Expression of 1-Cys peroxiredoxin in morphogenic and nonmorphogenic tatar buckwheat calli

Using two-dimensional gel electrophoresis and MALDI-TOF MS, we identified a protein with a mol wt of 24.5 kD and pI = 7.5 as 1-Cys peroxiredoxin. This protein was present among soluble proteins of morphogenic but not nonmorphogenic calli of tatar buckwheat (Fagopyrum tataricum (L.) Gaertn). Its expression was evidently related to the presence of proembryonal cell complexes in morphogenic calli.     

Comparative study of protein electrophoretic patterns during embryogenesis in Coffea arabica cv Catimor

Embryogenic and non-embryogenic calli from leaf sections of Coffea arabica cv. Catimor, were analyzed under denaturing conditions in one- and two-dimensions by polyacrylamide gel electrophoresis. The protein patterns revealed qualitative and quantitative differences in size and charge. In non-embryogenic calli, two dimensional analysis reveals seven distinctive polypeptides in the range of 15 to 70 kDa. Four of the polypeptides are acidic, three of 70 kDa and one of 15 kDa. Similarly, in embryogenic calli there are seven characteristic polypeptides with molecular weight from 23 to 35 kDa in a broad pI from acid to basic. Five of them are found in the neutral to acid pI, and are probably related to storage protein-like polypeptides detected in zygotic embryos and seeds of Coffea arabica cv. Catimor. Changes in the protein pattern appear to correlate with histological differences in embryogenic calli and with different stages of development of somatic embryos.Abbreviations SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - NEpHGE non-equilibrium pH gradient electrophoresis - TEMED N,N,N,N-tetramethylethylenediamine - 1D one dimensional gel electrophoresis - 2D two-dimensional gel electrophoresis - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - PVP polyvinylpyrrolidone - ME 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - NAA naphtaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine     

QUANTITATIVE MEASUREMENTS OF INTERACTIONS BETWEEN CROWN-GALL TUMORS AND THE PINTO BEAN HOST

Interactions between crown-gall tumors on the primary pinto bean leaf and the pinto bean seedling (Phaseolus vulgaris L. ‘Pinto‘) were estimated by quantitative measurements of tumor initiation and growth as affected by certain modifications of the host. Effects of the tumors on the host were estimated by measurements of host growth and correlation responses. The presence of crown-gall tumors was found to reduce the growth of the leaf in area but to nearly double the weight of the leaf 9 days after inoculation with Agrobacterium tumefaciens (Smith and Town.) Conn, strain B6. The presence of tumors on only one of the two primary leaves resulted in a decrease in the weight of the leaf without tumors, showing the tumors to be effective mobilization centers. Tumors also delayed the abscission of petiole explants and delayed the growth of the epicotyl bud, both reminiscent of auxin effects. The excision of the cotyledons, the epicotyl bud, or one of the pair of primary leaves at the time of inoculation increased the number of tumors initiated per leaf. Removing the epicotyl bud or one of the primary leaves, or placing a cytokinin on one of the leaves, altered leaf growth but failed to alter tumor growth, indicating that tumor growth is not affected by the changes responsible for the compensatory growth effects induced by these treatments. Tumor growth was shown to be generally correlated with leaf growth from day 2 through 8 after inoculation, suggesting that the factors normally limiting leaf growth in a determinate type leaf are also active in limiting tumor growth. The changes in the plant cell responsible for the enhanced rate of growth seen in crown-gall tumor cells, therefore, appear to occur in regulatory systems other than those normally limiting leaf growth. The regulatory systems that are affected may be identical with those activated in compensatory host growth effects.     

The concentration of selected cations in normal and crown-gall tumors obtained from potato discs

Crown-gall tumor tissue obtained from potato discs inoculatedwith virulent strains of Agrobacterium tumefadens containedhigher concentrations of K⁺, Mg²⁺ and Ca²⁺ than the correspondingnormal tissue. These tumors also contained higher concentrationsof these cations than normal tissue inoculated with an avirulentstrain of Agrobacterium tumefadens, or than normal tissue adjacentto the crown-gall tumors on the same potato disc. The concentrationof these cations remained significantly higher than controltissue regardless of the tumor age. 2,4-Dinitrophenol and myo-inositol,while affecting the concentration of Ca²⁺ in these tissues,had no effect on the Mg²⁺ and K⁺ concentrations. These resultssuggest increased concentrations of certain cations may be aspecific property of crown-gall tumors. (Received August 16, 1978; )     

Identification of proteins overexpressed in papillary thyroid tumors

A modified method of proteome comparative analysis based on preliminary removal of cell structural proteins by extraction using salt buffer and subsequent separation of extracts by two-dimensional gel electrophoresis was developed. Identification of differentially expressed proteins by mass spectrometry has revealed three proteins with noticeably increased level of synthesis in most samples of papillary thyroid tumors compared to normal tissues. An increase in ubiquitin content was found for the first time. Oncomarker search efficiencies by two-dimensional gel electrophoresis and bioinformatic search were compared.     

