Immunocytochemical localization of concanavalin A in developing jack-bean cotyledons

The lectin, concanavalin A (Con A), was localized in the cotyledon of developing jack beans (Canavalia ensiformis (L.) DC) by electron-microscope immunocytochemistry. In mature seeds, Con A was present in protein-storage vacuoles (protein bodies) of storage-parenchyma cells. Although protein bodies could be seen in other cell types, only protein bodies in storage-parenchyma cells contained Con A. During seed development, Con A was also localized on the endoplasmic reticulum and Golgi apparatus, presumably en route toward deposition within the protein bodies. The intensity of labeling of the endoplasmic reticulum was much greater during the developmental stage of protein-body filling (66% final seed weight) than in mature seeds.Abbreviations Con A concanavalin A - ER endoplasmic reticulum - IgG immunoglobulin G     

Development and regulation of three glyoxysomal enzymes during cotton seed maturation and growth

The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton (Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady-state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s).Abbreviations ABA abscisic acid - bp base pairs - DPA days post anthesis - HPI hours post imbibition - kb kilobase (pairs) - M _r relative molecular weight - S Svedberg unit (10^-13s)     

Crack and cook: A simple and quick process for elimination of concanavalin A (Con A) from Canavalia seeds

The most nutritionally important antinutritional factor in the Canavalia seeds is Concanavalin A (Con A) which takes at least 3 h of cooking at 96°C to inactivate. The effect of breaking the seeds into smaller pieces before cooking on the Con A content was investigated. Whole seeds of Canavalia ensiformis (jackbean) and Canavalia ensiformis seeds that were broken into smaller pieces (3–7 parts per seed) were cooked at 96°C for 30, 60, 90 and 120 min, respectively. Some were cooked for 15, 30 and 45 min, respectively, under pressure using household pressure cooker. Con A content of the products was determined by the hemagglutination of rabbit fresh erythrocytes by the extracted protein samples serially diluted in PBS buffer in 96-microwell plates. Hemagglutinating activity was expressed as minimal concentration of protein inducing hemagglutination of cells. Hemagglutinating activity of the cracked beans was completely eliminated within 1 h of ordinary cooking and 15 min of pressure cooking. The cooked materials took 5–6 h of sunshine to dry to 90% dry matter. The hemagglutinating activity of the whole seeds was eliminated in 45 min of pressure cooking but not in 2 h of ordinary cooking. They took 3 days of same sunshine to dry. The crack and cook process (CAC), the name being proposed for the method, is commercially applicable in view of its relative ease of execution.     

Synthesis of Canavalin and Concanavalin A in Maturing Canavalia gladiata Seeds

In successive stages of seed development of Canavalia gladiata,seeds were harvested, and time-course changes of the accumulationof canavalin and concanavalin A (Con A) were investigated bySDS-gel electrophoresis and protein blot analysis. Results showedthat the synthesis of both seed proteins started as early as30 days after flowering (DAF) and that they had different accumulationpatterns during seed development. The synthesis and accumulationof canavalin were most active at SOSO DAF, whereas the contentof Con A continued to increase gradually until the seed maturationwas nearly completed (80 DAF). Next, an RNA fraction was preparedfrom seeds of 40 DAF, translated in a wheat germ system andits translation products analyzed by the immunoprecipitationand SDS-gel electrophoresis. The mol wt (about 48,000) of thein vitro product immunoprecipitated with the antiserum to canavalinwas very closed to that of canavalin. The mol wt (34,000) ofthe in vitro product immunoprecipitated with the antiserum toCon A was about 4,000 higher than that of Con A. Canavalin-mRNAwas present as early as at 30 DAF and its translational activitywas the highest at 35–40 DAF. Con A-mRNA was also detectedat 30 DAF and, in contrast to canavalin-mRNA, high activitieswere maintained until 75 DAF. (Received November 25, 1986; Accepted January 29, 1987)     

Paracoccidoides brasiliensis 30 kDa Adhesin: Identification as a 14-3-3 Protein,Cloning and Subcellular Localization in Infection Models

