A chemical and histochemical study of the lipides of the pyloric cecum of the starfish,Asterias forbesi

The lipides of the diverticula of Asterias forbesi have been studied by histochemical and biochemical means. Correlations between results obtained by histochemical examination of sections, and chemical analysis of isolated lipide have been made, particularly with respect to phosphatides, steroids, and aldehyde lipides. The results of the histochemical study were in good agreement with the chemical data as to the nature of the phosphatide fraction, the presence of acetone-soluble aldehyde lipides, and the composition of the free droplet fat. Homogenized diverticula were differentially centrifuged in order to establish the distribution of types of lipides in the various cellular components. In addition, data have been presented which demonstrate a direct correlation between the titer of alpha-glycerol ethers and that of acetone-soluble lipide acetals in the unsaponifiable fraction.     

EFFECTS OF THE PHYSICAL ENVIRONMENT ON SOME LIPOPROTEIN LAYER SYSTEMS AND OBSERVATIONS ON THEIR MORPHOGENESIS

Lipoprotein membrane systems such as chloroplasts and the endoplasmic reticulum exhibit a generalized swelling response. The initial effect is an increase in interlamellar spacing, but as swelling proceeds, the membranes are transformed into closed thin-walled spherical vesicles. Available evidence suggests that morphogenesis of the endoplasmic reticulum of Nitella and the lamellar system of the Zea chloroplasts involves fusion of small spherical vesicles to yield closed double membrane structures, which subsequently undergo further differentiation. It is suggested that the vesicles comprise a convenient "micellar" form by which lipides may be transported within the cell from the sites of lipide synthesis to regions of lamellar growth. The characteristic formation of vesicles in swelling and the apparent fusion of vesicles in morphogenesis appear to represent two aspects of a fundamental plasticity of lipoprotein layer systems.     

Partition of cytoplasmic lipides

By means of differential centrifugation three fractions: large granules (L₂), microsomes (M₁), and supernatant fluid (MS), have been isolated from the cytoplasm of mouse liver cells and the contained lipides have been extracted and characterized.The particulate fractions, large granules and microsomes, make up approximately 38% of the total solids of the cytoplasm, but they contain 62% of the total lipide and 85% of the phospholipide. The phospholipide of the particulates has an N:P ratio of 1 while the supernatant phospholipide has an N:P ratio of 1.3, suggesting that the supernatant fluid contains lipides of higher nitrogen content than that of either lecithins or cephalins.The fatty acids of the large granule fraction are highly unsaturated and contain 20% of fatty acids with four double bonds. The unsaturation of the fatty acids of microsomes and the supernatant fluid is comparable, as indicated by the iodine values, but the supernatant fluid contains much more of the fatty acids with one double bond and less of the fatty acids with four double bonds than do the microsomes.     

HISTOCHEMICAL STAINING AND CHARACTERIZATION OF GLYCOPROTEINS IN ACID-SECRETING CELLS OF FROG STOMACH

Glycoproteins were histochemically localized in oxyntic cells of the frog stomach by staining with periodic acid-silver methenamine. Reduction of silver was most intense on (a) the outer aspect of the apical plasmalemma, (b) within the tubular smooth membrane system characteristic of oxyntic cells, and (c) within cisternae and vesicles of the Golgi complex. Other membrane components such as those from the mitochondria, nucleus, junctional complex, lateral and basal cell membranes showed little or no stainability. Gastric mucosal homogenates were fractionated by centrifugation for further morphological and chemical analysis. The staining reaction of the microsomal fraction (40,000 g x 60 min) was similar to that of the tubular membranous components of intact oxyntic cells. Carbohydrate analyses showed that all cell fractions are extremely low in acidic sugars, uronic and sialic acids, while neutral sugars and hexosamines are relatively abundant. The microsomal fraction contains the largest proportion of carbohydrates, ca. 9% of the fat-free dry weight. Another distinguishing feature is that glucosamine is the only detectable hexosamine in the microsomal fraction. These histochemical and chemical data indicate that neutral glycoproteins are associated with membranous components which have been implicated in the process of HCl secretion by oxyntic cells. The staining pattern within the cells supports the hypothesis of interrelationships between the Golgi membranes, tubular smooth membranes, and apical surface membrane.     

