The separation and comparison of albumin complexes of seed proteins in three cultivars ofPhaseolus vulgaris

Complexes of water soluble proteins (albumins) were investigated in three cultivarsof Phaseolus vulgaris, viz: Yeltruská Saxa, Vainica Saavegra B, and Krupnaya sakharnaya. The first two cultivars exhibit haemagglutinating activity against rabbit erythrocytes, but have different elution profiles on Sephadex G-100. Their individual peaks have a different subunit composition, as revealed by SDS gel electrophoresis, as well as a different immunoelectrophoretic pattern, although proteins I and II of the specificity Veltruská Saxa are present in both cultivars. The cultivar Krupnaya sakharnaya expressively differs from the preceding lectin cultivars; it has no erythroagglutinating activity, its albumin complex has a high-molecular component, absent in the preceding ones, and has no lectin peak in the region of molecular mass of 100 000 to 200 000. Immunoelectrophoresis gave no evidence of protein I and II of the specificity Veltruská Saxa.     

The comparison of lectins from albumin complexes of certain representatives of the speciesPhaseolus vulgaris

Using biospecific chromatography on D-Glc-Separon-fetuin, lectins were isolated from seed albumin complexes of four cultivars ofPhaseolus vulgaris (Veltruská Saxa, Vainica Saavegra, Krupnaya sakharnaya, Olympia) andPhaseolus vulgaris ssp.aborigineus (wild form, considered to be one of the ancestors of cultivated beans). In the lectins isolated the agglutinating activity against human erythrocytes of the A, B, and O groups was estimated, as well as against trypsin-treated and non-treated rabbit erythrocytes. Further analyses involved their mitogenic activity against lymphocytes of murine spleen, their isoelectric points by isoelectric focusing in polyacrylamide gels, and eventually their immunospecific similarity with the lectins of the standard cultivar ofPhaseolus vulgaris, Veltruská Saxa. The lectins of all taxa were mitogenic, but differed from one another in their agglutinating activity and in the number and isoelectric points of the zones, as revealed by both isoelectric focusing and immunoelectrophoresis. In the case of the cultivar Vainica Saavegra, which is a seggregating population, even the lectins of individual seed groups were different.     

Proteins found during maturation and germination of seeds ofPhaseolus vulgaris L.

Seeds of the beanPhaseolus vulgaris L. (Veltruská Saxa cultivar) were gathered gradually at different stages of development, starting at fertilization up to full maturity. Seeds were freeze-dried and the dry solid used for preparing extracts which were analyzed by immunoelectrophoresis for the presence of proteins resembling those contained in the cotyledons of a mature seed. Proteins from cotyledons of the first stages of development of bean seedlings were analyzed similarly. After a preparatory period, approximately from the second—third seed development stage, there is a period of intense protein synthesis that characterizes cotyledons of a mature seed. These proteins increase in quantity and are differentiated in quality up to maturity when a single antiserum detected a total of 12. After germination both the quantity and number of these proteins decreases. It was found that some proteins are metabolically more active, both during synthesis and cleavage. This holds e.g. for phaseolin during maturation, as well as during germination. In addition, phaseolin changes its electrophoretic mobility, which is apparently due to proteolytic hydrolysis of phaseolin molecules. During the last phase of maturation, viz. dehydration of seeds, some new proteins suddenly appear, apparently synthesized from pre-formed peptide chains. In the discussion the possibility is taken up that the beginning on the synthesis of specific proteins characteristic for mature seeds is the cause underlying the disturbances in the embryonal development of distant hybrids.     

Protein characters and relationship betweenPhaseolus vulgaris ssp.Aborigineus Burk. and related taxons of the genusPhaseolus

Both quantitative and qualitative immunochemical methods were used for studying the mutual relationships of several spocies and the subspecies of the genusPhaseolus: Ph. vulgaris L. ssp.vulgaris, Ph. vulgaris L. ssp.aborigineus Burk.,Ph. coccineus L.,Ph. acutifolius A. Gray,Ph. lunatus L. (American endemites) andPh. aureus L. (a typical Asian bean). Protein characters of cotyledons (i.e., ?storage” proteins) of the above species were compared with the aid of antisera prepared against seed (cotyledon) proteins ofPh. vulgaris L. ssp.vulgaris, cv. Veltruská Saxa, using

1. (a)
   the whole complex of cotyledon protein,     
   
   

Atypical composition of seed proteins in cultivars ofPhaseolus vulgaris L.

