STUDIES OF MEMBRANE FORMATION IN TETRAHYMENA PYRIFORMIS : III. Lipid Incorporation into Various Cellular Membranes of Logarithmic Phase Cultures

When 1-¹⁴C-palmitic acid is used to pulse label logarithmic cultures of Tetrahymena pyriformis, radioactivity appears in lipids of the various membrane types at vastly differing rates. The microsomes and postmicrosomal supernatant attain a high specific radioactivity within 1 min, while the membranes enveloping the cilia require several hours to reach the microsomal level. A similar pattern is obtained when the tracer is sodium 1-¹⁴C-acetate or 8,9-³H-hexadecyl glycerol. In all fractions the phosphonolipid incorporates radioactivity from ¹⁴C-palmitate much less rapidly than do the other major phospholipids. The patterns of labeling suggest that new lipids are transported from a cytoplasmic site of synthesis to points of membrane fabrication throughout the cell.     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : II. Isolation of Macro- and Micronuclei

This report describes a rapid, efficient method for isolating macronuclei from Tetrahymena. The macronuclear fraction contains only small amounts of micronuclear material and little detectable whole cell or cytoplasmic contamination. A method is also described for preparing a "micronuclear fraction" which contains 20–40 micronuclei for every macronucleus present. Electron microscope observations indicate that the ultrastructure of the nuclei in the macronuclear fraction closely resembles that of nuclei in situ. The presence of ribosomes on the outer membrane of micronuclei and of pores in the micronuclear envelope is also described.     

四膜虫S1有性生殖的电镜观察Ⅱ.接合区的形态与配子核的交换

四膜虫接合膜上的小孔是两接合体细胞质相连的通道。配子核形成后,按合膜由于增生而出现装有细胞器等的囊状折叠,并可脱离下来而落入另一细胞中,这可能是胞质交流的又一途径。配子核的交换不是从膜上原有小孔通过的,而是在溶酶体等作用下、使膜破裂,由核后方的微管推动进入对侧细胞中。 本文记述了四膜虫S1有性生殖过程中接合区的形态和配子核的交换。     

CILIARY MEMBRANE DIFFERENTIATIONS IN TETRAHYMENA PYRIFORMIS : Tetrahymena Has Four Types of Cilia

We have examined thin sections and replicas of freeze-fractured cilia of Tetrahymena pyriformis. The ciliary necklace located at the base of all freeze-fractured oral and somatic cilia has been studied in thin sections. Since electron-dense linkers have been found to connect both microtubule doublets and triplets to the ciliary membrane at the level of the necklace, the linkers and the associated necklace seem to be related to the transition region between the doublets and triplets of a cilium. Plaque structures, consisting of small rectangular patches of particles located distal to the ciliary necklace, are found in strain GL, but are absent in other strains examined in this study. In freeze-cleaved material, additional structural differentiations are observed in the distal region of the ciliary membranes of somatic and oral cilia. Somatic cilia contain many randomly distributed particles within their membrane. Oral cilia can be divided into three categories on the basis of the morphology of their freeze-fractured membranes: (a) undifferentiated cilia with very few randomly distributed particles: (b) cilia with particles arranged in parallel longitudinal rows spaced at intervals of 810–1080 Å that are located on one side of the cilium; and (c) cilia with patches of particles arranged in short rows oriented obliquely to the main axis of the cilium. The latter particles, found on one side of the cilium, seem to serve as attachment sites for bristles 375–750 Å long and 100 Å wide which extend into the surrounding medium. The particles with bristles are located at the tips of cilia in the outermost membranelle and may be used to detect food particles and/or to modify currents in the oral region so that food particles are propelled more efficiently into the buccal cavity. Examination of thin-sectioned material indicates that the particles in oral cilia which form the longitudinal rows could be linked to microtubule doublets. Linkage between microtubule doublets and adjacent membrane areas on one side of the cilium could modify the form of ciliary beat by restricting the sliding of the microtubules. It is suggested that membrane-microtubule interactions may form the basis for the various forms of ciliary beat observed in different organisms.     