Paracoccidoides brasiliensis adhesion to lung epithelial cells is considered an essential event for the establishment of infection and different proteins participate in this process. One of these proteins is a 30 kDa adhesin, pI 4.9 that was described as a laminin ligand in previous studies, and it was more highly expressed in more virulent P. brasiliensis isolates. This protein may contribute to the virulence of this important fungal pathogen. Using Edman degradation and mass spectrometry analysis, this 30 kDa adhesin was identified as a 14-3-3 protein. These proteins are a conserved group of small acidic proteins involved in a variety of processes in eukaryotic organisms. However, the exact function of these proteins in some processes remains unknown. Thus, the goal of the present study was to characterize the role of this protein during the interaction between the fungus and its host. To achieve this goal, we cloned, expressed the 14-3-3 protein in a heterologous system and determined its subcellular localization in in vitro and in vivo infection models. Immunocytochemical analysis revealed the ubiquitous distribution of this protein in the yeast form of P. brasiliensis, with some concentration in the cytoplasm. Additionally, this 14-3-3 protein was also present in P. brasiliensis cells at the sites of infection in C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells for 72 h (acute infections) and 30 days (chronic infection). An apparent increase in the levels of the 14-3-3 protein in the cell wall of the fungus was also noted during the interaction between P. brasiliensis and A549 cells, suggesting that this protein may be involved in host-parasite interactions, since inhibition assays with the protein and this antibody decreased P. brasiliensis adhesion to A549 epithelial cells. Our data may lead to a better understanding of P. brasiliensis interactions with host tissues and paracoccidioidomycosis pathogenesis.     

Differential Changes in Levels of mRNAs during Maturation of Wheat Seeds That Are Susceptible and Resistant to Preharvest-Sprouting

Seed dormancy is strongly related to the physiological conditions,especially as they relate to responsiveness to ABA, of embryocells during maturation of seeds. In this study, seeds of Triticumaestivum L. cv. Chihoku, which showed nondormancy at harvest,and line Kitakei-l354 (referred to as Kitakei), which showedpost-harvest dormancy, were collected 30 days after anthesis(DPA 30) and at the mature stage (DPA 60). Poly(A)⁺RNA was extractedfrom the embryos of the seeds and translated in a wheat germsystem. The majority of products of translation from the twogenotypes migrated to the same positions in two-dimensionalgels. Levels of six (for polypeptides h, i, k, m, n, and o)out of 14 Chihoku-specific mRNAs decreased dramatically duringseed maturation, concurrently with the loss of dormancy. Bycontrast, levels of 3 (for polypeptides c, e and f) out of 6Kitakei specific mRNAs were maintained during maturation andduring a 48-h imbibition of the dormant seeds but decreasedat germination. Polypeptides n of Chihoku and e of Kitakei hadthe same molecular size and slightly different pI values. Thesetwo polypeptides may be encoded by the same gene and may playsome role in the maintenance of seed dormancy. Levels of mRNAsfor 10 polypeptides, found in both Chihoku and Kitakei embryosat DPA 30, changed to different extents during maturation. Outof the 10 mRNAs, the relative abundance of 4 mRNAs of Kitakeidid not change dramatically during seed maturation, while inChihoku these mRNAs decreased in level or disappeared duringthe same maturation period. In addition, levels of 2 of these4 mRNAs did not decrease significantly during imbibition ofthe dormant Kitakei seeds but disappeared upon treatment forbreaking of dormancy. The maintenance of these mRNAs in thedormant seeds during maturation and imbibition suggests thatthe respective gene products are involved in the maintenanceof dormancy in wheat seeds. (Received December 5, 1991; Accepted April 1, 1992)     

Quantification of ABA and its metabolites in sweet cherries usingdeuterium-labeled internal standards

Abscisic acid (ABA), phaseic acid (PA), dihydrophaseic acid (DPA), and epi-dihydrophaseic acid (epi-DPA) were quantified in developing fruit and seeds of sweet cherry using each deuterium-labeled internal standard. ABA concentrations in the pulp were low at the early stage of fruit development, reached to the maximum before maturation, and subsequently declined during maturation. The significant increase of ABA after 29 days after full bloom (DAFB) coincides with the softening suggests that ABA may play a role to induce fruit maturation in sweet cherries. ABA metabolite levels were high at the immature stage and decreased with fruit maturation. This fact suggests that fruit may not need ABA in the early stage of fruit development. It was considered that DPA may be the major metabolite of ABA since the concentrations were higher than PA and epi-DPA at all stages of fruit development. ABA concentrations increased at the beginning of seed maturation and then decreased toward harvest. This decrease may be necessary to end seed dormancy. DPA in seeds changed similarly with ABA but its concentrations were always higher than those of ABA.     

