HPLC Analysis of Neurophysin Proteins from Neurosecretory Granules

Abstract: Neurosecretory granules were obtained from neurolobes of porcine pituitary glands. From the granules, highly purified neurophysins were prepared by HPLC. According to polyacrylamide gel electrophoresis, isoelectric focusing, N- and C-terminal amino acid residue determination, and amino acid composition, the neurophysins I₁ I₂, and II were identical to the neurophysins obtained from whole posterior lobes. Since degradation could not have occurred, we conclude that neurophysin I₁ and I₂ originated in the neurosecretory granules.     

Low temperature spectrophotometry of the phototransformation of Pfr to Pr in pelletable pea phytochrome

Manabe, K. 1987. Low temperature spectrophotometry of the phototransformation of P_fr to P_r, in pelletable pea phytochrome.
Low temperature spectrophotometry was used to study the phototransformation of P_fr to P_r in 1000–7000 g pelletable fractions extracted from dark grown pea ( Pisum sativum L. cv. Alaska) epicotyls which had been irradiated with red and then far-red light. At -170°C, far-red irradiation of the pelletable phytochrome which had been pre-irradiated with saturating fluence of red light before freezing caused formation of an intermediate (named I₆₆₀), the difference spectrum of which showed a marked ab-sorbance decrease at 740 nm and a concomitant small increase at about 660 nm. The inermediate I₆₆₀ was converted to another intermediate (I₆₆₀) when it was warmed above -80°C. The difference spectrum of this intermediate showed a positive peak at 670 nm. This intermediate was photoconverted to P_fr by red irradiation and also underwent dark reversion to P_fr at -60°C. I₆₆₀ formed P_r if the temperature was above -10°C. The basic features of the phytochrome intermediates resemble those obtained in vivo and in degraded purified phytochrome.     

The Use of Herr Four-and-a-Half Clearing Fluid for the Rapid Microscopic Examination of Thick Sections of Normal and Neoplastic Tissues

We have demonstrated that Herr's 4¹/₂ clearing fluid, developed for use with plant tissues, can be successfully used for the microscopic examination of thick sections of normal and neoplastic mammalian tissues. Rat Novikoff hepatoma, rat liver, and human colon and skin samples were fixed in Bouin's, stained with iron hematoxylm, treated with Herr's 4¹/₂ clearing fluid and examined by phase contrast miaoscopy. Tissue architecture and cytological detail were easily observed by focusing through tissue Sections as thick as 70μ. The method permits rapid microscopic examination of mammalian tissues and enables the investigator to detect readily morphological abnormalities within a tissue.     

Seasonal changes in leaf lipids of Diapensia lapponica, with special reference to storage lipid bodies

Seasonal fluctuations in storage lipids in the cushion plant Diapensia lapponica , growing in Northern Finland (70°N 27°E), were studied by microscopy and chemical analysis. Lipid bodies in the mesophyll cells were stained with Sudan Black for quantitative observation by light microscope. Electron microscope observations were made using aldehyde prefixed and osmium tetroxide postfixed sections of leaf blades. Thin layer and gas capillary chromatographic techniques were used to analyse total lipids and total fatty acids in green shoots of Diapensia . Free sugars and starch were extracted separately and determined by the anthrone method.
A mesophyll cell was characterized by a large lipid body (storage lipid) in summer but by several small spherules in winter. Total surface area of the cross-sectioned lipid globules was at its lowest from April to September; the maximal value was in March. The amount of total lipids in the leafy tops of D. lapponica was 91–200 mg g^-1 dry weight. Values were lowest at the end of June, when the total carbohydrate level was at its highest. Accordingly, the decrease in the total lipid level in the early growing season, when new leaves were developing, can be attributed primarily to the increase in the level of carbohydrates, particularly starch. The amount of total fatty acids varied from 21 to 30 mg g^-1 dry weight. The level increased in the early growing season and remained elevated throughout the summer. Like the total lipids, the total fatty acids are derived from structurally different parts of the sclerophyllous leaves, including the well-developed cuticle and epicuticular wax layer. The discrepancies in the results obtained from microscopic and chemical analyses are discussed.     

