Ultrastructure of the plasmodial slime moldPerichaena vermicularis

Summary The development of the peridium ofPerichaena vermicularis has been examined using light and electron microscopy and acid phosphatase localization. A newly formed fruiting body consists of undifferentiated protoplasm which is enveloped by a slime coat. Almost immediately after formation of the plasmodiocarp, the protoplasm differentiates into autolytic and fruiting regions. The autolytic region is located at irregular intervals between the slime coat and the fruiting region and separated from both of them by membranes.Soon after the autolytic region has formed, additional signs of degeneration appear in the autolytic region including unusual appearance of nuclei, increase in autophagic vacuoles, and the presence of clear areas in the ground substance. The plasma membrane, which once completely separated the slime coat from the autolytic region, is no longer continuous. Electron micrographs of the autolytic region from later developmental stages show formation of extensive channels which contain protoplasm in various stages of degradation. Acid phosphatase is present in the channels of the autolytic region. The morphological evidence and the presence of hydrolytic enzyme suggest the region is being digested and re-adsorbed.After the autolytic region has been digested, an even layer of peridial wall material is laid down, and at regular intervals additional wall material is produced. The additional wall material forms the reticulation on the inside of the peridial wall.This work was supported by National Sciences Foundation grants (GB-5883 and GB-8537) to Dr.Ian K.Ross and an NSF Traineeship (GZ 445 and 796) to I.Charvat.This constitutes a portion of a thesis presented to the Regents of the University of California by the first author in partial fulfillment of the requirements for the Ph. D. degree.     

Ultrastructural and cytochemical studies of salivary gland regression in Chironomus tentans

Summary The ultrastructural localization of acid phosphatase (AcPase) activity in regressing salivary gland cells of Chironomus tentans was studied with Gomori's lead method. In last instar intermolt larvae AcPase activity is restricted to Golgi vesicles, to small electrondense bodies of about 0.25 diameter, and to larger, more electron-lucid bodies which are considered to be lysosomes. The smaller bodies apparently arise from Golgi vesicles. The average frequency of lysosomes increases as development proceeds. Until the end of the pupal molt, only very few of them contain degenerating fragments of other cellular components.Overt cell regression begins in young pupae. At this stage practically all lysosomes contain degenerating cell components. In addition, cellular breakdown seems to occur outside of these organelles. Regressing cellular areas show in addition free AcPase reaction products (lead deposits), the amount of which closely parallels the degree of regression of the particular area.Possible genetic relationships between the various AcPase-containing cell organelles and the role of lysosomes in the control of gland cell breakdown are discussed.Supported by NSF Grant GB-2639 to U. Clever. The technical assistance of Mr. Hermann Bultmann in part of these studies is gratefully acknowledged.     

The fine structural localization of acid phosphatase in the gut epithelial cells of the slug,Avion ater (L.)

Summary Acid phosphatase, generally thought of as a lysosomal enzyme and indeed widely employed as a lysosomal marker, has been found associated with the Golgi complex of all cell types from the crop, intestine and digestive gland ofArion ater. Reaction product was also detected within the multivesicular bodies and cytoplasm of columnar cells from the crop and the multivesicular bodies of mucous cells from the intestine. A vacuolar localization was obtained in the digestive cells of the intestine and digestive gland. Secretory protein granules in the calcium cells of the same gland and apical vacuoles in the so-called thin cells also showed a positive reaction.This work was undertaken as part of a slug research project under the direction and co-ordination of Dr. D. K.Roach, supported by A.R.C. Assistance was given by Mr. T. R.Mainwaring in the preparation of tissue for electron microscopy.I would like to thank Professor J.Brough and Professor D.Bellamy for providing facilities and encouragement.     

Gleichzeitiger Nachweis von phenolarmen und phenolreichen Phasen in den Blattepidermiszellen vonSalix alba L.