Storage Protein Synthesis during Oat (Avena sativa L.) Seed Development

Oat (Avena sativa L.) seeds harvested at 2-day intervals from anthesis to maturity were tested for their ability to incorporate [³⁵S]sulfate into protein. Incorporation of [³⁵S]sulfate into TCA-insoluble material began 2 to 4 days postanthesis (DPA), reached a peak 14 to 16 DPA, and was barely detectable by 24 DPA. Incorporation of label into globulin was parallel to total protein accumulation, and averaged about 85% of the total protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein extracted from developing seeds indicated that some polypeptides coinciding with the α and β globulin subunits were present 2 to 4 DPA, but the full complement of globulin polypeptides was not present until 10 DPA. Immunoprecipitation of in vivo labeled seed extracts showed that globulin polypeptides and the 59 kilodalton precursor were present at early stages of development (4 DPA). Quantitation of dot blot analysis, using an oat globulin cDNA clone as a probe, indicated that one species of oat globulin mRNA was most abundant 15 DPA, which is during the peak time of storage protein synthesis.     

Reduced protein secretion and glycosylation induced by ammonium stress inhibits somatic embryo development in pumpkin

Extracellular proteins and glycoproteins secreted by ammonium- or auxin-induced somatic embryogenic cultures of pumpkin were analyzed. Despite an overall similarity in developmental characteristics between these embryogenic cultures, distinct expression patterns of extracellular proteins and glycoproteins were observed. Ammonium, when supplied as the sole source of nitrogen, caused acidification of the culture medium and significantly reduced protein secretion. Buffering pH in the ammonium-containing medium restored extracellular protein secretion and glycosylation and an enhanced cell aggregation but not the development of later embryo stages. As revealed by Concavalin A (Con A) immunodetection, extracellular glycoproteins containing α-D-mannose and α-D-glucose were most abundant in proembryogenic cultures grown in a buffered ammonium-containing medium and in a medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). We assume that extracellular proteins (Mr 28, 31, and 44 kDa) and Con Abinding glycoproteins (Mr 26, 30, 40, 53, and 100 kDa) found in both proembryogenic cultures may have a role during somatic embryogenesis induction. The glycan components of proteins were further characterized by affinity blotting with different lectins. Binding patterns of mannose-specific lectin from Galanthus nivalis partially correlated with those detected with Con A, whereas no signal was observed with lectins from Datura stramonium and Arachis hypogea regardless of the treatment applied. Results indicate that complex N- or O-glycans are not typical for early phases of pumpkin embryo development. The accumulation of extracellular glycoproteins with high-mannose-type glycans from 30 to 34 kDa, observed after the transfer from the ammonium- or 2,4-D-containing media into a maturation medium, appeared to be associated with development of later embryo stages. This study also revealed the presence of EP-3-like endochitinases in pumpkin embryogenic cultures, particularly in cultures grown in the buffered ammonium-containing medium, however, these proteins should be examined further.     

Proteins in development and germination of a desiccation sensitive (recalcitrant) seed species

In the recalcitrant seeds of Avicennia marina, protein content and the rates of protein synthesis increase during histodifferentiation. This is similar to the situation in desiccation tolerant seeds. During the stage of reserve accumulation the protein content and rates of synthesis remain constant and there is no de novo synthesis of proteins which might qualify as storage proteins. There is also no change in the nature of proteins present in either axis or cotyledonary tissues during development or germination. Similarly, fluorographs of axis proteins show only very limited changes in the patterns of protein synthesis during development and germination, at least until the onset of root growth. Heat-stable proteins are present from an early developmental stage. However, no late embryogenic abundant (LEA) proteins are synthesised during the late stages of development, indicating that seedling establishment is independent of such maturation proteins. It is suggested that the lack of desiccation tolerance of A. marina seeds might be related to the absence of desiccation-related LEAs. Although the rate of protein synthesis increases during germination, protein metabolism appears to remain qualitatively the same as that occurring during development. The present results suggest that in these desiccation sensitive seeds, protein metabolism characterising development changes imperceptibly into that of germination.     

