Changes in protein composition of the shoot meristem during floral evocation in Sinapis alba

Shoot apical meristcms of vegetative and induced plants of Sinapis alba L. were labelled with [³⁵S] methioninc for 2 h and the proteins were then separated by isoelectric focussing and polyacrylamide gel electrophoresis. Quantitative and possibly qualitative changes in the complement of proteins being synthesised during evocation were detected in the meristem, distal to the primordia, 50 to 52 h after the beginning of the inductive long day. This was before morphological changes in the meristem, and before the initiation of flower bud primordia.     

Resolution ofFusarium sporotrichioides proteins by two-dimensional polyacrylamide gel electrophoresis and identification by sequence homology comparison in protein data base

Proteins fromFusarium sporotrichioides M-1-1, a T2-toxin-producing strain, were separated by two-dimensional polyacrylamide gel electrophoresis. One thousand two hundred and forty-four protein spots were resolved and 103 protein spots were subjected to N-terminal sequencing. Fifty-eight protein spots were sequenced and 48 proteins were observed to have blocked N termini. Forty out of 58 sequenced proteins were identified by homology search against the PIR protein sequence data base and protein superfamily data base, while the residual 18 sequences were not identified. Twenty-seven of the N-terminal-blocked proteins were subjected to mild anhydrous hydrazine vapor deblocking. Twenty-four spots were not deblocked indicating the presence of acyl groups at the N termini, while 3 proteins were deblocked showing the blocked group to be pyrroglutamyl carboxylic acid residues. The results can provide a more global view of cellular genetic expression than any other technique. The created data may offer a unique opportunity to link information with DNA sequence data.     

Isolation and characterization of microorganisms able to produce 1,3-propanediol under aerobic conditions

Microbial fermentation under strictly anaerobic conditions has been conventionally used for the production of 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate (PTT) and other polyester fibers. In the current study, we have identified eight strains of microorganism which are able to produce 1,3-propanediol under aerobic condition. Those strains were isolated from garden soil, which were enriched by culturing in LB medium with glycerol added under aerobic condition. The identities of those strains were established based on their 16S rRNA sequences and physiological characteristics. Results indicated 6 strains are Citrobacter freundii and 2 strains are Klebsiella pneumoniae subsp Penumoniae. One of Klebsiella pneumoniae subsp Penumoniae strains, designated as TUAC01, demonstrated comparable levels of 1,3-propanediol oxidoreductase, glycerol dehydratase and glycerol dehydrogenase activity to the anaerobic microorganisms described in the literature. Accordingly, in larger scales (5 l) fed-batch culture the TUAC01 strain showed a remarkable 1,3-propanediol producing potency under aerobic conditions. 60.1 g/l of 1,3-propanediol was yield after 42 h incubation in an agitating bioreactor; and in air-lift bioreactor 66.3 g/l of 1,3-propanediol was yield after 58.5 h incubation. The aerobic ferment process, reduced the product cost and made the biological method of 1,3-propanediol production more attractive.     

The stress-70 protein family in diplopods: induction and characterization

To induce stress-70 proteins (hsp70), adults of the millipede Julus scandinavius (Diplopoda) were exposed to leaf litter contaminated with different concentrations of Cd²⁺ (10, 30, 50 and 60 mg·kg^-1 as CdCl₂). The expression of hsp70 was investigated by semiquantitative and qualitative biochemical methods. After SDS-gel electrophoresis and Western blotting a subsequent digital image analysis showed that increasing dietary concentrations of Cd²⁺ resulted in elevated levels of hsp70, which in turn indicated proteotoxic condition. Qualitative results were obtained by two-dimensional gel electrophoresis. A stress-70 protein family, similar to that of other arthropods, was detected in Julus scandinavius: at least five different proteins with an approximate molecular weight of 68, 69, 70, 77, and 78 kDa could be distinguished after heat shock as well as after Cd²⁺ exposure.Abbreviations IEF isoelectric focusing - hsp heat shock protein(s) - grp glucose regulated protein(s) - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Use of two-dimensional protein-pattern analysis for the characterization of Arabidopsis thaliana mutants