Zusammenfassung In vielen Epidermiszellen der Blattoberseite vonSalix alba L. wurden in den Vakuolen stark und schwach lichtbrechende Phasen nebeneinander beobachtet. Mit dem Vitalfarbstoff Brillantcresylblau und verschiedenen mikrochemischen Nachweisen konnten sekundäre Vakuoleninhaltsstoffe innerhalb einer Zelle getrennt voneinander nachgewiesen werden. Während in den stark lichtbrechenden Gebilden alle Reaktionen auf Gerbstoffe (Gemische mit vielen phenolischen OH-Gruppen) positiv verliefen, wiesen die Reaktionen in den schwach lichtbrechenden Phasen auf Flavonoide und ihre Derivate (Gemische mit weniger phenolischen OH-Gruppen) hin.

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Simultaneous demonstration of separate phases with different phenol content in the leaf epidermis cells ofSalix alba L.

Summary Strongly and weakly refractive phases occur in many vacuoles of the upper leaf epidermis ofSalix alba L. Different secondary vacuolar substances could be demonstrated separately in the interior of single cells by vital staining with brillant cresyl blue as well as by various microchemical assays. Only flavonoids (substances containing few phenolic OH-groups) could be detected in the weakly refractive phase whereas tannins (substances rich in phenolic OH-groups) could be demonstrated in the strongly refractive phases.

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Frau Prof. Dr.Maria Luhan zum 60. Geburtstag gewidmet.

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Die Autorin dankt Herrn Prof. Dr.Helmut Kinzel, Pflanzenphysiologisches Institut der Universität Wien, und Herrn Doz. Dr.Hanno Richter, Botanisches Institut der Universität für Bodenkultur, Wien, für ihre freundliche Diskussionsbereitschaft und Herrn Dr.Robert Kartusch für die Anfertigung der Abbildungen.     

Notes on the ultrastructure and sporoblast development in fish parasitizing myxosporidian of the genusSphaeromyxa

Summary The ultrastructure of trophic stage (plasmodium), spore and changes in fine structure during morphogenesis of the spore were studied by electron microscopy in two representatives of the genusSphaeromyxa. Plasmodium has a highly differentiated structure; there is an outer layer of homogeneous plasm, the endoplasm consisting of a vacuolated mass in which float generative cells and sporoblasts in different degree of development. Generative cells have well developed pseudopodia. Sporoblasts arise from the union of two cells, out of which the inner one forms all cells of the spore, the outer one has only an enveloping function. Polar capsule develops in a way identical with other myxosporidia; the wide filament, however, has a longitudinally folded structure and is located within the capsule in two loose loops. The mitochondria of an early sporoblast are characterized by a high content of DNA. The identity of polar capsule development with the nematocyst morphogenesis is too conspicuous to be taken for a mere convergence. Studies on ultrastructure give no evidence for a protozoan character of myxosporidia; together with other findings, they are in favor of the nonprotozoan nature of this group of organisms.The essential part of this work was carried out in the Department of Biology, University of Illinois at Chicago Circle, where this study was aided by a grant from the National Science Foundation (NSF GB-2800) to Dr.J. Corliss. The author is also indebted to Dr.R. Fernald, University of Washington, for the use of all facilities at the Friday Harbour Laboratories of the University of Washington.     

The comparative ultrastructure and possible function of eyespots: Euglena granulata and Chlamydomonas eugametos

