bluuebunny/crossref_metadata_2025 huggingface dataset:
SOME OBSERVATIONS ON THE NUCLEAR ENVELOPE
Authors: Whaley W. Gordon , Mollenhauer Hilton H. , Leech James H.
Published: May 2004

In maize root meristem cells, fixed in KMnO4, embedded in epoxy resin, ultrathin sectioned, and studied with an electron microscope, the nuclear envelope is demonstrated to be a double membrane structure. In the nuclear envelope there are: pores of the sort reported in many species of animals and plants; different types of openings associated with extensions of both nuclear membranes into the cytoplasm; and also, often, large discontinuities. The nuclear envelope is a component of the general vesicular reticulum. The reticula of neighboring cells including the nuclear envelopes make up, at certain stages at least, a "systemic" structure. The status of the nuclear envelope as a component of the general cellular reticulum is recognized to change during differentiation. The existence of several types of discontinuities in the nuclear envelope and the extent of nuclear-cytoplasmic surface relationships indicated suggests alteration in concepts of transport and exchanges between nucleus and cytoplasm.
THE MICROFIBRILLAR STRUCTURE OF THE CELL WALLS OF THE FILAMENTOUS FUNGUS, Allomyces
Authors: Aronson Jerome M. , Preston R. D.
Published: May 2004

Cell walls of the fungus, Allomyces, were isolated by chemical procedures, using either potassium permanganate oxidation or glacial acetic acid-hydrogen peroxide treatment followed by dilute mineral acid. The structure of the treated walls was investigated by means of electron microscopy and electron diffraction analysis which showed that rhizoidal walls were especially suitable for observation. Chitin microfibrils exist in the extreme tips of rhizoidal walls, and tend to lie in a preferred longitudinal orientation. Older rhizoidal wall segments show a crossed fibrillar structure under a thin layer of short randomly arranged microfibrils. In the possession of systems of crossed fibrils these walls are like the cell walls of certain green algae. Walls of branch rhizoidal filaments were observed in the early stages of development, in which case the observed microfibrillar orientations are such that it is possible to envisage their origin from pre-existing fibrils that have passively reoriented. With respect to the continued growth of the filaments, however, it is difficult to explain the observed microfibrillar arrangements in terms of the "multi-net" theory.

Hyphal walls usually show two layers, the outer consisting of microfibrils arranged randomly, and the inner consisting of well oriented microfibrils running parallel with the longitudinal axis of the hypha. The oriented inner layer appears to be similar in structure to the secondary wall of the Phycomyces sporangiophore.
ELECTRON MICROSCOPE AND X-RAY DIFFRACTION STUDIES OF THE EFFECTS OF DEHYDRATION ON THE STRUCTURE OF NERVE MYELIN
Authors: Finean J. B.
Published: May 2004

The dehydration of rat optic nerve has been studied by allowing specimens to become partially or fully dried before fixation and preparation for electron microscopy. A correlation is established between electron micrographs of the myelin sheath and corresponding small-angle x-ray diffraction patterns. The modifications of the optic nerve myelin layers during drying were very similar to those described in more detail for the myelin of frog sciatic nerve. The most striking difference was that the system of fine layers characteristic of the fully dried myelin was much more extensive in the case of the optic nerve, and the layer thickness was significantly greater than the corresponding layer in the frog sciatic nerve preparation. The significance of these correlations is discussed.
EXTRACTION, HYDROLYSIS, AND ELECTROPHORETIC ANALYSIS OF RIBONUCLEIC ACID FROM MICROSCOPIC TISSUE UNITS (MICROPHORESIS)
Authors: Edström Jan-Erik
Published: May 2004

A procedure for the purine-pyrimidine analysis of RNA in the 100- to 1000 µµg. range is presented. It includes hydrolysis and electrophoretic analysis of RNA, which is extracted from single isolated tissue units, like single cells. The quantitative determination of the separated compounds is carried out by a photographic-photometric procedure in ultraviolet light. The determined values show a coefficient of variation of about ±7 per cent on test substance. Microelectrophoretic analyses of RNA from different sources have been performed and are compared to macrochemical analyses. The agreement is good in those cases in which it is possible to get any information at all through macrochemical analyses.
COMPOSITION OF RIBONUCLEIC ACID FROM VARIOUS PARTS OF SPIDER OOCYTES
Authors: Edström Jan-Erik
Published: May 2004