In only one cultivar out of 1200 investigated cultivars ofPhaseolus vulgaris L. could we find an extreme change in the pattern of reserve proteins on the cathodic side: one of the proteins, called protein I, is completely absent in the cultivar ‘Krupnaya sakharnaya’ and is replaced on the same site by another protein,i.e. a protein completely different in its immunochemical specificity. The case is of interest from both the phylogenetic and systematic viewpoints and deserves further attention.     

Analysis of acid extractable bean leaf proteins induced by mercuric chloride treatment and alfalfa mosaic virus infection. Partial purification and characterization

Mercuric chloride treatment of Phaseolus vulgaris (var. ‘Saxa’) leaves, induces the synthesis of four new soluble proteins extractable at pH 2.8. The molecular weights of these proteins were found to be 17 000 for pathogenesis related (PR) 1 and PR 2 proteins, 28 000 for PR 3 protein and 32 000 for PR 4 protein, when determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In Alfalfa Mosaic Virus (AMV)-infected bean leaves only three new soluble proteins were found, corresponding to the mercuric chloride-induced PR 1, PR 3 and PR 4 proteins. The four mercuric chloride-induced proteins were purified by a technique including an ammonium sulfate fractionation and a preparative polyacrylamide gel electrophoresis. Some biochemical and serological properties of these proteins have been studied.     

Electrophoretic studies of the relationship of peroxidases,polyphenol oxidase,and indoleacetic acid oxidase to cold tolerance of alfalfa

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to investigate the relationship of soluble proteins and enzyme activity and solubility characteristics to cold tolerance.Evaluations of cold-tolerance levels developed in crown and root samples were compared with results of soluble protein analyses and were in agreement with previously reported observations. Soluble protein content was associated with increases in cold tolerance and related to the environment from which samples were obtained; however, the degree of protein differences within samples of the same cultivar as well as between the two cultivars seemed to be influenced by the type of extractant used.Polyacrylamide disc gel electrophoresis of the extracted soluble proteins was performed on the basis of equal dry weights and equal quantities of protein. Amido black straining of gels indicated mainly quantitative changes and slight qualitative differences in component bands influenced by environment and extractant.Gels assayed for peroxidase, polyphenol oxidase, and indoleacetic acid oxidase enzymes exhibited mainly quantitative differences in constitutive isoenzyme components of both cultivars which were associated with environmental changes. Enzyme activities generally increased in winter, as cold tolerance and soluble protein content increased, and decreased during dehardening. The few qualitative differences in isoenzyme bands that were detected, appeared to be influenced by cultivar, environment, extractant, or substrate specificity differences.Variation in isoenzyme components between cultivars was maximum in summer samples, and minimum in winter samples, suggesting that overall reaction rates or activities of individual isoenzymes, preceding or during hardening, could be a limiting factor in cold-tolerance development.     

Can interaction specificity in the fungus-farming termite symbiosis be explained by nutritional requirements of the fungal crop?

Fungus-growing termites are associated with genus-specific fungal symbionts, which they acquire via horizontal transmission. Selection of specific symbionts may be explained by the provisioning of specific, optimal cultivar growth substrates by termite farmers. We tested whether differences in in vitro performance of Termitomyces cultivars from nests of three termite species on various substrates are correlated with the interaction specificity of their hosts. We performed single-factor growth assays (varying carbon sources), and a two-factor geometric framework experiment (simultaneously varying carbohydrate and protein availability). Although we did not find qualitative differences between Termitomyces strains in carbon-source use, there were quantitative differences, which we analysed using principal component analysis. This showed that growth of Termitomyces on different carbon sources was correlated with termite host genus, rather than host species, while growth on different ratios and concentrations of protein and carbohydrate was correlated with termite host species. Our findings corroborate the interaction specificity between fungus-growing termites and Termitomyces cultivars and indicate that specificity between termite hosts and fungi is reflected both nutritionally and physiologically. However, it remains to be demonstrated whether those differences contribute to selection of specific fungal cultivars by termites at the onset of colony foundation.     

