RESOLUTION IN ELECTRON MICROSCOPE RADIOAUTOGRAPHY : II. Carbon14

Experimental resolution values, half distances (HD), were determined for electron microscope radioautography with ¹⁴C as the source of radioactivity. These were about a factor of 1.5–2 times higher than for tritium. Grain distributions normalized in units of HD were found to fit the "universal" curves previously obtained for tritium.     

QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS : I. The Number of Acetylcholinesterase Molecules and of Other DFP-Reactive Sites at Motor Endplates, Measured by Radioautography

Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-³²P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 10⁷ sites per endplate, in the diaphragm, about 3 x 10⁷. Reaction with DFP-³²P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 10⁷ and 1.1 x 10⁷per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF THIN SECTIONS: THE GOLGI ZONE AS A SITE OF PROTEIN CONCENTRATION IN PANCREATIC ACINAR CELLS

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections (~ 600 A) of OsO₄-fixed, methacrylate-embedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days at 4°C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H³ injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.     

Studies on the Fragmented Shoot Apex of Grapevine: III. A SCANNING ELECTRON MICROSCOPE STUDY OF ADVENTITIOUS BUD FORMATION IN VITRO

Scanning electron microscopy (SEM) was used to provide directevidence that shoots produced in vitro from fragmented shootapices of grapevine were adventitious in origin. The effectof temperature on the formation of the adventitious buds wasalso examined using SEM. At 27°C, shoot buds were initiatedby 31 d following fragmentation of the apex, while at 35°Cshoot initiation and multiplication were already well-advancedat only 18 d after the start of culture. At 38°C, apicalfragments quickly browned and died. After 25 d at 35°C,structures resembling inflorescence primordia were also visible.These did not occur in cultures at 27°C. The primordia laterdeveloped into multiple-branched tendrils, structures whichappear to be intermediate between tendrils and inflorescencesand have not been previously described.     

AN ELECTRON MICROSCOPIC STUDY OF PINOCYTOSIS IN AMEBA : II. The Cytoplasmic Uptake Phase

This report is devoted principally to a consideration of the fate of the pinocytotic vacuole and its content in the ameba Pelomyxa carolinensis (Chaos chaos). High resolution micrographs of the plasmalemma have shown it to consist of three layers, i.e., an outermost filamentiferous zone, a middle homogeneous zone, and an inner zone which appears to be a unit membrane. The three zones can be identified in the membranes lining the pinoyctotic tunnels and vacuoles of amebas fixed shortly after pinocytosis occurred. The first apparent change in the pinocytotic vacuole is an increase in the surface-to-volume ratio which occurs during the 1st hour of its existence. Within 24 hours the marker substance commonly collects in defecation vacuoles which can be identified by the profiles of bacteria usually found in the lumen. Occasionally, however, thorotrast can be seen in the lumen of the contractile vacuole. The thorotrast appears to enter the two excretory organelles by the coalescence of vesicular fragments of the pinocytotic vacuoles with the limiting membranes of the excretory organelles.     

THE COMBINATION OF ENZYME AND SUBSTRATE : I. A METHOD FOR THE QUANTITATIVE DETERMINATION OF PEPSIN. II. THE EFFECT OF THE HYDROGEN ION CONCENTRATION.

1. A quantitative method for the determination of pepsin is described depending on the change in conductivity of a digesting egg albumin solution. 2. The combination of pepsin with an insoluble substrate has been followed by this method. 3. The amount of pepsin removed from solution by a given weight of substrate is independent of the size of the particles of the substrate. 4. There is an optimum zone of hydrogen ion concentration for the combination of enzyme and substrate corresponding to the optimum for digestion. 5. It is suggested that the pepsin combines largely or entirely with the ionized protein.     

PHYSIOLOGICAL ONTOGENY : A. CHICKEN EMBRYOS. IX. THE IODINE REACTION FOR THE QUANTITATIVE DETERMINATION OF GLUTATHIONE IN THE TISSUES AS A FUNCTION OF AGE.

The iodine reaction to determine—SH groups in tissues according to the technique of Tunnicliffe showed that the percentage concentration of such compounds in the dried substance of chicken embryos declines with age chiefly during the third quarter of the incubation period.     

MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS : II. The Role of Nucleating Sites in Shape Development

The proposal made in the preceding paper that the species-specific shape of Ochromonas is mediated by cytoplasmic microtubules which are related to two nucleating sites has been experimentally verified. Exposure of cells to colchicine or hydrostatic pressure causes microtubule disassembly and a correlative loss of cell shape in a posterior to anterior direction. Upon removal of colchicine or release of pressure, cell shape regenerates and microtubules reappear, first in association with the kineto-beak site concomitant with regeneration of the anterior asymmetry, and later at the rhizoplast site concomitant with formation of the posterior tail. It is concluded that two separate sets of cytoplasmic tubules function in formation and maintenance of specific portions of the total cell shape. On the basis of the following observations, we further suggest that the beak and rhizoplast sites could exert control over the position and timing of the appearance, the orientation, and the pattern of microtubule distribution in Ochromonas. (a) the two sites are accurately positioned in the cell relative to other cell organelles; (b) in regenerating cells microtubules reform first at these sites and appear to elongate to the cell posterior; (c) microtubules initially reappear in the orientation characteristic of the fully differentiated cell; (d) the two sets of tubules are polymerized at different times, in the same sequence, during reassembly or resynthesis of the microtubular system. Experiments using cycloheximide, after a treatment with colchicine, have demonstrated that Ochromonas cannot reassume its normal shape without new protein synthesis. This suggests that microtubule protein once exposed to colchicine cannot be reassembled into microtubules. Pressure-treated cells, on the other hand, reassemble tubules and regenerate the normal shape in the presence or absence of cycloheximide. The use of these two agents in analyzing nucleating site function and the independent processes of synthesis and assembly of microtubules is discussed.     

OPTICAL DIFFRACTION STUDIES OF CRYSTALLINE STRUCTURES IN ELECTRON MICROGRAPHS : II. Crystalline Inclusions in Mitochondria of Human Hepatocytes

Unit cell dimensions of mitochondrial crystals were determined by optical diffraction analysis of electron micrographs of human liver biopsy specimens. Identical unit cells were found in pathologic material obtained from six patients with Wilson's disease, from one patient with sickle-cell hepatitis, and from two normal subjects. These measurements led to the conclusion that the crystals observed in patients and in normal subjects were probably chemically identical. Furthermore, the relatively large size of the unit cell limits the choices for its constituents to phospholipid micelles or to relatively large protein molecules.     

QUANTITATIVE STUDIES ON ENZYMES IN STRUCTURES IN STRIATED MUSCLES BY LABELED INHIBITOR METHODS : II. Confirmation of Radioautographic Measurement by Liquid-Scintillation Counting

Fragments of mouse diaphragm and sternomastoid muscles were incubated in diisopropyl-fluorophosphate (DFP)-³H in conditions known to saturate all the available DFP-sensitive reaction sites. After being extensively washed, the enzyme acetylcholinesterase (AChase) was specifically reactivated by treatment with pyridine-2-aldoxime methiodide (2-PAM). The radioactive DP-groups released into solution by 2-PAM were measured by liquid scintillation counting, and related to the known number of motor endplates present. Considerable difficulty was encountered in reducing the excess, adsorbed radioactivity to acceptable levels: long washing routines, extraction with organic solvents, and removing excess muscle fiber by microdissection were necessary. Six experiments gave a mean value of 2.4 x 10⁷molecules AChase per sternomastoid endplate, in reasonable agreement with the previously reported measurements by radioautography.     

ELECTRON MICROSCOPE STUDIES ON SALIVARY GLAND CELLS : I. The Nucleus of Bradysia mycorum Frey (Sciaridae), with Special Reference to the Nucleolus

Salivary glands were fixed in cold 1 per cent osmium tetroxide in veronal-acetate buffer containing sucrose and embedded in methacrylate mixture or Araldite. The salivary gland nuclei of sciarids show a continuous production of nucleoli, which remain multiple and not consolidated into a single structure. The earliest recognizable nucleoli, which we call "elementary nucleoli," are aggregations of a few paired 40 A fibrils and a few 150 A particles, at many points within chromosome bands. Further development consists of the detachment of the elementary nucleoli from their points of origin and their subsequent mutual coalescence. As a result, dense patches of nucleolar material are formed which become large nucleoli at the surface of chromosomes, either attached to the band or free. The fully formed nucleoli have a characteristic dual structure with a narrow dense periphery and a broader less dense internum. Fibrils and particles are present in both regions, and the difference in density reflects differences in the packing of the two structural elements. The duality in structure is lost in later stages. The nucleolar fibrils appear to be similar to the chromosomal fibrils. The 150 A particles in nucleoli, chromosomes, and nuclear sap seem identical. The significance of these observations is discussed for nucleologenesis in general.     

