Identification of catecholamines in the rat brain and mollusk tissues using semithin slices and retrograde staining

The suggested technique allows revealing the transport-specific dye (primulin) and catecholamine fluorescence simultaneously in the same cell of brain. Intense fluorescence is observed when brain tissue is quickly dehydrated and embedded in the epoxy resin. The same method is suggested for the identification of catecholamines in the embryonal and juvenile tissues of gastropod Lymnaea stagnalis (Mollusca Pulmonata) without using of primulin dye.     

Secretion of organic acids and bases by the kidneys of marine teleosts

In the experiments performed on kidneys of 5 species of marine Teleostei, morphological peculiarities in secretion of 8 fluorescent organic acids (uranin, primulin, tripaphlavin, erythrosin etc.) and in 5 organic bases (rhodamin C, auramin etc.) have been studied. At a very low concentration in the incubation medium--about 0.005 mg/ml--the substances mentioned penetrate into the epithelial cell of the canaliculus; its weak fluorescence appears, and soon they begin to be excreted in great amount through the apical part of the plasmolemma and accumulated in the canalicular lumen. All the substances studied accumulate in the cell and only some of them (uranin, primulin, titanic yellow, etc.) are secreted into the canalicular lumen. Penicillinum and probenecide inhibit penetration of the organic acids into the cell through the basal membrane. Uranin secretion into the canalicular lumen is inhibited in the presence of furocemid; amilorid, magnium and sulfate ions do not influence secretion of the organic acids. Secretion of the organic bases does not change when paraaminohippuric acid and furocemid are added to the medium, but it decreases when concentration of magnium ions increases.     

Accumulation of label from radioactive precursors into dry matter by wheat endosperm cultured in vitro

The capacity of in vitro cultured common wheat ( Triticum aestivum L.) endosperms to incorporate starch and protein precursors was investigated. Isolated 2–3 week-old endosperms were cultured up to 2 weeks in a liquid medium containing labelled (¹⁴C)-sucrose and (³H)-glutamine. Cultured endosperms were separated into ethanolsoluble, starch and protein fractions and the incorporation of the label into each of these fractions was assessed at different times after commencement of culture. The same medium was introduced through the peduncle into normally-developing grains, which were then similarly analyzed. Accumulation of both ¹⁴C and ³H in the ethanol-soluble fraction occurred, at a decreasing rate, only during the first 3 days, and then ceased. The accumulated label in the starch fraction, which originated mainly as ¹⁴C sucrose, proceeded at a relatively constant rate for one week and reached only about 1/5 of the expected in vivo starch production. Incorporation of both isotopes into the protein fraction reflected similar utilization of sucrose and glutamine from the medium (molar base), decreasing in rate with time. Culturing beyond one week produced deteriorated endosperms. Compared to cultured endosperms, normally-developing grains incorporated proportionally less precursors into the ethanol-soluble and more into the insoluble fraction. It is suggested that the reduced starch and protein synthesis in cultured grains stems from impaired capacity of the biosynthetic machinery rather than from low availability of precursors.     

Intravital study of the hydrophobic interactions in protein fibrils by a polarization-fluorescence method. I. Collagen fibrils

It has been shown earlier that anisotropy of extrinsic fluorescence (AEF) of ordered structures in living cell may serve as a measure of the free structure energy. The experimental study of AEF changes of myelin at varying temperatures (0-30 degrees C) revealed cold denaturation. This phenomenon occurs only in cases when the structure in question is stabilized through hydrophobic interactions. The purpose of this work was to show that not only membranes but also some native ordered protein structures might be analysed by fluorescence polarization to detect hydrophobic interactions. The fluorescence anisotropy of primulin adsorbed by collagen fibrils from rat tail has been investigated. The fluorophore orientation decreases with temperature, i.e. here the phenomenon of cold denaturation is observed. When the medium humidity falls down to 80%, no cold denaturation occurs. AEF of collagen fibrils depends to a considerable extent on organic substances (ethanol, ethylene glycol) added in small concentrations to the incubation medium. It is concluded that the dependence of the whole collagen molecular structure in fibrils on the content and structure of the solvent shell is of critical nature.     

