AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

THE DEVELOPMENT OF PIGMENT GRANULES IN THE EYES OF WILD TYPE AND MUTANT DROSOPHILA MELANOGASTER

The eye pigment system in Drosophila melanogaster has been studied with the electron microscope. Details in the development of pigment granules in wild type flies and in three eye color mutants are described. Four different types of pigment granules have been found. Type I granules, which carry ommochrome pigment and occur in both primary and secondary pigment cells of ommatidia, are believed to develop as vesicular secretions by way of the Golgi apparatus. The formation of Type II granules, which are restricted to the secondary pigment cells and contain drosopterin pigments, involves accumulation of 60- to 80-A fibers producing an elliptical granule. Type III granules appear to be empty vesicles, except for small marginal areas of dense material; they are thought to be abnormal entities containing ommochrome pigment. Type IV granules are characteristic of colorless mutants regardless of genotype, and during the course of development they often contain glycogen, ribosomes, and show acid phosphatase activity; for these reasons and because of their bizarre and variable morphology, they are considered to be autophagic vacuoles. The 300-A particles commonly found in pigment cells are identified as glycogen on the basis of their morphology and their sensitivity to salivary digestion.     

EFFECT OF DEHYDROASCORBIC ACID ON THE ISLETS OF LANGERHANS OF THE RAT PANCREAS

Rat pancreatic islets have been studied following successive daily administration of dehydroascrobic acid (DHA) and during the recovery phase following 3 daily injections. One injection of DHA produces degranulation of B cells seen in the light microscope as a loss of aldehyde fuchsin positivity. In the electron microscope the B cells appear to have secretory granules accumulated subjacent to the plasma membranes. Following 2 and 3 daily injections, B cells evidence alterations in the organization of the granular endoplasmic reticulum and mitochondria, and secretory granules are scant but when present are subjacent to the plasma membrane. After 5 to 7 days' recovery few secretory granules remain in B cell cytoplasm, but the cells have prominent granular ER and a Golgi apparatus with numerous prosecretory granules. The primary effect of DHA is an exaggerated secretory response of B cells, which is intensified with subsequent injections. Necrosis of B cells as produced by alloxan is not seen.     

A CORRELATED LIGHT AND ELECTRON MICROSCOPE STUDY OF THE NUCLEOLAR MATERIAL DURING MITOSIS IN VICIA FABA

Root meristematic cells of Vicia faba were examined, with both light and electron microscopes, in order to study the behaviour of the nucleolar material during the mitotic process. Under light microscopy, the preprophase nucleolus is seen to consist of a densely stained material in which are embedded several unstained vacuole-like structures of varying size. The electron microscope reveals that the dense nucleolar material is formed of two structurally distinct components, each segregated into irregularly shaped zones blending with one another. One of these components is represented by 150 A granules which, in places, are arranged into thread-like structures approximately 0.1 µ in diameter; the other component apparently consists of fibrils 60 to 100 A in diameter. The large and medium sized intranucleolar vacuoles contain loosely scattered granules and fibrils similar to those just described. The granular and fibrillar components of the denser portion of the nucleolus persist as such during prophase and disperse throughout the nuclear cavity at the time of nucleolar disintegration. After nuclear membrane breakdown, these granules and fibrils, as well as those of the nucleoplasm, mix freely with similar elements already present within the forming spindle. No evidence has been obtained that, during or after nucleolar disintegration, the structural components of the nucleolus become associated as such with the chromosomes to form an external or internal matrix. Our observations suggest the existence, of a matrix substance within late prophase, metaphase, and anaphase chromosomes, the fine structure of which bears strong resemblance to that of their constituent coiled chromonemata. Data are presented, moreover, that indicate that part of this matrix substance, presumably formed at some time during prophase, is released from the chromosomes during their anaphasic movement. A number of observations indicate that the main bulk of the next nucleolus is derived from a prenucleolar fibrillogranular material, arranged into thread-like structures some 0.1 µ in diameter, which collect in the interchromosomal spaces during early and midtelophase. Finally, our data would seem to favour the view that most of this prenucleolar material results from a resumption of the synthetic activity of the early and midtelophase chromosomes rather than from a mere shedding of a preexisting matrix substance.     

