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.     

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

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.     

Leishmania chagasi: in vitro differentiation of promastigotes monitored by flow cytometry

A sequential development from a less infective to an infective stage of Leishmania promastigotes growing in culture has been previously reported. The aim of this work was to investigate whether freeze-fracture electron microscopy and flow cytometry would be able to provide some reliable morphological markers of in vitro differentiation of Leishmania chagasi promastigotes. The flow cytometry technique discriminates between the L. chagasi promastigotes from the different stages of their in vitro differentiation. The "forward scatter" intensity of the parasite, very high 15 hr after seeding when the parasites were very condensed and with a high DNA content per particle, strongly decreased during the culture course. Parallel experiments have shown a striking correlation between forward scatter intensity, growth curves, and infectivity of promastigote populations. By contrast, freeze-fracture techniques showed that in either less infective or infective promastigote plasma membranes, the intramembrane particles density in protoplasmic fracture faces (about 2800/micron 2) and in exoplasmic fracture faces (about 1000/micron 2) was independent of the time of cultivation. The amount of filipin lesions, which reflects the cholesterol content within the plasma membrane, was also constant throughout the culture course. Both data suggest that the architecture of the plasma membrane is an intrinsic characteristic of the promastigote stage. This study shows that whereas freeze-fracture electron microscopy does not provide markers for the differentiation of Leishmania promastigotes, flow cytometry may on the other hand be of value as a screening test for promastigote populations allowing the characterization of their developmental stages in in vitro cultures.     

Allelopathic effects of nicotine on maize

Nicotine treatment of maize seeds at 5.0 and 7.5 mM concentrations caused an increase of 6.2 and 18.7% in amylase activity, respectively. It is being suggested that the positive effect of nicotine on maize is, at least, partially due to increased solubilization of the stored starch through induction of the synthesis or the activity of amylase.     

Glucoamylase production byAspergillus awamori on rice flour medium and partial characterization of the enzyme

Summary Aspergillus awamori ATCC 22342 was selected from 12 strainsof Aspergillus spp.and Rhizopus spp. as the best producer of amylase. Optimal growth conditions for the enzyme production in shake flasks were provided by: a medium containing 60 g/1 rice flour, 0.075% (w/v) NaNO₂ and 0.075% (v/v) corn-steep liquor, a temperature of 30° C and initial pH value of 6.5. The enzyme was characterized as a glucoamylase with a molecular weight of 49,000. Maximum enzyme activity occurred at 45 C and pH 5.8. The enzyme was stable at 40° C and lost 70 and 90% of activity when heated for 30 min at 50 and 60°C, respectively. Thermal inactivation was slowed in the presence of starch. Michaelis-Menten constants for soluble starch and dextrin were estimated as 12.5 and 33.3 mg/ml, respectively. This enzyme may be used for the production of glucose-rich syrups from rice starch.

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Producción de glucoamilasa por Aspergillus awamori en harina de arroz y caracterización parcial del enzima

Resumen Aspergillus awamori ATCC 22342 se seleccionó entre 12 cepas deAspergillus spp. y deRhizopus spp. como el mejor productor de amilasa. La condiciones óptimas de crecimiento para la producción del enzima en frascos de agitación fueron las siguientes: un medio con la composicion siguiente: 60 g/1 de harina de arroz, 0.075% (m/v) NaNO₂ y 0.075% (v/v) de extracto de maíz (corn steep liquor); una temperatura de 30°C y un pH inicial de 6.5. El enzima fue caracterizado como una glucoamilasa de peso molecular 49,000. La máxima actividad enzimática se obtuvo a 45°C con un pH de 5.8. El enzima era estable a 40 C pero perdió un 70 y un 90% de su actividad cuando se calentó durante 30 min a 50 y 60° C respectivamente. La inactivación térmica fue más lenta en presencia de almidón. Las constantes de Michaelis-Menten para almidón soluble y para dextrina se estimaron como 12.5 y 33.3 mg/ml respectivamente. Este enzima puede utilizarse para la producción de jarabes ricos en glucosa a partir de almidón de arroz.

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Production de glucoamylase par Aspergillus awamori cultivé sur milieu à la farine de riz et caractérisation partielle de l'enzyme

Résumé Aspergillus awamori ATCC 22342 a été sélectionné parmi 12 souches d'Aspergillus spp. et deRhizopus spp. comme étant le meilleur producteur d'amylase. Les conditions optimales de croissance pour la production d'enzyme en fioles agitées sont: un milieu contenant 60 g/1 de farine de riz, 0.075% (w/v) de NaNO₂ et 0.075% (v/v) de liqueur de corn steep, une température de 30° C et un pH initial de 6.5. L'enzyme a été caractérisé comme étant une glucoamylase de poids moléculaire 49,000. L'activité maximum de l'enzyme se situe à 45°C et pH 5.8. L'enzyme est stable à 40°C et perd 70 et 90% de son activité par chauffage pendant 30 min à 50 et à 60°C, respectivement. L'inactivation thermique est ralentie en présence d'amidon. Les constantes de Michaelis-Menten pour l'amidon soluble et pour la dextrine ont été éstimées, respectivement, à 12.5 et 33.3 mg/ml. Cet enzyme peut être utilisé pour la production de sirops riches en glucose à partir d'amidon de riz.

     

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.