ULTRASTRUCTURE OF THE LAMELLAE AND GRANA IN THE CHLOROPLASTS OF ZEA MAYS L

The fine structure of the chloroplasts of maize (Zea mays L.) has been investigated by electron microscopic examination of ultrathin sections of leaves fixed in buffered osmium tetroxide solutions. Both the parenchyma sheath and mesophyll chloroplasts contain a system of densely staining lamellae about 125 A thick immersed in a finely granular matrix material (the stroma), and are bounded by a thin limiting membrane which often appears as a double structure. In the parenchyma sheath chloroplasts, the lamellae usually extend the full width of the disc-shaped plastids, and grana are absent. The mesophyll chloroplasts, however, contain numerous grana of a fairly regular cylindrical form. These consist of highly ordered stacks of dense lamellae, the interlamellar spacing being ca. 125 A. The grana are interlinked by a system of lamellae (intergrana lamellae) which are on the average about one-half as numerous as the lamellae within the grana. In general, this appears to be due to a bifurcation of the lamellae at the periphery of the granum, but more complex interrelationships have been observed. The lamellae of the parenchyma sheath chloroplasts and those of both the grana and intergrana regions of the mesophyll chloroplasts exhibit a compound structure when oriented normally to the plane of the section. A central exceptionally dense line (ca. 35 A thick) designated the P zone is interposed between two less dense layers (the L zones, ca. 45 A thick), the outer borders of which are defined by thin dense lines (the C zones). Within the grana, the C zones, by virtue of their close apposition, give rise to thin dense intermediate lines (I zones) situated midway between adjacent P zones. A model of the lamellar structure is proposed in which mixed lipide layers (L zones) are linked to a protein layer (P zone) by non-polar interaction. Chlorophyll is distributed over the entire lamellar surface and held in the structure by van der Waals interaction of the phytol "tail" with the hydrocarbon moieties of the mixed lipide layers. The evidence in favour of the model is briefly discussed.     

THE CYTOCHEMICAL LOCALIZATION OF ASCORBIC ACID IN ROOT TIP CELLS

The intracellular distribution of ascorbic acid was studied in frozen-dried root tips of Allium cepa and Vicia faba by the silver nitrate procedure. The sites of the ascorbic acid as indicated by the deposited silver appear as spherical (0.2 to 0.6 µ in diameter) cytoplasmic particles. The site appears to have small amounts of lipides and to be rich in ribonucleic acid. These particles are concluded to be submicroscopic in size and associated, in the elongating cell, with the cell surface. In the meristematic cells they appear fewer in number and are distributed throughout the cytoplasm.     

THE HISTOCHEMICAL DEMONSTRATION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ACTIVITY

A histochemical method for demonstration of glyceraldehyde-3-phosphate dehydrogenation by tissues is described. The method utilizes Nitro BT as an indicator, glyceraldehyde-3-phosphate obtained from hydrolysis of commercially obtainable glyceraldehyde-3-phosphate diethylacetal (monobarium salt) as substrate, and (ethylenediamine)tetraacetic acid acid disodium as an activating agent in a medium buffered to pH 7.2 by 0.2 M sodium phosphate. The heat lability, substrate and coenzyme specificity, and sulfhydryl and phosphate dependence of the tissue component catalyzing this reaction indicate that glyceraldehyde-3-phosphate dehydrogenase activity is being demonstrated. The disparity between the known pH optimum of this enzyme and that determined histochemically, and the anomalous histochemical localization to mitochondria of this enzyme which has been found in the soluble fraction by differential centrifugation, are thought to result from the diaphorase dependence of the tetrazolium methods and to emphasize the need for caution in the interpretation of histochemically determined intracellular localization of dehydrogenating enzymes. The evidence gathered by previous workers concerning the feasibility of demonstrating specific dehydrogenases with Nitro BT, and the correspondence of the distribution of glyceraldehyde-3-phosphate dehydrogenase determined histochemically with available quantitative data, suggest that at the cellular level the histochemical results accurately reflect the distribution of this enzyme.     

Metal Interrelationships in Plant Nutrition: 2. THE RELATION OF METAL TOXICITY, MOLYBDENUM AND NITROGEN SOURCE TO CHLOROPHYLL AND MAGNESIUM CONTENT OF BEET IN SAND CULTURE

