Pea Xyloglucan and Cellulose: VI. Xyloglucan-Cellulose Interactions in Vitro and in Vivo

Since xyloglucan is believed to bind to cellulose microfibrils in the primary cell walls of higher plants and, when isolated from the walls, can also bind to cellulose in vitro, the binding mechanism of xyloglucan to cellulose was further investigated using radioiodinated pea xyloglucan. A time course for the binding showed that the radioiodinated xyloglucan continued to be bound for at least 4 hours at 40°C. Binding was inhibited above pH 6. Binding capacity was shown to vary for celluloses of different origin and was directly related to the relative surface area of the microfibrils. The binding of xyloglucan to cellulose was very specific and was not affected by the presence of a 10-fold excess of (1→2)-β-glucan, (1→3)-β-glucan, (1→6)-β-glucan, (1→3, 1→4)-β-glucan, arabinogalactan, or pectin. When xyloglucan (0.1%) was added to a cellulose-forming culture of Acetobacter xylinum, cellulose ribbon structure was partially disrupted indicating an association of xyloglucan with cellulose at the time of synthesis. Such a result suggests that the small size of primary wall microfibrils in higher plants may well be due to the binding of xyloglucan to cellulose during synthesis which prevents fasciation of small fibrils into larger bundles. Fluorescent xyloglucan was used to stain pea cell wall ghosts prepared to contain only the native xyloglucan:cellulose network or only cellulose. Ghosts containing only cellulose showed strong fluorescence when prepared before or after elongation; as predicted, the presence of native xyloglucan in the ghosts repressed binding of added fluorescent xyloglucan. Such ghosts, prepared after elongation when the ratio of native xyloglucan:cellulose is substantially reduced, still showed only faint fluorescence, indicating that microfibrils continue to be coated with xyloglucan throughout the growth period.     

TRANSVERSE ELECTRIC IMPEDANCE OF NITELLA

Alternating current measurements have been taken on single Nitella cells over a frequency range from 30 to 2,500,000 cycles per second with the current flow perpendicular to the axis of the cell. The measuring cells were so constructed that electrolytes of any desired concentration could be circulated during the course of the measurements. The cellulose wall which surrounds the cell is found to play an important part in the interpretation of the results obtained. In a mature cell, this cellulose has a specific resistance of about 1000 ohm cm. which is independent of the medium in which the cell is suspended. The thickness of the wall is computed to be about 10 µ. The cell membrane is found to be virtually non-conducting, and to have a capacity of 0.94 µf./cm.² ± 10 per cent and a phase angle of 80° ± 4°. The specific resistances of the sap were difficult to compute from data on living cells and were unsatisfactory because they were very much dependent upon the medium, while measurements on extracted sap gave 58 ohm cm. ± 8 per cent which was independent of the medium. There are indications that the chloroplasts have impedance properties similar to those of living cells.     

Absorption and Screening in Phycomyces

In vivo absorption measurements were made through the photosensitive zones of Phycomyces sporangiophores and absorption spectra are presented for various growth media and for wavelengths between 400 and 580 mµ. As in mycelia, β-carotene was the major pigment ordinarily found. The addition of diphenylamine to the growth media caused a decrease in β-carotene and an increase in certain other carotenoids. Growth in the dark substantially reduced the amount of β-carotene in the photosensitive zone; however, growth on a lactate medium failed to suppress β-carotene in the growing zone although the mycelia appeared almost colorless. Also when diphenylamine was added to the medium the absorption in the growing zone at 460 mµ was not diminished although the colored carotenoids in the bulk of the sporangiophore were drastically reduced. Absorption which is characteristic of the action spectra was not found. Sporangiophores immersed in fluids with a critical refractive index show neither positive nor negative tropism. Measurements were made of the critical refractive indices for light at 495 and 510 mµ. The critical indices differed only slightly. Assuming primary photoreceptors at the cell wall, the change in screening due to absorption appears too large to be counterbalanced solely by a simple effect of the focusing change. The possibility is therefore advanced that the receptors are internal to most of the cytoplasm; i.e., near the vacuole.     