Proteomic map of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> CL Brener: the reference strain of the genome project

In this work two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of soluble proteins from epimastigote form of Trypanosoma cruzi CL Brener. This strain is a hybrid organism derived from two genotypes, T. cruzi I and T. cruzi II and was chosen for genome sequencing. The two-dimensional gel electrophoresis showed that most of proteins focused at 4–7 pH range. The identification demonstrated that several proteins were in multiple isoforms, such as tubulin and heat shock proteins. Potential targets for development of chemotherapeutic agents like arginine kinase, an enzyme absent from mammalian tissues that is involved in the energy supply of the parasite, were also detected.     

Analysis of the chloroplast protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE)

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a powerful procedure for the separation and characterization of the protein complexes from mitochondria. Membrane proteins are solubilized in the presence of aminocaproic acid and n-dodecylmaltoside and Coomassie-dyes are utilized before electrophoresis to introduce a charge shift on proteins. Here, we report a modification of the procedure for the analysis of chloroplast protein complexes. The two photosystems, the light-harvesting complexes, the ATP synthase, the cytochrome b ₆ f complex and the ribulose-bisphosphate carboxylase/oxygenase are well resolved. Analysis of the protein complexes on a second gel dimension under denaturing conditions allows separation of more than 50 different proteins which are part of chloroplast multi-subunit enzymes. The resolution capacity of the blue-native gels is very high if compared to 'native green gel systems' published previously. N-terminal amino acid sequences of single subunits can be directly determined by cyclic Edman degradation as demonstrated for eight proteins. Analysis of chloroplast protein complexes by blue-native gel electrophoresis will allow the generation of 'protein maps' from different species, tissues and developmental stages or from mutant organelles. Further applications of blue-native gel electrophoresis are discussed.     

Tumorous cultures of Crepis capillaris: chromosomes and growth

Different crown-gall culture strains of Crepis capillaris derived from haploid and diploid tumors were studied karyologically during the period of 18 transfers (18–20 months). The very low proportion of mitotic anomalies observed in the first transfers is in agreement with the generally accepted assumption that chromosomal changes are not involved in the initiation of tumors. However, it has been established that an intimate relationship exists between chromosome aberrations and the further development of the tumors. Morphological and physiological changes which occurred in some strains and substrains were invariably associated with cytologieal changes. The results are discussed in connection with similar phenomena described for animal tumors.     

INFLUENCE OF CERTAIN PLANT GROWTH REGULATORS AND CROWN-GALL RELATED SUBSTANCES ON BUD FORMATION AND GAMETOPHORE DEVELOPMENT OF THE MOSS PYLAISIELLA SELWYNII

Bud formation and gametophore development were studied in the moss Pylaisiella selwynii (Kindb.) Steere and Anderson grown from spores in a liquid medium consisting of inorganic salts. Indoleacetic acid and ethrel increased bud formation within a narrow concentration range. Copious bud formation was obtained with the five cytokinins tested at concentrations varying from 5 X 10⁻⁶ to 5 X 10⁻¹⁴ M. Except for about 10 % of the buds obtained with 6-γ, γ-dimethylallylaminopurine at 5 times 10⁻¹⁴ m, the cytokinin-induced buds failed to develop into normal gametophores. Octopine, lysopine, and octopinic acid, substances obtained from crown-gall tumors, increased bud formation at 10⁻³ m. On lysopine-treated plants these buds developed into typical gametophores. Gemma-like structures were obtained with octopine but no gametophores. l -arginine and l -lysine, the amino acids which respectively occur in octopine and lysopine, failed to induce gametophore formation although buds were obtained with 10⁻³ m lysine. γ-Guanidinobutyric acid induced bud formation at 10⁻³ m, but these buds developed into highly abnormal gametophores. The failure of buds obtained with many of these treatments to develop into gametophores appeared to result from the formation of new cell walls in other than the normal geometrical relationship during initial divisions of the pro-bud. The relevance of the findings to the crown-gall problem is discussed.     