Microphoretic purine-pyrimidine analyses of the ribonucleic acid (RNA) in nucleoli, nucleoplasm, cytoplasm, and yolk nuclei of spider oocytes have been carried out. The material necessary for the analyses was isolated by micromanipulation. Determinations of the amounts of RNA in the different parts of the cell were also performed. No differences between the composition of RNA in the nucleolus and the cytoplasm could be disclosed. Nucleoplasmic RNA was, on the other hand, distinctly different from that in the nucleolus and in the cytoplasm. The difference lies in the content of adenine, which is highest in nucleoplasmic RNA. The few analyses carried out on yolk nuclei showed their RNA to be variable in composition with a tendency to high purine values. The cytoplasm contains about 99 per cent of the total RNA in these cells, the nucleoplasm about 1 per cent, and the nucleolus not more than 0.3 per cent, although the highest concentrations are found in these latter structures. When considered in the light of other recent findings the results are compatible with the view that nucleolar RNA is the precursor of cytoplasmic RNA.
METABOLIC AND ULTRASTRUCTURAL CHANGES INDUCED IN ADIPOSE TISSUE BY INSULIN
Authors: Barrnett Russell J. , Ball Eric G.
Published: May 2004

The addition in vitro of insulin to rat adipose tissue (epididymal) produces marked metabolic changes which may be followed by measurement of the net gas exchange of the tissue. Using this method to monitor the metabolic action of insulin, concomitant observations with the electron microscope on the tissue have been made. These reveal that pronounced morphological changes are induced by insulin. The plasma membranes of the adipose cells become invaginated at many sites to form minute finger-like indentations. Numerous tiny, membrane-bounded vesicles are also present and arranged in relationship to the plasma membrane in such a way as to suggest that their formation occurred when a recessed fold was pinched off. Deeper in the cytoplasm, especially in specimens that had been incubated a longer time, numerous large, smooth, membrane-limited vesicles are seen. Finally, in these incubated specimens the cytoplasmic matrix has lost much of its granular nature, small lipid droplets are frequently found in the cytoplasm and suggestive changes have occurred in mitochondria. In control specimens, incubated without insulin for identical periods of time, indentations and vesicles in the plasma membrane are sparse at best and no vesicles or membrane-bound spaces appear deeper in the cytoplasm. The metabolic and morphologic changes induced by insulin seem to be interdependent events. Both changes appear to be initiated rapidly and concomitantly in the tissue. Both processes are initiated by insulin at concentrations considered to be physiological, 0.004 µg. (100 µunits) per ml. Insulin treated with alkali fails to initiate either process. It is concluded that insulin initiates pinocytosis in rat adipose tissue and the possible significance of this process in the mode of action of insulin is discussed.
THE ULTRASTRUCTURE OF CARDIAC DESMOSOMES IN THE TOAD AND THEIR RELATIONSHIP TO THE INTERCALATED DISC
Authors: Grimley Philip M. , Edwards George A.
Published: May 2004

The fine structure of desmosomes and intercalated discs in the toad heart is discussed. A definite relationship between the dense components of these structures and the dense region of the Z band is demonstrated. The dense region of the Z band characteristically widens at its approach to the plasma membrane, and often terminates beneath it in a distinct discoidal plaque. Cardiac desmosomes appear to be structures which result from the intimate apposition of plaques of Z band material. These desmosomes retain the Z band function as sites of attachment for myofilaments. The suggestion is made that rotation of a desmosome through 90° and splitting of filaments from the adjacent sarcomere could result in the formation of a simple step-like intercalated disc. Intermediate stages in this process are illustrated. Complex discs present in the toad probably represent the alignment of groups of simple discs produced by contractile forces. Possible physiologic functions of the disc and desmosome are discussed. Other morphologic features of toad cardiac cells include a distinct amorphous outer coat to the sarcolemma, a prominent N band, and a granular sarcoplasm with poorly developed reticulum.
PHASE CONTRAST OBSERVATIONS OF THE ENDOPLASMIC RETICULUM IN LIVING TISSUE CULTURES
Authors: Rose George G. , Pomerat C. M.
Published: May 2004

Cells from three human sources (two malignant and one fetal) were observed through phase contrast microscopy to contain unusual cytoplasmic images. These were photographed and are discussed as representing the endoplasmic reticulum in the living cell.
THE FINE STRUCTURE OF MEISSNER's TOUCH CORPUSCLES OF HUMAN FINGERS
Authors: Cauna Nikolajs , Ross Leonard L.
Published: May 2004

