N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.     

Alkyl-dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes.

Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross-linking. After cross-linking and immunoblot analysis alkyl-GrnP synthase could be detected in a 210-kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two-dimensional SDS/PAGE demonstrated that GrnP-acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can form a homotrimeric complex in the absence of GrnP-acyltransferase as was demonstrated by immunoblot analysis after cross-linking experiments with either GrnP-acyltransferase deficient human fibroblast homogenates or recombinant (His)6-tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthase interacts selectively with GrnP-acyltransferase in a heterotrimeric complex and in the absence of GrnP-acyltransferase can also form a homotrimeric complex.     

Lipid composition of the epidermal gel secretion from the Arabian Gulf catfish (Arius thalassinus Ruppell)

Lipids associated with a threat induced epidermal gel secretion from the catfish, Arius thalassinus, have been analyzed. Phospholipids, neutral lipids and glycolipids are all present and each of these subclasses has been analyzed by thin layer and gas chromatography with a general similarity with membrane lipids being noted. The epidermal gel lipids differed from total liver lipids of the catfish. Fatty acid analysis showed the gel lipid to be rich in the unsaturated fatty acids: oleate (omega 7, C18:1), arachidonate (omega 6, C20:4), and docosahexaenoate (omega 3, C22:6). Some prostaglandins were quantitated in lipid extracts from the epidermal gel.     

Immunocytochemical localization of the elongation factor Tu in E. coli cells

The localization of the elongation factor Tu (EF-Tu) in ultrathin cryosections of E. coli cells was determined with the electron microscope using a highly specific immunological labelling technique. EF-Tu is distributed almost homogeneously throughout the cytoplasm. Although it has often been suggested that EF-Tu could be part of a putative prokaryotic cytoskeleton, we did not find any evidence for supramolecular assemblies, such as fibres or filaments, containing a large amount of EF-Tu. EF-Tu was not observed in association with the outer cell membrane and periplasmic space. A topological relationship with the inner membrane is not apparent in our micrographs. In cells in which the EF-Tu level is raised significantly, the protein piles up in discrete cell regions.     

Flight loss linked to faster molecular evolution in insects

The loss of flight ability has occurred thousands of times independently during insect evolution. Flight loss may be linked to higher molecular evolutionary rates because of reductions in effective population sizes (N_e) and relaxed selective constraints. Reduced dispersal ability increases population subdivision, may decrease geographical range size and increases (sub)population extinction risk, thus leading to an expected reduction in N_e. Additionally, flight loss in birds has been linked to higher molecular rates of energy-related genes, probably owing to relaxed selective constraints on energy metabolism. We tested for an association between insect flight loss and molecular rates through comparative analysis in 49 phylogenetically independent transitions spanning multiple taxa, including moths, flies, beetles, mayflies, stick insects, stoneflies, scorpionflies and caddisflies, using available nuclear and mitochondrial protein-coding DNA sequences. We estimated the rate of molecular evolution of flightless (FL) and related flight-capable lineages by ratios of non-synonymous-to-synonymous substitutions (dN/dS) and overall substitution rates (OSRs). Across multiple instances of flight loss, we show a significant pattern of higher dN/dS ratios and OSRs in FL lineages in mitochondrial but not nuclear genes. These patterns may be explained by relaxed selective constraints in FL ectotherms relating to energy metabolism, possibly in combination with reduced N_e.     

Electromyogram and force during stimulated fatigue tests of muscles in dominant and non-dominant hands

Mechanical and electrical properties were studied for the first dorsal interosseous muscle of the dominant (d-FDI) and non-dominant hand (nd-FDI). Observations were made before, during and after a fatigue test, fatigue being evoked by percutaneous electrical stimulation of the ulnar nerve. The test consisted of 30 Hz bursts of ten supramaximal 0.1 ms pulses, repeated once a second for 5 min. The measurements included the amplitude of the first and fifth compound muscle action potentials (M-waves) within bursts, the peak burst force and the amplitude and time course of single twitches. At the end of the fatigue test, burst force had decreased to about the same extent in the FDI of both hands. The final decline in first M-wave amplitude was, however, significantly more pronounced for the nd-FDI than for the d-FDI. There were no longer any significant discrepancies between the two muscles after a subsequent recovery-period of 15 min. Comparisons among nd-FDI of various individuals demonstrated the presence of significant inter-individual differences in fatigue-related force-drop without any associated differences in M-wave decline. Intra-individual variability was similar for fatigue-related force-drop and M-wave decline.     

Detection of Infectious Astroviruses in Water

A method based on the infection of CaCo-2 cells and molecular hybridization with a specific cDNA probe has been developed for the detection of infectious astroviruses in environmental samples. By this procedure wild-type astroviruses have been detected in water from an area where a concurrent gastroenteritis outbreak was reported.     

A machine for sawing 80-micrometer slices of carious enamel

Design and construction of a machine that cuts 80-microns slices of sound and carious dental enamel and other calcified tissues is described. These slices can be used for quantitative microradiographic studies. Preparation takes minutes. Thickness for a given slice is uniform within 2 microns, mean thickness is within 4 microns of the intended value and roughness is about 0.1 micron. Commercial components have been used where possible. Information is provided to permit purchase of the components of the machine and its construction in the average university workshop.     

Diurnal variation in 5′-nucleotidase activity in rat liver

Summary The diurnal variation of 5-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.