Unwinding by Local Strand Separation Is Critical for the Function of DEAD-Box Proteins as RNA Chaperones

The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure.     

Cholinesterase activity in some species of theAllium genus

In partly purified protein complexes obtained from 22 species of theAllium genus and 6 cultivars ofAllium cepa the activity of cholinesterases was detected and measured using the method of Ellman et al. The degree of its inhibition with 10^-4 M neostigmine was also tested. It was found that the activity of cholinesterase differed in individual species up to two hundred times, while the differences in the inhibitory activity of 10^-4 M neostigmine occurred only in a few cases. Individual sections and cultivars could not be characterized on the basis of the differences in the activities of the cholinesterases. Of all the sections that ofPhyllodolon shows the highest average activity. In the case of the tested cultivars distinctly the lowest activity was observed in cv. Kastická. The values of the enzymatic activity measured by Ellman’s method in this plant material include the activity of specific and unspecific cholinesterases and the part uninhibitable by neostigmine.     

Serological characterization of a ribonuclease from Hordeum vulgare

Specific rabbit antisera against purified Hordeum vulgare seedling RNase I from two winter barley cultivars each formed a single precipitin band when reacted with the homologous crude tissue extract. RNase antigen from either cultivar was equally reactive with both antisera when evaluated by immunodiffusion and immunoelectrophoresis. A small but consistent difference in anti-RNase specificity between cultivars was shown by passive hemagglutination inhibition, suggesting that molecular differences may exist between the two RNase antigens. Immunodiffusion and rocket immunoelectrophoresis were used to qualitatively test the cross-reactivity of protein preparations from various members of the genus Hordeum and species from other related grass genera. Neither antiserum showed cross-reactivity with soluble protein preparation from species outside the genus Hordeum. A few species within the genus Hordeum were cross-reactive. A modification of rocket immunoelectrophoresis was developed to determine the amount of RNase in unpurified tissue extracts. The technique involved a template-reservoir which allowed detection of 250 ng RNase in tissue extract volumes of 50 μl. The amount of RNase in unpurified protein extracts from the two cultivars of barley was similar.     

The protein euphaseolin inPhaseolinae — A chemotaxonomical study

On the basis of immunochemical analyses of the main reserve protein ofPh. vulgaris, euphaseolin, in numerous cultivars ofPh. vulgaris, in additional 23Phaseolus species, and several representatives of further genera of Viciaceae and on the basis of the comparison of these data with morphological and genetical data the authors propose to separate the sectionEuphaseolus characterized by the presence of the protein euphaseolin. The species characterised by euphaseolin are closely related and capable of being crossed. The proposal requires an additional formal completion from the point of view of the conventions of classical systematies. Further the questions of the taxonomical extent of various protein characters and the problematies of the so-called large and small protein characters are discussed. Na základě imunochemických analys hlavní zásobní bílkovinyPh. vulgaris euphaseolinu u velkého po?tu kultivar?Phaseolus vulgaris, dal?ích 23 druh?Phaseolus a několika zástupc? dal?ích rod?Viciaceae a srovnáváním těchto údaj? s údajl morfologickými a genetickými auto?i navrhují vydělení sekceEuphaseolus, charakterisované p?ítomností bílkoviny euphaseolinu. Druhy charakterisované euphaseolinem jsou si blízce p?íbuzné, jsou k?i?itelné. Návrh vy?aduje je?tě formální doplnění z hlediska zvyklostí klasické systematiky. V ?lánku jsou dále diskutovány otázky taxonomické ?í?e r?zných bílkovinných znak?.     