STUDIES ON THE MAINTENANCE OF ORAL DEVELOPMENT IN TETRAHYMENA PYRIFORMIS GL-C : II. The Relationship of Protein Synthesis to Cell Division and Oral Organelle Development

The effects of puromycin on synchronized Tetrahymena pyriformis were investigated at two different concentrations, 43 µg per ml and 430 µg per ml. The rate of incorporation of histidine-¹⁴C into hot TCA-insoluble material was reduced by 30% at the low concentration and by 80–90% at the high concentration. The rate of oxygen uptake was lowered by only 10–20% at both concentrations. Cell division was prevented at both concentrations, if the drug was added prior to a "transition point" at about 45 min after the end of the synchronizing treatment. Development of "anarchic field" oral primordia was arrested, while primordia in early stages of membranelle differentiation were resorbed. Resorption began shortly after addition of the drug, and proceeded most rapidly at the lower concentration. If the drug was added after the "transition point," cell division and oral primordium formation were completed with only slight delay at the low concentration, and with considerable delay (in some cases complete arrest) at the high concentration. The results thus indicate that protein synthesis is involved in the later as well as the earlier stages of development; what specially characterizes the earlier stages, prior to the "transition point," is a dramatic response to partial inhibition of protein synthesis. It is suggested that this response involves the activation or release of a latent intracellular degradative system which is specific for developing structures.     

STUDIES ON HISTONE FRACTION F2A1 IN MACRO- AND MICRONUCLEI OF TETRAHYMENA PYRIFORMIS

Histone fraction F2A1 has been isolated and purified from macronuclei of the ciliate Tetrahymena pyriformis. It migrates as a single species on sodium dodecyl sulphate-acrylamide gel electrophoresis, with a molecular weight indistinguishable from that of calf thymus F2A1. The solubility properties of Tetrahymena F2A1 are also similar to those of calf thymus F2A1. Electrophoretic analyses on urea-acrylamide gels indicate that Tetrahymena F2A1 consists of four or five subspecies, the two fastest having electrophoretic mobilities identical with those of the two major electrophoretically separable forms of calf thymus F2A1. High resolution (long gel) electrophoresis coupled with incorporation of radioactive acetate both in vivo and in vitro suggest that, as in the case of calf thymus F2A1, differentical acetylation of a parent molecule can explain the observed electrophoretic heterogeneity of Tetrahymena F2A1. Electrophoretic analysis of histones isolated from the micronucleus, which is genetically less active than the macronucleus, indicates that it contains largely the relatively unacetylated (parent) form of histone F2A1.     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : I. RNA Synthesis in Macro- and Micronuclei

Tetrahymena in the log phase of growth were pulse labeled with uridine-³H, fixed in acetic-alcohol, extracted with DNase, and embedded in Epon. 0.5-µ sections were cut, coated with Kodak NTB-2 emulsion, and developed after suitable exposures. Grains were counted above macronuclei, above 1000 micronuclei, and above 1000 micronucleus-sized "blanks" which were situated next to micronuclei in the visual field by means of a camera lucida. An analysis of grain counts showed that micronuclei were less than ½₀₀₀ as active as macronuclei on the basis of grains per nucleus. Since micronuclei contained, on the average, about ½₀ as much DNA as macronuclei, micronuclear DNA had less than 1% of the specific activity of macronuclear DNA in RNA synthesis. However, even this small amount of apparent incorporation was not significantly different from zero. Comparisons of the frequency distributions of labeled micronuclei with those of micronuclear "blanks" showed no evidence of a small population of labeled nuclei such as might be expected if micronuclei synthesized RNA for only a brief portion of the cell cycle. We conclude from these studies that there is no detectable RNA synthesis in Tetrahymena micronuclei during vegetative growth and reproduction.     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : III. Comparison of the Histones of Macro- and Micronuclei by Quantitative Polyacrylamide Gel Electrophoresis

Histones were extracted from isolated macro- and micronuclear fractions and from nucleohistone fibers which were prepared from the isolated macronuclear fraction. Analysis of these histones by polyacrylamide gel electrophoresis indicated that there are electrophoretic differences between the histones of macro- and micronuclei.     