Total proteins extracted from wild-type plants of Arabidopsis thaliana Heyhn, an etiolated mutant, a de-etiolated mutant and a mutant affected in cotyledon morphology, were analyzed by two-dimensional gel electrophoresis. Computer analysis of two-dimensional gels allowed the characterization of the mutants by a set of proteins showing a differential expression when compared with the wild-type plant grown under the same conditions. The overlap between comparisons of the diferent mutants with the wild-type allowed the identification of groups of polypeptides which, since their expression is altered in several mutants, might be involved in certain physiological functions. For example, this approach showed a possible involvement of actin in the elongation process. The simultaneous analysis of the two-dimensional protein patterns of different mutants seems, therefore, to be a promising approach to characterize proteins involved in various physiological functions.Abbreviations 2-D two-dimensional - IEF isoelectrofocusing - M_r relative molecular mass - E17.1 E17 mutant grown in light - E22.1 E22 mutant grown in light - E44.d E44 mutant grown in darkness - WT.d wild-type plants grown in darkness - WT.1 Wild-type plants grown in light We would like to thank our laboratory colleagues J.A. Traas, H. Höfte and D. Bouchez (all from INRA) for useful discussions throughout this work. Also grateful thanks to Mr Zivy (Lab. Génétique des Sytèmes Végétaux. INRA. La Ferme du Moulon, Gif-sur-Yvette, France) for helpful discussions concerning 2-D gel analysis and to I. Small (Lab. Biologie Cellulaire. INRA, Versailles, France) for English text corrections.     

Investigation of granulation of a mixture of suspended anaerobic and aerobic cultures under alternating anaerobic/microaerobic/aerobic conditions

This is the first granulation study except Ferguson [Ferguson LN. Anaerobic codigestion of aircraft deicing fluid and microaerobic studies. M.S. Thesis. Milwaukee, WI, USA: Marquette University; 1999] to develop coupled granules by using a mixture of suspended anaerobic and aerobic cultures exposed to alternating cyclic anaerobic/microaerobic/aerobic conditions. Coupled granules with median sizes of 1.28–1.86 mm and settling velocities of 31–39 m/h were developed, which were comparable to those of both anaerobic and aerobic granules. Coupled granules displayed noteworthy specific methanogenic activity (SMA) and specific oxygen uptake rate (SOUR) as 14–42 mL CH₄/g VSS h and 6–47 mg DO/g VSS h, respectively, indicating that they were composed of both anaerobic and aerobic cultures.     

Studies on embryonic diapause in thepnd mutant of the silkworm,Bombyx mori

Summary Two-dimensional gel electrophoresis has been used to analyse patterns of proteins synthesized in the eggs from theBombyx mutantpnd, whose homozygous embryo never enters diapause owing to a genetic defect. At the middle to late stage of gastrulation the diapause type of the heterozygous embryo, derived from a homozygouspnd female mated to a wild-type male, synthesizes eight proteins which are not detected in the homozygouspnd embryo. To examine the relationship between embryonic diapause and the appearance of the heterozygote-specific proteins, the pattern of proteins synthesized in the heterozygotes of the diapause type was compared with that in heterozygotes which were artificially altered so that they would continue development. Only one of the eight heterozygote-specific proteins was constitutively synthesized according to the embryonic genome, irrespective of their developmental state, whereas appearance of the remaining seven proteins was exclusively dependent on their developmental nature. This finding strongly suggests that the unique protein might result from the expression of thepnd ⁺ gene, and the other proteins might be synthesized along with diapause initiation in the heterozygotes. The possible role of the putativepnd ⁺ gene-specific protein at the onset of embryonic diapause is discussed in relation to the action of the diapause factor, which predetermines embryonic diapause by affecting the developing oocytes.     

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments

Despite recent progress in sequencing the complete genome of rice (Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60–98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Jürgens     

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide     

Changes in cell morphology and polyamine composition during growth of Campylobacter jejuni

Resting cells of Campylobacter jejuni were spherical whereas growing cells were mainly spiral. Content of cadaverine increased with the decrease in spherical forms prior to growth commencing but production of spermidine increased in early log phase. Cadaverine and spermidine are possibly involved in changes in cell morphology and growth, respectively.S. Suzuki is with the Faculty of Agriculture, Kochi University, Nankoku, Kochi 783, Japan; Y. Horikoshi and K. Takama are with the Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, Japan.     

Long-term oxidant deficiency in Pseudomonas aeruginosa PAO303 results in cells which are non-culturable under aerobic conditions

Abstract The effect of long-term energy starvation (lack of electron acceptor in respiration) on the culturability of Pseudomonas aeruginosa PAO303 was studied by subsequent incubations for growth on aerobic medanaerobic media. A batch culture was grown on O₂-free citrate minimal medium containing NO₃⁻ as oxidant. Stationary phase was reached when NO₃⁻ was exhausted. This was followed by a rapid loss of cell culturability as tested by aerobic growth on agar plates (colony forming units, cfu) or on 0.2 μm membrane filters (epifluorescence technique) using the citrate minimal medium. However, energy-starved cells could form ten times more colonies when incubated anaerobically with NO₃⁻ (denitrifying conditions) than when incubated aerobically. Hence the energy starvation resulted in a subpopulation of cells, which were detectable under denitrifying, but not under aerobic growth conditions.     

