Peptide maps of T mutant of influenza virus

Peptide maps were prepared by high-voltage electrophoresis and chromatography for t 30 degrees C mutant and the original strain A Port Chalmers 1/73 H3N2 of influenza virus. Clear differences in the distribution and number of spots between the mutant and the original strain were found. The differences are due presumably to changes in the amino-acid sequences.     

Leucine incorporation in the ganglia of praying mantids during a learning process

Leucine incorporation into four ganglia (‘brain’. B; prothoracic ganglion, P; mesothoracic ganglion, M; and metathoracic ganglion, T) was studied in mantids (Stagmatoptera biocellata) trained not to attack a black star figure in movement. There were two experimental groups, i.e. LM and WH experimental groups, and one control group. The LM and WH groups differed in the experimental conditions of training in such a way that both had similar motor activity and sensorial stimulation but only one of them evinced learning (Maldonado and Tablante, 1975).After training, incorporation of |¹⁴C| leucine into P and B was greater in experimental than in control animals. This result is not necessarily related to learning because no difference was found between LM and WH experimental groups. The metabolic gradient of the four ganglia of the experimental animals was P→B→M→T, whereas that found for the control group was M→T→P→B. The authors interpret these findings as supporting a hypothesis previously set forth that correlates the metabolic gradient in the four ganglia with differences regarding control of motor activity and/or sensorial input.Experiments involving double labelling and electrophoresis indicated that the P and B changes after training involved all the proteins, and were not restricted to one or a few protein species.     

Origin of small RNA in von Magnus particles of influenza virus.

A clone of recombinant virus obtained from the cross between WSN and Hong Kong strains of influenza virus gave rise to progeny containing predominantly von Magnus particles. In the electropherogram of virus RNA, the P3 gene was markedly diminished, and a new species of RNA (extra RNA) was present in addition to eight gene segments. The origin of the extra RNA was studied by two-dimensional gel electrophoresis of T1 RNase-generated oligonucleotides. Four out of five large oligonucleotide spots present in the extra RNA matched to those contained by the P3 gene. It was concluded that the extra RNA was derived from the P3 gene probably by deletion. The possible origin of the spot which was present in the extra RNA but not in eight gene segments including P3 was discussed.     

Changes in brain peptides and memory consolidation in the praying mantis

A praying mantis (Stagmatoptera biocellata) faced with a bird (Padda orzyvora) displays a frightening reaction called a deimatic reaction (DR). Habituation of this response takes place after repeated presentation of the bird (no-DR training). The purpose of the present paper is to: (a) attempt an analysis of the effect of anoxia at various times with different types of no-DR training; (b) study the dynamics of those types of training that cause habituation but not consolidation, in order to use these groups of animals as controls of groups that show both habituation and consolidation; and (c) test a correlation between the no-DR memory consolidation and the appearance of specific amine-containing components in high-voltage electrophoregrams obtained from brains of the mantids.     

Preliminary analysis of the cauliflower mitochondrial proteome

Purified cauliflower (Brassica oleracea var. botrytis) mitochondrial proteins fractionated into soluble, membrane, integral membrane and peripheral membrane samples were analyzed by 2D- PAGE (isoelectric focusing/ SDS polyacrylamide gel electrophoresis). 2D gels patterns were compared using the Imager Master 2D Elite software. 561 silver stained protein spots were resolved after electrophoresis under standard conditions of a whole protein extract. In the soluble fraction a prevalent number of more intense protein spots was observed. The cauliflower protein 2D patterns resembled Arabidopsis thaliana 2D patterns. The two protein spots selected which occupied a similar isoelectric point positions on both gels represented the same proteins as revealed by ESI-MS analysis of cauliflower proteins. The third selected spot belongs to unidentified proteins. The comparative analysis of mitochondrial suborganellar fractions proved the usefulness of this approach.     

Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.

Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons.     