Summary The structure of Euglena granulata and Chlamydomonas eugametos has been studied using polarization and electron microscopy, cinematography, and chemical extraction procedures, with the main focus on the structure of the eyespot.The 50–60 granules which form the extrachloroplastic eyespot of E. granulata are large bodies, up to 1200 m in diameter. They are found in the cytoplasm near the base of the reservoir and are associated with the parabasal body which contains a large crystal. The eyespot granules are contained within membranes having a unit membrane structure; 2 or 3 are usually present in a single eyespot packet; microtubules are also contained within the packet. The eyespot granules have the structure of a positive spherite and clearly exhibit birefringence; this structure is modified by fixation.The granules of the chloroplastic eyespot of C. eugametos are about 75 m in diameter and are contained within the chloroplast in an ordered array. Occasionally, the eyespot contains elongate or helical bodies mixed with the granules. Extraction with organic solvents caused the removal of materials which formed the eyespot granules as well as that of the osmophilic globules in the chloroplasts.Several hypotheses which concern the function of the eyespots in these and other species are discussed in the light of our results. the possible origin and demise of the eyespot granules are also discussed.Supported in part by NSF Grant GB-313. We thank Dr. Harold C. Bold and Dr. W. Gordon Whaley for their support and encouragement, Dr. R.M. Brown, jr. and Dr. Tom Kantz for aid in cinephotography, Dr. Peter Sitte for his help with polarization microscopy, and Mrs. Virginia Stork for her excellent technical assistance.A preliminary report of this research was presented at the Annual Meeting, Phycological Society of America, American Institute of Biological Sciences, College Park, Maryland, August, 1966.Contribution No. 286 from the Department of Botany, The University of Tennessee, Knoxville.     

Zur Kenntnis vonTetrasporopsis fuscescens

The macroscopic colonies ofTetrasporopsis fuscescens consist of a tegument of irregularly folded bag-like mucilage. It contains isolated ellipsoidal cells arranged in a regular peripheral stratum and rather sparsely and irregularly beneath. They show polarity, 4 parietal chromatophors when fully developed, have a wall containing cellulose, and no contractile vacuoles. Naked protoplasts are released from them through circular openings oriented towards the surface. Flagellated cells and cysts were not observed.T. reticulata may be closely related toT. fuscescens, but probably no other species is allied.

Herrn Univ.-Prof. Dr.Walter Leinfellner zum 70. Geburtstag gewidmet.     

Some studies on the fluorescence of mast cells after paraformaldehyde treatment

Summary Air dried peritoneal fluid and tissue spreads from rat, mouse, hamster, rabbit, guinea pig, cat, chicken, and frog were treated with paraformaldehyde (pCHO) at 80 ° C for 1 hr. Only rat and mouse mast cells fluoresced. In the rat, mast cells fluoresced whether present in vascular or avascular areas of mesentery, in fat depots, or in peritoneal fluid. Photographs were obtained by fluorescence microscopy, the preparations were then stained and the same fields rephotographed in white light. Comparison of the photographs showed that every fluorescent rat mesentery mast cell also stained with acidified toluidine blue and with Astrablau; a few fluorescent cells did not stain with toluidine blue and an occasional cell that did not fluoresce stained with this dye or with Astrablau. Paraformaldehyde depressed somewhat toluidine blue, inhibited strongly Astrablau, and abolished Alcian blue binding. It had no effect on the staining of purified heparin in model experiments.Reserpine administration in the rat did not prevent visualization of mast cells by the pCHO method.These experiments indicate that all rat mast cells contain serotonin, regardless of cell size (age ?) or site and suggest that no massive, cyclic release of this amine occurs either physiologically or in response to reserpine treatment.Some of the experiments for this paper were carried out in the laboratory of Dr. G. Bloom, Department of Cell Research and Genetics, Karolinska Institutet, Stockholm, Sweden, (September and October, 1963). I wish to acknowledge the help and cooperation of the members of his staff, and especially Dr. Martin Ritzén, whose open, warm, efficient and enthusiastic manner enabled me to accomplish much in a relatively short time. Availability of darkroom facilities to pursue much of the work in the laboratories of Dr. W. Bargmann, Anatomisches Institut, Neue Universität, Kiel, Germany and of Dr. R. Robineaux, Hôpital St. Antoine, Paris, is also gratefully acknowledged. Grant support was furnished by the American Heart Association (62 G 118) and NSF (GB-4166) (to the author), by NIH 5 T 1 GM 102, and by the Swedish Medical Research Council (to Prof. B. Uvnäs and Dr. G. Bloom).This paper is dedicated to Dr. Berta Scharrer on the occasion of her 60th birthday.     