Calcium, Sulfhydryls and the Mitotic Apparatus

The in vivo mitotic apparatus (MA) of clam, worm and sea urchineggs may be augmented or dispersed by application of specificantimitotic agents. Glycols, which are antimitotic at concentrationsof 1–3% in sea water, cause a rapid massive increase inMA volume and retardation as seen in the polarizing microscope.Caffeine and dinitrophenol (DNP) cause a rapid disappearanceof the MA by shrinkage. Glycol effects can be balanced by DNPor caffeine if the agents are applied at the proper time andconcentration although normal cleavage does not ensue. Analysisof DNP and caffeine shrinkage suggests that they act indirectlyby causing release of calcium from intracellular stores, calciumcausing inactivation of polymerizable tubulin. DNP could causerelease of calcium either from mitochondria (if egg mitochondriahave a calcium uptake system) or by causing a decrease in ATPlevels which would inactivate calcium uptake systems such asthe Petzelt Ca⁺² ATP-ase or Kinoshita Ca⁺² uptake vesicles. Caffeine, while an inhibitor of the phosphodiesterase for cyclicAMP does not cause inhibition of the MA through the cyclic nucleotide.It is found that caffeine (and other methyl xanthines) causean inhibition of glutathione reductase activity in treated eggs.It is postulated that the calcium ATP-ases in the egg may becontrolled by reversible oxidation and reduction of their sulfhydrylsthus regulating calcium concentration in the cytosol. The possiblemodes of action of this system are discussed.     

Investigations of Carbon Transport in Plants: I. THE USE OF CARBON-11 TO ESTIMATE VARIOUS PARAMETERS OF THE TRANSLOCATION PROCESS

A technique is described which enables several components ofthe translocation system to be measured routinely, in vivo andsimultaneously. Plants are exposed to a 1-min pulse of ¹¹CO₂and the movement of the ¹¹C pulse through the plant followedusing an array of scintillation counters. It is possible toestimate the time taken for the ¹¹C to enter the sieve tubes.In addition, the speed of translocation can be obtained frommeasurements of the arrival of the ¹¹C pulse at different positionsalong the translocation pathway. The increasing half-width ofthe pulse as it enters and moves through the sieve tubes givessome indication of the extent of the delays in the differentparts of the translocation system. The short half-life of the¹¹C allows the measurements to be repeated on the same plantseveral times per day. A technique is discussed whereby dataon the movement of ¹¹C could be combined with simultaneous measurementsof the rate of uptake of carbon dioxide to provide an estimateof the rate of movement of carbon.     

THE INFLUENCE OF ENVIRONMENT ON THE SHELL STRUCTURE OF STARCH GRANULES

It is well known from light microscope studies of potato starch that the granules formed in a constant environment (of light and temperature) have a ring formation indistinguishable from that of granules formed under field conditions. Electron microscope studies have confirmed that normal potato starch granules have a fine shell structure not usually resolved by the light microscope, and also that shells do not develop in barley granules grown in a constant environment. The paper presented here reports a further study of the dependence of shell formation on environment. Potatoes were grown in a constant environment and starch granules from the newly formed tubers were examined in the light microscope, and in the electron microscope after corrosion by acid. No difference between these granules and normal granules was observed; both wide (light microscope) rings and fine lamellae developed in both granules. Parallel studies were made on wheat starch granules. In this case, shells were not differentiated in granules that developed in a constant environment, but they could be produced at will by imposing a dark period. Thus, shell formation in potato granules must be controlled by an endogenous rhythm, whereas in wheat granules it must be controlled by external environment.     