Thin slices of the finger pads of six individuals were fixed in buffered 1 per cent osmic acid, embedded in deaerated, nitrogenated methacrylate, and cut into thin sections for electron microscopic study. Before embedding, the slices were trimmed so as to include several digital tactile corpuscles. Some thin sections were stained in 10 per cent aqueous phosphotungstic acid solution. The principal part of Meissner's corpuscle is made up of flattened laminar cells stretching across the corpuscle in irregular layers. The perinuclear cytoplasm of these cells contains numerous small mitochondria, a sparse granular endoplasmic reticulum, and a large number of small vesicles. Nerve fibers enter the side or base of the corpuscle, lose their myelin sheaths, and follow a meandering course between the laminar cell plates. The nerve endings enter into a close appositional relationship with the flattened portions of the laminar cells. In some areas the apposed axolemma and cell membranes are slightly thickened with small vesicles located along the cell membrane or on both surfaces. These regions are interpreted as synapses. The most prominent feature of the nerve endings is an extraordinary accumulation of small mitochondria which vary in size and internal density. The nerve endings also contain vacuoles, groups of dense concentric membranes, and small dense vesicles of irregular distribution. The laminar cells are separated from one another by a dense intercellular substance of uniform thickness which also envelops the entire corpuscle. This material contains randomly oriented collagen fibers and fine fibrils bound together by a dense material at nodal points recurring at regular intervals of approximately 120 mµ. These findings are discussed in relation to the problems of the function of Meissner's corpuscle, neural material loss and replacement, and the presence of synapses.
PARTICIPATION OF THE CYTOPLASMIC MEMBRANE IN THE GROWTH AND SPORE FORMATION OF BACILLI
Authors: Fitz-James Philip C.
Published: May 2004

A polyester embedding technique was used to study the early stages of spore formation in members of the genus Bacillus in order to investigate further the origin and nature of the initial spore septum and the resulting forespore envelope. Whereas previously, with a methacrylate procedure, this layer had appeared to be continuous with the cell wall, this study reveals it as a double layer of cytoplasmic membrane. Perisporal, membranous organelles connected both to the developing forspore envelope and to the cytoplasmic membrane were encountered in the four species studied. Similar organelles were prominent during growth at the sites of transverse septa formation. These were connected to, or continuous with, the cytoplasmic membrane and often adherent to the chromatin bodies of the dividing bacilli.
IDENTIFICATION OF GLYCOGEN IN ELECTRON MICROGRAPHS OF THIN TISSUE SECTIONS
Authors: Revel Jean Paul , Napolitano Leonard , Fawcett Don W.
Published: May 2004

The electron microscopic appearance of glycogen has been studied in the organs of several animal species. Glycogen almost always appears as roughly circular granules from 150 to 400 A in diameter. The intrinsic electron density of glycogen varies from tissue to tissue; however, treatment with lead hydroxide as described by Watson deeply stains the granules. Glycogen pellets were isolated from some of the tissues studied by centrifugation. Such pellets were shown to be glycogen by chemical and histochemical criteria. When thin sections of the pellet are examined under the electron microscope they can be seen to consist of densely packed granules similar to those found in the intact tissues. Such pellets are also stained for electron microscopy by short exposure to lead hydroxide.
THE STAINING OF THIN SECTIONS OF MOUSE PANCREAS PREPARED BY THE FERNÁNDEZ-MORÁN HELIUM II FREEZE-SUBSTITUTION METHOD
Authors: Bullivant Stanley
Published: May 2004

Small pieces of mouse pancreas were rapidly frozen in helium II, substituted in methanol at -75°C., and embedded in methacrylate by ultraviolet polymerization in the cold. The unstained cells show a structure similar to that after OsO4 fixation, except that the RNP particles have little or no contrast and the mitochondria and Golgi zones appear as grey areas without internal structure. After staining the sections by floating them on solutions of lead acetate or osmium tetroxide, there is an increase in contrast of RNP particles, ergastoplasmic membranes, and zymogen granules. Mitochondrial and Golgi membranes, zymogen granule membranes, and a membrane along the outside of the ergastoplasmic cisterna appear in negative contrast. The structure of the ergastoplasm, the existence of RNP particles, and the production of negative contrast are discussed. A modification of Gomori's method for acid phosphatase produces a lead deposit around the periphery of the zymogen granules. Possibly this deposit does not represent the true site of the enzyme, but the results show the feasibility of histochemistry at the level of resolution of the electron microscope.
HEMOGLOBIN ABSORPTION BY THE CELLS OF THE PROXIMAL CONVOLUTED TUBULE IN MOUSE KIDNEY
Authors: Miller Fritz
Published: May 2004

The formation of protein absorption droplets in the cells of the proximal convolution was studied in mouse kidney. Ox hemoglobin was administered intraperitoneally and kidney specimens were collected at intervals of 30 minutes to 4 days after injection. In the lumen of the nephron, hemoglobin was concentrated to an opaque mass whose relations with the brush border and the epithelium could be easily followed. It was found that hemoglobin passes through the brush border in between the microvilli, enters the channels of tubular invaginations at the bases of the brush border, and is transported in bulk into vacuoles in the intermediate cell zone. These vacuoles increase in size and are transformed through further concentration into dense absorption droplets. Using the opaque hemoglobin content of the nephron as a tracer, functional continuity of the system of the tubular invaginations with the lumen on one side and the vacuoles on the other was demonstrated. Mitochondria lie closely apposed to vacuoles and droplets, but are not primarily involved in droplet formation. 15 hours after injection and later, ferritin and systems of layered membranes become visible in the droplets as their density decreases. These membranes are interpreted as lipoprotein membranes; similar membranes are found in the lumen of the tubuli. It is suggested that phospholipids enter into the vacuoles together with hemoglobin from the tubular lumen and form membrane systems of lipoproteins in the droplets. At 3 to 4 days the droplets contain aggregates of ferritin, and the iron reaction becomes positive in the tubule cells. No significant changes were found in the Golgi apparatus or in the microbodies during hemoglobin absorption. At all time points investigated, the terminal bars seal the intercellular spaces against penetration by hemoglobin in the proximal and distal convolutions and in the collecting ducts.
THE FINE STRUCTURE OF THE NUCLEAR MATERIAL OF A BLUE-GREEN ALGA, ANABAENA CYLINDRICA LEMM
Authors: Hopwood David A. , Glauert Audrey M.
Published: May 2004

