Isolation and enzymatic digestion of body complex of soybean seed

The body complex of the soybean seed (BCSS) was isolated from the single cells (27.2%) by a sequential procedure of autoclaving with water, cellulase digestion for the primary cell wall, pectinase digestion for the secondary cell wall, and defatting with hexane washing. Its characteristics were then investigated. The defatted BCSS (DBCSS) consisted of protein (76.5%) and mannose-rich carbohydrates (3.2%). Screening of the food-processing protease for the digestion of DBCSS was carried out, and a kind of alkaline protease was selected. The inner protein of DBCSS was easily extracted with 0.1 M sodium carbonate buffer, pH 10, and the insoluble shell of the body complex (SDBCSS) was left. SDBCSS consisted of hydrophobic amino acid-rich protein. SDBCSS was easily digested by the selected alkaline protease. SDBCSS was dissolved by boiling with sodium dodecyl sulfate-mercaptoethanol, and it was found to consist of a protein of approximately 3 kDa. The high enzymatic digestion including the selected protease for soybean seed and defatted soybean meal was carried out; both were extracted and digested with a yield of >99.5%. The final indigestible residue was found as paired hexagonal and filamentous organs of the soybean cells.     

Energy-dispersive x-ray analysis of phytin globoids in aleurone particles of developing rice grains

Abstract

In this study energy-dispersive X-ray analysis was used to discover changes in the composition of phytin globoids in aleurone particles of developing rice grains.

At early milky stage (the 7th to 10th day after flowering) many aleurone particles were observed as electron lucent vacuole-like particles in aleurone cells, some of which contained a small electron-dense inclusion (phytin globoid). The major mineral elements present in phytin globoids were phosphorus (P) and magnesium (Mg). Potassium (K) was also detectable but its concentration was extremely low relative to these two. Calcium (Ca) and zinc (Zn) were found as minor components. At early dough stage (the 17th to 19th day after ftowering) P, Mg and K were observed as the major mineral elements. The composition of mineral elements in phytin globoids tend to be constant in the late staae of ripening. On the other hand, minor elements, i.e. Ca and Zn, were only detectable in the early stage of ripening, suuesting that these elements accumulated in the aleurone particles only during the early stage, and in later stage their accumulation was either complete or at very low levels compared with those of K and Mg. The relative amount of Mg existing in phytin globoid remained roughly constant throughout the ripening periods, while that of K varied.

Energy-dispersive X-ray analysis of phytin globoids in developing rice aleurone particles confirmed that the accumulation of P, Mg and K in rice grains was closely related to the formation of phytin globoids. This analysis suggested further that the mechanism of accumulation of P into aleurone particles was very similar to that of Mg, while that of K differed from both. The results obtained suggested that Ca and Zn might be required in the formation of phytin globoid at the early ripening stage. EDX analysis of a protein body in the starchy endosperm revealed that there was no significant element detectable in an EDX spectrum other than sulfur. This suggests that lamellar concentric structures in protein bodies are not due to phytin.     

Comparison of the early response to aluminum stress between tolerant and sensitive wheat cultivars: Root growth,aluminum content and efflux of K+

In order to characterize the mechanism of Al tolerance (Atlas 66) and Al sensitivity (Scout 66) in two cultivars of wheat (Triticum aestivum L.), the early responses to Al stress under acidic conditions were investigated. Marked inhibition of root elongation of Scout was observed upon treatment with 10 μM AlCl₃ for less than 3 h. The inhibition of root elongation of Scout was reversed within 3 days when the treated samples were transferred to a solution without Al. However, treatment for 6 h with AlCl₃ repressed root elongation almost completely and irreversibly. Root elongation of Atlas was only partially inhibited by the treatment with 10 μM AlCl₃ for more than 6 h. Levels of Al in two portions of roots, namely, portions 0–5 mm and 5–10 mm from the tip, were lower in Atlas than those in Scout. In Atlas the levels of Al on a fresh weight basis in both portions were very similar, while the level of Al in the portion 0–5 mm from the tip was almost double than that in the 5–10 mm portion in Scout. A distinct increase in levels of Al in the 0–5 mm portion over that in the 5–10 mm portion of Scout was observed even after 3 h of treatment with AlCl₃.