ELECTRON MICROSCOPY OF THE OXYNTIC CELL IN THE GASTRIC GLANDS OF THE BULLFROG, RANA CATESBIANA : II. The Acid-Secreting Gastric Mucosa

The oxyntic cell in the gastric glands of the bullfrog was examined in lead hydroxide-stained sections of gastric mucosae fixed in buffered osmium tetroxide and embedded in n-butyl methacrylate. During gastric acid secretion (pH 1–2) induced by histamine administration in cannulated frogs, the pattern of fine structure in the oxyntic cell differs strikingly from that in the oxyntic cell of the non-acid-secreting stomach. The relative number of smooth surfaced profiles decreases and a greater concentration of these elements is associated with the apical region of the oxyntic cell facing the lumen of the gastric gland. Similar concentrations of these elements are found in those regions of cytoplasm surrounding intercellular canaliculi which lie between adjacent cells and communicate with the lumen of a gastric gland. In these regions, the smooth surfaced profiles (35 to 65 mµ in width) characteristically form a tubular network. The membrane-bounded contents appear to be continuous with the extracellular medium, both on the capillary side and at the apical surface of the cell adjoining the lumen of the gastric gland. Mitochondria are distributed randomly in the cytoplasmic matrix of the oxyntic cell.     

The Sodium-Potassium Exchange Pump: II. Analysis of Na+-Loaded Frog Sartorius Muscle

A model for the Na-K exchange pump was applied to data on Na⁺-loaded frog sartorius muscle, and was used to relate the rate of adenosine triphosphate (ATP) hydrolysis to the electrical properties of the cell membrane. Membrane hyperpolarization was considered to arise from an electrical current which was produced by the hydrolysis reaction coupled to ion movements, and which flowed across the membrane. The reaction rate, as calculated from hyperpolarization, agreed with direct measurements of ATP hydrolysis and with the rate estimated from Na⁺ tracer efflux studies. Although Na⁺ is actively extruded, the model showed that K⁺ is inwardly transported if the potassium permeability of the membrane is less than about 6.6 × 10^-6 cm/sec, as is suggested by resistance data. Calculations indicated that the reaction conductance L_rr was relatively constant when compared with the reaction rate and reaction free energy for large changes in internal and external ionic concentrations. Its value agreed with the value obtained from the dependence of Na⁺ tracer efflux on external K⁺. A set of experiments was suggested which would provide a more complete interpretation of the data.     

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : II. Cytochemistry and Electron Microscopy of Bone Marrow Cells

In the previous paper we presented findings which indicated that enzyme heterogeneity exists among PMN leukocyte granules. From histochemical staining of bone marrow smears, we obtained evidence that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appeared to be restricted to one of the two types. Clear results were obtained with alkaline phosphatase, but those with a number of other enzymes were suggestive rather than conclusive. Since the approach used previously was indirect, it was of interest to localize the enzymes directly in the granules. Toward this end, we carried out cytochemical procedures for five enzymes on normal rabbit bone marrow cells which had been fixed and incubated in suspension. The localization of reaction product in the granules was determined by electron microscopy. In accordance with the results obtained on smears, azurophil granules were found to contain peroxidase and three lysosomal enzymes: acid phosphatase, arylsulfatase, and 5'-nucleotidase; specific granules were found to contain alkaline phosphate. Specific granules also contained small amounts of phosphatasic activity at acid pH. Another finding was that enzyme activity could not be demonstrated in mature granules with metal salt methods (all except peroxidase); reaction product was seen only in immature granules. The findings confirm and extend those obtained previously, indicating that azurophil granules correspond to lysosomes whereas specific granules represent a different secretory product.     

The Viscoelastic Properties of Passive Eye Muscle in Primates. II: Testing the Quasi-Linear Theory

We have extensively investigated the mechanical properties of passive eye muscles, in vivo, in anesthetized and paralyzed monkeys. The complexity inherent in rheological measurements makes it desirable to present the results in terms of a mathematical model. Because Fung''s quasi-linear viscoelastic (QLV) model has been particularly successful in capturing the viscoelastic properties of passive biological tissues, here we analyze this dataset within the framework of Fung''s theory.We found that the basic properties assumed under the QLV theory (separability and superposition) are not typical of passive eye muscles. We show that some recent extensions of Fung''s model can deal successfully with the lack of separability, but fail to reproduce the deviation from superposition.While appealing for their elegance, the QLV model and its descendants are not able to capture the complex mechanical properties of passive eye muscles. In particular, our measurements suggest that in a passive extraocular muscle the force does not depend on the entire length history, but to a great extent is only a function of the last elongation to which it has been subjected. It is currently unknown whether other passive biological tissues behave similarly.     

THE CONVERSION OF FAT TO CARBOHYDRATE IN THE GERMINATING CASTOR BEAN : II. THE COMBUSTION RESPIRATORY QUOTIENT AS DETERMINED BY A MODIFIED OXYCALORIMETER

The combustion respiratory quotients of castor beans germinated to various stages, depending upon the length of the hypocotyl, were determined by means of a modified oxycalorimeter. After germination was well started, the respiratory quotient of the combusted germinated seed increased as the stage of germination increased, indicating a change from an oxygen-poor to an oxygen-rich substance, probably fat to sugar. The accuracy of the method was checked by organic combustions. The seat of formation of the oxygen-rich substance is in the endosperm.