The chromatinic material of the blue-green alga Anabaena cylindrica has complex configurations in the central regions of the cells. The distribution of the chromatin within the cells varies in different filaments, probably in response to variations in the disposition of other cellular components. In electron micrographs of thin sections of organisms fixed by the method of Kellenberger, Ryter, and Séchaud (1958) the centroplasm contains fibrillar and possibly granular components which can be identified as the nuclear material by comparison with stained preparations viewed in the light microscope. The fibrils in the nuclear regions have diameters in the range of 5 to 7 mµ and are embedded in a matrix of lower density. The nuclear regions are not greatly different from the cytoplasm in their electron density. Reducing the calcium content of the fixative results in coagulation of the fibrils to form coarser structures. The significance of the observations is discussed in relation to observations on the fine structure of other classes of algae and of bacteria.
Ribonucleoprotein staining of centrioles and kinetochores in newt lung cell spindles.
Authors: Rieder C L
Published: May 2004

The distribution of ribonucleoprotein (RNP) within the mitotic spindle of newt lung epithelial cells was studied with the high voltage electron microscope (HVEM) using Bernhard's uranyl-EDTA-lead staining of thick sections in conjunction with the ribonuclease digestion of fixed cells. The results indicate that aside from ribosomes, the major RNP-containing components of the spindle are the kinetochores and centrioles, both of which stain electron-opaque after EDTA treatment. In both cases, the electron-opaque material associated with these microtubule organizing centers (MTOC's) can be removed by RNAse digestion and cold perchloric acid (PCA) extraction under conditions which leave the spindle microtubules (Mts) centrioles, and kinetochores intact. The staining reaction is not abolished by cold PCA extraction alone or by substituting other positively charged proteins (i.e., cytochrome c or lysozyme) for RNAse. The RNP component of the kinetochore is closely associated with the bases of the kinetochore microtubules. The RNP component of the centriole can be seen to surround the microtubules of the triplet blades. No evidence was found to indicate the presence of RNP in the pericentriolar material. The possible function of both kinetochore and centriolar RNP is discussed.
Fibronectin expression is determined by the genotype of the transformed parental cells in heterokaryons between normal and transformed fibroblasts.
Authors: Laurila P , Wartiovaara J , Stenman S
Published: May 2004

The expression of fibronectin, a cell surface-associated transformation-sensitive glycoprotein, was studied in hetero- and homokaryons of normal and SV40-transformed human fibroblasts. In immunofluorescence, fibroblast homokaryons had an intense surface-associated and intracelluar fibronectin fluorescence similar to that of normal fibroblasts. Transformed cells and their homokaryons had a minimal surface-associated and a weak intracellular fibronectin fluorescence. In heterokaryons formed between transformed and normal fibroblasts, the expression of fibronectin fell within 24 h to the level of the transformed cell homokaryons. The change was detectable already at 3 h after fusion and was gene-dose dependent. These results show that the transformed genotype determines fibronectin expression in the heterokaryons.
Membrane movements and fluidity during rotational motility of a termite flagellate. A freeze-fracture study.
Authors: Tamm S L
Published: May 2004

Freeze-fracture electron microscopy was used to examine the structure of a region of plasma membrane that undergoes continual, unidirectional shear. Membrane shear arises from the continual clockwise rotation of one part (head) of a termite flagellate relative to the rest of the cell. Freeze-fracture replicas show that the lipid bilayer is continuous across the shear zone. Thus, the relative movements of adjacent membrane regions are visible evidence of membrane fluidity. The distribution and density of intramembrane particles within the membrane of the shear zone is not different from that in other regions of the cell membrane. Also, an additional membrane shear zone arises when body membrane becomes closely applied to the rotating axostyle as cells change shape in vitro. This suggests that the entire membrane is potentially as fluid as the membrane between head and body but that this fluidity is only expressed at certain locations for geometrical and/or mechanical reasons. Membrane movements may be explained solely by cell shape and proximity to rotating structures, although specific membrane-cytoskeletal connections cannot be ruled out. The membrane of this cell may thus be viewed as a fluid which adheres to the underlying cytoplasm/cytoskeleton and passively follows its movements.
Copurification of actin and desmin from chicken smooth muscle and their copolymerization in vitro to intermediate filaments.
Authors: Hubbard B D , Lazarides E
Published: May 2004