Membranous localization and properties of ATPase of rat liver lysosomes

Summary Lysosomes isolated from rat liver were found to have ATPase activity (EC No. 3.6.1.3). Subfractionation of the lysosomes revealed a membranous localization of ATPase activity. The enzyme has half maximal activity at 0.2mm ATP and is inhibited by high concentrations of ATP. The apparentK _m for divalent metal is 0.2mm, and either ca²⁺ or Mg²⁺ give maximal activity.The ATPase activity has latency when lysosomes are isolated from rats treated with Triton WR-1339. This latency may be due to the presence of internalized sucrose because the activity ofL fraction lysosomes is much less latent and Triton WR-1339 itself is not inhibitory. The latency of glucosamindase, a marker enzyme for lysosomes, contrasts with the low latency of the ATPase and points to an ATPase with an exposed active site in intact lysosomes.     

On the nonidentity of several carnivore hemoglobins

Antibodies were prepared in rabbits to the hemoglobin of the dog (Canis familiaris). Each of the major adult hemoglobins of several canoid and pinniped species was compared to that of the dog using these antibodies in the micro-complement fixation (MC'F) procedure. Each non-Canis hemoglobin tested reacted less well than that of the dog, and each cross-reaction value was unique. The hypothesis of Seal (1969) of identity of primary structure among these hemoglobins is thus made highly suspect.This research was supported by NSF Grants GB-20750 to the author and GB-13119 to Dr. A. C. Wilson.     

The reticular substance of the medulla oblongata of the albino rat

Summary The region of the reticular giganto-cellular nucleus, perfused with formalin and postfixed in osmium tetroxide, was studied with histochemical and electron microscopic techniques. The perikarya of the neurons have two zones. The peripheral cytoplasm contains Nissl bodies, mitochondria, and free RNP particles. The juxtanuclear cytoplasm contains the Golgi complex, mitochondria, RNP particles and dense bodies. The nucleus is indented and has a prominent nucleolus and a paranucleolar body. Dense bodies are found along the axon and dendrites as well. Three different types of synapses are described and two types of synaptic vesicles (spherical and ellipsoidal) are shown.The capillary endothelium shows microvilli and marginal flaps. The endothelial cytoplasm contains vacuoles, micropinocytotic vesicles, and a few dense bodies. Processes of pericapillary cells, surrounded by a basement membrane, also contain dense bodies. The dense bodies found in the neurons and endothelial cells show acid phosphatase activity. On the basis of their morphology and their enzymatic activity these bodies are identified as lysosomes.Partially supported by a school grant No RF 62051 from the Rockefeller Foundation, New York, USA, and Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.Fellows of the Consejo National de Investigaciones Científicas y Técnicas, Argentina. The authors wish to thank Dr. Mario H. Burgos for his constant interest and criticism, and to Dr. Eduardo Rodriguez Echandia and Dr. Fabio L. Sacerdote for their valuable assistance.     

The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii

Summary The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii, at different times after feeding, has been examined by cytochemical techniques at electron microscope level and by measurement of yeast viability. The measurement of viability, combined with cytochemistry is presented as a novel method of examining the progress of digestion. Particular attention has been given to the temporal development of digestion.Vacuoles, probably primary lysosomes, have been identified containing acid phosphatase activity within minutes of feeding and these accumulate around and fuse with phagocytic vacuoles. Acid phosphatase levels in the digestive vacuoles appeared highest at 20 to 40 minutes. Yeast digestion was observed and yeast viability began to decline at this time. Mixing of autophagic and heterophagic material was also observed. At least half of the yeast population was still viable after 90 minutes.Our method (p-nitrophenyl phosphate) of enzyme localization has demonstrated plasma membrane associated acid phosphatase activity.     