Antimony Trichloride as A Test Reagent for Steroids, Especially Diosgenin and Yamogenin, in Plant Tissues

A saturated solution of SbCl₃ in 60% HClO₄ was used to detect steroids in plant tissue, using (a) cut sections of fresh material (b) dried and powdered material and (c) dried aqueous ethanolic extracts of microquantities of tissues. The utility of this reagent as a microchemical test for diosgenin and yamogenin has been established by application to 55 plant specimens from 35 species in 12 families, including plant parts which hold steroidal sapogenin (spirostans) as well as those that do not. The presence or absence of spirostans in the plant parts was confirmed by infrared analysis after processing the material on a macro scale. Colours obtained with the reagent and pure sapogenins, some of their common derivatives and their glycosides are given for reference.     

Flat, Adherent, Well-Contrasted Semithin Plastic Sections for Light Microscopy

Semithin (0.5-2.0 μm) sections of plastic embedded specimens have long been used for identifying and orienting structures destined for electron microscopic observation. Improved staining methods and the development of more versatile plastics have increased the use of semithin plastic sections for histochemical and autoradiographic studies. The principal advantage of plastic over paraffin sections is the possibility of increased resolution. This advantage is often compromised, however, by problems arising during processing and staining. Wrinkles are common in sections containing tissues of different consistencies or when the hardness of the tissue does not match that of the surrounding plastic (Millonig 1980). Unfortunately, many of the methods designed to eliminate wrinkles (e.g., Alsop 1974, Sommer et al. 1979) require prolonged staining or repeated handling of the sections. Section adhesion problems usually arise during staining, particularly if the protocol requires alkaline or oxidizing reagents. Adhesives such as Mayer's albumen or chrome alum-gelatin (Hayat 1981) work well but may contribute to undesirable background staining or trapping of debris. A more complicated problem, inadequate stain contrast for photomicrography, usually can be traced to inability of the stain to penetrate the plastic, staining of the plastic, or nonspecific staining of the tissue. Alkaline staining solutions and chemicals which etch plastic can increase penetration, but may also cause section loss or staining of the plastic. The following is a simple method to eliminate these processing problems. It exploits the solvent properties and low surface tension of glycerol to aid in softening, flattening, and adhering semithin plastic sections to microscope slides.     

高水分、富含淀粉植物组织的扫描电镜制备技术优化

应用常规高真空扫描电子显微镜观察生物样品必须经过脱水和干燥处理,但无论采用临界点干燥还是冷冻干燥方法,都存在样品表面不同程度失真的问题。植物高水分、富含淀粉组织样品经处理后,容易出现淀粉流失、细胞壁变形等现象,从而造成扫描图像粗糙,无法获得真实的细胞内部结构。本文通过对CO_2临界点干燥、化学固定样品冷冻干燥和新鲜样品冷冻干燥3种扫描电镜样品制备技术中后期制样进行机械断裂和液氮脆断改进,优化出两种植物高水分、富含淀粉组织的扫描电镜样品制备方法:(1)样品首先进行FAA化学固定,经冷冻干燥后用液氮脆断,对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞结构完整,细胞壁整齐,淀粉粒和蛋白轮廓明确,可用于分析淀粉粒和蛋白颗粒在细胞内的分布。(2)新鲜样品直接进行冷冻干燥,经液氮脆断后对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞壁整齐,淀粉粒轮廓更清晰,并且无蛋白颗粒干扰,用于分析淀粉粒在细胞内的分布更加理想。     

Trifluoperazine-Sensitive Step during Sea Urchin, Echiuroid and Pelecypod Egg Activation

Trifluoperazine, a calmodulin-antagonist, is shown to inhibit egg activation by ionophore A 23187 in sea urchin (I₅₀: 43 μM), by trypsin in echiufoids (I₅₀: 22 μM) and by KCl in bivalves (I₅₀: 34 μM). In each case the inhibition could be reversed by washing the eggs and the trifluoperazine-sensitive period was clearly limited. In Barnea and Urechis , trifluoperazine inhibits calcium uptake. A common trifluoperazine-sensitive step, possibly involving calmodulin, may thus be shared by a variety of animal groups during egg activation.     