Comparative studies of four protein preparation methods for proteomic study of the dinoflagellate Alexandrium sp. using two-dimensional electrophoresis

Alexandrium is a wide-spread genus of dinoflagellate causing harmful algal blooms and paralytic shellfish poisoning around the world. Proteomics has been introduced to the study of Alexandrium, but the protein preparation method is still unsatisfactory with respect to protein spot number, separation and resolution, and this has limited the application of a proteomic approach to the study of dinoflagellates. In this study we compared four protein preparation methods for the two-dimensional electrophoresis (2DE) analysis of A. tamarense: (1) urea/Triton X-100 buffer extraction with trichloroacetic acid (TCA)/acetone precipitation; (2) direct precipitation with TCA/acetone; (3) 40 mM Tris (hydroxymethyl) aminomethane (Tris) buffer extraction; and (4) 50 mM Tris/5% glycerol buffer extraction. The results showed that, among the four protein preparation methods, the method combining the urea/Triton X-100 buffer extraction and TCA/acetone precipitation allowed detection of the highest number and quality of protein spots with a clear background. Although the direct TCA/acetone precipitation method also detected a high number of protein spots with a clear background, the spot number, separation and intensity were not as good as those obtained from the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method. The 40 mM Tris buffer and 50 mM Tris/5% glycerol buffer methods allowed the detection of fewer protein spots and a pH range only from 4 to 7. Subsequently, the urea/Triton X-100 buffer extraction with TCA/acetone precipitation method was successfully applied to profiling protein expression in A. catenella under light stress conditions and the differential expression proteins were identified using MALDI TOF–TOF mass spectrometry. The method developed here appears to be promising for further proteomic studies of this organism and related species.     

Isolation and characterization of thermotolerant bacterium utilizing ammonium and nitrate ions under aerobic conditions

A thermotolerant bacterium, identified as Bacillus licheniformis, completely utilized 0.1% (w/v) NH₄NO₃ at 30 and 50°C under aerobic condition. The addition of 0.5 mM Fe²⁺ to the NH₄NO₃ medium markedly promoted the utilization of NH₄⁺ and NO₃⁻. At 50°C, of total nitrogen originally provided, 24% was taken up into the cells and 20% remained in the culture supernatant. Residual nitrogen (56%) was probably removed into the atmosphere. The cell extracts contained enzymes involved in denitrification. GC-MS demonstrated that NH₄ ¹⁵NO₃ had been converted to ¹⁵N₂O. These results indicate that the strain has denitrification ability under aerobic condition.     

Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions.     

Ageing of theSilene coeli-rosa L. shoot apex under non-inductive conditions: Changes in morphology,mitotic index,and polypeptide composition

Summary Ageing was studied in the shoot apex of the long day plant,Silene coeli-rosa by maintaining it in non-inductive short day conditions for 170 days. The dimensions, the zonation, and the polypeptidic pattern of the shoot apex, and the rate of leaf initiation were altered in 170-day-old plants compared with young plants grown under the same conditions (28 days). In aged plants, the number of cells increased in all shoot apical zones and, notably, the mitotic index increased in the axial zone; however, the rate of leaf initiation slowed down. These changes showed some similarities to those during the intermediate phase in quantitative photoperiodic species. The two-dimensional mini-gel electrophoretic study of protein extracts from shoot apices of young and ageing plants maintained in non-inductive conditions revealed 489 common polypeptidic spots, 13 unique to the young state and 24 new ones specific to aged plants. The spots characteristic for each state represented only 3.6% of the total identified polypeptides, but apical development under non-inductive conditions was characterized by qualitative changes in the polypeptide complement.Abbreviations C nuclear DNA complement - 1D gel first dimension gel - LD long day - NEPHGE non equilibrium pH gradient electrophoresis - SD short day - SDS sodium dodecyl sulfate - TCA trichloracetic acid     

Proteomics Analysis of Drought Stress-Responsive Proteins in <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis> L.