Manipulation Through Liquid Culture in the Contents of Sucrose and Glutamine and Their Transformation to Starch and Protein in Wheat Grain

Detached ears of wheat were cultured in liquid medium manipulated for sucrose and glutamine contents, and the accumulation of starch and protein in relation to the activities of sucrose cleaving—, ammonia assimilating—, and transaminating enzymes was studied in the grain. With an increase in the concentrations of sucrose from 44 to 176 mM and glutamine from 6.4 to 25.7 mM (keeping their ratio at a constant value of 7:1), the contents of starch and protein increased in the grains. However, when the grains were cultured in the medium containing 8.5 to 34 mM glutamine and a fixed concentration of 117 mM sucrose, there was a gradual increase in protein and decrease in starch content in the grain. By such manipulation in the liquid medium, the content of free amino acids also increased in the grain up to 12 days culturing. Amongst sucrose cleaving enzymes, the activities of sucrose-UDP glucosyl transferase and soluble alkaline invertase were much lower than the activity of soluble acid invertase. At high concentration (34 mM) of glutamine in the medium, containing 117 mM sucrose, there was drastic decrease in the activities of soluble acid invertase and UDPG pyrophosphorylase but the activities of ADPG pyrophosphorylase, alkaline inorganic pyrophosphatase, glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase increased in the grain with increase in glutamine concentration in the culture medium. Evidently, an increase in the level of amino nitrogen, coupled with an optimum sucrose concentration in the grain raised through liquid culturing enhances the conversion of sucrose to protein at the cost of starch accumulation in wheat.     

Grain size,sucrose level and starch accumulation in developing rice grain

Five rices (Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K⁺ to the medium did not improve dry matter accumulation in the developing grain.     

Regulation of Starch and Protein Synthesis in Wheat Grains by Feeding Sucrose and Glutamine to Detached Ears Cultured in vitro

Three experiments of in vitro ear culture were conducted to evaluate how the substrates of C (carbon) and N (nitrogen) supply in liquid medium regulate the grain growth and synthesis of protein and starch in two winter wheat cultivars. Increasing glutamine supply with constant sucrose concentration increased the contents of total protein and protein components of albumin and globulin in grain, and the activity of glutamate pyruvate transaminase (GPT) across most treatments, while markedly reducing the contents of total starch and components of amylose and amylopectin as well as the activities of soluble starch synthase (SSS) and granule bounded starch synthase (GBSS). The opposite patterns were observed in the experiment of increasing sucrose supply at constant glutamine concentration. When simultaneously increasing sucrose and glutamine supply at constant ratio, the contents of total protein, albumin and globulin in grain were slowly enhanced, whereas the contents of total starch, amylose and amylopectin and the activities of SSS, GBSS and GPT increased only to a certain extent and then decreased. Negative correlations were found between the contents of protein or protein components in grains and the relative ratio of sucrose to glutamine concentrations in the culture medium, while positive correlations were seen between the contents of total starch or starch components and the ratio of sucrose to glutamine. These results implied that the composition of protein and starch in wheat grain could be readily manipulated by varying the concentrations of sucrose and glutamine and their ratio in the culture medium.     

Calcium-mediated conversion of sucrose to starch in relation to the activities of amylases and sucrose-metabolizing enzymes in sorghum grains raised through liquid culture

Detached ears of sorghum (Sorghum vulgare) were cultured in complete liquid medium containing Ca2+(0, 3, 10 and 30 mM) and effect of this ion on the conversion of sucrose to starch with respect to the activities of amylases, sucrose synthase, sucrose phosphate synthase and soluble invertases were studied in developing grains. Presence of 3 mM Ca2+ in culture medium enhanced both accumulation of starch and activity of alpha-amylase in grain but without having any influence on the activity of beta-amylase. However, with 10 and 30 mM Ca2+, the accumulation of starch and activities of both amylases decreased and with advancement in culturing period, starch accumulation was further decreased. Irrespective of its concentration, Ca2+ enhanced the activities of sucrose synthase (synthesis), sucrose-phosphate synthase, soluble acid invertase and soluble-neutral invertase. Increase in the concentration of Ca2+ in culture medium was concomitant with an elevation in relative proportion of sucrose in the grain reflecting a net balance in per cent increase with Ca2+ in the activities of sucrose-synthesizing enzymes over sucrose-hydrolysing ones. Based on the results, it is suggested that assimilation of Ca2+ by grain is essential for maintaining high activity of alpha-amylase to generate starch primers required for the conversion of sucrose to starch during grain filling in sorghum.     

Location and orientation of DODCI in lipid bilayer membranes: effects of lipid chain length and unsaturation.