Contractile apparatus and sarcoplasmic reticulum function: effects of fatigue, recovery, and elevated Ca2+

Williams, Jay H. Contractile apparatus and sarcoplasmicreticulum function: effects of fatigue, recovery, and elevated Ca²⁺. J. Appl.Physiol. 83(2): 444-450, 1997.This investigationtested the notion that fatiguing stimulation induces intrinsic changes in the contractile apparatus and sarcoplasmic reticulum (SR) and thatthese changes are initiated by elevated intracellularCa²⁺ concentration([Ca²⁺]_i).Immediately after stimulation of frog semitendinosus muscle, contractile apparatus and SR function were measured. Despite a largedecline in tetanic force (P_o),maximal Ca²⁺-activated force(F_max) of the contractileapparatus was not significantly altered. However,Ca²⁺ sensitivity was increased. Inconjunction, the rate constant ofCa²⁺ uptake by the SR wasdiminished, and the caffeine sensitivity ofCa²⁺ release was decreased. Duringrecovery, P_o, contractileapparatus, and SR function each returned to near-initial levels.Exposure of skinned fibers to 0.5 µM freeCa²⁺ for 5 min depressed bothF_max andCa²⁺ sensitivity of thecontractile apparatus. In addition, caffeine sensitivity ofCa²⁺ release was diminished.Results suggest that fatigue induces intrinsic alterations incontractile apparatus and SR function. Changes in contractile apparatusfunction do not appear to be mediated by increased[Ca²⁺]_i.However, a portion of the change in SRCa²⁺ release seems to be due toelevated[Ca²⁺]_i.

     

THE ULTRASTRUCTURE AND FUNCTION OF THE OESOPHAGUS OF PATELLA VULGATA LINNAEUS

The oesophagus of Patella vulgata Linnaeus is divisible intotwo histologically and functionally distinct regions, a centralfood channel and a series of lateral pouches forming the oesophagealglands: both are lined by a glandular ciliated columnar epithelium.Subepithelial mucous cells are restricted to the epitheliumlining the food channel and posterior section of oesophagus,whilst amylase-secreting cells occur only in the oesophagealglands. The activity of the ciliated cells lining the food channelmixes ingested food with saliva and mucus to form a food stringthat is carried towards the stomach. Amylase-secreting cellsproduce an extracellular amylase in the form of proteinaceoussecretory granules, which are released by apocrine secretionwithin cytoplasmic blebs. Release is correlated with periodsof immersion when feeding is possible. The free blebs are carriedout of the pouches and into the food channel where the amylaseinitiates carbohydrate digestion. The oesophageal epitheliumabsorbs tritiated D-glucose by a Na⁺-dependent mechanism inhibitedby 2,4-DNP and phloridzin and the amylase-secreting cells pinocytoseferritin. It is suggested that the oesophagus absorbs smallsolute molecules derived from ruptured algal cells and theirpartially digested polysaccharide storage products. There alsoappears to be pinocytosis of dissolved organic matter. ^*Current address: The R L. Weston Institute for NeurologicalStudiet, University College & Middlesex Medcal School, MortimerSt., London Wl. (Received 8 March 1988; accepted 29 April 1988)     

DEGRADATION AND FORMATION OF SULFOLIPID OCCURRING CONCURRENTLY WITH DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES

1. It has been demonstrated that when the cells of Chlorella protothecoidesare grown mixotrophically under illumination in a medium richin nitrogen source (urea) and poor in glucose, the normal greencells are obtained, while in a medium rich in glucose and poorin the nitrogen source, entirely chlorophyll-less cells withprofoundly degenerated plastids ("glucose-bleached" cells) areproduced, irrespective of whether in the light or in darkness.The "glucose-bleached" cells turn green with regeneration offully organized chloroplasts when incubated in a nitrogen-enrichedmedium in the light ("light-greening"), while in the dark theybecome pale green with formation of only partially organizedchloroplasts ("dark-greening"). When, on the other hand, thegreen cells are transferred into a medium enriched with glucose,they are bleached fairly rapidly with degeneration of chloro-plastsin the light as well as in darkness ("bleaching"). Using ³⁵Sas a tracer, investigations were made on the changes of contentsof the algal cells in sulfolipid and other sulfur compoundsduring the processes of the greening and bleaching.
2. By determiningthe radioactivities of chromatographically separatedsulfur-containingcompounds of the uniformly ³⁵S-labeled green("G") and "glucose-bleached"("W") cells, it was found thatthe concentration of a speciesof sulfolipid (discovered byBENSON et al.) as well as thoseof glutathione, sulfotriosesand most of the other sulfur-containingcompounds were at least5 times higher in the "G" cells thanin the "W" cells, whilesulfoquinovosyl glycerol was presentin approximately equalamounts in the two types of cells.
3. Phospholipidcontents and compositions in the two types of algalcells werefound to be practically identical.
4. The sulfolipid contentof algal cells increased and decreasedalmost in parallel withthe processes of greening and bleaching,respectively.
5. Studyingthe mode of incorporation of radiosulfate into varioussulfurcompounds of algal cells during the processes of "light-anddark-greening" and "bleaching" (lasting about 70 hr), itwasfound that active ³⁵S-incorporation into sulfolipid occurredthroughout the process of "light-greening," while in the "dark-greening"and "bleaching" the active incorporation abruptly ceased afterthe initial 24 hr period of experiments. It was suggested thatthe biosynthesis of the sulfolipid is closely related to theformation of photosynthetic apparatus in chloroplast.
6. Whenthe ³⁵S-labeled green cells were bleached in a medium containingno radiosulfate, the ³⁵S-sulfolipid and most of other ³⁵S-sulfurcompounds decreased markedly but the ³⁵S-sulfoquinovosyl glycerolincreased considerably. It was inferred that the deacylationof the sulfolipid, a surfactant lipid, with formation of watersoluble sulfoquinovosyl glycerol may be a cardinal event ofbleaching process, causing a disintegration of the intact architechtureof photosynthetic apparatus.
7. Based on these observations itwas concluded that the sulfolipidis an integral component ofphotosynthetic structure.