EFFECTS OF DIPEPTIDES CONTAINING THE AMINO ACID,PROLINE ON THE CHEMOTAXIS OF TETRAHYMENA PYRIFORMIS. EVOLUTIONARY CONCLUSIONS ON THE FORMATION OF HORMONE RECEPTORS AND HORMONES

Our investigations demonstrate that proline-containing dipeptides can provoke a chemosensory response from the unicellular Tetrahymena pyriformis The chemotactic effects of the dipeptides have a close relationship with the side chain and the lipophilicity of the amino-terminal amino acid. Comparison of ‘mirror’ variants of proline-containing dipeptides points to the fact that dipeptides with small side chain and non-polar character amino acids (Gly-Pro, Ala-Pro) are preferred on the amino-terminal end. In the case of amino acids with very variable side chains, small (Pro-Gly) and the large side chain and non-polar character amino acids (Pro-Leu, Pro-Phe) on the carboxyl-terminal end can induce significant chemotactic responses. With valine on any terminus the proline-containing dipeptide induced a weak repellent effect.     

MORPHOLOGICAL AND CHEMICAL STUDIES OF COLLAGEN FORMATION : II. Metabolic Activity of Collagen Associated with Subcellular Fractions of Guinea Pig Granulomata

Electron micrographs of thin sections of nuclear, microsomal, and mitochondrial fractions obtained from a carrageenin-induced granuloma showed considerable contamination of the heavier by the lighter fractions. Striated collagen fibrils could be identified in the nuclei + debris fraction. Only a few striated fibrils occurred in the mitochondrial fraction; very fine filaments (diameter 50 A) could be seen in this fraction, but could not be distinguished with certainty from fibrillar material derived from broken nuclei. 35 per cent of the mitochondrial and 80 per cent of the microsomal collagen was extractable by 0.2 M NaCl and could be purified by the standard methods of solution and reprecipitation. The amino acid composition of these collagen fractions determined by ion exchange chromatography was within the range normally found for collagen and gelatin from other mammalian species, allowing for 10 to 20 per cent of some non-collagenous contaminant of the microsomal collagen. Hydroxyproline and proline were isolated by chromatography on paper from hydrolysates of the nuclear, mitochondrial, and microsomal collagen fractions, after incubation of tissue slices with L-¹⁴C-proline. The specific activities of the hydroxyproline from these collagens were in the approximate ratio 1:2:6, while that of bound hydroxyproline derived from the supernatant was only 1, indicating primary synthesis of collagen in the microsomes. Attempts to demonstrate incorporation of L-¹⁴C-proline into collagen or into free hydroxyproline in cell free systems were unsuccessful, nor was it possible to demonstrate non-specific incorporation of L-¹⁴C-valine into TCA-insoluble material by various combinations of subcellular fractions.     

STUDIES OF LUNG METABOLISM : I. Isolation and Properties of Subcellular Fractions from Rabbit Lung

The isolation and partial characterization of subcellular particles from rabbit and rat lung are described. Detailed methods for separating a purified, active mitochondrial fraction are outlined and evaluated in terms of enzymatic, chemical, and morphological criteria. Mitochondrial preparations from rabbit and rat liver were used as comparative indices. The lung mitochondrial fraction was identified by its ability to oxidize succinate with a P/O ratio of 1.7 by a process sensitive to 2,4 dinitrophenol and antimycin A. The adenosine triphosphatase activity of the lung mitochondrial fraction is stimulated by magnesium ions, but this stimulation is not augmented by 2,4 dinitrophenol. In the absence of magnesium ions, the specific activity of the adenosine triphosphatase increases with increasing protein concentration. The presence of lysosomes in the mitochondrial fraction is suggested by acid phosphatase and cathepsin activities and by electron microscope observations.     

NEW MEMBRANE FORMATION DURING CYTOKINESIS IN NORMAL AND CYTOCHALASIN B-TREATED EGGS OF XENOPUS LAEVIS : II. Electrophysiological Observations

The electrical membrane potential (E_m) and electrical membrane resistance (R_m) were measured continuously during the first cleavage of Xenopus eggs, using intracellular microelectrodes. A sharp hyperpolarization of E_m and decrease in R_m can be observed from 6 to 8 min after the onset of cleavage. This moment coincides with the onset of the insertion of new membrane (Bluemink and de Laat, 1973) leading to the formation of the interblastomeric membrane during normal cleavage. Removal of the vitelline membrane or exposure to cytochalasin B (CCB) leads to exposure of the entire surface area of the membrane newly formed during cleavage. These conditions allow for a direct measurement of the permeability properties of the new membrane. It was found that under these conditions E_m reaches values about 3 times more negative and R_m reaches values about 1.5–3 times smaller than during normal cleavage. The extent of reduction of R_m can be correlated with the surface area of the newly formed membrane. We conclude that the new membrane has different ionic permeability properties than the pre-existing membrane (most probably a relatively high permeability for K⁺ ions). Its mean specific resistance is 1–2 kΩ·cm², as against 74 kΩ·cm² for the pre-existing membrane. No influence of CCB on the permeability properties of the pre-existing or new membrane could be detected.     

