments, which could in turn create different gateways for social learning and vocal development.     

STUDIES OF PALEOZOIC FUNGI. III. FUNGAL PARASITISM IN A PENNSYLVANIAN GYMNOSPERM

Evidence of fungal parasitism is found in the Pennsylvanian gymnospermous cone, Lasiostrobus polysacci Taylor. Indication of fungal activity is found in the outer cortical region of the axis of the cone and in the fleshy microsporophylls. Specimens exhibit severe tissue disruption, thick-walled, branched, septate hyphae, and possible reproductive structures. Parenchymatous cortical cells may also contain rounded bodies which are continuous with the cell wall. Similar structures are formed in many extant taxa in response to fungal invasion, and are termed wall appositions or callosities. Although their role in extant plants is disputed, they are clearly the product of a living host cell. Such spherical bodies, however, are not restricted to the cell periphery but in some cases occlude the cell lumen. In appearance they resemble resinous remains similar to those found in other coal ball plants. The blockage of entire cells or groups of cells may have served to retard hyphal growth or isolate infected cells. The occurrence of such structures in a Carboniferous plant provides the best evidence to date of parasitism during the Paleozoic.     

Aquatic Snails, Passive Hosts of Mycobacterium ulcerans

Accumulative indirect evidence of the epidemiology of Mycobacterium ulcerans infections causing chronic skin ulcers (i.e., Buruli ulcer disease) suggests that the development of this pathogen and its transmission to humans are related predominantly to aquatic environments. We report that snails could transitorily harbor M. ulcerans without offering favorable conditions for its growth and replication. A novel intermediate link in the transmission chain of M. ulcerans becomes likely with predator aquatic insects in addition to phytophage insects. Water bugs, such as Naucoris cimicoides, a potential vector of M. ulcerans, were shown to be infected specifically by this bacterium after feeding on snails experimentally exposed to M. ulcerans.     

Cartilage is a metazoan tissue; integrating data from nonvertebrate sources

Connective tissues are responsible for much of the variation in morphology that we see today. Cartilage is a type of connective tissue that is often considered to be restricted to vertebrates, however, cartilaginous tissues are also found within invertebrates. Unfortunately, most definitions and classification schemes for cartilages suffer from a strong vertebrate bias, severely hampering the efforts of those who have attempted to include invertebrate tissues as cartilage. To encompass all types of cartilage, current classification systems need to be expanded. Here we present vesicular cell‐rich as a new cartilage classification. Invertebrate cartilages, comparable to vertebrate cartilages at both cell and tissue levels, are composed of similar molecules, yet the extent to which they may be homologous is unknown. One option for studying the evolution of tissues is to adopt molecular phylogenetic approaches. However, the paucity of published molecular data makes addressing the evolution of cartilage using molecular phylogenetic approaches unrealistic at this time. Cartilage likely evolved from a chondroid connective tissue precursor, and may have been independently derived many times. The appearance of cartilaginous tissues of unknown phylogenetic affinities in such a wide diversity of animal groups warrants further investigation.     

MONOMORPHISM,REDUCED GENE FLOW,AND CLEISTOGAMY IN RARE AND COMMON SPECIES OF LESPEDEZA (FABACEAE)

Population genetic structure was analyzed in the rare, native prairie legume Lespedeza leptostachya Engelm. and in the widespread L. capitata Michx. Both species produce a mixture of chasmogamous and cleistogamous flowers. Allozymes were analyzed for 32 loci from 224 individuals from 12 populations of L. leptostachya and for 34 loci in 291 individuals from 12 populations of L. capitata. L. leptostachya is entirely monomorphic at all loci studied, while L. capitata shows strong among-population differentiation for the limited variation that occurs in that species. Allozyme data suggest that the level of gene flow among populations of L. capitata is very low, and that very low levels of outcrossing are effected by the chasmogamous flowers in L. capitata.     

Structure, function, and motility of vacuoles in filamentous fungi

Current information on the structure and function of motile tubular vacuoles in Pisolithus tinctorius and other fungi is reviewed. The use of fluorochromes to label the vacuole lumen is evaluated and observations on the structure and motility of vacuoles in P. tinctorius are differentiated from possible artifacts. The styryl dyes FM4-64 and MDY-64, used in yeast to demonstrate endocytosis, show little or no labeling of internal membranes in undamaged P. tinctorius cells. This agrees with our data showing that other probes for endocytosis such as Lucifer yellow CH are not taken up by hyphal tip cells. Overall, the observations do not support endocytosis in hyphal tips. It has been suggested that tubular vacuole systems carry out longitudinal transport, and evidence in favor of this hypothesis is evaluated. New data are presented to show that many of the large vacuoles in subapical cells are attached to the plasma membrane and are relatively immobile, while video sequences show movement of fluorochrome in pulses along a series of several large vacuoles, all interconnected via tubules. Tubular vacuoles from thick sections of hyphae processed under anhydrous conditions are shown by X-ray microanalysis to contain relatively high levels of P and K, as seen previously in the larger vacuoles. These results provide further evidence for a role of the tubular vacuoles in longitudinal transport of P. Copyright 1998 Academic Press.