CENTRIFUGAL,BIOCHEMICAL, AND ELECTRON MICROSCOPIC ANALYSIS OF CYTOPLASMIC PARTICULATES IN LIVER HOMOGENATES

A combined centrifugal, biochemical, and electron microscopic study of the cytoplasmic particulates present in 0.88 M sucrose homogenates of rat liver has been carried out. Size distribution analyses of particles containing pentose nucleic acid (PNA) and exhibiting several types of enzymatic activity revealed three major size groups within the range of particle radius between 10 and 500 mµ. A different array of biochemical properties was associated with each size group. The largest particles, with an average radius (assuming spherical shape) in the region of 220 to 260 mµ, contained all of the succinic dehydrogenase activity of the cytoplasmic extract, 29 per cent of the diphosphopyridine nucleotide (DPN)-cytochrome c reductase activity, and minor amounts of PNA and acid phosphatase activity. Cytologically, this group of particles was identified with the mitochondria. All of the uricase activity, 58 per cent of the acid phosphatase activity, and 26 per cent of the PNA was apparently associated with a second size group of particles (average radius 120 mµ) which were tentatively identified by electron microscopy with vesicular structures derived from the ergastoplasm of the intact cell. The third particle group demonstrated by centrifugation exhibited a major size distribution peak at 25 mµ and a second smaller peak at 55 mµ. Over 50 per cent of the total cytoplasmic PNA and DPN-cytochrome c reductase activity was associated with particles in this size group. Electron microscopy revealed a morphologically heterogeneous population of particles within this size range.     

THE PIGMENT-PROTEIN COMPOUND IN PHOTOSYNTHETIC BACTERIA : II. THE ABSORPTION CURVES OF PHOTOSYNTHIN FROM SEVERAL SPECIES OF BACTERIA

Absorption curves have been obtained in the spectral region of 450 to 900 mµ for the water soluble cell juice of four species of photosynthetic bacteria, Spirillum rubrum (strain S1), Rhodovibrio sp. (strain Gaffron), Phaeomonas sp. (strain Delft), and Streptococcus varians (strains C11 and orig.). These curves all show maxima at 790 and 590 mµ due to bacteriochlorophyll, whose highest band, however, occurs at 875, 855, or 840 mµ depending on the species. The bacteria that appear red rather than brown have a band at 550 mµ due to a carotinoid pigment. An absolute absorption curve of bacteriophaeophytin has maxima at 530 and 750 mµ. The extraction of cell juice by supersonic vibration does not change the position of the absorption bands or of the light absorbing capacity of the pigment.     

THE PHOTOTROPIC SENSITIVITY OF PHYCOMYCES AS RELATED TO WAVE-LENGTH

Under the circumstances of experimentation described, the sporangiophores of Phycomyces are found to be most sensitive to stimulation by light in the violet between 400 and 430 mµ. Toward the red, sensitivity falls to nearly zero near 580 mµ, while in the near ultra-violet around 370 mµ, sensitivity is still high. The previous experiments of Blaauw had placed the point of greatest sensitivity some 80 mµ nearer the red end of the spectrum. Because of the known presence in the sporangiophores of Phycomyces of "accessory" pigments, care must be taken in identifying such results with the absorption spectrum of the photosensitive substance.     

Action spectra for photoreactivation of ultraviolet-irradiated Escherichia coli and Streptomyces griseus

Action spectra for photoreactivation (light-induced recovery from ultraviolet radiation injury) of Escherichia coli B/r and Streptomyces griseus ATCC 3326 were determined. The spectral region explored was 365 to 700 mµ. The action spectrum for S. griseus differed from that for E. coli, indicating that the chromophores absorbing reactivating energy in the two species were not the same. Reactivation of S. griseus occurred in the region 365 mµ (the shortest wave length studied) to about 500 mµ, with the most effective wave length lying near 436 mµ. This single sharp peak in the spectrum at 436 mµ suggested the Soret band typical of porphyrins. Reactivation of E. coli occurred in the region 365 to about 470 mµ, with the most active wave length lying near 375 mµ. The single, non-pronounced peak near 375 was probably not due to a Soret band, and the identification of the substance absorbing reactivating light in E. coli is uncertain. In neither species was the region 500 to 700 mµ active. The implications of these action spectra and their differences are discussed.     