Separation and determination of cross-linking amino acids of elastin by high-voltage paper electrophoresis

A method is described for the separation and quantitative determination of the weakly basic (cross-linking) amino acids of elastin. A 6 N HCl hydrolyzate is submitted to high-voltage electrophoresis at pH 3.8. At least eight spots can be identified in the weakly basic region and quantitated by the ninhydrin-photodensitometric method. Some of these are spot 3 for desmosine + isodesmosine, and 7 for lysinonorleucine. Quantitative data given for two typical elastin preparations are in agreement with direct amino acid analysis.     

Analysis of grape berry cell wall proteome: a comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.     

How many spots with missing values can be tolerated in quantitative two-dimensional gel electrophoresis when applying univariate statistics?

Quantitative proteomic comparisons require a sufficient number of samples to reach an acceptable level of significance. But 2D gel electrophoresis commonly results in incomplete data sets due to spots with missing values reducing thereby the number of parallel measurements for individual proteins. Here we investigated how many missing values per spot can be tolerated. The number of spots in common between all gels was found to decrease with the number of parallel gels in a non-linear fashion. Increasing numbers of missing values were associated with a moderate increase in the quantitative variation of spot volumes. Based on the missing value pattern in 20 gels we performed an analysis of the multiple testing power for the hypothetical scenario of a comparative 2DE study with six or twelve parallel gels. The calculation considered the statistical power of the individual spot as well as the number of spots included in the analysis. The power increased with inclusion of spots with higher number of missing values and showed an optimum at a specific minimum number of spot replicates. The results suggest that proteins with missing values can be included in a univariate analysis as long as a sufficient number of parallel gels are made.     

Mnemonic factors in a learning process of praying mantids

Adult female mantids (Stagmatoptera biocellata) learn not to attack a black star figure in movement. The influence of different mnemonic factors in the aquisition, the speed, and/or the retention of this learning was tested. Results demonstrate that learning depends on the number of trials, the span of the intersession period, the presence or absence of a load on the mantid's head, and the greater or smaller constancy in the stimulus presentation. On the other hand, the presence or absence of an intertrial interval seems to have no influence on the process. Mantids without a load on their heads, i.e. without an experimental trick that facilitates learning, can only overcome their strong tendency to attack when two conditions are fulfilled: a massive number of presentations (80 trials, at least) and great constancy in the stimulus presentation. Three principal points are discussed concerning these results: (1) The survival value of these findings. (2) The importance of mnemonic factors to minimize in an experimental design the difference in the learning concomitants between a group that learns and another group which does not learn. (3) The classification of this learning process as a case of negative reinforced conditioning and not one of simple habituation, because there is a punishment effect during every strike due to the load on the mantid's head.     

Visual learning in walking blowflies,Lucilia cuprina

Summary The Australian sheep blowfliesLucilia cuprina were trained by presenting droplets of sugar solution on a light spot of blue (460 nm wavelength) or green (520 nm wavelength). During the test, the searching behaviour was elicited by sugar stimulation. Then, the flies were allowed to walk in the arena where four coloured spots (two blue and two green) with light intensities similar to the training light were exhibited. Visits at these coloured spots were recorded. The flies visited preferably the light spot of the colour to which they had been trained. Next, the flies were trained to a light spot of blue or green displayed in various intensities, and later tested to discriminate between these two colours displayed in fixed intensities. The flies preferred the trained colour over the untrained one irrespective of the intensity used during training. It was only at the lowest intensity that they showed random orientation. These results suggest that the flies can learn to visit a coloured spot, and that they can discriminate between colours on the basis of wavelength rather than intensity. Training caused the flies not only to increase the probability of visiting the trained colour, but also to extend the proboscis and to elicit a characteristic searching behaviour once they had reached the coloured spot.     