MITOCHONDRIAL AND CYTOPLASMIC RIBOSOMES FROM TETRAHYMENA PYRIFORMIS : Correlative Analysis by Gel Electrophoresis and Electron Microscopy

Mitochondrial and cytoplasmic ribosomes from Tetrahymena pyriformis have been isolated and studied by the techniques of polyacrylamide gel electrophoresis and electron microscopy used in conjunction. Although the two ribosome types show the same coefficient of sedimentation (80S) in sucrose gradients, they can be distinguished by gel electrophoresis: mitoribosomes migrate in a single band, considerably slower than the cytoribosome band. Electron microscope observations of negatively stained cytoribosomes show typical rounded or triangular profiles, about 275 x 230 Å; mitoribosome profiles are much larger and clearly elongate, about 370 x 240 Å. An electron-opaque spot delimits two nearly equal size subunits. In mixtures of mito- and cytoribosomes, each type can be recognized by its characteristic electrophoretic mobility and by its distinctive fine structure. Cytoribosomal 60S and 40S subunits each produce a distinct electrophoretic band. On the contrary, neither electrophoretic analysis, using a variety of conditions, nor electron microscopy is able to discern two different subunit types in the single 55S mitoribosomal subunit peak. Electrophoretic analysis of RNA shows that both ribosomal RNA species are present in the mitoribosomal subunit fraction. These results establish that mitoribosomes from T. pyriformis dissociate into two subunits endowed with the same sedimentation coefficient, the same electrophoretic mobility, and a similar morphology.     

CYTOCHEMICAL STUDIES OF MITOCHONDRIA : II. ENZYMES ASSOCIATED WITH A MITOCHONDRIAL MEMBRANE FRACTION

1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained ~ 12 per cent of the protein N and ~ 35 per cent of the phospholipide of the whole mitochondria and accounted for ~ 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.     

STUDIES ON BLOOD COAGULATION : II. THE FORMATION OF FIBRIN FROM THROMBIN AND FIBRINOGEN

Although calcium is essential for the formation of thrombin, it can be recovered quantitatively from formed horse thrombin without affecting its coagulating activity. Citrate also has no significant effect. As stated in the text, this does not exclude the possibility that thrombin is actually a calcium compound present in minute concentration; but confirming the results of Hammarsten, it does show that fibrin cannot be a calcium-protein compound unless one assumes molecular weights for fibrinogen greater than 1,000,000. Although the available experimental data concerning the properties of thrombin, the kinetics of its reaction with fibrinogen, and the quantitative relationships between the two do not allow a definite decision as to whether thrombin is an enzyme analogous to rennin, or whether it combines with fibrinogen to form an insoluble compound, fibrin, the weight of evidence does favor the enzyme theory. A given quantity of thrombin can form at least 200 times its weight of fibrin, and in view of the crudeness of the preparation this ratio is probably many times greater. There is no apparent stoichiometric relationship between thrombin and fibrinogen, and thrombin does not disappear from a mixture of the two until the moment of coagulation; the quantity which then disappears is many times the minimal quantity necessary to form the amount of fibrin produced.     

ELECTRON MICROSCOPE STUDIES ON SPERM DIFFERENTIATION IN MARINE ANNELID WORMS. II. SPERM FORMATION IN ARENICOLA BRASILIENSIS

The course of spermiogenesis in arenicola brasiliensis was observed with the electron microscope. The spermatogonia floating in the body cavity seem to proliferate and differentiate to mature spermatozoa in the coelomic fluid. More than a hundred spermatids are connected to one large central mass of cytoplasm and spermiogenesis proceeds synchronously in one cluster, which changes into a sperm-disc during maturation. The pre-acrosomal vesicle originates from the Golgi-body and gradually changes into the acrosomal vesicle of peculiar structure like a cup upside down. In the process of differentiation of the acrosome, a part of the material in the acrosomal vesicle is transferred into the space between the vesicle and the nucleus. The posterior one-third of the cylindrical nucleus is surrounded by four middle-piece mitochondria. The flagellar axoneme originates from one of the centrioles, which is located near a posterior pit in the nucleus.