Seperation of proteins using cetyltrimethylammonium bromide discontinuous gel electrophoresis

The gel electrophoresis system presented here allows the separation of proteins with the concomitant retention of detectable native activities. The system, referred to as CAT gel electrophoresis. uses the detergent cetyltrimethylammonium bromide in combination with a discontinuous gel matrix to resolve protein mixtures into discrete bands. Many proteins retain detectable levels of native activity after CAT electrophoresis, and gel bands can be easily identified using assays based on specific enzymatic activities or binding characteristics. The ability to identify protein bands based on BothMnr and activity in a single gel makes the CAT system a powerful adjunct to existing biochemical techniques.     

Comparative proteome analysis of breast cancer and normal breast

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p<0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.     

Differential Expression of Specific Proteins during In Vitro Tomato Organogenesis1

The cotyledons of tomato (Lycopersicon esculentum L., cv. Jia Fen-10) seedlings were induced to produce calli and regenerate plants via organogenesis. Utilizing this system, the composition and content of stage-specific proteins associated with organogenesis were analyzed. Moreover, a comparison of the protein composition and content between embryogenic and nonembryogenic calli was conducted. The SDS-PAGE results and laser densitometric scanning maps showed that there were different specific proteins expressed at different stages. Among them, six proteins (61, 54, 38, 37, 35, and 23 kD) were associated with the morphogenesis of organs, and two proteins (39 and 24 kD) were related to the morphogenesis of calli. Although no distinctive difference in protein components of embryogenic calli was noted, there were different trends of changes, both for the content of the proteins 39 and 24 kD, and for the content of the total proteins, at different developmental stages of embryogenic calli. The results obtained from the embryogenic and nonembryogenic calli indicated that these two materials were distinct in the protein components as well as in its content; for example, the protein 54 kD was detected in nonembryogenic but not in embryogenic calli. The total protein content in nonembryogenic calli was lower than that in the embryogenic calli.     

Basic nuclear proteins in the Oömycete fungus Achlya bisexualis

Summary A comparison was made of the basic proteins extracted from the chromatin and nuclei of Achlya bisexualis, Blastocladiella emersonii, and Pisum sativum. Extraction of purified chromatin and nuclei of the Chytridiomycete, B. emersonii followed by gel electrophoresis produced no detectable protein bands. Extractions of purified nuclei and chromatin by either mineral acid or CaCl₂ from the Oömycete A. bisexualis resulted in several protein bands following separation by disc gel electrophoresis. Monitoring the nucleic acids during the nuclear isolation procedure as well as comparing electropherograms of basic nuclear proteins with basic ribosomal proteins suggests no significant contamination of the nuclear preparations with ribosomes. Carboxymethyl cellulose chromatography of the A. bisexualis nuclear basic proteins resolved three distinct fractions. Gel electrophoresis of the CM cellulose fractions indicated heterogeneity of each fraction. Amino acid analysis of the CM fractions showed that they were all lysine-rich and meet the basisity requirements of histones.     

Influence of RET/PTC1 and RET/PTC3 oncoproteins in radiation-induced papillary thyroid carcinomas on amounts of cytoskeletal protein species

Radiation-induced human papillary thyroid carcinomas (PTCs) show a high prevalence of fusions of the RET proto-oncogene to heterologous genes H4 (RET/PTC1) and ELE1 (RET/PTC3), respectively. In contrast to the normal membrane-bound RET protein, aberrant RET fusion proteins are constitutively active oncogenic cytosolic proteins that can lead to malignant transformation of thyroid epithelia. To detect specific tumor-associated protein changes that reflect the effect of RET/PTC fusion proteins, we analyzed normal thyroid tissues, thyroid tumors of the RET/PTC1 and RET/PTC3 type and their respective lymph node metastases by a combination of high-resolution two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-mass spectrometry. PTCs without RET rearrangements served as controls. Several cytoskeletal protein species showed quantitative changes in tumors and lymph node metastases harboring RET/PTC1 or RET/PTC3. We observed prominent C-terminal actin fragments assumedly generated by protease cleavages induced due to enhanced amounts of the active actin-binding protein cofilin-1. In addition, three truncated vimentin species, one of which was proven to be headless, were shown to be highly abundant in tumors and metastases of both RET/PTC types. The observed protein changes are closely connected with the constitutive activation of RET-rearranged oncoproteins and reflect the importance to elucidate disease-related typical signatures on the protein species level.     

