Block-Surface Staining for Differentiation of Starch and Cell Walls in Wheat Endosperm

A staining technique for differentiating starch granules and cell walls was developed for computer-assisted studies of starch granule distribution in cells of wheat [Triticum aestivum L.] caryopses. Blocks of embedded caryopses were sectioned, exposing the endosperm tissue, and stained with iodine potassium iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining was avoided by using stains prepared in 80% ethanol and using short staining times. The IKI quenched background fluorescence which facilitated the use of higher concentrations of Calcofluor White. Cell wall definition was improved with the IKI-Calcofluor staining combination compared to Calcofluor alone. The high contrast between darkly stained starch granules and fluorescent cell walls permitted computer assisted analysis of data from selected hard and soft wheat varieties. The ratio of starch granule area to cell area was similar for both wheat classes. The starch granule sizes ranged from 2.1 μm³ to 22,000 μm³ with approximately 90% of the granules measuring less than 752 μm³ (ca. 11 μm in diameter). Hard wheat samples had a greater number of small starch granules and a lower mean starch granule area compared to the soft wheat varieties tested. The starch size distribution curve was bimodal for both the hard and soft wheat varieties. Three-dimensional starch size distribution was measured for four cells near the central cheek region of a single caryopsis. The percentage of small granules was higher at the ends than at the mid-section of the cells.     

Elimination of Fat and Protein Prior to the Selective Staining of Bone

The technic of staining skeletal systems previously described is often unsatisfactory for fetal specimens of Aves, because of the large amount of fat and protein. The writer avoids this by introducing two preliminary steps: (1) The specimen is placed in equal parts of glycerin, 95% alcohol and distilled water, and 10% aqueous pepsin (with a drop of 6N HC1 added) injected into the yoik sac, with 2-3 hours incubation at 40^oC. (2) While in 5% aqueous KOH (with a few drops of 2% H₂O₂), the fat areas are injected with cellosolve; and the specimen is left in this solution until skeletal elements become clearly visible. Staining in alizarin red S then follows.     

Selective Axonal Argentaffin Staining in Rat Central Nervous System after Protein Mercuration

The mercury-silver (Hg-Ag) argentaffin technique, known to stain specifically proteins in the lateral components of triads/diads in striated muscle cells, was applied to the central nervous system of adult rats. Following fixation in glutaraldehyde, axons in white and gray matter were selectively stained, but not perikarya or their proximal axon and dendrites. Neural tissues were postfixed 24 hr in 5% (w/v) mercuric acetate in 2% (v/v) acetic acid in distilled water, stained for 12-24 hr in darkness at 37-43 C with ammoniacal silver nitrate solution, freshly prepared by adding concentrated ammonia to 60% (w/v) silver nitrate solution until a small amount of silver oxide precipitate remained undissolved. Samples were then washed with freshly prepared 5% (w/v) sodium sulfite and distilled water. All steps were carried out using dark-colored glass flasks. Samples were dehydrated with ethanol and embedded in Paraplast or Poly Bed. Electron microscopy showed the silver-reducing protein inside the axons. Methylation abolished Hg-Ag axonal reactivity indicating that carboxyl groups were necessary for silver staining. Proteins with solubility properties characteristic of neurofilament proteins were involved in Hg-Ag staining. In the cerebellum the plexus of parallel fibers in the molecular layer were not stained, while basket cell axonal processes reacted intensely. The method appears to distinguish neuronal protein variants related to cytotypic differences in cytoskeletal neurofilaments.     

The Selective Staining of Mitochondria

A critical analysis of extant selective mitochondrial stains has elucidated certain empirical criteria for the adoption of dyes for trial. These criteria include a specific triphenylmethane structure for the dye, with sulfonation and the use of aniline and heat as adjuvants. By the application of these characteristics the dyes fast green FGF and light green SF yellowish were chosen for study and proved to be highly selective mitochondrial stains. They are applicable to both tissue slices and homogenate studies and permit examination of the internal structure of mitochondria.     

谷氨酰胺供应水平对离体培养小麦穗籽粒淀粉和蛋白质及其组分含量的影响

选用籽粒蛋白质含量不同的小麦(Triticum aestivum)品种,运用离体穗培养技术,研究了谷氨酰胺供应水平对小麦穗籽粒重、淀粉和蛋白质及其组分积累的影响.实验结果表明,随着培养基中谷氨酰胺供应水平的提高,粒重、籽粒淀粉和蛋白质积累量均呈单峰曲线,在6 g·L-1谷氨酰胺水平时达到最大值;籽粒氨基酸和蛋白质含量随谷氨酰胺浓度的提高呈上升趋势,而淀粉含量呈下降趋势.籽粒清蛋白和球蛋白含量的变化与总蛋白质含量变化趋势一致,而醇溶蛋白和麦谷蛋白含量在2～6 g·L-1谷氨酰胺浓度范围内呈上升趋势,此后则无明显变化.     