A Comparative Analysis of Embryo and Endosperm Proteome from Seeds of Jatropha curcas

Jatropha curcas is an important economic plant for biodiesel, which is extracted mainly from the endosperm of its mature seeds. Despite the morphological and functional differences between the embryo and endosperm, proteomic characteristics of the two tissues are not yet known. Similar proteomic profiles were observed in the two-dimensional gel electrophoresis maps from the two tissues. There were 380 and 533 major protein spots in the embryo and endosperm, respectively. Fourteen identical spots, showing a notable change, were selected and identified by tandem mass spectrometry. Among these proteins, dihydrolipoamide acetyltransferase (spot 27) participates in tricarboxylic acid cycle, which is an amphibolic pathway. The two parts both included proteins related to stress (spots 8, 115, 118, 125, 130) and signal transduction (spots 7, 100, 108). According to the volume percentage of proteins in embryo and endosperm, the proteins in endosperm (spots 54, 61, 73) were catabolism-related enzymes and reserves to provide the nutrition for seed germination; the proteins in embryo (spots 27, 62, 122) were inclined to anabolism and utilized the nutrition from the endosperm to generate a new life.     

Effects of electromagnetic fields on biological systems

The effect of power-frequency electric fields on invertebrate movement and behavior was studied. Data was obtained in the vicinity of an operating 500-kV powerline, and from underneath a simulated powerline (where the electric field could be controlled). Electric fields greater than 8-10 kV/m affected the behavior of flying insects; small insects could not fly, and large insects avoided the high-field region. Electric fields lowered both the activity of mosquitoes (to attack the host) and insect flower attendance. The prevalence of most non-flying insect species was not altered by the electric field, probably because of screening of the electric fields by vegetation and the ability of the insects to migrate. The existing Soviet safety standards for high-voltage powerlines seem sufficient to protect invertebrates.     

Eusociality and the success of the termites: insights from a supertree of dictyopteran families

Sociality in insects may negatively impact on species richness. We tested whether termites have experienced shifts in diversification rates through time. Supertree methods were used to synthesize family‐level relationships within termites, cockroaches and mantids. A deep positive shift in diversification rate is found within termites, but not in the cockroaches from which they evolved. The shift is responsible for most of their extant species richness suggesting that eusociality is not necessarily detrimental to species richness, and may sometimes have a positive effect. Mechanistic studies of speciation and extinction in eusocial insects are advocated.     

On invariants of a 2-D proteome map derived from neighborhood graphs

We consider the problem of the construction of invariants for characterization of 2-D maps, such as 2-D proteome maps, 2-D NMR spectral maps, etc., that in addition to facilitating cataloguing such maps, can be used for comparison of maps and numerical evaluation of their degree of similarity. A novel approach, based on the concept that the nearest neighborhood of points (spots) on a map are sufficiently flexible to allow one not only to vary the number of points used for characterization of the map but also the density of information on their relative positions, is put forward. The method is illustrated with the Coomassie brilliant blue stained 2-D gel electrophoresis patterns of the proteomes from liver cells of healthy male Fisher F344 rats and the rats treated with four peroxisome proliferators.     

A Comparison of the Evolutionary Distribution of the Two Neuroendocrine Markers, Neurone-Specific Enolase and Protein Gene Product 9.5

Abstract: One- and two-dimensional polyacrylamide gel electrophoresis followed by immunoblotting has been used to examine the phylogenetic distribution of the two neuronal and neuroendocrine proteins, neurone-specific enolase and protein gene product 9. 5 , in animal brains. A new immunoblotting procedure was used in which complex two-dimensional patterns of brain proteins were transferred to nitrocellulose paper simultaneously with the Coomassie Blue stain. This produced a copy of the blue spot pattern against which brown protein spots reacting in a specific antibody-immunoperoxidase procedure could be identified unequivocally. Extracts of human, bovine, sheep, rabbit, rat, guinea-pig, chicken, trout, and frog brains were examined. Proteins cross-reacting with antisera to the human forms of both proteins could be demonstrated in all species examined. This suggests that proteins corresponding to neurone-specific enolase and protein gene product 9.5 could have evolved at least 400 million years ago and have been highly conserved throughout evolution.     