The location and orientation of a linear dye molecule, DODCI, in lipid bilayer membrane were determined by the effect of viscosity and refractive index of the aqueous medium on the fluorescence properties of the dye bound to the membrane. The membrane-bound dye is solubilized in two sites, one near the surface (short fluorescence lifetime) and another in the interior of the membrane (long lifetime). The ratio of the dye in the two locations and the orientation of the dye (parallel or perpendicular to the membrane) are sensitive to the lipid chain length and unsaturation in the alkyl chain. The fraction of the dye in the interior region is higher for short alkyl chains (C12>C14>C16>C18C20) and in unsaturated lipids (C14:1>C14:0, C16:1>C16:0). These experimental results are consistent with the general principle that the penetration of an amphiphilic organic molecule in the interior region of the membrane is more when the structure of th bilayer is more fluid-like.     

Activities of key enzymes for starch synthesis in relation to growth of superior and inferior grains on winter wheat (Triticum aestivum L.) spike

Weight of individual grains is a major yield component in wheat. The non-uniform distribution of single grain weight on a wheat spike is assumed to be closely associated with starch synthesis in grains. The present study was undertaken to determine if the enzymes involved in starch synthesis cause the differences in single grain weight between superior and inferior grains on a wheat spike. Using two high-yield winter wheat (Triticum aestivum L.) varieties differing in grain weight and three nitrogen rates for one variety, the contents of amylose and amylopectin, and activities of enzymes involved in starch synthesis in both superior and inferior grains were investigated during the entire period of grain filling. Superior grains showed generally higher starch accumulation rates and activities of enzymes including SS (sucrose synthase), UDPGPPase (UDP-glucose pyrophosphorylase), ADPGPPase (ADP-glucose pyrophosphorylase), SSS (soluble starch synthase) and GBSS (starch granule bound starch synthase) and subsequently produced much higher single grain weight than inferior grains. Nitrogen increased enzyme activities and starch accumulation rates, and thus improved individual grain weight, especially for inferior grains. The SS, ADPGPPase and SSS were significantly correlated to amylopectin accumulation, while SS, ADPGPPase, SSS and GBSS were significantly correlated to amylose accumulation. This infers that SS, ADPGPPase and starch synthase play key roles in regulating starch accumulation and grain weight in superior and inferior grains on a wheat spike.     

Conversion of sucrose to starch in the developing Pennisetum typhoides grain

Developing grains of pearl millet ( Pennisetum typhoides Burm. S & H cv. PIB 155) were sampled and analyzed for starch and its free-sugar precursors. The activities of invertase, sucrose-ADP (UDP) glucosyl transferase and of α-amylase and β-amylase in relation to the rate of starch accumulation in the developing grain were assayed. By culturing detached ears, the incorporation of ¹⁴C from free sugar precursors to starch was studied. The starch content gradually increased until grain maturity. The rate of starch accumulation was maximum around 12 days after anthesis. Around this period, the activities of sucrose-ADP(UDP) glucosyl transferase and α-amylase, β-amylase were also at a peak. Invertase activity was high during the early period of grain development but gradually declined as the grains matured. In the most actively metabolising milky grains, incorporation of ¹⁴C from [¹⁴C]-sugars to starch was maximum in the mid mid-milky grains. Addition of 20 m M K⁺ to the culture solution did not affect the incorporation of ¹⁴C from supplied sucrose to the free sugar pool and to the starch of the grain, but Mg²⁺ supply at 20 m M concentration lowered ¹⁴C incorporation from exogenous sucrose to grain free sugars, although the utilization of the latter for starch synthesis was enhanced.     

A new type of two-color fluorescence staining for cytology specimens.

A new two-color fluorescence staining technique for cervical cytology specimens is described. To permit application of this staining in automated cytology, techniques for specimen collection and cell preparation giving a sufficient number of well-separated cells on slides were used. The staining consists of a combination of a modified Feulgen-acriflavine procedure for DNA and a primulin or stilbene isothiocyanate staining for protein. This results in a bright yellow nuclear fluorescence and a blue cytoplasmic fluorescence. The staining procedure can be completed in about 90 min and is therefore suitable for routine application. Sequential inspection of the yellow nuclear and blue cytoplasmic fluorescence can be done with the two-wavelength excitation method used in fluorescence microscopy. For the application of this method, special vertical illuminators are now available. These illuminators are provided with quickly interchangeable filter sets permitting consecutive visualization of, for example, only the nuclei in the first image and the whole cell in the second image. This procedure opens new possibilities for rapid image-analysis systems.     