The Cellulose System in the Cell Wall ofMicrasterias

The cellulose system of the cell wall ofMicrasterias denticulataandMicrasterias rotatawas analyzed by diffraction contrast transmission electron microscopy, electron diffraction, and X-ray analysis. The studies, achieved on disencrusted cell ghosts, confirmed that the cellulose microfibrils occurred in crisscrossed bands consisting of a number of parallel ribbon-like microfibrils. The individual microfibrils had thicknesses of 5 nm for a width of around 20 nm, but in some instances, two or three microfibrils merged into one another to yield larger monocrystalline domains reaching up to 60 nm in lateral size. The orientation of the cellulose ofMicrasteriasis very unusual, as it was found that in the cell wall, the equatorial crystallographic planes of cellulose having ad-spacing of 0.60 nm [(110) in the Iβ cellulose unit cell defined by Sugiyamaet al.,1991,Macromolecules24, 4168–4175] were oriented perpendicular to the cell wall surface. Up to now, such orientation has been found only inSpirogyra,another member of the Zygnemataceae group. The unusual structure of the secondary wall cellulose ofMicrasteriasmay be tentatively correlated with the unique organization of the terminal complexes, which in this alga occur as hexagonal arrays of rosettes.     

STUDIES OF NATIVE GLYCOGEN ISOLATED FROM SYNCHRONIZED TETRAHYMENA PYRIFORMIS (HSM)

Native glycogen was isolated from Tetrahymena pyriformis (HSM) by isopycnic centrifugation in cesium chloride density gradients. A density of 1.62 to 1.65 was isopycnic for glycogen. Most of the banded glycogen existed as 35 to 40 mµ particles which had a sedimentation coefficient of 214. These particles were composed of aggregates of 2 to 3 mµ spherical particles. Extraction of glycogen with hot alkali reduced the sedimentation coefficient of native glycogen from 214 to 64.7 and the particle diameter from approximately 40 to 20 mµ and smaller. Cell division was synchronized by a repetitive 12-hour temperature cycle, and glycogen was measured at several times during the cell cycle. The temperature cycle consisted of 9.5 hours at 12°C and 2.5 hours at 27°C. Approximately 90 per cent of the cells divided during the last 1.5 hours of the warm period. The carbohydrate/protein ratio of cells at the end of the cold period was 0.27 and was reduced slightly during the warm period. Glucose was incorporated into glycogen during both periods, although the rate of incorporation was greater during the warm period. No preferential incorporation on the basis of particle size was noted. Incorporation was measured in both native glycogen and KOH-extracted glycogen. Tetrahymena glycogen is compared with rat liver glycogen previously isolated by similar procedures, and the significance of using combined rate-zonal and isopycnic centrifugation for isolating native glycogen is discussed.     

The spectral sensitivities of the dorsal ocelli of cockroaches and honeybees; an electrophysiological study

The spectral sensitivities of the dorsal ocelli of cockroaches (Periplaneta americana, Blaberus craniifer) and worker honeybees (Apis mellifera) have been measured by electrophysiological methods. The relative numbers of quanta necessary to produce a constant size electrical response in the ocellus were measured at various wave lengths between 302 and 623 mµ. The wave form of the electrical response (ERG) of the dark-adapted roach ocellus depends on the intensity but not the wave length of the stimulating light. The roach ocellus appears to possess a single photoreceptor type, maximally sensitive about 500 mµ. The ERG's of bee ocelli are qualitatively different in the ultraviolet and visible regions of the spectrum. The bee ocellus has two types of photoreceptor, maximally sensitive at 490 mµ and at about 335 to 340 mµ. The spectral absorption of the ocellar cornea of Blaberus craniifer was measured. There is no significant absorption between 350 and 700 mµ.     