Combined light and electron microscope immunocytochemical localization of scattered peptidergic neurons in the central nervous system

A pre-embedding immunocytochemical technique is described for combined light and electron microscope study of peptidergic neurons in the central nervous system. The protocol is especially designed to overcome the sampling problems inherent in electron microscope study of structures, such as luteinizing hormone-releasing hormone (LHRH) neurons, that are scattered individually across large brain regions. The fixation methods outlined for several mammalian species include immersion and vascular perfusion with acrolein. Fine-structural preservation and LHRH immunoreactivity obtained with this fixative are compared to results with more conventional fixatives. Vibratome sectioning and a "pretreatment" regime, which prepare the tissues for immunocytochemistry, are described. Immunocytochemical labeling is done with free-floating sections and the peroxidase-antiperoxidase unlabeled antibody enzyme technique. Techniques are also described for the subsequent processing of immunoreacted sections for electron microscopy. These methods ensure that the processed sections are readily scanned by light microscopy, so that regions containing immunoreactive structures can be specifically chosen for electron microscope analysis. Sample electron micrographs are shown that illustrate some fine structural features of LHRH neurons in rats, bats, ferrets, and monkeys, as revealed with the techniques described.     

Malt Diastase And Ptyalin In Place of Saliva in The Identification of Glycogen

Digestion in 1% U. S.P. malt diastase or in 1% ptyalin at 37°C. for 1 hour is an effective substitute for the salivary digestion test used by Bauer, by Bensley and others for the identification of glycogen. Actually the test is not specific for glycogen, since diastase, ptyalin and amylopsin digest other polysaccharides than glycogen, notably starch. In animal tissues this should produce no confusion. Of the two samples tested, the malt diastase proved somewhat more effective than ptyalin and can be fully recommended for sharpness of results.

The enzymes should be dissolved in a buffered neutral saline solution consisting of 8g. NaCl, 1.3 g. Na₂HPO₄ and 0.8 g. NaH₂PO₄-H₂O in 1 liter of water. This solvent by itself does not remove glycogen from liver sections in 1 hour at 37°C.

Enzyme tests should be done on uncollodionized sections. Since collodionization permits demonstration of larger amounts of glycogen by both the Best and Bauer methods, this step should be interposed after the digestion test and before the specific glycogen stain.     

Ovary starch reserves and pistil development in avocado (Persea americana)

In avocado, only a very small fraction of the flowers are able to set fruit. Previous work in other woody perennial plant species has shown the importance of carbohydrates accumulated in the flower in the reproductive process. Thus, in order to explore the implications of the nutritive status of the flower in the reproductive process in avocado, the starch content in the pistil has been examined in individual pollinated and non‐pollinated flowers at anthesis and during the days following anthesis. Starch content in different pistilar tissues in each flower was quantified with the help of an image analysis system attached to a microscope. Flowers at anthesis were rich in highly compartmentalized starch. Although no external morphological differences could be observed among flowers, the starch content varied widely at flower opening. Starch content in the ovary is largely independent of flower size because these differences were not correlated with ovary size. Differences in the progress of starch accumulation within the ovule integuments between pollinated and non‐pollinated flowers occurred concomitantly with the triggering of the progamic phase. The results suggest that starch reserves in the ovary could play a significant role in the reproductive process in avocado.     

Green plastids, maximal PSII photochemical efficiency and starch content of inner stem tissues of three Mediterranean woody species during the year

Wood and pith of 1–2-year-old stems of three woody species with different life strategies common in the Mediterranean basin were studied during the year regarding (i) the occurrence of green plastids, (ii) their maximal photosystem II photochemical efficiency (F_v/F_m) and (iii) their starch content. Green plastids were identified from the red chlorophyll auto-fluorescence under epi-fluorescence microscope, F_v/F_m was estimated using imaging-PAM fluorometry and starch content was recorded under bright field microscope after iodine staining. The evergreen sclerophyll Nerium oleander, the summer deciduous Euphorbia acanthothamnos and the winter deciduous species Platanus orientalis were selected for the study.

