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.     

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.     

Postdoctoral Researchers in the UK: A Snapshot at Factors Affecting Their Research Output

Postdoctoral training is a typical step in the course of an academic career, but very little is known about postdoctoral researchers (PDRs) working in the UK. This study used an online survey to explore, for the first time, relevant environmental factors which may be linked to the research output of PDRs in terms of the number of peer-reviewed articles per year of PDR employment. The findings showed reliable links between the research output and research institutions, time spent as PDR, and parental education, whereas no clear links were observed between PDRs'' output and research area, nationality, gender, number of siblings, or work environment. PDRs based in universities tended to publish, on average, more than the ones based in research centres. PDRs with children tended to stay longer in postdoctoral employment than PDRs without children. Moreover, research output tended to be higher in PDRs with fathers educated at secondary or higher level. The work environment did not affect output directly, but about 1/5 of PDRs were not satisfied with their job or institutional support and about 2/3 of them perceived their job prospects as “difficult”. The results from this exploratory study raise important questions, which need to be addressed in large-scale studies in order to understand (and monitor) how PDRs'' family and work environment interact with their research output—an essential step given the crucial role of PDRs in research and development in the country.     

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.     

Sectoring of fungal colonies induced by low concentrations of fernasan

An inhibitor of Aspergillus flavus has been isolated from peanuts and identified as 5,7-dimethoxyisoflavone. Authentic 5,7-dimethoxyisoflavone, synthesized from benzyl-2-hydroxy-4,6 dimethoxyphenyl (3), and the isolated compound display identical ultraviolet spectra in methanol, in methanol+sodium methoxide, and in methanol+AlCl₃; similar behavior in solvent extraction, column chromatography, TLC, and fungal inhibition.     

Isolation of aflatoxin from Acacia and the incidence of Aspergillus flavus in the Sudan

A series of environmental and clinical isolates of Sporothrix schenckii being studied in our laboratories under standard conditions of temperature, humidity and media, displayed, in some of the isolates, large dematiaceous hyphae. The large hyphae could be seen to produce the fine hyphae associated with S. schenckii on which typical microconidia developed. Typical microconidia also developed occasionally on the large hyphae, and strands of the unusually large hyphae also were seen to form arthroconidia. Some strains formed large, thin-walled cells on thin and/or thick hyphae resembling the balloon cells seen in mature colonies of Trichophyton tonsurans.All strains which have demonstrated these various and unusual structures histopathologically produced typical sporotrichosis in laboratory animals. When reisolated from these animals, the cultures again exhibited the unusual structures, as well as those typical of S. schenckii.     

Identification and characterization of three genes and two pseudogenes on chromosome 13

A study was conducted on the feasibility of isolating genes and pseudogenes that map to chromosome 13 by a hybridization-based approach using a 13-specific library and pools of repeat-free cDNA clones. Five pairs of cDNA and chromosome 13 genomic clones were identified and characterized. Partial or full-length sequence was derived from all cDNAs, and database searches were performed for putative gene identification. Partial sequence was also obtained from the chromosome 13 genomic clones for comparison with those of the hybridizing cDNAs. As a result of these analyses we identified three genes, a putative homologue of a porcine mRNA encoding an unidentified hepatic protein, a putative homologue of a yeast integral membrane protein, and a gene for a translationally controlled tumor protein, and two processed pseudogenes, ribosomal proteins L23a and S3a. The latter was formerly identified as the v-fos transformation effector gene, Fte-1, and recently cited as a possible candidate for the BRCA2 gene on chromosome 13. All genes and pseudogenes were localized to cytogenetic bands by in situ hybridization of metaphase chromosomes with probes derived from the chromosome 13 genomic clones.     

Actions of platelet-derived growth factor isoforms in mesangial cells

Platelet-derived growth factor (PDGF) occurs as homodimers or heterodimers of related polypeptide chains PDGF-BB, -AA, and -AB. There are two receptors that bind PDGF, termed alpha and beta. The beta receptor recognizes PDGF B chain and is dimerized in response to PDGF BB. The alpha receptor recognizes PDGF B as well as A chains and can be dimerized by the three dimeric forms of PDGF AA, AB, and BB. To characterize PDGF receptor signaling mechanisms and biologic activities in human mesangial cells (MC), we explored the effects of the three PDGF isoforms on DNA synthesis, phospholipase C activation, and PDGF protooncogene induction. PDGF-BB homodimer and AB heterodimer induced a marked increase in DNA synthesis, activation of phsopholipase C, and autoinduction of PDGF A and B chain mRNAs, whereas PDGF-AA homodimer was without effect. The lack of response to PDGF AA could be accounted for by down regulation of the PDGF-alpha receptor since preincubation of MC with suramin restored PDGF AA-induced DNA synthesis. Ligand binding studies demonstrate specific binding of labeled PDGF BB and AB and to a lower extent PDGF AA isoforms to mesangial cells. These results are consistent with predominant expression of PDGF beta receptor in MC, which is linked to phospholipase-C activation. The potent biologic effects of PDGF-AB heterodimer in cells that express very few alpha receptors and do not respond to PDGF AA are somewhat inconsistent with the currently accepted model of PDGF receptor interaction and suggest the presence of additional mechanisms for PDGF isoform binding and activation. © 1994 Wiley-Liss, Inc.