Sugar beet and spinach beet were grown in sand culture withdifferent sources of nitrogen or iron and different levels ofmolybdenum and heavy metals. Chlorophyll and some magnesiumfractions were determined. Extra molybdenum accentuated chlorosis caused by excess of Mn⁺⁺,Cr⁺⁺⁺, Zn⁺⁺, and Cu⁺⁺ and decreased it in the presence of CrO₄^––with nitrate. In the presence of these ions urea reduced chlorophyll in youngand increased it in old leaves of sugar beet. Effects of ureaand molybdenum were additive and independent. Ammonium sulphatecaused increased chlorosis of spinach beet with Mn⁺⁺ and Zn⁺⁺. The existence of an acetone-soluble magnesium fraction otherthan chlorophyll was shown. Excess Cu⁺⁺ and Zn⁺⁺ decreased totalmagnesium; Mn⁺⁺ did not. Mn⁺⁺ and Zn⁺⁺ reduced chlorophyll,increased the non-chlorophyll acetone-soluble magnesium, butreduced other fractions. Ferrous iron ipcreased total magnesiumcontent and also increased the acetone-soluble fractions inthe presence of Mn⁺⁺ and Zn⁺⁺. Extra molybdenum decreased theacetone-soluble magnesium fractions but did not affect the totalmagnesium content. Significant interactions between Mo, N, Fe, and heavy metalswere also disclosed.     

ELECTRON MICROSCOPE AND LOW-ANGLE X-RAY DIFFRACTION STUDIES OF THE NERVE MYELIN SHEATH

1. A close correlation has been obtained between high resolution electron microscopy and low-angle x-ray diffraction studies of the myelin sheath of frog and rat peripheral and central nerves. Extensive studies were performed by application of both techniques to the same specimens, prepared for examination by OsO₄ or KMnO₄ fixation, and embedding either in methacrylate or in gelatin employing a new procedure. Controlled physical and chemical modifications of the myelin sheath prior to fixation were also investigated. 2. A correspondence was established between the layer spacings observed in electron micrographs and the fundamental radial repeating unit indicated by the low-angle x-ray diffraction patterns. The variations in relative intensities of the low-angle x-ray reflections could be related to the radial density distributions seen in the electron micrographs. 3. An analysis of the preparation procedures revealed that OsO₄ fixation introduces a greater shrinkage of the layer spacings and more pronounced changes in the density distribution within the layers than KMnO₄ fixation. The effects of methacrylate and gelatin embedding are described, and their relative merits considered in relation to the preservation of myelin structure by OsO₄ fixation. 4. The experimental modifications introduced by freezing and thawing of fresh whole nerve are described, particularly the enhancement of the intermediate lines and the dissociation of the layer components in the myelin sheath. A characteristic collapsing of the radial period of the sheath is observed after subjecting fresh nerve trunks to prolonged and intense ultracentrifugation. 5. Controlled extraction of fresh nerve with acetone at 0°C., which preferentially removes cholesterol, produces characteristic, differentiated modifications of the myelin sheath structure. Electron microscopy reveals several types of modifications within a single preparation, including both expanded and collapsed layer systems, and internal rearrangements of the layer components. Alcohol extraction leads to a more extensive structural breakdown, but in certain areas collapsed layer systems can still be observed. The components of the lipide extracts could be identified by means of x-ray diffraction. These modifications emphasize the importance of cholesterol in the myelin structure, and disclose a resistance of the dense osmiophilic lines to lipide solvents. 6. The significance of these structures is discussed in relation to present concepts of the molecular organization of myelin. The available evidence is consistent with the suggestion that the primary site of osmium deposition is at the lipoprotein interfaces and that the light bands probably represent regions occupied by lipide chains. The electron microscope and x-ray diffraction data also indicate the possibility of a regular organization within the plane of the layers, probably involving units of 60 to 80 A. The myelin sheath is regarded as a favourable cell membrane model for detailed analysis by combined application of x-ray diffraction and electron microscopy.     

Silver methenamine and phosphotungstic acid staining of the acrosome of Mytilus edulis.

The Mytilus acrosome was investigated by histochemical methods combined with electron microscopy, using silver methenamine (SM) and phosphotungstic acid (PTA) staining, as well as some chemical and enzymatic pretreatments followed by the staining. As one of two major components in the Mytilus acrosome, the egg-membrane lysin was conspicuously stainable with PTA and susceptible to pronase digestion. The other component, that occupies the space between the acrosomal membrane and the axially located strand containing lysin, was stained with SM very specifically. This staining property was not affected by pronase digestion or treatment that blocked aldehyde and SH groups.     