Epi-fluorescence microscopy revealed that (i) all species possess abundant green plastids in their wood ray and pith cells throughout the year. In the winter deciduous species chlorophyll fluorescence was found to be strong during the leafless period. By contrast, in the evergreen and the summer deciduous species chlorophyll fluorescence was found uniformly bright during the year; (ii) F_v/F_m value variation during the year seems to be species-specific: in the wood of N. oleander it remains unchanged whereas in the pith it is low during spring–summer; in both tissues of E. acanthothamnos F_v/F_m value reaches maximal value during spring and in P. orientalis during autumn; (iii) in N. oleander and E. acanthothamnos starch is accumulated during spring, whereas in P. orientalis starch content is high during winter.

The scanning electron microscopy (SEM) investigation revealed that the stem epidermis of all three species lacks stomata and formation of lenticels is delayed. Provided that gas exchange is therefore minimized and that PSII photochemical efficiency of inner stem tissues is relatively high, it is further supported that green plastids of wood ray and pith cells may help toward the re-fixation of the internally respired CO₂.     

Effects of mitochondrial complex III disruption in the respiratory chain of Neurospora crassa

In mitochondria from most organisms, including Neurospora crassa , dimeric complex III was found associated with complex I. Additional association of complex IV with this core structure leads to the formation of a respirasome. It was recently described for bacteria and mammals that complex III is needed for the assembly/stability of complex I. To elucidate the role of complex III in the organization of the respiratory chain of N. crassa , we analysed strains devoid of either the Rieske iron-sulphur or the COREII polypeptide subunits. The mutants display reduced growth, are female sterile and lack active complex III. The supramolecular organization of the oxidative phosphorylation system was characterized by electrophoretic analyses and the efficiency of the respiratory chain analysed by oxygen consumption measurements. The results obtained indicate that absence of complex III activity is not associated with the absence of complex I or complex IV, and leads to the induction of alternative oxidase. Complex III mutant mitochondria are devoid of respirasomes but contain significant amounts of dimeric complex I (I₂) and of the supercomplex I₁IV₁. Moreso, for the first time the alternative oxidase was found associated with dimeric complex IV and with supercomplex I₁IV₁.     

Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4

A protease-sensitive antibacterial substance produced by Bacillus coagulans I₄ strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named coagulin. The inhibitory spectrum included B. coagulans and unrelated bacteria such as Enterococcus , Leuconostoc , Oenococcus , Listeria and Pediococcus . Coagulin was stable at 60 °C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by α-amylase, lipase or organic solvents (10% v/v). Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells. The apparent molecular mass was estimated to be about 3–4 kDa by SDS-PAGE. The B. coagulans I₄ strain harbours a plasmid, pI₄, approximately 14 kb in size. Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance. Plasmid content of these two derivatives showed that one had lost pI₄,whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for coagulin production.     

Cytological Staining Procedures Applicable to Methacrylate-Embedded Tissues

Experiments indicate that osmic-fixed, plastic-embedded sections are suitable for examination in the light microscope. Nuclei, mitochondia, cellular membranes and cytoplasmic granules are readily demonstrable by phase microscopy. Connective tissue stains permit the identification of elastic and collagenous fibers. Glycogen and other carbohydrate-containing structures are demonstrable by the periodic acid-Schiff and the ammoniacal silver nitrate procedures. It is, therefore, possible to cross-check individual structures by comparing alternate thick and thin sections, examined in the light microscope and electron microscope respectively. Several other advantages pertain to plastic embedded tissues. The sections compare favorably in translucency and in their lack of distortion with material embedded in celloidin, yet the procedure is simpler and much more rapid. Sections of any desired thinness can be prepared, and alternate thick and thin sections are easily forthcoming. When examined in the phase-contrast microscope, mitochondrial preparations become routinely available without the uncertainties of most of the mitochondrial staining methods. It appears, therefore, that plastic embedding should find a useful place among the methods for light microscopy as well as in the armamentarium of the electron microscopist.     