Desmin is a 50,000-mol wt protein that is enriched along with 100-A filaments in chicken gizzard that has been extracted with 1 M KI. Although 1 M KI removes most of the actin from gizzard, a small fraction of this protein remains persistently insoluble, along with desmin. The solubility properties of this actin are the same as for desmin: they are both insoluble in high salt concentrations, but are solubilized at low pH or by agents that dissociate hydrophobic bonds. Desmin may be purified by repeated cycles of solubilization by 1 M acetic acid and subsequent precipitation by neutralization to pH 4. During this process, a constant nonstoichiometric ratio of actin to desmin is attained. Gel filtration on Ultrogel AcA34 in the presence of 0.5% Sarkosyl NL-97 reveals nonmonomeric fractions of actin and desmin that comigrate through the column. Gel filtration on Bio-Gel P300 in the presence of 1 M acetic acid reveals that the majority of desmin is monomeric under these conditions. A small fraction of desmin and all of the actin elute with the excluded volume. When the acetic acid is removed from actin-desmin solutions by dialysis, a gel forms that is composed of filaments with diameters of 120-140 A. These filaments react uniformly with both anti-actin and anti-desmin antiserum. These results suggest that desmin is the major subunit of the muscle 100-A filaments and that it may form nonstoichiometric complexes with actin.
alpha-Actinin localization in the junctional complex of intestinal epithelial cells.
Authors: Craig S W , Pardo J V
Published: May 2004

We have used antibody to chicken gizzard alpha-actinin to identify and localize this molecule in chicken intestinal epithelium. The antibody binds only to alpha-actinin when tested against a crude extract of chicken gizzard. Extracts of purified epithelial cells contain a molecule which has a subunit molecular weight of 100,000 on sodium dodecyl sulphate gels and which is able to inhibit the interaction of alpha-actinin antibody and 125I-labeled chicken gizzard alpha-actinin. By indirect immunofluorescence, alpha-actinin is localized in the apical portion of chicken intestinal epithelial cells. Ethanol-fixed cryostat sections of intestine taken through the apical portion of the epithelial cells and in a plane perpendicular to the long axis of the cells show that alpha-actinin is organized in a polygonal pattern which corresponds to the outlines of the polygonally packed epithelial cells. We interpret the data as indicating that alpha-actinin is a component of the tight junction (zonula occludens) and/or the belt desmosome (zonula adherens), both of which are membrane structures known to encircle the cell and to be confined to its apical portion.
The angular distribution of directional changes of guided 3T3 cells.
Authors: Albrecht-Buehler G
Published: May 2004

This paper reports that the angular distribution of directional change of 3T3 cells which followed guiding lines on a substrate and left the guidance at various angles shows a peculiar preference for angles between 30 degrees and 60 degrees. Regarding the phenomenon of cellular guidance itself, 3T3 cells faced with a choice between guiding lines toward different directions seem to explore various options before following one. This observation suggests that the outcome of a probing action and not substrate properties alone can explain the guidance behavior of 3T3 cells.
Distribution of cell surface saccharides on pancreatic cells
Authors: Maylie-Pfenninger M , Jamieson JD
Published: May 2004

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a "universal" affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.
Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.
Authors: Quaroni A , Wands J , Trelstad R L , Isselbacher K J
Published: May 2004

Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19--22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells.
Mechanisms of transcription in nucleoli of amphibian oocytes as visualized by high-resolution autoradiography.
Authors: Angelier N , Hemon D , Bouteille M
Published: May 2004

In oocytes of Pleurodeles waltlii, the method of Miller and Beatty has been combined with a method of high-resolution autoradiography especially suitable for the study of isolated molecules. In vitro labeling of RNA by tritiated precursors was carried out with increasing incubation times (1, 4, 15, 24, 48, and 72 h). Silver grains were present over ribonucleoprotein fibrils in amounts sufficient for quantitative analysis of nucleolar DNA transcription. Statistical analysis of the data revealed that: (a) The units of any one nucleolus exhibited a large degree of heterogeneity in their number of grains. (b) There was a parallelism between the increasing grain number and the ribonucleoprotein-fibril lengthening as observed along the transcription unit.
Cyclic 3',5' AMP relay in Dictyostelium discoideum. I. A technique to monitor responses to controlled stimuli.
Authors: Devreotes P N , Derstine P L , Steck T L
Published: May 2004