¹This work was partly reported at the Symposium on Biochemistryof Lipids, sponsored by the Agricultural Chemical Society ofJapan, Sapporo, July, 1964.     

THE EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.     

ELECTRON MICROSCOPY OF BASOPHILIC STRUCTURES OF SOME INVERTEBRATE OOCYTES : II. FINE STRUCTURE OF THE YOLK NUCLEI

RNA containing yolk nuclei from the surf clam Spisula solidissima have been studied with the light microscope and with the electron microscope. A variety of structures can be seen with both and a correlation can be made between bodies studied with the electron microscope and those studied with the light microscope. The electron microscope shows many of these structures to be composed of double walled lamellae arranged in space, in various ways. The variety of patterns seen with the electron microscope can be satisfactorily explained on the basis of a hypothetical model. The presence of yolk platelets within the yolk nuclei lends support to classical observations on the synthesis of yolk within yolk nuclei or yolk nuclei derivatives. Small granules (about 100 A) are included within the double walled lamellae and their presence probably accounts for the basophilic nature of the entire body. The presence of small granules attached to electron-dense layers relates the yolk nuclei described here to ergastoplasm discussed by others.     

ELABORATION OF THE MATRIX GLYCOPROTEIN OF ENAMEL BY THE SECRETORY AMELOBLASTS OF THE RAT INCISOR AS REVEALED BY RADIOAUTOGRAPHY AFTER GALACTOSE-3H INJECTION

The elaboration of enamel matrix glycoprotein was investigated in secretory ameloblasts of incisor teeth in 30–40-g rats. To this end, the distribution of glycoprotein was examined histochemically by the use of phosphotungstic acid at low pH, while the formation of glycoprotein was traced radioautographically in animals sacrificed 2.5–30 min after galactose-³H injection. Histochemically, the presence of glycoprotein is observed in ameloblasts as well as in the enamel matrix; in ameloblasts glycoprotein occurs within the Golgi apparatus in amounts increasing from the outer to the inner face of the stacks of saccules, and is concentrated in condensing vacuoles and secretory granules; in the enamel matrix, glycoprotein is observed within linear subunits. Radioautographs at 2.5 min after injection demonstrate the uptake of galactose-³H label by Golgi saccules, indicating that galactose-³H is incorporated into glycoprotein within this organelle. After 5–10 min, the label collects in the condensing vacuoles and secretory granules of the Golgi region. By 20–30 min, the label appears in the secretory granules of the apical (Tomes') processes, as well as in the enamel matrix (next to the distal end of the apical processes, and at the tips of matrix prongs). In conclusion, galactose contributes to the formation of glycoprotein within the Golgi apparatus. The innermost saccules then distribute the completed glycoprotein to condensing vacuoles, which later evolve into secretory granules. These granules rapidly migrate to the apical processes, where they discharge their glycoprotein content to the developing enamel.     