Effects of abscisic acid (ABA) on grain filling processes in wheat

The effect of in situ water stress on the endogenous abscisic acid (ABA) content of the endosperm and the in vitro application of ABA on some important yield regulating processes in wheat have been studied. Water stress resulted in a marked increase in the ABA content of the endosperm at the time close to cessation of growth. Application of ABA to the culture medium of detached ears reduced grain weight. Exogenously applied ABA, at the highest concentration (0.1 mM) reduced transport of sucrose into the grains and lowered the starch synthesis ability of intact grains. In vitro sucrose uptake and conversion by isolated grains was stimulated by low ABA concentrations (0.001 mM) in the medium but was inhibited by higher concentrations. ABA application had no effect on sucrose synthase (SS) and uridine diphosphate glucose pyrophosphorylase (UDP-Gppase) activities, whereas adenosine diphosphate glucose pyrophosphorylase (ADP-Gppase), soluble starch synthase (SSS), and granule-bound starch synthase (GBSS) activities were reduced. These results raise the possibility that water stress-induced elevated levels of endogenous ABA contribute to reduced grain growth.     

Differential Congo Red staining: The effects of pH, non-aqueous solvents and the substrate

Summary Congo Red is an acid-base indicator dye. In free solution the colour and absorption characteristics of Congo Red depend not only on the pH but are also governed by the nature of the solvent environment. In tissue sections stained by Congo Red, alteration of the pH and the use of non-aqueous solvents can effect differential colouring of the tissue components. Stained sections of unmodified and chemically substituted celluloses show that differential red or blue coloration reflects the acidic or basic character of the substrate. In stained tissue sections, secondary protein structure and porosity of the substrate may also influence their colour. The effect of non-aqueous solvents is probably to modify the ionization state of the dye- substrate complex, thus altering the colour of the Congo Red. Such solvents may also change the aggregation or solvation states of the dye, with consequent modification in the colour of tissue components.     

CHARACTERIZATION OF STARCH GRAINS IN THE NONARTICULATED LATICIFER OF EUPHORBIA PULCHERRIMA (POINSETTIA)

Starch grain morphology in laticifer amyloplasts of Euphorbia pulcherrima Willd. (poinsettia) was examined for evidence of starch metabolism in vegetative and flowering plants. Laticifer starch grains in vegetative plants were rod shaped with lengths ranging from 3 to 60 μm. Average grain size was significantly larger in stems than leaves, and in older than younger tissues. Starch grain length frequency was unimodal and approximated a normal probability distribution in stems, but was skewed positively toward smaller grains in leaves. Frequency distributions were shifted toward larger grains in older tissues. Under short-day photoperiod (flowering) conditions, round starch grains formed in latex of stems, and the average length of rod-shaped grains decreased in latex of stems and leaves. Round grains did not occur in laticifers of leaves or bracts. Round starch grains often occurred in aggregates of two or more subunits. Changes in size and shape of latex starch grains indicate that amyloplasts in fully differentiated laticifers metabolize starch. Identification of metabolically active amyloplasts in differentiated laticifers suggests that the function of these organelles may involve starch mobilization under certain physiological conditions.     

Initial Effects of Differently Treated Biogas Residues from Municipal and Industrial Wastes on Spring Barley Yield Formation

Soil application of biogas residues (BGRs) is important for closing nutrient cycles. This study examined the efficiency and impact on yields and yield formation of solid-liquid separated residues from biodegradable municipal and industrial wastes (bio-waste) in comparison to complete BGRs, nitrification inhibitor, agricultural BGRs, mineral fertilizer and unfertilized plots as control. The experiment was set up as a randomized block design on silt loam Cambisol. Biogas residues from four biogas plants were evaluated. Plants per m², ears per plant, grains per ear and thousand grain weight (TGW) were measured at harvest. Fertilization with BGRs resulted in similar biomass yields compared with mineral fertilizer. Mineral fertilizer (71 dt/ha) and plots fertilized with liquid fraction (59–62 dt/ha) indicated a trend to higher yields than solid fraction or complete BGR due to its high ammonia content. Liquid fractions and fraction with nitrification inhibitor induced fewer plants per m² than corresponding solid and complete variants due to a potential phytotoxicity of high NH₄-N concentration during germination. However, barley on plots fertilized with liquid fraction compensated the disadvantages at the beginning during the vegetation period and induced higher grain yields than solid fraction. This was attributable to a higher number of ears per plant and grains per ear. In conclusion, BGRs from biodegradable municipal and industrial wastes can be used for soil fertilization and replace considerable amounts of mineral fertilizer. Our study showed that direct application of the liquid fraction of BGR is the most suitable strategy to achieve highest grain yields. Nevertheless potential phytotoxicity of the high NH₄-N concentration in the liquid fraction should be considered.     