Both Atlas and Scout were preloaded with K⁺ at pH 5.5 and transferred to distilled water at various pH values to monitor the efflux of K⁺. A reduction in the pH induced increases in the efflux of K⁺ in both cultivars, and the rate of efflux in Scout was twice that in Atlas at pH 4.2. AlCl₃ at concentrations as low as 5 μM markedly repressed K⁺efflux at pH 4.2 and this effect was more pronounced in Scout. Ca²⁺ also had a repressive effect on K⁺ efflux, while EGTA increased K⁺ efflux. Vanadate increased K⁺ efflux, a result that suggests the involvement of a H⁺ pump in K⁺ efflux. Ca²+ failed to repress the increased efflux of K⁺ caused by vanadate while Al repressed the K⁺ efflux even in the presence of vanadate. These results suggest that a low extracellular pH may cause an increase in the cytoplasmic concentration of H⁺ that is followed by depolarization of the plasma membrane, which may be modified by the efflux of K⁺ and H⁺. The characteristic difference in terms of K⁺ efflux between Atlas and Scout at low pH may be caused by differences associated with plasma membrane potentials, as follows. The net influx of H+ at low pH, which causes depolarization of the plasma membrane, is higher in Scout than in Atlas. The difference in the net influx of H⁺ may be regulated in part by Ca2⁺, that either repress the influx of H⁺ or the activate of the H⁺ pump. Inhibition of K⁺ efflux by Al, which tends to depolarize the plasma membrane at low pH, may be an important factor in determining sensitivity and/or tolerance to Al.     

Enhancement of nitrate efflux and the plasma membrane hydrogen ion‐transport activity of barley roots by nitrate treatment

The effect of nitrate (NO₃) on the activities of hydrogen (H⁺)‐ATPase and H⁺‐transport in the plasma membrane vesicles isolated from barley (Hordeum vulgare L. cv. Kikaihadaka) roots was investigated. After treatment with 10 mM NO₃ for longer than 24 h, both activities of H⁺‐ATPase and H⁺‐transport assayed in the presence of chloride (Cl) were stimulated less than 20%. While H⁺‐transport activity was approximately two times higher in NO₃‐grown roots than in control roots when assayed in the presence of NO₃ instead of Cl. This result suggests that NO₃ is permeable in the plasma membrane vesicles isolated from NO₃‐grown roots rather than that of the control, and thereby stimulate H⁺‐transport activity through the collapse of positive potentials established by H⁺‐ATPase. Furthermore, NO₃ efflux from roots was markedly enhanced after 48 h of the exposure to NO₃. Taken together, NO₃ efflux may be mediated by the permeability to NO₃ which is induced by NO₃.     

RNA silencing as a tool to uncover gene function and engineer novel traits in soybean

RNA silencing refers collectively to diverse RNA-mediated pathways of nucleotide-sequence-specific inhibition of gene expression. It has been used to analyze gene function and engineer novel traits in various organisms. Here, we review the application of RNA silencing in soybean. To produce soybean lines, in which a particular gene is stably silenced, researchers have frequently used a transgene that transcribes inverted repeats of a target gene segment. Suppression of gene expression in developing soybean embryos has been one of the main focuses of metabolic engineering using transgene-induced silencing. Plants that have enhanced resistance against diseases caused by viruses or cyst nematode have also been produced. Meanwhile, Agrobacterium rhizogenes-mediated transformation has been used to induce RNA silencing in roots, which enabled analysis of the roles of gene products in nodulation or disease resistance. RNA silencing has also been induced using viral vectors, which is particularly useful for gene function analysis. So far, three viral vectors for virus-induced gene silencing have been developed for soybean. One of the features of the soybean genome is the presence of a large number of duplicated genes. Potential use of RNA silencing technology in combination with forward genetic approaches for analyzing duplicated genes is discussed.     