Vatairea macrocarpa Lectin (VML) Induces Depressive-like Behavior and Expression of Neuroinflammatory Markers in Mice

Lectins are proteins capable of reversible binding to the carbohydrates in glycoconjugates that can regulate many physiological and pathological events. Galectin-1, a β-galactoside-binding lectin, is expressed in the central nervous system (CNS) and exhibits neuroprotective functions. Additionally, lectins isolated from plants have demonstrated beneficial action in the CNS. One example is a lectin with mannose-glucose affinity purified from Canavalia brasiliensis seeds, ConBr, which displays neuroprotective and antidepressant activity. On the other hand, the effects of the galactose-binding lectin isolated from Vatairea macrocarpa seeds (VML) on the CNS are largely unknown. The aim of this study was to verify if VML is able to alter neural function by evaluating signaling enzymes, glial and inflammatory proteins in adult mice hippocampus, as well as behavioral parameters. VML administered by intracerebroventricular (i.c.v) route increased the immobility time in the forced swimming test (FST) 60 min after its injection through a carbohydrate recognition domain-dependent mechanism. Furthermore, under the same conditions, VML caused an enhancement of COX-2, GFAP and S100B levels in mouse hippocampus. However, phosphorylation of Akt, GSK-3β and mitogen-activated protein kinases named ERK1/2, JNK1/2/3 and p38^MAPK, was not changed by VML. The results reported here suggest that VML may trigger neuroinflammatory response in mouse hippocampus and exhibit a depressive-like activity. Taken together, our findings indicate a dual role for galactose binding lectins in the modulation of CNS function.     

Differential elimination of mercury during maturation of leguminous seeds

The pods of Acacia koa, Canavalia cathartica, Leucaena leucocephala, Samanea saman and six other leguminous species lose as much as 75–85 % of their tissue water during maturation, but show no loss in tissue mercury content. Unlike fruit, seeds not only lose most of their water content, but also at least 75 % of their mercury during the maturation process. It is suggested that selective elimination is more likely to take place by bio-volatilization rather than by translocation, and that the phenomenon allows for detoxification of a cytogenetically hazardous chemical substance.     

Protein Synthesis during Natural and Precocious Soybean Seed (Glycine max [L.] Merr.) Maturation

Protein synthesis was studied during precocious and natural soybean seed (Glycine max [L.] Merr.) maturation. Developing seeds harvested 35 days after flowering were precociously matured through controlled dehydration. Total soluble proteins and proteins labeled with [³⁵S]methionine were extracted from control, developing seeds and from precociously and naturally matured seeds and were analyzed by one-dimensional PAGE and fluorography. The results demonstrated that several polypeptides which were designated “mature polypeptides,” were synthesized de novo during precocious and natural seed maturation. Two of these polypeptides, 31 and 128 kilodalton in mass, also stained intensely with Coomassie blue, suggesting their abundant accumulation during seed maturation. Results from in vitro translation experiments showed that the mRNAs corresponding to these “maturation polypeptides” accumulated during precocious maturation and in naturally matured seeds, but not in seeds freshly harvested 35 days after flowering (control). The role of the “maturation polypeptides” is currently unknown; however, their presence and that of their corresponding mRNAs was coincident with the ability of matured seeds to establish seedling growth. This study has demonstrated that precocious seed maturation treatments may be extremely useful for investigations of metabolic events and molecular control mechanisms affecting soybean seed maturation.     