A perfusion technique was developed to deliver [14C]adenosine 3',5'-cyclic monophosphate (cAMP) stimuli of well-defined magnitude and duration to tritium-labeled Dictyostelium discoideum amoebae and simultaneously monitor the elicited secretion of [3H]cAMP (i.e., the relay response). The tritiated compounds secreted in response to [14C]cAMP stimuli were highly enriched in [3H]cAMP and reflected an increase in intracellular cAMP accompanying stimulation rather than the release of a preexisting store or bulk cellular contents. The secretory response (per 10(6) cells) to 2-min stimuli increased during differentiation from about 0.2 pmol at 0.5 h to approximately 5 pmol of cAMP at 7 h. Without adequate perfusion, amoebae altered the level of cAMP in their environment in two ways: phosphodiesterases destroyed cAMP stimuli under some conditions so as to attenuate the relay response; under other circumstances, secreted cAMP magnified minimal exogenous stimuli into maximal responses. Amoebae, furthermore, would respond to their basal secretion of cAMP autocatalytically if its removal or destruction were interrupted. The perfusion system minimized these cell-induced modifications, allowing control of the level of the stimulus and response in quantitative studies.
Cyclic 3',5' AMP relay in Dictyostelium discoideum. II. Requirements for the initiation and termination of the response.
Authors: Devreotes P N , Steck T L
Published: May 2004

The secretion of 3H-cyclic adenosine 3',5'-monophosphate (cAMP) by prelabeled and suitably differentiated Dictyostelium discoideum amoebae was elicited in a perfusion apparatus by 10(-10) to 10(-5) M [14C]cAMP stimuli of defined magnitude and duration. Exogenous stimuli evoked an immediate increase in the rate of [3H]cAMP secretion which accelerated continuously to reach a peak of up to 100 times the unstimulated rate after 2--3 min of stimulation. Withdrawal of the stimulus at any time during the response led to a rapid decline to basal levels. Furthermore, a spontaneous decline in secretion rate was observed during prolonged cAMP stimulation, with a return to basal levels after 3--8 min of stimulation. After the initial secretory event, cells did not respond further to the continued presence of external [14C]cAMP unless (a) it was interrupted by a brief recovery period or (b) the level of the stimulus was increased sufficiently. Since the second increment could follow the first at any time, continuous secretion of [3H]cAMP could be sustained for up to 30 min by progressively increasing the stimulus between 10(-10) and 10(-5) M cAMP. The total magnitude of spontaneously terminated responses depended on the size of the increment in applied cAMP, larger stimuli evoking both a more rapid acceleration and a slower deceleration in [3H]cAMP secretion rate. The integrated response to a given increment in stimulus level was apparently independent of its "shape" - i.e., the duration, magnitude, and number of sub-steps in the increment. These data support two mechanistic inferences: that amoebae respond in proportion to relative increases in extracellular cAMP concentration, but adapt to the concentration of cAMP itself. The data further suggest that the initiation and termination of the response are mediated by cellular component(s) beyond cAMP-occupied receptors.
The dynein electrophoretic bands in axonemes naturally lacking the inner or the outer arm.
Authors: Baccetti B , Burrini A G , Dallai R , Pallini V
Published: May 2004

Two unconventional sperm models (all motile) have been studied. The first one has only the outer arm on the doublets (the gall midge, Diplolaboncus); the second one, has only a well-developed inner arm (the eel, Anguilla). Both are devoid of central tubules and radial spokes. The gall midge sperm yields a single electrophoretic band migrating similarly to the sea urchin dynein band A; a major high-molecular-weight band is obtained from eel sperm which co-migrates with the sea urchin dynein band B. The present picture is consistent with the localization of dynein in the axoneme--namely, of an A-like band in the outer arm, and of the B band in the inner arm. Moreover, the D band is present only in the eel, where gamma-links are present. ATPase activity was localized histochemically and found to be associated with both inner and outer arms, as well as with the gamma-links.
Studies on the organization and localization of actin and myosin in neurons.
Authors: Kuczmarski E R , Rosenbaum J L
Published: May 2004

The organization of actin in mouse neuroblastoma and chicken dorsal root ganglion (DRG) nerve cells was investigated by means of a variety of electron microscope techniques. Microspikes of neuroblastoma cells contained bundles of 7- to 8-nm actin filaments which originated in the interior of the neurite. In the presence of high concentrations of Mg++ ion, filaments in these bundles became highly ordered to form paracrystals. Actin filaments, but not bundles, were observed in growth cones of DRG cells. Actin was localized in the cell body, neurites, and microspikes of both DRG and neuroblastoma nerve cells by fluorescein-labeled S1. Myosin was localized primarily in the neurites of chick DRG nerve cells with fluorescein-labeled anti-brain myosin antibody. This antibody also stained stress fibers in fibroblasts and myoblasts but did not stain muscle myofibrils.
Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study.
Authors: Bergeron J J , Sikstrom R , Hand A R , Posner B I
Published: May 2004

Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.
Quantitative conservation of chromatin-bound RNA polymerases I and II in mitosis. Implications for chromosome structure.
Authors: Matsui S I , Weinfeld H , Sandberg A A
Published: May 2004

RNA synthesis almost ceases in mitosis. It is ambiguous whether this temporal, negative control of RNA synthesis is solely because of the nature of chromosomes per se, (i.e., their condensed state), or to a physical loss of RNA polymerases along with other nuclear proteins which have been shown to pass into the cytoplasm in mitosis, or to their combined feature. Aside from such regulatory considerations, a question has also been raised as to whether RNA polymerases are constituents of metaphase chromosomes. To clarify these aspects of RNA polymerase-chromatin interaction in mitosis, the enzymes in chromosomes were quantitated and their levels compared to those in interphase nuclei and cells at various phases of the cell cycle. The results show that the amounts of form I, form II, and probably form III enzymes bound to a genome-equivalent of chromatin stay constant during the cell cycle. Thus, the mechanism for the negative control of RNA synthesis in mitosis appears to exist in the chromosomes per se, but not to be directly related to the RNA polymerase levels. This quantitative conservation of chromatin-bound RNA polymerases implies that they may persist as structural components of the chromosomes in mitosis.
Domains of receptor mobility and endocytosis in the membranes of neonatal human erythrocytes in the membranes of neonatal human erythrocytes and reticulocytes are deficient in spectrin
Authors: Tokuyasu KT , Schekman R , Singer SJ
Published: May 2004

It has previously shown (Schekman, R., and S.J. Singer, Proc. Natl. Acad. Sci. U.S.A. 73:4075-4079) that receptors in the membranes of neonatal human erythrocytes show a restricted degree of lateral mobility, whereas in adult human erythrocytes the receptors are essentially immobile. This restricted mobility is exhibited, for example, when concanavalin A (Con A) induces a limited clustering of its receptors in the neonatal erythrocyte membrane, resulting in the formation of invaginations and endocytic vesicles. This does not happen with adult cells. By the use of indirect immunoferritin labeling of ultrathin frozen sections of Con A-treated neonatal blood cells, we now show that the invaginations and endocytotic vesicles do not stain for spectrin, whereas the adjacent unperturbed membrane is heavily stained. The reticulocytes in the neonatal cell population undergo substantially more Con A-induced invagination and endocytosis than do the erythrocytes. These results lend strong support to the hypothesis that specialized discrete domains exist, or are induced, in the membranes of these neonatal cells, in which receptors are laterally mobile, whereas in the remaining (and predominant) part of the membrane the receptors are immobile. Such mobile domains are characterized by an absence of spectrin. During the maturation of the neonatal reticulocyte to erythrocyte, it is proposed that these domains are in large part, but not completely, eliminated.
Motility of the microtubular axostyle in Pyrsonympha.
Authors: Langford G M , Inoué S
Published: May 2004

The rhythmic movement of the microtubular axostyle in the termite flagellate, Pyrsonympha vertens, was analyzed with polarization and electron microscopy. The protozoan axostyle is birefringent as a result of the semi-crystalline alignment of approximately 2,000 microtubules. The birefringence of the organelle permits analysis of the beat pattern in vivo. Modifications of the beat pattern were achieved with visible and UV microbeam irradiation. The beating axostyle is helically twisted and has two principal movements, one resembling ciliary and the other flagellar beating. The anterior portion of the beating axostyle has effective and recovery phases with each beat thereby simulating the flexural motion of a beating cilium. Undulations develop from the flexural flipping motion of the anterior segment and travel along the axostyle like flagellar waves. The shape of the waves differs from that of flagellar waves, however, and are described as sawtooth waves. The propagating sawtooth waves contain a sharp bend, approximately 3 micron in length, made up of two opposing flexures followed by a straight helical segment approximately 23 micron long. The average wavelength is approximately 25 micron, and three to four sawtooth waves travel along the axostyle at one time. The bends are nearly planar and can travel in either direction along the axostyle with equal velocity. At temperatures between 5 degrees and 30 degrees C, one sees a proportionate increase or decrease in wave propagation velocity as the temperature is raised or lowered. Beating stops below 5 degrees C but will resume if the preparation is warmed. A microbeam of visible light shone on a small segment of the axostyle causes the typical sawtooth waves to transform into short sine-like waves that accumulate in the area irradiated. Waves entering the affected region appear to stimulate waves already accumulated there to move, and waves that emerge take on the normal sawtooth wave pattern. The effective wavelengths of visible light capable of modifying the wave pattern is in the blue region of the spectrum. The axostyle is severed when irradiated with an intense microbeam of UV light. Short segments of axostyle produced by severing it at two places with a UV microbeam can curl upon themselves into shapes resembling lockwashers. We propose that the sawtooth waves in the axostyle of P. vertens are generated by interrow cross-bridges which are active in the straight regions.
Electrophysiological recordings from spontaneously contracting reaggregates of cultured smooth muscle cells from guinea pig vas deferens.
Authors: McLean M J , Pelleg A , Sperelakis N
Published: May 2004