PRODUCTION OF MUCOUS GRANULES BY THE TERRESTRIAL SLUG ARION ATER L.

Mucous granules were obtained from mature Arion ater. Granulesfrom the dorsal body, in modified slug Ringer solution, didnot differ significantly in length from those taken from theanimals ventral surface (mean length 6.75µm ± 0.047,mean ± SEM, n = 180) but their widths were significantlysmaller (1.18µm ± 0.007 < 2.70µm ±0.036, n = 90). Granules removed from the anterior, middle andposterior parts of each surface did not differ significantlyin length or width. Individual granules burst in distilled waterto absorb approximately 300 times their own volume of water.Regression analysis on swelling experiments using variable numbersof granules demonstrated that the number of granules was a significantpredictor of the weight of swollen mucus. Mucus swollen fromgranules could be dried then reswollen in distilled water toacieve approximately 60% of its original swollen weight. Theserelationships were used to quantify granule incorporation intoslug mucus trails. Active adult Anon ater in high humidity useapproximately 0.6 x 10⁶ granules to produce 1cm² of slug mucoustrail with a fully swollen weight of approximately 3mg Slugweight was a significant predictor of the number of granulesincorporated into the slug trail. (Received 21 April 1995; accepted 15 November 1995)     

THE REACTION OF RAT MAST CELLS TO POLYLYSINE

The effects of a single intraperitoneal injection of polyamino acids (lysine, glutamic, aspartic) on mast cells of the rat are described. In vitro interaction of mast-cell components with these polyamino acids is also explored. Poly-DL-lysine (but not the acidic amino acids) has both immediate (minutes-hours) and long-term (days-weeks) effects on mast cells. At the dosage selected, some cells evidence rapid fusion of granules and degranulation, but without concomitant swelling; most display intracellular changes only. Neither degranulation nor granule fusion appears to be lethal. Rather, these spur the cell to greater synthetic activity which involves first the Golgi apparatus and subsequently also the endoplasmic reticulum. Early involvement of macrophages and eosinophils is described. Sequential studies after polylysine injection support the following concepts: (a) mast-cell granules exist as "physiological sets," several being confined to a common membranous "sac;" (b) each set can respond independently to applied stimuli; (c) each set can connect directly to the extracellular milieu; (d) poly-DL-lysine binds directly to the granules and stabilizes them; it is not readily digested; (e) mast-cell granules are produced directly; they do not arise by intake of exogenous polysaccharides. It is hypothesized that mast-cell granules are topologically outside the cell while held intimately within extensive cytoplasmic folds and recesses. Mast cells may function by causing intercellular connective tissue fluids to percolate over their granules which may process this fluid in some as yet undefined way(s).     

ON THE SITE OF SULFATION IN THE CHONDROCYTE

As observed autoradiographically in the cartilage of embryonic rats, radiosulfate is bound and concentrated only in vesicles of the juxtanuclear Golgi apparatus of secreting chondrocytes within 3 minutes of its presentation. From this area, vacuoles migrate peripherally and lodge in the subcortex; their sulfated contents are thence discharged via stomata to the extracellular matrix. The label, apparently often associated with microvesicles at 10 and 20 minutes, is subsequently localized in the dense contents of the larger vacuoles. Bound radiosulfate is not detectable in other organelles. It is concluded that the vesicular component of the Golgi apparatus is the actual site of sulfation. Intracellular hyaluronidase-sensitive metachromatic granules are found chiefly at the cell periphery or mantle, rarely juxtanuclear in the main Golgi zone.     