Electron Microscopy of the Centrifuged Sea Urchin Egg, with a Note on the Structure of the Ground Cytoplasm

Centrifuged, unfertilized eggs of the sea urchin, Arbacia punctulata, have been studied with the electron microscope. Subcellular particles were stratified by centrifuging living cells, known to be normally fertilizable, for five minutes at 3,000 g. The layered subcellular particles, including cortical granules, 16 mµ RNP particles, pigment, yolk, mitochondria, and oil droplets, possess characteristic ultrastructural features by which they may be identified in situ. The clear zone contains 16 mµ particles, most of them freely dispersed, scattered mitochondria, and a few composite structures made up of annulate lamellae in parallel layers or in association with dense, spherical aggregates of the RNP particles. Free 16 mµ particles are found, in addition, throughout the cell, in the interstices between the stratified larger particles. They show a tendency to form ramifying aggregates resulting from certain types of injury to the cell. A few vesicular structures, found mainly in the clear zone, have attached RNP particles, and appear to be related to the ER of tissue cells. Other vesicles, bounded by smooth membranes, are found throughout the cell. These are extremely variable in size, number, and distribution; their total number appears to depend upon conditions of fixation. It is suggested that limited formation of such structures is a normal property of the ground cytoplasm in this cell, but that fixed cells with very large numbers of smooth surfaced vesicles have produced the latter as a response to chemical injury. A model of the ground cytoplasm is proposed whose aim is to reconcile the rheological behavior of the living cell with the ultrastructural features observed.     

CYTOPLASMIC MICROTUBULE AND WALL MICROFIBRIL ORIENTATION IN ROOT HAIRS OF RADISH

The fine structure of young root hairs of radish was studied, with special attention to cytoplasm-wall relationships. Hairs up to 130 µ in length were examined after fixation of root tips in glutaraldehyde followed by osmium tetroxide. Microtubules occur axially aligned in the cytoplasm just beneath the plasmalemma, and extend from the base of the hair to within 2 to 3 µ of the tip. Poststaining with uranyl acetate and lead citrate clearly reveals in thin sections the presence of the two layers of cellulose microfibrils known from studies on shadowed wall preparations: an outer layer of randomly arranged microfibrils arising at the tip, and a layer of axially oriented microfibrils deposited on the inside of this layer along the sides. The youngest microfibrils of the inner, oriented layer first appear at a distance of about 25 µ from the tip. Although the microfibrils of the inner layer and the adjacent microtubules are similarly oriented, the oriented microtubules also extend through the 20- to 25-µ zone near the tip where the wall structure consists of random microfibrils. This suggests that the role of microtubules in wall deposition or orientation may be indirect.     

Photoreversal of nuclear and cytoplasmic effects of short ultraviolet radiation on Paramecium caudatum

1. Irradiation with three short ultraviolet (UV) wave lengths, 226, 233, and 239 mµ rapidly immobilizes Paramecium caudatum, the dosage required being smaller the shorter the wave length. 85 per cent of paramecia immobilized with wave length 226 mµ recover completely. Recovery from immobilizing doses is less the longer the wave length. 2. Irradiation continued after immobilization kills the paramecia in a manner which is markedly different for very short (226, 233, and 239 mµ) and longer (267 mµ) wave lengths. 3. An action spectrum for immobilization in P. caudatum was determined for the wave lengths 226, 233, 239, 248, and 267 mµ, and found to resemble the absorption of protein and lipide in the wave length region below 248 mµ. Addition of these data to those of Giese (1945 b) gives an action spectrum resembling the absorption by albumin-like protein. 4. Division of P. caudatum is delayed by doses of wave lengths 226, 233, and 239 mµ which cause immobilization, the longest wave length being most effective. 5. Immobilization at any of the wave lengths tested (226, 233, 239, 248, 267 mµ) is not photoreversible when UV-treated paramecia are concurrently illuminated. 6. Division delay resulting from immobilizing doses of 226, 233, and 239 mµ is photoreversible by exposure to visible light concurrently with the UV. 7. Division delay induced by exposure to wave length 267 mµ is reduced by exposure to visible light applied concurrently with UV or immediately afterwards. 8. The data suggest that the shortest UV wave length tested (226 mµ) affects the cytoplasm selectively, because it is absorbed superficially as indicated by unilateral fluorescence in UV. Consequently it immobilizes paramecia rapidly but has little effect on the division rate because little radiation reaches the nucleus. 9. The data support the view that nuclear effects of UV are readily photoreversed but cytoplasmic effects are not.     