Proteomic analysis of early germs with high-oil and normal inbred lines in maize

High-oil maize as a product of long-term selection provides a unique resource for functional genomics. In this study, the abundant soluble proteins of early developing germs from high-oil and normal lines of maize were compared using two-dimensional gel electrophoresis (2-DGE) in combination with mass spectrometry (MS). More than 1100 protein spots were detected on electrophoresis maps of both high-oil and normal lines by using silver staining method. A total of 83 protein spots showed significant differential expression (>two-fold change; t-test: P < 0.05) between high-oil and normal inbred lines. Twenty-seven protein spots including 25 non-redundant proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Functional categorization of these proteins was carbohydrate metabolism, cytoskeleton, protein metabolism, stress response, and lipid metabolism. Three such proteins involved in lipid metabolism, namely putative enoyl-ACP reductase (ENR), putative stearoyl-ACP desaturase (SAD) and putative acetyl-CoA C-acyltransferase (ACA), had more abundant expressions in high-oil lines than in normal. At the mRNA expression level, SAD, ENR and ACA were expressed at significantly higher levels in high-oil lines than in normal. The results demonstrated that high expressions of SAD, ENR and ACA might be associated to increasing oil concentration in high-oil maize. This study represents the first proteomic analysis of high-oil maize and contributes to a better understanding of the molecular basis of oil accumulation in high-oil maize.     

Cadmium speciation studies in the intestine of Lumbricus terrestris by electrophoresis of metal proteins complexes

Cadmium speciation of the intestinal compartment of the earthworm species, Lumbricus terrestris, has been investigated using polyacrylamide gel electrophoresis under non-denaturing conditions. Worms exposed to Cd(NO₃)₂ supplemented soils have been studied and compared to control samples. Prior to electrophoresis, the worm intestines were removed and dissected. Proteins in the crude intestinal extracts were separated using polyacrylamide gel electrophoresis. The cadmium distribution in the proteins has also been described. In a second set of experiments, cadmium bound to proteins was first isotopically exchanged with labelled cadmium (¹⁰⁹Cd) and then cadmium speciation was performed using gel electrophoresis. Autoradiography of this gel shows an intense band in the contaminated sample whereas this band was absent in the control sample. These results show that one type of major protein has a strong affinity for cadmium in the worm intestinal extract. This type of protein had a migration close of that of rabbit liver metallothionein used for comparison.     

Characteristics of Membrane Protein Phosphorylation in Plasmodium berghei-Infected Mouse Erythrocytes1

ABSTRACT. Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (³²P)orthophosphate and incubating isolated membrane with (γ-³²P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with M, 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.     

Cellulases from Neocallimastix frontalis EB188 synthesized in the presence of protein glycosylation inhibitors: measurement of protein molecular weights and isoelectric focusing values

Summary Protein and cellulase secreted in the presence of glycosylation inhibitors by Neocallimastix frontalis EB188 were studied using gel electrophoresis. Tunicamycin and 2-deoxy-D-glucose added to established cultures inhibited the production and secretion of proteins and cellulases. Schiff reagent staining of proteins after denaturing polyacrylamide gel electrophoresis confirmed the presence of extracellular glycoproteins. Intracellular or extracellular cellulases from cultures treated with inhibitors possessed distinct isoelectric focusing values and native gel R _f values. In N. frontalis EB188, glycosylation of protein occurred and was important for the production, secretion and activity of cellulases. Offprint requests to: R. E. Calza     

Micropropagation through excised root culture of Clitoria ternatea and comparison between in vitro–regenerated plants and seedlings

An efficient protocol has been developed for high‐frequency shoot regeneration and plant establishment of Clitoria ternatea – a potential medicinal legume. Adventitious shoots were regenerated from young excised root segments of aseptic seedlings on Murashige and Skoog (MS) medium supplemented with various concentrations of 6‐benzyladenine (BA), kinetin, α‐naphthalene acetic acid (NAA) or 2,4‐dichlorophenoxy acetic acid (2,4‐D) either singly or in various combinations. The highest frequency (100%) of shoot regeneration and maximum number (16.4 ± 0.24) of shoots per explant was obtained on MS medium supplemented with 20 μm BA and 2.0 μm NAA. Organogenic calli were produced on a medium containing 2,4‐D (10 or 20 μm ) and BA (5.0 μm ). The calli were differentiated into multiple shoots on MS medium supplemented with 2.5–10 μm BA and 2.0 μm NAA. The microshoots were rooted on half‐strength MS medium supplemented with 5.0 μm indole‐3‐butyric acid and transplanted successfully in field conditions. After 12 months of transfer to ex vitro conditions, the performance of micropropagated plants were evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo–grown plants of the same age. The sodium dodecyl sulphate polyacrylamide gel electrophoresis protein profile was same between regenerated and naturally growing shoots. Total soluble protein in aerial part as well as in seeds of in vitro–regenerated and in vivo–grown plants was almost the same. The mitotic study showed normal chromosomal movement and numbers (2x = 16).