THE CYTOLOGY OF THE NORMAL PARATHYROID GLANDS OF MAN AND VIRGINIA DEER : A Light and Electron Microscopic Study with Morphologic Evidence of Secretory Activity

The normal parathyroids of six humans and a Virginia deer were studied by light and electron microscopy. The parenchyma of the deer parathyroid is composed of uniform chief cells, which contained 100 to 400 mµ electron-opaque, membrane-limited granules, presumed to be secretory granules, in addition to the usual cytoplasmic organelles. Desmosomes are present between adjacent cells, and rare cilia are observed protruding from the chief cells into the intercellular space. The human parathyroids contain chief cells in two phases—active and inactive—as well as oxyphil cells. Active chief cells have a large Golgi apparatus, sparse glycogen, numerous secretory granules, and rare cilia. Inactive chief cells contain a small Golgi apparatus, abundant glycogen, and few secretory granules. Both forms have the usual cytoplasmic organelles and, between adjacent cells, desmosomes. Oxyphil cell cytoplasm is composed of tightly packed mitochondria and glycogen granules, with rare secretory granules. Cells with cytoplasmic characteristics intermediate between chief and oxyphil cells, possibly representing transitional cells, have been observed. Secretory granules of both man and deer are composed of 100 to 200 A particles and short rods, and the granules develop from prosecretory granules in the Golgi region of the cell. The human secretory granules are smaller and more variable in shape than those of the deer. The granules are iron and chrome alum hematoxylin-positive, argyrophilic, and aldehyde fuchsin-positive, permitting light microscopic identification. They are also found in the capillary endothelial cells of the parathyroid and in its surrounding connective tissue. The secretory granules of the parathyroid cells can thus be followed from their formation in the Golgi apparatus almost to their extrusion into the blood stream.     

THE DISTRIBUTION OF FORMYLTETRAHYDROFOLATE SYNTHETASE IN PLANTS, AND THE PURIFICATION AND PROPERTIES OF THE ENZYME FROM PEA SEEDLINGS

1. Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was foundto be widely distributed in higher plants and the high enzymeactivity was observed in green leaves of Brassica and Alliumspecies, spinach, and in pea seedlings. In pea seedlings, theenzyme activity changed during the course of germination, andmost of the enzyme activity was located in a soluble fractionof the cytoplasm.
2. The enzyme was labile and lost the activityrapidly, even whenstored at 5 in the presence of 0.1 M mercaptoethanol.It was,however, found that ammonium sulfate was very effectivein stabilizingthe enzyme activity.
3. The enzyme has been purifiedapproximately 500-fold from extractsof pea seedlings by treatmentswith ammonium sulfate, protaminesulfate, hydroxylapatite, calciumphosphate gel, and DEAE-cellulosecolumn chromatography.
4. Thepurified enzyme was specific for formate, ATP and FAH₄,andthe Michaelis constants for these reactants were 2.1 10^–2M, 5.1 10^–4 M, and 5.6 10^–3 M, respectively.
5. The optimum pH was found to be 8.0, and the optimal temperaturewas observed at 37. Both NH₄^$ and a divalent cation (Mg^SS orMn^SS) were required for the optimal activity.

¹ Studies on the Enzymatic Synthesis and Metabolism of FolateCoenzymes in Plants. II. (For the previous paper see reference(8)) A part of this paper was presented at the Meeting of theKansai Division of the Agricultural Chemical Society of Japan,Kyoto, January 29, 1966.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells I. Intracellular Site of Synthesis and Transport

The distribution of arabinose-containing macromolecules in suspension-culturedtobacco cells was examined using sucrose density gradients.Exogenously applied ¹⁴Carabinose was scarcely converted intoother sugars, and concentrated in the Golgi-rich fraction (1.15g/cm³) and then secreted to the cell wall. ¹⁴C-Arabinose wasalso incorporated in a lower sucrose density fraction (1.11g/cm³), which contains small vesicles presumably originatedfrom the Golgi apparatus. The arabinose-containing macromoleculesin this fraction was more easily solubilized in water than thosein the Golgi-rich fraction. Alkaline hydrolysis of the macromoleculesindicated that cell-wall glycoprotein is a major component ofthe macromolecules and that the degree of glycosylation is slightlygreater in the lower density fractions than in the Golgi-richfraction. Based on these results, a scheme is suggested in whichthe glycoproteins and polysaccharides are glycosylated in theGolgi apparatus and secreted to the cell wall via secretionvesicles in the low density fraction. The possibility of ¹⁴C-arabinose-containingmacromolecules, in the early phase of synthesis, being a markerof the plant Golgi apparatus is also proposed. (Received September 21, 1980; Accepted January 27, 1981)     

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.