Benzylideneacetone, an eicosanoid biosynthesis inhibitor enhances baculovirus pathogenicity in the diamondback moth, Plutella xylostella

Benzylideneacetone (BZA) is a monoterpenoid compound produced by an entomopathogenic bacterium, Xenorhabdus nematophila. BZA inhibits phospholipase A₂ to suppress biosynthesis of eicosanoids that mediate immune responses in insects. In response to per os infection of Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), the diamondback moth, Plutella xylostella, developed red spots on the midgut epithelium. The midgut exhibiting red spot formation suffered abnormal cell integrity, such as genomic DNA fragmentation and condensed spots in the nucleoplasm. The number of red spots increased with viral dose and incubation time after the viral treatment. BZA inhibited the formation of the midgut red spots in a dose-dependent manner. However, the inhibitory effect of BZA on the red spot formation was reversed by addition of arachidonic acid, suggesting that the red spot response may be mediated by eicosanoids. BZA treatment resulted in significant enhancement of AcMNPV occlusion body (OB) pathogenicity to P. xylostella.     

Hierarchical grid transformation for image warping in the analysis of two-dimensional electrophoresis gels

Two-dimensional electrophoresis is a widely used method for separating a large number of proteins from complex protein mixtures and for revealing differential patterns of protein expressions. In the computer-assisted proteome research, the comparison of protein separation profiles involves several heuristic steps, ranging from protein spot detection to matching of unknown spots. An important prerequisite for efficient protein spot matching is the image warping step, where the geometric relationship between the gel profiles is modeled on the basis of a given set of known corresponding spots, so-called landmarks, and the locations of unknown spots are predicted using the optimized model. Traditionally, polynomial functions together with least squares optimization has been used, even though this approach is known to be incapable of modeling all the complex distortions inherent in electrophoretic data. To satisfy the need of more flexible gel distortion correction, a hierarchical grid transformation method with stochastic optimization is presented. The method provides an adaptive multiresolution model between the gels, and good correction performance in the practical cross-validation tests suggests that automatic warping of gel images could be based on this approach. We believe that the proposed model also has significance in the ultimate comparison of corresponding protein spots since the matching process should benefit from the closeness of the true spot pairs.     

A proteomics study of in vitro cyst germination and appressoria formation in Phytophthora infestans

A proteomics study using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed on Phytophthora infestans. Proteins from cysts, germinated cysts and appressoria grown in vitro were isolated and separated by 2-DE. Statistical quantitative analysis of the protein spots from five independent experiments of each developmental stage revealed significant up-regulation of ten spots on gels from germinated cysts compared to cysts. Five spots were significantly up-regulated on gels from appressoria compared to germinated cysts and one of these up-regulated spots was not detectable on gels from cysts. In addition, one spot was significantly down-regulated and another spot not detectable on the gels from appressoria. The corresponding proteins to 13 of these spots were identified with high confidence using tandem mass spectrometry and database searches. The functions of the proteins that were up-regulated in germinated cysts and appressoria can be grouped into the following categories: protein synthesis (e.g. a DEAD box RNA helicase), amino acid metabolism, energy metabolism and reactive oxygen species scavenging. The spot not detected in appressoria was identified as the P. infestans crinkling- and necrosis-inducing protein CRN2. The identified proteins are most likely involved in the establishment of the infection of the host plant.     

大豆雄性不育系与其保持系不同器官蛋白质比较

采用双向凝胶电泳技术对大豆质核互作雄性不育系NJCMS2A及其保持系NJCMS2B的种子、叶片和花药等不同器官蛋白质进行比较分析.结果显示,不育系NJCMS2A与其保持系NJCMS2B的花药2-DE图谱间存在较多差异表达蛋白点,种子2-DE图谱间仅有少量差异表达蛋白点,而叶片2-DE图谱间基本没有差异表达蛋白点.结果表明,不育基因表达具有时空性和器官特异性,与育性有关的蛋白主要在花药中表达.