Development of a PCR-based DNA marker for <Emphasis Type="Italic">Glu-1By</Emphasis> alleles in the old Hungarian Bánkúti wheat

Based on sequence alignment, phylogenetic, and dotplot analyses, primers were designed in order to distinguish the wheat high-molecular-weight glutenin subunit alleles By8 and By9. The primers were tested on 26 lines of Bánkúti wheat, an old Hungarian variety, and a number of other varieties. Consistency was observed between their known By protein subunit and the obtained DNA marker. Comparison of the B subunit content and the By alleles of the Bánkúti lines was also in agreement with the previous prediction that the By8 and By9 subunits are linked to the Bx7 and Bx7? subunits, which are responsible for dough quality in Bánkúti wheat, respectively. Thus, the developed molecular marker would be appropriate for marker assisted selection of the dough quality trait in the introgressive breeding of Bánkúti lines into modern cultivars.     

Activation of type I and type II cyclic AMP-dependent protein kinases by 2,8-disubstituted derivatives of cyclic AMP

Derivatives of cyclic AMP with substituents in both the 2-position (methyl or butyl) and the 8-position (bromo, benzylthio, p-chlorophenylthio or azido) and their singly modified parent compounds were examined for their abilities to activate type I isozymes of cyclic AMP-dependent protein kinases from rabbit and porcine muscle and type II isozymes of cyclic AMP-dependent protein kinases from bovine brain and heart. The specificity of 2-n-butyl-cyclic AMP for type II was substantially reduced or eliminated by the addition of 8-substituents. The lack of specificity of 2-methyl-cyclic AMP for either type I or II was not changed by the addition of 8-substituents.     

Changes in the Proteins of Bean Leaves Infected With Tobacco Necrosis or Alfalfa Mosaic Viruses

Acidic extracts from TNV and AMV infected “Saxa” bean leaves were electrophoretically examined for protein content. In native conditions of resolution (PAGE) at least three protein bands (PS_1–3) not present in the control were found. In denaturing conditions (SDS–PAGE) at least one (PS_a), but often two or three such proteins (PS_{b, c}) were found in the same extracts. Chromatographic resolution of proteins on Sephadex G-100 column resulted in partial purification of the PS-proteins. Additional, not known before, slow-migrating protein (PS₀) induced by hypersensitive viral infection was discovered in some of the eluted fractions. Both PS_0–3 and PS_a–c proteins were present in the same fractions. This fact suggests their similarity in molecular weights and/or shapes.     

Vacuolar localization of ethylene-induced chitinase in bean leaves

The localization of ethylene-induced endochitinase was studied in bean (Phaseolus vulgaris L. cv Saxa) leaves. The specific activity of chitinase in mesophyll protoplasts isolated from the leaves was as high as in tissue homogenates, indicating that most of the enzyme was located intracellularly. Vacuoles isolated and purified from the protoplasts were found to contain most of the intracellular chitinase activity.     

Pokeweed antiviral protein region Gly209-Lys225 is critical for RNA N-glycosidase activity of the prokaryotic ribosome

Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209–225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209–225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209–225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.     

Response of oat cultivars to <Emphasis Type="Italic">Fusarium</Emphasis> infection with a view to their suitability for food use

Oats as a source of antioxidants and complex polysaccharides are currently an important component in human nutrition. Producing healthy, safe and high-quality grain for this purpose depends upon growing oat cultivars with improved resistance to diseases caused by Fusarium spp. producing mycotoxins. Thirteen cultivars of naked (Ábel, Detvan, Izák and Avenuda) and covered (Zvolen, Auron, Atego, Flämingsstern, Kanton, Viktor, Zla?ák, Euro and Ardo) oats were inoculated with conidial suspensions of F. culmorum isolate in the field at flowering in 2006 and 2007. After harvest, reduction in thousand-kernel weight (R-TKW), reduction in panicle-kernel weight (R-PKW), and deoxynivalenol (DON) content in grain and hulls were determined. The ELISA immunochemical method was employed for the quantitative analyses of DON. Values of yield components (R-TKW; R-PKW) were 35.4% and 31.1% lower in dehulled covered oats than in naked oat cultivars. The DON accumulation was highest in hulls as compared with DON content in kernels of naked and covered oat cultivars. Accumulation of DON in dehulled covered cultivars was 34.4% lower than the average contamination in naked cultivars. When the cultivars were compared, there were positive correlations between R-TKW and R-PKW and also between DON content and R-PKW. With a view to growing oat cultivars for production of cereal foods, it was shown that dehulling of covered oat grain resulted in substantially reduced DON content.