小麦籽粒发育及淀粉晶体特性对花后高温胁迫的响应

选用2个品质类型和成熟期不同的新疆主栽小麦品种‘新春11号’和‘新春39号’,分别进行花后灌浆早期高温(花后5~8d,32℃,T_1)和中期高温(花后15~18d,38℃,T_2)处理,分析花后高温对小麦籽粒发育及淀粉晶体的影响。结果显示:(1)T_1处理明显降低了两品种籽粒长度和粒重,而T_2处理显著影响籽粒宽度和厚度;高温处理虽然降低了籽粒灌浆速率,但两品种灌浆最大峰值出现时间均在花后18d。(2)T_1处理对小麦籽粒A型淀粉粒形态的影响较大,中熟品种‘新春11号’的A型淀粉粒表面在花后10d时可观察到微孔,在花后15~20d时其粒径明显小于同期对照,在花后20~25d时淀粉粒表面压痕增多且A、B型淀粉粒表面出现明显缢缩;而早熟品种‘新春39号’淀粉粒形态和粒径大小受花后高温的影响相对较小。(3)两品种在不同高温处理下,其淀粉粒晶体特性衍射峰出现的位置相同,但淀粉粒的尖峰强度不同,表明高温胁迫不影响淀粉粒的晶体类型,但可能改变了淀粉粒内部的层状结构。研究表明,花后早期高温不仅对小麦籽粒外部形态有较大的影响,同时也影响到籽粒内部淀粉粒的形态和晶体的特性。     

Sucrose and starch catabolism in the anther of <Emphasis Type="Italic">Lilium</Emphasis> during its development: a comparative study among the anther wall,locular fluid and microspore/pollen fractions

In order to better understand the various pathways of sucrose and starch catabolism in the anther of lily (Lilium hybrida var. “Enchantment”), invertase (EC 3.2.1.26) and amylase (EC 3.2.1.1, EC 3.2.1.2) activities were measured separately in different fractions (anther wall, locular fluid and microspore/pollen) and correlated with the sugar content during anther development. Our findings showed significant differences among the fractions analyzed, suggesting that the regulation of sucrose and starch catabolism could follow distinct pathways in each fraction. Glucose and fructose amounts progressively decreased from anther wall to fluid and from fluid to microspore/pollen. Thus, the developing pollen could act as a sink for the carbohydrates that reach the anther. In this sense, cell wall-bound invertases seem to play a major role in soluble sugar partitioning in the different fractions of the anther. Sucrose concentration was found to be substantially higher in the locular fluid than in the other fractions, indicating a probable site for storage. On the other hand, the anther wall tissues could have a buffering function, storing nutrient surplus in starch grains and thus regulating the availability of soluble sugars in the whole anther. All these results proved the advantages of the experimental model proposed here, as well as its usefulness to investigate sugar metabolism in Lilium anthers.     

Membrane electropermeabilization effects of frequency and membrane surface order on liposome leakage

Liposomes containing fluorescence marker were exposed to an alternating electric field of 80 V peak to peak square electric waves at different frequencies 0.01, 1, and 100 kHz to perturb the liposome permeation. The efflux of fluorescence dye after application of the electric field was measured by recording the fluorescence emission due to the complex formation reaction between the fluorescence dye and calcium ions in the bulk medium solution. Two independent sets of experiments were conducted: 1) calcium ions were present during electropulsation; and 2) they were added after electric field application. Two parameters, fluorescence emission intensity and increment of temperature of the solution in the chamber, were studied. The effect of membrane surface order on the fluorescence dye leakage from the liposomes was studied by addition of urea at threshold concentration before the liposomes sealed. The data demonstrate the existence of frequency dependency window at 1 kHz. Furthermore, the data were interpreted according to the theory of interactions of electromagnetic fields with highly polarized and deformed materials such as liposome particles. The urea caused an enhancement of the fluorescence dye leakage at frequency of 100 kHz. This effect could be explained as a decrease of the membrane binding rigidity due to the disordering effect of urea on the membrane lipid surface. Our conclusion is that the frequency and the membrane surface order are additional parameters that influence the processes of membrane electropermeabilization.