Concanavalin A induces germinal vesicle breakdown in Barnea candida (Mollusca,Pelecypoda) oocytes

Treatment of Barnea candida oocytes with 5 μg/ml Con A or above elicits germinal vesicle breakdown (GVBD), the timing for this event being dose dependent. At 100 μg/ml, GVBD occurs within 20 to 30 min, a lag time corresponding to that observed after fertilization. Con A-induced GVBD requires the presence of 2 mM external calcium during all the treatment period while at 10 mM external Ca²⁺, the calcium-dependent period is slightly reduced. It is sensitive to low pH Na-acetate sea water, 50 μM trifluoperazine, 20μM D-600 or 2,4-dinitrophenol, as well as to 10 μg/ml cytochalasin B. A straightforward interpretation of these data would be that Con A-induced maturation is sustained by an energy-requiring effector mechanism involving intracellular contractile proteins.     

Change of thiamin-binding protein and thiamin levels during seed maturation and germination in sesame

Thiamin-binding proteins (TBPs) occur in many types of plant seeds. The biochemical and structural properties such as subunit structure and affinity for thiamin of the proteins have been characterized. However, the change of TBP and thiamin during seed maturation and germination is little known. Sesame (Sesamum indicum L.) seeds have unique albumin TBPs, because the other TBPs from plant seeds are generally globulins. In this study, we studied the change of the TBP and thiamin levels in sesame seeds. The protein content and thiamin-binding activity of the seeds increased with seed development after flowering. Immunological analysis using an antibody against the TBP of sesame seeds showed that the protein was accumulated in seeds during maturation. The thiamin content of the seeds increased with seed development after flowering. On the other hand, the thiamin-binding activity decreased during seed germination when TBP was degraded. The thiamin content of the seeds decreased during the germination. However, the amount of thiamin phosphate in the seeds during germination was little changed. These results suggested that thiamin was accumulated and stored as a complex with TBP in sesame seeds.     

Lectin fromCanavalia brasiliensis (MART.). isolation,characterization and behavior during germination

A lectin was isolated fromCanavalia brasiliensis Mart. seeds by combining solubility fractionation with affinity chromatography on Sephadex G-50. The lectin showed a carbohydrate specificity for D-mannose (D-glucose) binding and a requirement for Ca²⁺ and Mn²⁺. All the hemagglutinating activity was found in the cotyledons and the presence of the lectin was followed during the first 15 days of plant germination, through the activity against rabbit erythrocytes, the presence of the “lectin peak” in Sephadex G-50 affinity chromatography, presence of the “lectin bands” in SDS-polyacrylamide gel electrophoresis and the “lectin arcs and rockets” in immunoelectrophoresis in agarose gel. On application of all these methods the lectin showed a differentiated metabolism, disappearing more slowly than the other high molecular weight proteins of the seed.     

Macrophage Interaction with Paracoccidioides brasiliensis Yeast Cells Modulates Fungal Metabolism and Generates a Response to Oxidative Stress

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.     

Changes in Level and Activity of Phospholipid Transfer Protein during Maturation and Germination of Maize Seeds

The variations of the amounts of phospholipid transfer proteins (PLTP), determined by ELISA and immunoblotting methods, were followed during the maturation and germination of maize (Zea mays L.) seeds. Changes of the amounts of PLTP occur during seed maturation. The levels of PLTP, low in the first 3 weeks after fecondation, strongly raised 3 to 5 weeks after, then reached and maintained a high value (10% of total soluble proteins) during the last steps of maturation. These variations, determined by ELISA, are in accordance with the observations made by immunoblotting. Changes in phospholipid transfer activity were also found when protein extracts prepared from seeds at different stages of maturation were assayed for transfer activity. The levels of PLTP were also determined during the germination of maize seeds and the early growth of the plantlets, both in the endosperm and the aerial parts. While no major change was observed in the endosperm, a high increase in PLTP level was found in the aerial part of the plantlet, both by ELISA and immunoblotting. An enhancement of the phospholipid transfer activity was parallely observed in the protein extracts of plantlets at various stages of germination. These results are consistent with an in vivo correlation between the synthesis of phospholipid transfer protein, observed during the maturation and germination of maize seeds, and the biogenesis of membranes which involves intracellular movements of phospholipids.