The effect of mutant genes at the r, rb, rug3, rug4, rug5 and lam loci on the granular structure and physico-chemical properties of pea seed starch

The granular structure and gelatinisation properties of starches from a range of pea seed mutants were studied. Genes which affect the supply of substrate during starch synthesis (rb, rug3, rug4) affected the total crystallinity and possibly increased the content of A polymorphs in the starch. Conversely, genes directly affecting the synthesis of starch polymers (r, rug5, lam) increased the content of B polymorphs, but had a minimal effect on total crystallinity. During gelatinisation, starches from the rb, rug3, rug4 and lam mutants had narrow endothermic peaks which were similar to starch from the wild-type, although all the starches had different peak temperatures and enthalpy changes. Starches from r and rug5 mutants were very different to all other starches, having a very wide transition during gelatinisation. In addition, the amylopectin in starch from these mutants had altered chain lengths for those parts of the polymer which form the ordered structures in the granule.     

Cell specialization within the parenchymatous bundle sheath of barley

Abstract. Structural and physiological aspects of the parenchymatous bundle sheath (PBS) were studied in cultivars of Hordeum distichum L. The PBS of intermediate, lateral and midrib veins consisted of a single layer of cells closely appressed to the mestome sheath. These cells were large, vacuolate and approximately cylindrical in shape, extending parallel to the vein. Mean PBS cell volume was 4 × 10⁻⁵mm³ compared to 1.23 × 10⁻⁵mm³ for mesophyll cells. Transverse sections revealed three cell types within the PBS, cells with small chloroplasts (S-type), cells with large chloroplasts (L-type) and structural cells. The majority of cells were S-type, containing chloroplasts of approximately a third of the volume of mesophyll chloroplasts; they were able to reduce tetranitro blue-tetrazolium and synthesize starch. Structural cells interrupted the phloem and xylem are of the sheath in lateral veins and the midrib, whilst between one and four PBS cells within the phloem are of each vein type contained chloroplasts similar in volume and starch content to those of the mesophyll. Only these L-type cells contained noticeable starch grains at the end of an 8-h dark period, a further 4 h darkness being required for complete mobilization of starch. Starch deposition within S-type and structural cells was detectable after 4 h illumination but was only appreciable in leaves excised from the plant and illuminated for 9–12 h. The role of S-type PBS cells in assimilate transport is discussed in relation to these findings.     

Respiration, cyanide-insensitive oxygen uptake and oxidative phosphorylation in cyanobacteria

Cyanide-insensitive oxygen uptake in the dark of 9 species of cyanobacteria was 6–20% of the total oxygen uptake of intact cells. In Phormidium , no cyanideinsensitive oxygen uptake was observed. In intact cells, the I₅₀ value for cyanide was significantly lower in cyanobacteria of the taxonomic sections I to III (1–9 μ M ) than in those from section IV and V (10–60 μ M ). Cyanide-insensitive oxygen uptake in the cell-free system of Anabaena variabilis was not affected by typical inhibitors of the alternative pathway of plants. Cell-free oxidation of cytochrome c was completely inhibited by cyanide with an I₅₀ value of 0.5–1 μ M . Electron transport of intact cells without cyanide present yielded P/O ratios of 0.7–3.0. The data on oxidative phosphorylation using intact cells and the cell-free system, indicate that cyanide-insensitive oxygen uptake is not coupled to ATP formation.     

Accumulation and Disintegration of Starch in the Chloroplasts of Stellaria media Grown under Constant Illumination

The persent paper contains a study un the starch content of the chloroplasts of the leaves of Stellaria media, performed by hsitochemical methods. Three leaves were collected at short intervals, fixed and cut in 10 μ sections, and stained according to the PAS-procedure. The starch content of single chloroplasts was measured by a microscope photometer. The chemical composition of the stainable material was demonstrated by enzyme specific experiments. At the end of a 12 hours' dark period the chloroplasts contained only traces of starch. Light caused the starch accumulation to begin and after a time chloroplasts seemed to be filled with starch. Subsequently, however, a sudden decrease in the starch content of the chloroplasts took place, in spite of constant illumination. A rather high level of starch content was restored in a few hours. The author puts forward some ideas about the nature of the factors causing the transitory disintegration of starch in illuminated leaves. The induction or the activation of certain enzymes seems to be the most probable explanalion.     

Localization of Plant Lipids for Light Microscopy Using P-Phenylenediamine in Tissues of Arachis Hypogaea L.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need o further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.