Reference values of hematological and biochemical parameters for the world smallest microminipigs

In this study, we demonstrated growth curves and reference values for hematological and serum biochemical parameters of Microminipigs, the world smallest experimental minipigs. In both male and female animals, the body weights (BWs) at 3 and 6 months of age were <5 kg and <10 kg, respectively, and growth curve revealed almost plateau (approximately 20 kg BW) after 18 months of age. Major hematological and serum biochemical parameters showed no gender differences and the values were very similar to those in G?ttingen and Yukatan minipigs. The values obtained in this study can serve as fundamental reference, and thereby facilitate the use of Microminipig in life science research.     

Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet

Insulin secretion from intact mouse pancreatic islets was investigated with two-photon excitation imaging. Insulin granule exocytosis occurred mainly toward the interstitial space, away from blood vessels. The fusion pore was unusually stable with a lifetime of 1.8 seconds. Opening of the 1.4-nanometer-diameter pore was preceded by unrestricted lateral diffusion of lipids along the inner wall of the pore, supporting the idea that this structure is composed of membrane lipids. When the pore dilated to 12 nanometers, the granules rapidly flattened and discharged their contents. Thus, our methodology reveals fusion pore dynamics in intact tissues at nanometer resolution.     

Sex and age differences in meat composition of Yeso sika deer (Cervus nippon yesoensis) reared for a short period after capture in the wild

Yeso sika deer captured in winter around Lake Akan in Hokkaido were reared for 8–10 months at Tokyo University of Agriculture in Abashiri. Six 1‐year‐old females and males and six 2‐year‐old or older (adult) females and males were slaughtered and their carcasses were processed. The chemical composition, mineral contents and fatty acid composition of the loin were measured. No marked influence of gender or age was noted in the chemical composition of loin. In the mineral contents, significant differences were noted. The potassium and sulfur contents were lower and the sodium content was higher in adult deer meat (P < 0.05, respectively) and the potassium content was higher in male deer meat (P < 0.05). Arsenic, cadmium or lead were not detected. In the unsaturated fatty acid, a significant interaction was detected (P < 0.05), and it was high in 1‐year‐old female deer meat and low in 1‐year‐old male deer meat. Significant gender or age differences were noted only in the mineral contents in the loin of deer reared for a short period after capture.     

Neuroanatomical distribution of disease-associated prion protein in cases of bovine spongiform encephalopathy detected by fallen stock surveillance in Japan

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle characterized by accumulation of the disease-associated prion protein (PrP(Sc)) in the central nervous system (CNS). The immunohistochemical patterns and distribution of PrP(Sc) were investigated in the CNS, brains, and spinal cords of 7 naturally occurring BSE cases confirmed by the fallen stock surveillance program in Japan. No animals showed characteristic clinical signs of the disease. Coronal slices of 14 different brain areas in each case were immunohistochemically analyzed using an anti-prion protein antibody. Immunolabeled PrP(Sc) deposition was widely observed throughout each brain and spinal cord. Intense PrP(Sc) deposition was greater in the thalamus, brainstem, and spinal cord of the gray matter than in the neocortices. The topographical distribution pattern and severity of PrP(Sc) accumulation were mapped and plotted as immunohistochemical profiles of the different brain areas along the caudal-rostral axis of the brain. The distribution pattern and severity of the immunolabeled PrP(Sc) in the CNS were almost the same among the 7 cases analyzed, suggesting that the naturally occurring cases in this study were at the preclinical stage of the disease. Immunohistochemical mapping of the PrP(Sc) deposits will be used to clarify the different stages of BSE in cattle.