Ultrastructural localization of some phosphatases in the prothoracic gland of the insect Leucophaea Maderae

Summary The cytochemical localization of acid phosphatase and thiamine pyrophosphatase activity was studied by light and electron microscopy in prothoracic gland cells of the cockroach Leucophaea moderae. Nymphal and young adult animals were used.Prominent sites of acid phosphatase activity included large membrane-bounded dense bodies or lysosomes, and certain cisternae of the Golgi apparatus. The results suggest a possible difference in the enzymatic activity toward glycerophosphate and aromatic phosphates as substrates.Thiamine pyrophosphatase activity was localized in elements of the Golgi apparatus and endoplasmic reticulum, and in lysosome-like dense bodies. This latter activity was abolished by sodium fluoride treatment, whereas the phosphatase activity in the Golgi apparatus and endoplasmic reticulum is unaffected by such inhibition.The cytochemical results confirm through direct evidence the suggestions of Scharrer (1964), that the large dense bodies present in the prothoracic gland cells are lysosomes, and that their activity may be related to stages in the life history of the glands. Furthermore, the lysosomes or their derivative structures may play an essential role in the autolysis of the prothoracic glands toward the end of their active period.The enzymatic activity of the endoplasmic reticulum may indicate the involvement of this organelle in the metabolism of steroid-like precursor materials necessary for the synthesis of ecdysone.This study was supported by U.S.P.H.S. grants 5 T1-MH-6418 and NB-05219, and grant RO 1-AM-3984 to Dr. Berta Scharrer. I would like to express my appreciation to Dr. Scharrer for her encouragement and assistance during this study. I also wish to thank Mrs. Sarah Wurzelmann for her competent technical aid.     

GROWTH AND DEVELOPMENT OF THE MYXOMYCETE PERICHAENA VERMICULARIS. II. CHROMOSOME NUMBERS AND NUCLEAR CYCLES

Chromosome counts of dividing nuclei of the myxomycete Perichaena vermicularis indicate a number of 25 ± 2 in the amoebae and 50 ± 4 in the Plasmodia, confirming earlier reports that amoebae are haploid and plasmodia diploid. Chromosome numbers obtained from nuclei during sporangial development indicate a fluctuation in the location of meiosis influenced by environmental conditions. The implications of these observations are discussed in reference to past conflicting evidence of the location of meiosis.     

Gammarus desperatus,a new species from New Mexico (Crustacea : Amphipoda)

A new species of the Amphipoda (Gammarus desperatus) is described from North Spring in Roswell, Chaves County, New Mexico. Apparently this is the same species reported erroneously as G. fasciatus Say from nearby Lander Springbrook by Noel (1954).Supported in part by NSF Grants GB-2461 and GB-6477X to W. L. MinckleySupported in part by NSF Grants GB-2461 and GB-6477X to W. L. Minckley     

Antigenic relationships between Aracoccidioides loboi and other pathogenic fungi determined by immunofluorescence

Summary The fluorescent antibody technique was used to study antigenic relationships betweenParacoccidioides loboi and other pathogenic fungi. The findings suggest thatP. loboi is more closely related antigenically to certainP. brasiliensis strains than to others and that it has antigens in common with the yeast form ofHistoplasma capsulatum, H. duboisii, Blastomyces dermatitidis, Candida albicans and also the mycelial form ofCoccidioides immitis. Serum globulins from 3 cases of keloidal blastomycosis were labelled with fluorescein isothiocyanate. These conjugates showed slight or no reactivity withP. loboi, the yeast forms ofP. brasiliensis, H. capsulatum, H. duboisii andB. dermatitidis, However, they stained brightlyC. albicans, serotypes A and B, the tissue form ofC. immitis and the yeast form ofSporotrichum schenckii. Adsorption of these reagents withC. albicans eliminated all staining except that forS. schenckii. These patients had no history of clinical sporotrichosis.Deceased. Last address: Fundacão Gonçalo Moniz, Salvador, Bahia, Brazil. Requests for reprints should be sent to Dr.William Kaplan.Dr.Miranda is in private practice in Rio de Janeiro, Brazil.     