The Relation between Protein Synthesis and Lipide Accumulation in L Strain Cells and Ehrlich Ascites Cells

It has long been known that fat accumulates in old injured cells both in tissue culture and in many mammalian disease states. The use of L cells grown in suspension tissue culture permitted the opportunity to study conditions in which lipide accumulation could be retarded or accelerated. These cultures exhibit a three-phase growth curve which is similar to that previously found with bacteria and consists of a lag period, logarithmic growth period, and stationary period. Daily aliquots were removed from cultures going through these phases and protein and cholesterol content correlated with cell division. It was found that L cells gradually accumulated lipide in the cell concurrent with retardation of cell division and protein synthesis. Conversely old lipide-laden cells, placed in fresh media and encouraged to active division with net protein synthesis progressed from a high to a low lipide/cell ratio over a period of 2 to 4 days. An amino acid analogue p-fluorophenylalanine and a mitotic inhibitor, colchicine, also markedly increased the lipide/cell ratio. Similar results were found in in vitro experiments with Ehrlich ascites cells.     

ACTIVITY OF ALDEHYDE DEHYDROGENASE, ALDEHYDE REDUCTASE, AND ACETYLCHOLINE ESTERASE IN STRITATUM OF RATS BEARING ELECTROLYTIC LESIONS OF THE MEDIAL FOREBRAIN BUNDLE

A number of enzymes have been measured in the striatum of rats in which the dopamine-containing nerve terminals had been unilaterally destroyed by means of unipolar electrolytic lesions of the medial fore-brain bundle. Fourteen and 28 days after such lesions the tyrosine hydroxylase activity of the striatum was reduced to immeasurably low values, but neither aldehyde dehydrogenase, aldehyde reductase, nor acetylcholine esterase was affected when compared with the striatum from the intact side of the same rat or with those from control rats. These results indicate that in the rat the 3 enzymes are not localized with tyrosine hydroxylase, in the dopaminergic nerve terminals of the striatum. This conclusion is supported by a study of the subcellular localization of aldehyde dehydrogenase in rat brain. This enzyme is distributed between the cytosol and the particulate fraction of brain homogenates separated by centrifugal techniques. with no exceptionally high concentration of the enzyme in the synaptosomal fraction. Because neither of the enzymes of post-deaminative catabolism of dopamine is concentrated in the dopaminergic nerve terminals of the striatum of the rat, it is proposed that in this species the amine is not necessarily taken up by the nerve terminals prior to catabolism.     

Qualitative analysis of products formed during the acid catalyzed liquefaction of bagasse in ethylene glycol

Bagasse was liquefied in ethylene glycol (EG) catalyzed by sulfuric acid at 190 degrees C under atmospheric pressure. The compositions of the crude products obtained were analyzed after separating them into three fractions: a water-soluble fraction, an acetone-soluble fraction and a residue. With infrared, gel permeation chromatography and elemental analyses, the residue mainly included undissolved cellulose and lignin derivatives and the acetone-soluble fraction mainly contained lignin degradation products with high molecular weights. The water-soluble fraction, after further analyzed by GC-MS and HPLC, showed EG, diethylene glycol, EG derivatives, saccharides, alcohols, aldehydes, ketones, phenols, especially some acids such as formic acid, levulinic acid, acetic acid, oxalic acid and 2-hydroxy-butyric acid and their esters. The Higher Heating Value (HHV) of the residue and the acetone-soluble fractions were higher than that of bagasse. The results showed that some useful chemicals and biofuels could be obtained by this process.     

FORMALIN FIXATION IN THE CYTOCHEMICAL DEMONSTRATION OF SUCCINIC DEHYDROGENASE OF MITOCHONDRIA

A variety of established methods for protecting mitochondria were tested on rat duodenal epithelium during the histochemical assay for succinic dehydrogenase. The use of sucrose at isotonic or hypertonic concentrations, 7.5 per cent polyvinylpyrrolidone, divalent cations, physiological salt solutions, phenazine methosulfate, coenzyme Q₁₀, and menadione failed to improve the quality of the histochemical preparation once fresh frozen sections were prepared. However, preservation of mitochondrial integrity with little diminution in succinic dehydrogenase activity was obtained by fixing tissue slices (less than 1 mm. in thickness) in 8 per cent unneutralized, aqueous formaldehyde from 8 to 16 minutes at from 5° to 10°C. prior to freezing. To offset the inhibition of enzymatic activity it was necessary to extend the incubation period by 10 to 15 minutes. Two-micron-thick sections were easily obtained from the frozen blocks of such fixed tissue and incubated in the unmodified Nitro—BT-succinate medium. Once the optimum conditions for fixation of intestinal epithelium were determined, many other tissues were subjected to the same procedure. From the morphological standpoint the appearance of the mitochondria in these histochemical preparations compares favorably with the results obtained using the classical Regaud iron-hematoxylin staining procedure. With most tissues, the results are superior to those with fresh frozen sections. However, results with muscle, sperm, and kidney tubular epithelium are not as strikingly improved as with gut and liver.     

THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.     

ELECTRON MICROSCOPIC AND HISTOCHEMICAL COMPARISON OF THE TWO TYPES OF ELECTROPLAQUES OF NARCINE BRASILIENSIS

The torpedine electric fish Narcine brasiliensis has two morphologically distinct electric organs (main and accessory) which also differ with respect to a number of electrophysiological properties. The fine structure of the electroplaques of these organs has been examined by electron microscopy and by a histochemical method for localizing esterase activity with a high degree of resolution. In both kinds of electroplaques the innervated surface (ventral in those of the main organ, dorsal in those of the accessory) is the only site of esterase activity. The latter is further confined to the regions of synaptic contact between vesicle-containing axon terminals and the electroplaque membrane. The synaptic apparatus is similar to, but less elaborate than, that of neuromuscular junctions. The axon terminals and electroplaque membranes are free of connective tissue envelopments. The membrane of the uninnervated surfaces forms a continuum with a dense canalicular network which penetrates deeply into the 7 µ thick electroplaques of the main organ. The canalicular network has about the same thickness in the 20 µ electroplaques of the accessory organ. Except for this difference, the two kinds of cells appear to have the same fine structure. This finding is discussed in relation to the electrophysiological data on functional differences.     

THE ROLE OF WATER IN THE STRUCTURE OF PERIPHERAL NERVE MYELIN

In the study of the drying kinetics of nerve fibres, at least five "phases" of water evaporation can be distinguished. A consideration of the accompanying changes in low-angle x-ray diffraction patterns permits a tentative identification of the "phases" and a quantitative interpretation of the data in terms of the water distribution in nerve fibres. These results suggest that the myelin sheath of frog sciatic nerve contains 40 to 50 per cent water, and it is suggested further that the greater part of this water is "organised" in relation to the hydrophilic groups of the lipide and protein components.     

A STUDY OF THE FINE STRUCTURE OF THE EPIDERMIS OF RANA PIPIENS

The epidermis of adult Rana pipiens has been studied by electron microscopy and histological and histochemical methods. It was found that the epidermis is engaged in the production of both keratin and mucus. The basal cells are mainly filled with tonofilaments, whereas the cells located in the mid-portion of the epidermis contain both tonofilaments and mucous granules. Golgi vesicles and endoplasmic reticulum are found in relative abundance in the mucus-producing cells and seem to be involved in the production of mucous granules. The mucus seen was partly retained within the cells and partly secreted into the intercellular spaces. The outermost keratinized cells contain mainly filaments and a few remnants of cell constituents.     

THE DISTRIBUTION OF SELECTED DIAGNOSTIC CHARACTERS IN THE LECANORALES

Traditional classification concepts in lichenology are often, but not always, supported by molecular results. Molecular data should be compared and correlated with micro-morphological and ultrastructural information before systematic rearrangements are undertaken. Visualization of the distribution of morphological and other characters in specified groups is considered as a desirable resultper se, but it is also important to discover whether correlating characters are dependent on each other or not; and if not, whether their distribution in a group might support existing classification concepts. A data set for lecanoralean and other lichenized and lichenicolous genera, comprising 90—mostly multi-state—characters was used to store morphological, chemical and ecological data, and to test character correlations. Several examples of such analyses are presented. The following pairs of characters show some degree of dependence: ascospore septation and number per ascus, ascospore wall type and pigmentation, ascospore and epihymenium pigmentation. Several authors postulated that ascus types are good phylogenetic markers. Ascus types have been widely used for classification concepts of the Lecanorales. Two-dimensional correlation queries of ascus types with the following morphologcal characters were made: substratum preference, thallus growth form and ascospore septation. These correlations supply characteristic profiles for the various ascus types, which have to be compared with forthcoming phylogenetic hypotheses based on molecular data.     

THE CHEMICAL NATURE OF KERATOHYALIN GRANULES OF THE EPIDERMIS

Keratohyalin granules were isolated in the native form from the epidermis of newborn rats by the use of citric acid and a detergent. The isolated granules revealed a fine granular substructure in the electron microscope similar to that seen in situ. Analyses of amino acids by automated column-chromatography showed that proline and cystine are present in large proportions whereas histidine is present in a small amount. Accordingly, it was concluded that keratohyalin represents a sulfur-rich amorphous precursor of the horny cell content, rather than a sulfur-poor side product of the keratinization process, or a unique histidine-rich protein as proposed by in situ histochemical and radioautographic studies.