The Photosensitive Retinal Pigment System of Gekko gekko

Retinal extracts of Gekko gekko were found to contain two retinene₁ photopigments, one with maximum absorption at about 521 mµ, the second with a maximum in the region of 478 mµ. These pigments were assayed by the method of partial bleaching and their spectral characteristics studied by examining their difference spectra. The 478 mµ pigment was present in the extracts as 8 per cent of the total photopigment concentration. The two pigment systems were shown to be biochemically independent and to have different properties. Unlike the 521 mµ pigment, for example, the 478 mµ pigment was found to resist the action of NH₂OH and, within the cells, to be unaffected by sucrose solutions. These solutions destroyed or altered the 521 system so that extracts of sucrose-treated retinae were found to contain significantly less 521 photopigment. In digitonin solution the 521 pigment was unaffected by sucrose treatment. Both pigments were extracted from separated, washed outer segments and so are considered to be visual pigments. This dual system accounts for the spectral sensitivity of this gecko as determined by Denton. A search was made, but no evidence was secured for the presence of a photopigment absorbing at longer wavelengths. Electoretinographic data suggest, however, that an elevated sensitivity at longer wavelengths occurs in some geckos so that a continued search is justified for a third photopigment.     

FURTHER ELECTRON MICROSCOPE STUDIES ON FIBRILLAR ORGANIZATION OF THE GROUND CYTOPLASM OF CHAOS CHAOS

Further evidence for fibrillar organization of the ground cytoplasm of Chaos chaos is presented. Fixations with osmium tetroxide at pH 6 or 8 and with glutaraldehyde at pH 6 or 7 were used on two preparations: (a) single actively streaming cells; (b) prechilled cells treated with 0.05% Alcian blue in the cold and returned to room temperature for 5–10 min. In addition, a 50,000 g pellet of homogenized cells was examined after fixation with glutaraldehyde-formaldehyde alone. In sections from actively streaming cells considerable numbers of filaments were observed in the uroid regions after glutaraldehyde fixation, whereas only traces of filaments were seen after osmium tetroxide fixation at either pH 6 or 8. Microtubules were not seen. In sections from dye-treated cells, filaments (4–6 mµ) and fibrils (12–15 mµ) were found with all three fixatives. The 50,000 g pellet was heterogeneous but contained both clumps of fibrils and single thick fibrils like those seen in the cytoplasm of dye-treated cells. Many fibrils of the same dimensions (12–15 mµ wide, 0.5 µ long) were also seen in the supernatant above the pellet. Negative staining showed that some fibrils separated into at least three strands of 4–6 mµ filaments.     

THE USE OF SHADOW-CASTING TECHNIQUE FOR MEASUREMENT OF THE WIDTH OF ELONGATED PARTICLES

A method is described for the estimation of the true width of fibrillar or rod-like structures from electron micrographs of metal-shadowed preparations. The method is based on variations in the image width as a function of the angle (β) between the long axis of the fibril and the direction of the shadow in the plane of the preparation. The image width when β = 0° practically represents the real width of the elongated particle but is often indistinguishable from the background. The fibril image width is conveniently measured at β values between 15° and 90°. The true width is obtained by plotting the image width versus sin β and extrapolating to β = 0°. Latex spheres are sprayed with the fibrils or rods to indicate the direction of shadow. Tobacco mosaic virus (TMV) was used as a model structure because of its known constant diameter of 150 A (5). The width (in the case of TMV equal to the diameter) found by the present method was 150 A ± 8 A.     

DETACHMENT OF BACTERIOPHAGE FROM ITS CARRIER PARTICLES

The active substance (phage) present in the lytic broth filtrate is distributed through the medium in the form of particles. These particles vary in size within broad limits. The average size of these particles as calculated on the basis of the rate of diffusion approximates 4.4 mµ in radius. Fractionation by means of ultrafiltration permits partial separation of particles of different sizes. Under conditions of experiments here reported the particles varied in the radius size from 0.6 mµ to 11.4 mµ. The active agent apparently is not intimately identified with these particles. It is merely carried by them by adsorption, and under suitable experimental conditions it can be detached from the larger particles and redistributed on smaller particles of the medium.     