Pleiotropic Effects of a Temperature-Sensitive Mouthless Mutation in Tetrahymena thermophila on the Excretion of Extracellular Enzymes

The mode of enzyme excretion was investigated during balanced growth in wild type Tetrahymena thermophila and in a temperature-sensitive mutant that did not form a mouth and food vacuoles at the restrictive temperature. The mutant was used to determine which of the extracellular enzymes are normally excreted by defecation. During balanced growth at the permissive temperature the excretion of enzymes was constant, and maximal in complex medium. No protease activity against azocasein was detected. Changes in the growth temperature of the wild type cells only affected the release of 3′-nucleotidase. For the mutant, however, the excretion changed markedly with increasing temperature: (a) ribonuclease, deoxyribonuclease, α-glucosidase, and β-glucosidase were detected in 0–10% of normal amounts; (b) 3′- and 5′-nucleotidase, not measured previously in Tetrahymena, were found in 10- to 14-fold of normal amounts; (c) excretion of acid phosphatase was unaffected. We therefore conclude that the extracellular enzymes (a) are released by defecation, that 3′- and 5′-nucleotidase are secreted through the membrane system, and that acid phosphatase is released by lysosomes emptied through pinocytosis. It is proposed that the lysosomes used for the formation of digestive vacuoles are different from those used for the formation of pinocytotic vacuoles and for autophagic vacuoles.     

Effect of prior vaccination on experimental Blastomycosis

Immunogens were found associated with particular fractions prepared from spherules ofCoccidioides immitis (Kong, Levine &Smith, 1963) and from yeast cells ofHistoplasma capsulatum (Salvin &Ribi, 1955). However,Blastomyces dermatitidis, another dimorphic systemic fungal pathogen was shown to elicit a minimal immunogenic response in experimental animals (Kong &Levine, 1967). It was therefore deemed pertinent to study factors which might enhance the resistance of mice to infection withB. dermatitidis.     

Die Stipeln derIrvingioideae undRecchioideae und ihre systematische Wertung nebst Bemerkungen zur Holzanatomie und Palynologie

TheSimaroubaceae generally have no true stipules. The stipule-like appendages of some genera proved to be pseudo- or metastipules (Weberling & Leenhouts 1965). There seem to be some exceptions, however: the generaCadellia (incl.Guilfoylia) andRecchia on the one hand, and theIrvingioideae on the other. As these taxa, with exception ofRecchia, have simple leaves, there are no indications that their stipule-like appendages might be pseudo- or metastipules. In regard to their position and ontogeny these appendages behave completely like true stipules. Assuming the view ofForman, one could conceive a morphological line from the long, broadly inserted axillary stipules of mostIrvingioideae to the small scaly triangular stipules ofIxonanthoideae. The similarities between the stipules ofIrvingioideae andErythroxylaceae (already emphasized byHallier and others), become even more evident when their ontogeny is investigated. TheIrvingioideae, therefore, might be regarded as a separate family (perhaps with some relation to theErythroxylaceae,Hallier) or as a subfamily ofIxonanthaceae (Forman).—In addition to data on stipules some results on the palynology and shoot anatomy of the generaCadellia (incl.Guilfoylia) andRecchia are reported. Their relationship with theSimaroubaceae also appears doubtful. If they are to be included, they represent a somewhat isolated group near the base of the family which otherwise has lost its stipules.

Herrn Univ.-Prof. Dr.Walter Leinfellner zum 70. Geburtstag gewidmet.     

The genetic control of sporulation in Saccharomyces. II. Dominance and complementation of mutants of meiosis and spore formation

Summary Heat-sensitive sporulation-deficient (spo) mutants ofS. cerevisiae may be either dominant or recessive. The number of loci which can mutate to thespo phenotype has been estimated to be 48±27 from complementation studies. Comparison of the wild type and mutants by light microscopy after exposure to sporulation medium at the restrictive temperature and Giemsa staining has shown that mutant populations can not complete the meiotic nuclear divisions.Supported by NSF grants GB-8564 and GB-27688, and the Wallace C. and Clara Abbott Memorial Fund from the University of Chicago.