Hippophae rhamnoides L. is uniquely capable of growing well under extreme environmental conditions such as water deficit, low temperature, and high altitude. Such tolerance invokes much interest in understanding the biology of this plant species and its utilization potential. In this study, analysis of drought stress-responsive proteins in H. rhamnoides was conducted wherein greenhouse-grown seedlings were subjected to drought stress. By using proteomic techniques, proteins, extracted from leaves, were analyzed using two-dimensional electrophoresis and MALDI-TOF MS. Altogether, 55 proteins exhibited changes in abundance under stress. Of these, 13 proteins were identified, including three that disappeared under drought (a putative ABC transporter ATP-binging protein, a heat shock protein HslU, and a hypothetical protein XP-515578), seven that were up-regulated (three large subunits of rubisco, a hypothetical protein DSM3645–23351, a putative acyl-CoA dehydrogenase, a nesprin-2, and a J-type co-chaperone HSC20), and three that were only detected under drought (a probable nitrogen regulation protein (NtrX), a 4-hydroxyphenylpyruvate dioxygenase, and an unnamed protein product). These proteins may function in β-oxidation pathways in mitochondria, across membranes transport, abnormal protein removal, or prevent protein aggregation arrest, cell division, cytoskeleton stabilization, iron–sulfur cluster assembly, nitrogen metabolism regulation, and antioxidant substance biosynthesis. Four proteins (J-type co-chaperone Hsc20, a putative ABC transporter ATP-binging protein, NtrX, and HslU) were deemed as new discoveries in higher plants, and their functions were predicted either from their conserved domains or homologies to other organisms. These results provide new insights into our understanding of the mechanism of drought tolerance in plants.     

Modulation of pigment profiles of Calothrix elenkenii in response to environmental changes

Cyanobacteria are versatile tetrapyrrole synthesizers that can regulate their tetrapyrrole content and composition in response to environmental signals. The present investigation analyses the interplay between light and dark regimes (continuous light, light-dark cycles (16:8) and continuous darkness) and aerobic, air-tight, and anaerobic environments (argon-enriched), on the relative composition of various pigments and growth attributes of Calothrix elenkenii as a prelude to exploiting C. elenkenii's bioindustrial potential as a source of pigments. Incubation in an anaerobic environment stimulated hormogonia formation and induced colouration/thickening of cells. Aerobically grown cultures of Calothrix, under continuous illumination produced the maximum amount of total phycobiliproteins and sugars, although chlorophyll accumulation and nitrogenase activity were highest in the light-dark environment. However, the beta-carotene content was observed to vary under anaerobic conditions with different light-dark regimes. This C. elenkenii strain can be a valuable source of pigments under optimized environmental conditions.     

Two-dimensional patterns of soluble proteins including three hydrolytic enzymes of mature pollen of tristylous <Emphasis Type="Italic">Lythrum salicaria</Emphasis>

Lythrum salicaria, now a widespread invasive species, exhibits tristyly, a form of heteromorphic selfincompatibility. In tristyly, each plant exhibits one (and only one) of three morphologically different floral forms. Moreover, each flower produces two types of stamens, and these two exhibit different incompatibility reactions. Differences between stamens of a single flower must be the result of epigenetic phenomena and for that reason, we performed two-dimensional gel electrophoresis (2-DE) to analyze fractions of soluble proteins derived from the pollen coat and protoplast including three hydrolytic enzymes from the six different stamen types (two from each of three floral forms). There were significant differences in the 2-D protein profiles both between pollen from the same flower and between the same type of pollen from two different flowers, in the pollen coat as well as in the protoplast extracts. In five of the six samples of pollen fractions, characteristic peptides were found. Quantitative differences between pollen from the same flower were observed in case of esterases. Furthermore, analysis of proteases and acid phosphatases revealed also qualitative differences between these enzymes in pollen from the same flower.     

乌龙岭’龙眼胚胎发育时期特异性蛋白质的变化

应用IEF-SDS-PAGE技术分析龙眼胚胎分化发育过程中蛋白质组分的变化。结果表明,在各发育阶段大多数蛋白质组分的电泳图谱基本一致,但也有变化。其中花后38d存在TE1(27．1kD、p,7．3),TE2(17．5kD、pI8．2)2个特异蛋白,45d存在TE3(11．4kD、pI7．6),TE4(13．2kD、pI9．9)2个特异蛋白,52d存在TE5(22．6kD、pI7．2),TE6(18．6kD、pI8．3),TE,(23．5kD、pI3．6)3个特异蛋白。31d胚胎电泳图谱中的蛋白质点数相对较多,表明此时蛋白质旺盛合成与积累,这与蛋白含量的变化基本一致。龙眼胚胎发育过程中特异蛋白的出现或消失．对胚胎的分化发育具有重要作用。     

Proteomic detection of changes in protein synthesis induced by NGX6 transfected in human nasopharyngeal carcinoma cells

In the previous study, we cloned a new gene, named NGX6, related to nasopharyngeal carcinoma (NPC) at 9p. To study its function in the pathogenesis of NPC, we have investigated changes in protein synthesis between NPC cell line HNE1 and that transfected with the gene. Using high-resolution two-dimensional electrophoresis, we found that 22 protein spots showed variations that were significant and reproducible. Analysis of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searches identified seven proteins that were upregulated and seven proteins that were downregulated. These proteins included Fas, zinc-finger protein (ZNF), RAB, and Ah receptor-interacting protein (AIP). The functional implications of the identified proteins are discussed.