Particle Deposition in a Child Respiratory Tract Model: In Vivo Regional Deposition of Fine and Ultrafine Aerosols in Baboons

To relate exposure to adverse health effects, it is necessary to know where particles in the submicron range deposit in the respiratory tract. The possibly higher vulnerability of children requires specific inhalation studies. However, radio-aerosol deposition experiments involving children are rare because of ethical restrictions related to radiation exposure. Thus, an in vivo study was conducted using three baboons as a child respiratory tract model to assess regional deposition patterns (thoracic region vs. extrathoracic region) of radioactive polydisperse aerosols ([d16–d84], equal to [0.15 µm–0.5 µm], [0.25 µm–1 µm], or [1 µm–9 µm]). Results clearly demonstrated that aerosol deposition within the thoracic region and the extrathoraic region varied substantially according to particle size. High deposition in the extrathoracic region was observed for the [1 µm–9 µm] aerosol (72%±17%). The [0.15 µm–0.5 µm] aerosol was associated almost exclusively with thoracic region deposition (84%±4%). Airborne particles in the range of [0.25 µm–1 µm] showed an intermediate deposition pattern, with 49%±8% in the extrathoracic region and 51%±8% in the thoracic region. Finally, comparison of baboon and human inhalation experiments for the [1 µm–9 µm] aerosol showed similar regional deposition, leading to the conclusion that regional deposition is species-independent for this airborne particle sizes.     

Structure of the Red Fluorescence Band in Chloroplasts

Using Weber's method of "matrix analysis" for the estimation of the number of fluorescent species contributing to the emission of a sample, it is shown that the fluorescence¹ band in spinach chloroplast fragments at room temperature originates in two species of chlorophyll a. Emission spectra obtained upon excitation with different wavelengths of light (preferentially absorbed in chlorophyll a or b) are presented. Upon cooling to - 196°C, the fluorescence efficiency increases about twentyfold. Two additional bands, that now appear at 696 and 735 mµ, suggest the participation of four molecular species. Emission spectra observed at different concentrations of chloroplast fragments with excitation in chlorophyll a and b and excitation spectra for different concentrations of chloroplast fragments and measurements at 685 and 760 mµ are presented. Two of the four emission bands may belong to pigment system I and two to system II. The 685, 696, and 738 mµ bands respond differently to temperature changes. In the -196°C to -150°C range, the intensity of the 685 mµ band remains constant, and that of the 696 mµ band decreases twice as fast as that of the 738 mµ band.     

PANCREATIC MICROSOMES : AN INTEGRATED MORPHOLOGICAL AND BIOCHEMICAL STUDY

The pancreatic exocrine cell of the guinea pig has a voluminous endoplasmic reticulum distinguished by extensive association with small, dense particles, and by its orderly disposition in the basal region of the cell. In addition to the small, (~15 mµ), dense particles attached to the limiting membrane of the endoplasmic reticulum, numerous particles of similar appearance are found freely scattered in the cytoplasmic matrix. The various cell structures of pancreatic exocrine cells can be satisfactorily identified in pancreatic homogenates. The microsome fraction consists primarily of spherical vesicles (80 to 300 mµ), limited by a thin membrane (7 mµ) which bears small (~15 mµ) dense particles attached on its outer surface. The content of the microsomal vesicles is usually of high density. Pancreatic microsomes derive by extensive fragmentation mainly from the rough surfaced parts of the endoplasmic reticula of exocrine cells. A few damaged mitochondria and certain dense granules (~150 mµ) originating probably from islet cells, contaminate the microsome fraction. Pancreatic microsomes contain RNA, protein, and a relatively small amount of phospholipide and hemochromogen. They do not have DPNH-cytochrome c reductase activity. In six experiments the RNA/protein N ratios were found grouped around two different means, namely 0.6 and 1.3. Pancreatic microsomes are more labile than liver microsomes but react in a similar way to RN-ase-(loss of the particulate component and RNA), and deoxycholate treatment (loss of the membranous component and of phospholipide, hemochromogen, and most of the protein). Postmicrosomal fractions consisting primarly of small (~15 mµ), dense particles of ribonucleoprotein (RNA/protein N ratio = 1 to 2) were obtained by further centrifugation of the microsomal supernatant. The small nucleoprotein particles of these fractions are frequently found associated in chains or clusters.