谷氨酰胺供应水平对离体培养小麦穗籽粒淀粉和蛋白质及其组分含量的影响

选用籽粒蛋白质含量不同的小麦(Triticam aestivum)品种, 运用离体穗培养技术, 研究了谷氨酰胺供应水平对小麦穗籽粒重、淀粉和蛋白质及其组分积累的影响。实验结果表明, 随着培养基中谷氨酰胺供应水平的提高, 粒重、籽粒淀粉和蛋白质积累量均呈单峰曲线, 在6 g.L-1谷氨酰胺水平时达到最大值; 籽粒氨基酸和蛋白质含量随谷氨酰胺浓度的提高呈上升趋势, 而淀粉含量呈下降趋势。籽粒清蛋白和球蛋白含量的变化与总蛋白质含量变化趋势一致, 而醇溶蛋白和麦谷蛋白含量在2~6 g.L-1谷氨酰胺浓度范围内呈上升趋势, 此后则无明显变化。     

Evaluation and Elimination of the Interference by Starch in the Biuret Determination of Wheat Protein

In the biuret determination of wheat protein, starch, color substances and lipids extractable with an alkaline solution such as biuret reagent were found to interfere with the biuret method. Comparative evaluation of their effects revealed that starch, a major component of wheat, has the most significant effect on the biuret method. In the presence of starch, the correlation between Kjeldahl protein and the optical density of biuret was poor.

It was found that several organic solvents effectively retarded solubilization of wheat starch in the biuret reaction mixture. By treatment of the wheat samples with these solvents prior to the biuret procedure, the effect of starch was completely eliminated and the optical density of biuret became closely correlated with Kjeldahl protein.     

Moisture Sorption Characteristics of Starchy Products: Oat Flour and Rice Flour

Moisture sorption isotherms for oat flour and rice flour were determined at 5, 23, and 45 °C using a gravimetric technique in an a _w range of 0.08–0.98. The results obtained showed that, for a _w values lower than around 0.75, in both flours, the sorption capacity decreased with increasing temperature, while the opposite behavior was observed at a _w greater than this value. It was found that the experimental data in the water activity range considered were better represented by the four-parameter Peleg equation. The monolayer water contents for both materials were determined by the BET and GAB models. The net isosteric heats of sorption were estimated using the Clausius–Clapeyron equation. For both materials, the monolayer water content and the isosteric heat of sorption were found to decrease with increasing temperature and increasing moisture content, respectively.     

The Selective Staining of Red Blood Cells

A method for the selective staining of red blood cells is described. Material is fixed in 10% neutral formalin in .85% NaCl and imbedded in paraffin or celloidin. Sections 6-10 μ are stained 1-5 minutes in chromotrope 2R. Basophilic and the less strongly acidophilic elements are decolorized with 5% phosphotungstic acid in 95% ethyl alcohol. Red blood cells and other strongly acidophilic elements that may be present in the preparation retain the chromotrope 2R. A counterstain of methyl blue may be used for staining the decolorized basophilic elements. As a result, erythrocytes are stained red by the chromotrope 2R, and basophilic elements blue, by the methyl blue. Less strongly acidophilic elements, having little affinity for either primary or secondary dye, are colorless or gray.     

The Uptake of Surcrose and its Conversion to Starch in Detached Ears of Wheat

Detached ears of wheat were cultured on solutions of ¹⁴C-sucroseand the distribution of carbon-14 in the ear was followed. Within 8 h radioactive sucrose was found in all the tissuesof the cultured ear, and considerable amounts of carbon-14 hadaccumulated in other ethanol-soluble compounds. Carbon-14 accumulatedrapidly in the starch deposited in the endosperm, but littlewas found in the starch of the pericarp, or in other materialinsoluble in ethanol in the vegetative organs. During 48 h the specific radioactivity of the sucrose in theendosperm increased in a hyperbolic pattern and was equal tothat of the starch produced. Carbon-14 in the glucose and fructoseaccumulated more slowly and in a Linear fashion. Experiments with sucrose containing carbon-14 in both moietiesequally, or in the fructosyl moiety exclusively, confirmed thatboth moieties are converted into starch and at about the samerate. As sucrose in endosperm provided with asymmetric sucroseretained a considerable degree of asymmetry, it seems as thoughinversion is not a necessary step in the transport of sucroseinto the grain. In ears provided with ¹⁴C-sucrose at 30 mg ml^–1 the rateof accumulation of ¹⁴C-sucrose in the culm, rachis, and floralorgans was about 0.6 times the value at 50 mg ml^–1. However,in the sucrose of the endosperm, and in the starch depositedthere, the rates of accumulation of carbon-14 from both levelswere identical. This finding supports the concept that the transportof sucrose is limited during the final stages of its passageinto the grain.     