Smooth muscle cells were enzymatically dispersed from vasa deferentia of adult male guinea pigs (250-400 g). These cells reassociated in vitro to form monolayers and small spherical reaggregates (0.05-0.3 mm in Diam). Within 48 h of being placed in culture, cells in both types of preparation began to contract spontaneously. The contractions were rhythmic and slow. Cells in the monolayers stopped contracting after approximately 1 wk in vitro, but the reaggregates continued to contract spontaneously for at least 3 wk. Electron microscopy of the reaggregates revealed the presence of thick and thin myofilaments. Overshooting action potentials were recorded in many of the cells penetrated (primarily in reaggregates), and were accompanied by visible contractions of the aggregate or monolayer. Quiescent cells could often be excited by intracellularly applied depolarizing and hyperpolarizing (anodal-break) current pulses. The resting potentials had a mean value of -58 +/- 2 mV. The action potentials were usually preceded by a spontaneous depolarization. The action potentials had slow rates of rise (1--4 V/s) which were unaffected by tetrodotoxin (TTX, 1 microgram/ml), a known blocker of fast Na+ -channels. Verapamil (1 microgram/ml) blocked the action potentials. The mean value of input resistance was 6.9 +/- 0.5 M omega (n = 12). These electrophysiological properties are similar to those of intact adult vas deferens smooth muscle cells. Thus, the cultured adult vas deferens smooth muscle cells retain their functional properties in vitro even after long periods.
Effects of divalent cations on dynein cross bridging and ciliary microtubule sliding.
Authors: Zanetti N C , Mitchell D R , Warner F D
Published: May 2004

We recently demonstrated that addition of the divalent cation Mg++ to demembranated cilia causes the dynein arms to attach uniformly to the B subfibers. We have now studied the dose-dependent relationship between Mg++ or Ca++ and dynein bridging frequencies and microtubule sliding in cilia isolated from Tetrahymena. Both cations promote efficient dynein bridging. Mg++-induced bridges become saturated at 3 mM while Ca++-induced bridges become saturated at 2 mM. Double reciprocal plots of percent bridging vs. the cation concentration (0.05-10 mM) suggest that bridging occurs in simple equilibrium with the cation concentration. When microtubule sliding (spontaneous disintegration in 40 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), 0.1 mM ATP at pH 7.4) is assayed (A350 nm) relative to the Mg++ or Ca++ concentration, important differential effects are observed. 100% Disintegration occurs in 0.5-2 mM Mg++ and the addition of 10 mM Mg++ does not inhibit the response. The addition of 0.05-10 mM Ca++ to cilia reactivated with 0.1 mM ATP causes a substantial reduction in disintegration at low Ca++ concentrations and complete inhibition at concentrations greater than 3 mM. When Ca++ is added to cilia reactivated with 2 mM Mg++ and 0.1 mM ATP, the percent disintegration decreases progressively with the increasing Ca++ concentration. The addition of variable concentrations of Co++ to Mg++-activated cilia causes a similar but more effective inhibition of the disintegration response. These observations, when coupled with the relatively high concentrations of Ca++ or Co++ needed to inhibit disintegration, suggest that inhibition results from simple competition for the relevant cation-binding sites and thus may not be physiologically significant. The data do not yet reveal an interpretable relationship between percent disintegration, percent dynein bridging, and percent ATPase activity of both isolated dynein and whole cilia. However, they do illustrate that considerable (sliding) disintegration (60%) can occur under conditions that reveal only 10-15% attached dynein cross bridges.
Effect of fasting and feeding on synthesis and intracellular transport of proteins in the frog exocrine pancreas.
Authors: Slot J W , Strous G J , Geuze J J
Published: May 2004

Frog exocrine pancreatic tissue was studied in vitro under conditions which maintain the differences between tissues from fasted and fed animals. Sodium dodecyl sulfate (SDS) gel electrophoresis after labeling with [14C]amino acids showed that feeding stimulated the synthesis of secretory proteins to the same relative degree as the overall protein synthesis. The intracellular transport of secretory proteins was studied by electronmicroscopy autoradiography after pulse-labeling with [3H]leucine. It was found that the transport route is similar under both feeding conditions. After their synthesis in the rough endoplasmic reticulum (RER), the proteins move through the peripheral elements and cisternae of the Golgi system into the condensing vacuoles. The velocity of the transport increases considerably after feeding. When frogs are fasted, the release of labeled proteins from the RER takes greater than 90 min, whereas after feeding, this happens within 30 min. Comparable differences were observed for transport through the Golgi system. The apparent differences between the frog and mammalian pancreas in the regulation of synthesis, intracellular transport, and secretion of proteins are discussed.