Selective Staining of Volutin In Corynebacterwm Diphtheriae

Stain air-dried, heat-fixed smears of the usual Loefner-medium cultures of C. diphtheriae 5 min in 0.5% aqueous chrysoidin solution. Wash with water and apply Albert's iodine for 1 min. Wash with water and air-dry. Volutin stains brownish black or black, the bacterial body weak brown or yellow. There is no staining of nuclear structures.     

Selective and Contrast Staining of Granules in the Juxtaglomerular Complex

A review of four methods for staining juxtaglomerular cells revealed that one method may be highly selective for juxtaglomerular granules (JGG) whereas another may stain general cytological features in addition to the granules. The kind of research undertaken would determine the particular method to be used. Harada's (1952) method, which uses a 1:400,000 solution of gentian violet is recommended as the highly selective stain, and the Masson-Goldner stain after a Ciaccio type fixation is best for cytological detail combined with clear tinctorial contrast of the JGG.     

Immunoelectrophoretic Analysis of Wheat Flour Albumins

Albumin preparations from four kinds of wheat flour (Durum, Manitoba No. 2, Western White and Norin No. 26) were analyzed by Immunoelectrophoresis. Seven to eleven components were detected for each preparation. They were classified and designated on the basis of the electrophoretic mobility (R) and the character of precipitin lines formed by antigen-antibody reactions.     

Rheological Studies of Wheat Flour Dough

Dynamic rigidity G′, dynamic viscosity η′ and the relaxation spectrum L(t) of dough under periodic shear stress have been obtained. At first, G′ and η′ decreased rapidly with rest time, but maintained a constant value after 60 minutes. Values of both G′ and η′ decreased, and L(t) became flat with the decrease in water absorption and with the temperature. G′ increased slightly with the increase in salt content, but η′ decreased in a low frequency range.     

小麦地方品种淀粉颗粒结合蛋白及淀粉含量差异研究

淀粉颗粒结合蛋白包含了多种淀粉生物合成的关键酶,对作物淀粉品质有重要影响。本研究利用1D-SDS-PAGE,分离了74份四川、西藏及云南毗邻地区小麦的淀粉颗粒结合蛋白,对突变材料进行了分子标记检测,对总淀粉和直链淀粉含量差异进行了比较。发现供试材料中,在分子量57～130 kDa区域共有9种不同的蛋白条带。其中,2个条带可能为新的淀粉颗粒结合蛋白;存在12份Wx-B1缺失的自然突变体和3份稀有的SGP-B1缺失突变体;筛选到直链淀粉含量超过30%的材料2份,直链淀粉含量为15%左右的材料10份。这些材料为小麦淀粉品质改良及淀粉生化合成机理研究提供了基础。     

Chemical and Nutritional Properties of Hypoallergenic Wheat Flour

The chemical and nutritional properties were investigated of hypoallergenic wheat flour (HWF) prepared by the cellulase-actinase treatment. HWF was composed mainly of oligopeptides and free amino acids, and its average molecular weight was lower than 1,000. Feeding tests on rats showed that, with respect to the PER, GOT and GPT activities and other nutritional indices, the HWF diet was almost equivalent to the control diet which had been prepared from normal wheat flour (NWF). No abnormality was apparent in the main organs after the HWF diet had been fed for 3 weeks. The small intestinal absorption of the HWF diet was found normal by measuring the free amino acid concentration in the intestinal tract and in the portal vein plasma. These data suggest that te absorption of amino acids from the HWF diet was comparable with or more efficient than that from a simulated free amino acid diet.     

小麦面粉Puroindoline蛋白的提取与纯化

Puroindoline蛋白是小麦面粉中一种非常重要的蛋白质,不仅影响和决定了籽粒的硬度,而且有抗G^＋、G^-菌以及抗真菌的作用。用含4％TritonX-114、100mmol／L pH7．8Tris-HCl缓冲液处理小麦面粉来分离Puroindoline蛋白。经处理后得到的蛋白质混合溶液首先用分子筛葡聚糖G-75纯化,每个收集管内的组分经SDS-PAGE分析,分子量小于31kD的蛋白质组分被回收和集中,回收的蛋白质组分经PEG20000浓缩后,再用离子交换柱羧甲基纤维素（CM-23）进行纯化。其洗脱液分别是双蒸水和NaCl,梯度为0．05～0．7mol／L、8mmol／L pH5．5的MES缓冲液,回收只含15kD的蛋白质的组分,接着用PEG20000浓缩。最后冷冻干燥得到Puroindoline蛋白。